ALBERT

Description: As previously reported (Colombat, Blood2001;97:101), rituximab (4 weekly doses of 375mg/m²) can lead to high response rates (RR) and prolonged remissions with minimal toxicity as 1st line therapy for low tumor burden FL. We report the final analysis of a trial evaluating long term efficacy and safety of rituximab in untreated low tumor burden FL (GELF criteria). 49 patients (pts) were included in the initial trial (median age 52 yrs), 2 refused consent for the extended F/Up period, and 1 pt died at M12. Molecular bcl2-JH rearrangement was assessed throughout the study. The median F/up was 83.8 mths. Overall best RR, complete/unconfirmed RR and partial RR at D78 were 74%, 50% and 24% respectively. Median PFS was 23.5 mths for the study population. Median duration of response (34 responders at D78, i.e 6 weeks after the last rituximab dose) was 28.6 mths, but response was still maintained without any further treatment in 11 pts after 5 years (24%) and in 7 pts after 7 years (15%). 31/46 pts were bcl2 positive in blood and/or marrow samples before rituximab: 11 (35%) became negative at D50, and 20 remained positive (65%). Median PFS was 37 mths for bcl2-negative pts at D50, and 12 mths for patients remaining positive (p=0.018 Log-rank). Of the 7 pts with sustained response after 7 years, 5 were bcl2 positive at D0, 2/5 became negative at D50, and 5/5 were still negative at M84. At year 7, 4/46 pts have died (1 from myelodysplasia, 3 from NHL), 35/42 have progressed, and 7 have never progressed without any other treatment than the initial rituximab therapy. Time to progression was significantly longer in the bcl2-negative population at D50 (p= 0.018, Log-rank). Duration of response was not correlated with bcl2 status at D50, but was associated with ‘Best response CR/Cru’ (p=0.007 Log-rank). Long-term tolerance was good, with only 13 SAE observed in 13 pts during the additional 4 years of F/Up (4 surgeries for non NHL-related pathologies, 1 node biopsy, 1 sleep apnea syndrome, 1 ischemic cardiopathy, 2 deaths from NHL, 1 depression, 1 pneumonia, 1 erysipela, 1 bronchitis). This long-term update confirms that a single 4-dose rituximab treatment yields durable benefits without the toxicity of chemotherapy for pts with low burden FL : Median PFS of 23.5 mths for the cohort, 28.6 mths for responders and 37 mths for pts turning bcl2-negative at D50, 15% of pts have maintained their response after 7 years, (2bis) the quality (CR/Cru) of the initial response was associated with a longer response duration high overall survival is observed with 4 deaths/46 pts (8.6%).

Description: Recent studies have demonstrated that in peripheral lymphoid tissues of normal mice and healthy humans, 1% to 5% of αβ T-cell receptor–positive (TCR+) T cells are CD4−CD8− (double-negative [DN]) T cells, capable of down-regulating immune responses. However, the origin and developmental pathway of DN T cells is still not clear. In this study, by monitoring CD4 expression during T-cell proliferation and differentiation, we identified a new differentiation pathway for the conversion of CD4+ T cells to DN regulatory T cells. We showed that the converted DN T cells retained a stable phenotype after restimulation and that furthermore, the disappearance of cell-surface CD4 molecules on converted DN T cells was a result of CD4 gene silencing. The converted DN T cells were resistant to activation-induced cell death (AICD) and expressed a unique set of cell-surface markers and gene profiles. These cells were highly potent in suppressing alloimmune responses both in vitro and in vivo in an antigen-specific manner. Perforin was highly expressed by the converted DN regulatory T cells and played a role in DN T-cell–mediated suppression. Our findings thus identify a new differentiation pathway for DN regulatory T cells and uncover a new intrinsic homeostatic mechanism that regulates the magnitude of immune responses. This pathway provides a novel, cell-based, therapeutic approach for preventing allograft rejection.

Description: The role of the intrinsic coagulation system on the risk of myocardial infarction is unclear. In the Study of Myocardial Infarctions Leiden (SMILE) that included 560 men younger than age 70 with a first myocardial infarction and 646 control subjects, we investigated the risk of myocardial infarction for levels of factor XI (factor XIc) and factor XII (factor XIIc). Furthermore, the risks for factor VIII activity (factor VIIIc) and factor IX activity (factor IXc) were assessed. Factor XIc was 113.0% in patients compared with 109.8% in control subjects (difference, 3.2%; 95% CI, 1.1%-5.4%). The risk of myocardial infarction adjusted for age for men in the highest quintile compared with those in the lowest quintile was 1.8-fold increased (ORadj, 1.8; 95% CI, 1.2-2.7). In contrast, factor XIIc among patients with myocardial infarction was lower than in control subjects, respectively, 93.0% and 98.6% (difference, 5.6%; 95% CI, 3.3%-7.9%). The odds ratio of myocardial infarction for men in the highest quintile versus those in the lowest quintile was 0.4 (ORadj, 0.4; 95% CI, 0.2-0.5). The highest risk was found among men with both high factor XIc and low factor XIIc (analyses in tertiles: ORadj, 6.4; 95% CI, 2.2-18.0). Factor VIIIc increased the risk of myocardial infarction although not dose dependently. Factor IXc increased the risk; odds ratio of myocardial infarction for men in the highest quintile versus those in the lowest quintile was 3.2 (ORadj, 3.2; 95% CI, 2.0-5.1). Thus, factors XIc and XIIc have opposite and synergistic effects on the risk of myocardial infarction in men; factor VIIIc and factor IXc increase the risk.

Description: Zinc deficiency is common in adult sickle-cell disease (SCD) patients, due to continued hemolysis and hyperzincuria. Growth retardation, hypogonadism, and immune dysfunctions due to zinc deficiency have been described in SCD patients. Our studies show that zinc has not only anti-inflammatory functions, but is also an antioxidant. We have previously shown that zinc supplementation to adult SCD patients decreased the incidences of infection and hospital admissions. We hypothesize that zinc supplementation improves T-helper cell and vascular endothelial cell activation, and decreases oxidative stress and NF-κB activation in SCD patients. To test this hypothesis, we recruited 36 ambulatory SCD (homozygous) patients (ages 18–47 years, 11 males and 7 females in each group) and randomly divided these into 2 groups. One group (n=18) received 25 mg zinc as acetate orally thrice a day for 3 months. The other group (n=18) received placebo. All these patients were free of pain crisis for 3 months and were not receiving hydroxyurea. The results indicate that zinc supplemented group had decreased incidence of infection in comparison to the placebo group (Chi square analysis: p=0.017). After 3 months of zinc supplementation, the plasma zinc level increased. The anti-oxidant power increased and the plasma levels of NO, lipid peroxidation products (MDA+HAE), DNA oxidation product (8-OHdG), and sVCAM-1 decreased in the zinc supplemented group, compared to the placebo group (p

Description: The incidence of follicular lymphoma (FL) in industrialized countries has been increasing since the 1950s. Polymorphisms in genes encoding key enzymes controlling folate-methionine metabolism, including methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS or MTR), serine hydroxymethyltransferase (SHMT), and thymidylate synthase (TS or TYMS), modify the risk of various cancers and possibly FL. This study specifically looks for an association between MTHFR, MTR, TYMS, and SHMT polymorphisms and the risk of FL. We carried out a case-control study with 172 patients diagnosed with FL and 206 control subjects. We report that the risk of FL was doubled by the association of one mutant allele at both MTHFR polymorphisms. Individuals with MTR 2756AA had 2-fold higher risk of FL, and subjects not having at least one TYMS 2R allele showed a 2-fold higher risk of FL. The MTR 2756AA genotype conferred a greater multivariate-adjusted relative risk of FL, and the risk was multiplied by almost 5 in the TYMS2R(-)/MTR 2756AA combination. In conclusion, common polymorphisms in key enzymes of the folate-methionine metabolism pathway result in an increased risk of FL and suggest that inadequate intake of dietary folate and other methyl donor nutrients may contribute to the development of this malignancy. (Blood. 2006;108:278-285)

Description: Leukostasis, a life-threatening complication of acute leukemia, occurs when leukemia cells obstruct the circulation of vital organs like the brain and lungs leading to intracranial hemorrhage or respiratory failure. Although the pathophysiology of leukostasis is poorly understood, an elevated concentration of circulating leukemia cells, pathologic adhesion, and decreased cell deformability are thought to play significant roles. Clinical deterioration can occur soon after chemotherapy is initiated, suggesting that chemotherapy itself may be a risk factor for leukostasis. To investigate the effects of chemotherapy on cell stiffness, we performed serial single cell deformability measurements with an atomic force microscope (AFM), a commonly used tool in nanoscience for imaging and characterizing mechanical properties of materials on a submicron level, and modified the AFM to operate in cell culture conditions at 37°C. Leukemia cells from patients with acute lymphoblastic leukemia and acute myeloid leukemia as well as leukemia cell lines were incubated with chemotherapeutic agents, and changes in cell stiffness were tracked over time with AFM as the cells underwent chemotherapy-induced cell death. In the presence of dexamethasone or daunorubicin, leukemia cells exhibited increases in stiffness by as much as two orders of magnitude. Cell stiffness appeared to increase before caspase activation and peaked after completion of cell death, and the rate at which cell stiffness increased was dependent on chemotherapy type. Stiffening with cell death was found to occur for all cell types and chemotherapies investigated and is due, at least in part, to dynamic changes in the actin cytoskeleton. This observed correlation between cell death and cell stiffening may partially explain why some leukemia patients develop leukostasis shortly after starting chemotherapy, and it suggests that leukocytoreduction should remain an important treatment for hyperleukocytosis in acute leukemia. Figure 1. Average apparent stiffness of dead (dark gray) leukemic cells exposed to chemotherapy is significantly higher compared to untreated (light gray) cells (n 〉 15, p 〈 0.05 for all comparisons of dead/untreated populations). (A) Primary ALL cells and lymphoid leukemic cell lines exposed to 1 μM dexamethasone (B) Primary AML and myeloid leukemic cell lines exposed to 1μM daunorubicin. Error bars are standard error. Figure 1. Average apparent stiffness of dead (dark gray) leukemic cells exposed to chemotherapy is significantly higher compared to untreated (light gray) cells (n 〉 15, p 〈 0.05 for all comparisons of dead/untreated populations). (A) Primary ALL cells and lymphoid leukemic cell lines exposed to 1 μM dexamethasone (B) Primary AML and myeloid leukemic cell lines exposed to 1μM daunorubicin. Error bars are standard error. Figure 2. Apparent stiffness of leukemic cells increases with progression of cell death. (A) A typical stiffness trace of a single M5 AML cell exposed to 1μM daunorubicin (circles). The apparent stiffness of a typical control cell remains relatively constant (triangles) and does not undergo apoptosis or cell death during the course of the experiment. Transition from open to filled shapres represents onset of cell death. Early apoptosis is defined as caspase 3 or 7 postivie staining and late apoptosis/dead is defined as Sytox Green (marker for cell membrane integrity loss) positive staining. (B) From the same patient sample, the average apparent stiffness of a population of late apoptotic/dead AML cells was significantly stiffer than early apoptopic cells and controls (n = 15, p〈 0.05). Error bars are standard error. Figure 2. Apparent stiffness of leukemic cells increases with progression of cell death. (A) A typical stiffness trace of a single M5 AML cell exposed to 1μM daunorubicin (circles). The apparent stiffness of a typical control cell remains relatively constant (triangles) and does not undergo apoptosis or cell death during the course of the experiment. Transition from open to filled shapres represents onset of cell death. Early apoptosis is defined as caspase 3 or 7 postivie staining and late apoptosis/dead is defined as Sytox Green (marker for cell membrane integrity loss) positive staining. (B) From the same patient sample, the average apparent stiffness of a population of late apoptotic/dead AML cells was significantly stiffer than early apoptopic cells and controls (n = 15, p〈 0.05). Error bars are standard error.

Description: FTY720, a potent immunomodulatory drug in phase 2/3 clinical trials, induces rapid and reversible sequestration of lymphocytes into secondary lymphoid organs, thereby preventing their migration to sites of inflammation. As prerequisite for its function, phosphorylation of FTY720 to yield a potent agonist of the sphingosine-1-phosphate receptor S1P1 is required in vivo, catalyzed by an as-yet-unknown kinase. Here, we report on the generation of sphingosine kinase 2 (SPHK2) knockout mice and demonstrate that this enzyme is essential for FTY720 phosphate formation in vivo. Consequently, administration of FTY720 does not induce lymphopenia in SPHK2-deficient mice. After direct dosage of FTY720 phosphate, lymphopenia is only transient in this strain, indicating that SPHK2 is constantly required to maintain FTY720 phosphate levels in vivo.

Description: The incorporation of blood-borne forms of tissue factor (TF) into a growing blood clot is necessary for normal fibrin generation and stabilization of the blood clot. Tissue factor pathway inhibitor (TFPI) is the primary physiologic inhibitor of tissue factor and is present within platelets. Expression of TFPI on the platelet surface may be the optimal location for it to abrogate blood-borne TF activity that incorporates within the blood clot, balancing the need for adequate hemostasis while preventing development of occlusive thrombosis. TFPI is produced by megakaryocytes but is not expressed on the platelet surface. Activation of platelets with thrombin receptor activation peptide does not cause release or surface expression of TFPI, demonstrating that TFPI is not stored within platelet α granules. TFPI is expressed on the platelet surface following dual-agonist activation with convulxin plus thrombin to produce coated platelets. In association with its expression on the surface of coated platelets TFPI is also released in microvesicles or as a soluble protein.

Description: Following cord blood (CB) transplant and bone marrow (BM) protracted thrombocytopenia remains a serious clinical problem. Platelet production following transplant depends on the availability of adequate numbers of cytokine responsive stem and megakaryocyte progenitor cells (MK-p). Thrombopoietin (TPO), had no clinical impact on thrombopoiesis when given to patients post BMT due to the paucity of MK-p in the grafts. If expanded, Mk-p would supply the appropriate target cells to maximize the effect of TPO and provide efficient earlier platelet engraftment. We propose a novel strategy to facilitate thrombopoiesis, by expanding MK-p from CB mononuclear cells (MNC) prior to transplantation in short term cultures. While CB CD34+ cells can be expanded by several reported methods, isolation of CD34+ cells from the fresh CB is not practical due to the limited number of stem and progenitor cells in the CB units. Additionally, MK expansion from purified stem cells requires long culture periods which are inappropriate for transplantation. We aimed to improved techniques for enrichment and ex-vivo expansion of MK-p and hematopoietic stem cells, from small aliquots of whole CB, using 7–10 days cultures and new growth conditions. CB progenitors were enriched by separation of MNC from RBC on gelatin followed by centrifugation on ficoll, as we previously reported (1). MNC were expanded on fibronectin (FN) coated dishes in the presence of autologous plasma with various new cytokine combinations. These included r-hu-TPO (10 ng/ml), b- FGF (10 ng/ml), r-hu-SCF (50 ng/ml) and ARP a peptide derived from the stress variant of acetylcholinesterase (AChE-R) recently discovered to have potent hematopoietic stem cell and MK growth factor activity (2). The cell populations, MK and MK-p were characterized by high resolution flow cytometry on day 0 and 10 of culture using SSC, CD41 and CD34. True MK expansion was assessed by appropriate gating out of granulocyte and monocytes, which acquire CD41+ adherent platelets in culture. FN alone, without any other growth supplement increased the viability of cells in culture and expansion of MK-p (CD41high, SSClow and FSClow) by 2.8±1.1 (P 〈 0.05) fold. The combination of FN with TPO enhanced MK-p number by 4.8±2.7 and the addition of either SCF or b-FGF or ARP further stimulated the expansion of MK-p all producing about a 6 fold increase (P 〈 0.05). Further analysis was performed on the maturing MKs which were characterized as CD41high, CD45low/negative, CD34negative. Increased Mk ploidy was found when either b-FGF or ARP were added to cultures containing TPO, grown on FN coated plates. Significant MK maturation, as measured by GPIIb/IIIa expression using real time quantitative PCR, was also found. The combination of FN and TPO increased the MK colony forming progenitors in culture by 9 fold and up to 35 fold when other supplements were added. We demonstrate that short term expansion of enriched MK-p from a small fraction of the CB unit is feasible and easy to perform and can comply with GTP regulations. This approach may lead to the development of more effective cell therapy modalities to facilitate platelet production and decrease the time of thrombocytopenia in severely myelosuppressed patients during the extended nadir before platelet engraftment.

Description: Natural interferon (IFN)-producing cells are the primary cell type responsible for production of type I IFN in response to viruses. Herein we report the identification of the first molecular marker of mouse natural interferon-producing cells (IPCs), a novel member of the sialic acid-binding immunoglobulin (Ig)-like lectin (Siglec) family termed Siglec-H. Siglec-H is expressed exclusively on IPCs and is unique among Siglec proteins in that it associates with the adaptor protein DAP12. Moreover, we show that DAP12 modulates the type I IFN response of IPCs to a Toll-like receptor 9 (TLR9) agonist. This observation explains our previous finding that stimulation of IPCs with 440c, a Siglec-H-specific antibody, reduces IPC secretion of type I IFN. Moreover, it supports a model in which engagement of DNAX-activation protein 12 (DAP12)-associated receptors with antibodies or low avidity endogenous ligands interferes with TLR-mediated cellular activation. (Blood. 2006;107:2474-2476)

Description: Background: Platelet transfusion practice in neonates is not evidence-based and there is a lack of data relating transfusion to clinical outcome. To inform the design of clinical trials, we conducted a prospective multicentre observational study of platelet transfusion in thombocytopenic neonates to describe: transfusion practice, including reason for transfusion; clinically-related outcomes, including minor and major bleeding and mortality. Methods: Neonates with platelets

Description: Stem cell gene therapy has long been limited by low gene transfer efficiency to hematopoietic stem cells. Recent years have witnessed clinical success in select diseases such as X-linked severe combined immunodeficiency (SCID) and ADA deficiency. Arguably, the single most important factor responsible for the increased efficacy of these recent protocols is the fact that the genetic correction provided a selective in vivo survival advantage. Since, for most diseases, there will be no selective advantage of gene-corrected cells, there has been a significant effort to arm vectors with a survival advantage. Two-gene vectors can be used to introduce the therapeutic gene and a selectable marker gene. Efficient in vivo selection strategies have been demonstrated in clinically relevant large-animal models. Mutant forms of the DNA repair-enzyme methylguanine methyltransferase in particular have allowed for efficient in vivo selection and have achieved sustained marking with virtually 100% gene-modified cells in large animals, and with clinically acceptable toxicity. Translation of these strategies to the clinical setting is imminent. Here, we review how in vivo selection strategies can be used to make stem cell gene therapy applicable to the treatment of a wider scope of genetic diseases and patients.

Description: Rapid loss of blood, in the operating room or trauma, necessitates a need for hastening coagulation Attempts to hasten coagulation include electrocautery based on thermal plasma discharges. Although there have been other effective attempts to prevent further loss of blood via coagulation, tissue damage and dessication can occur as a result of the high temperatures 2. Our group has developed a method to initiate rapid coagulation with dielectric plasma discharge (cold). Initial experiments were performed using fresh blood from volunteers to compare time for coagulation of whole blood exposed to plasma, one minute versus 10 minutes. We tried same experiments on cut cadaver organs such as spleen and placenta which showed evidence of rapid coagulation compared to control without evidence of tissue damage. Our research team has developed a novel method using non-thermal dielectric barrier discharge plasma (DBD plasma) to coagulate blood and sterilize tissues without causing thermal damage. This treatment would be safe to patients because no exposed electrodes are involved and high frequency current (under 10 KHz) is kept below mili-ampere. Our experiments have shown that such plasma treatment hastens blood coagulation and causes simultaneous wound sterilization via a large concentration of chemically active species in plasma that are ions, radicals (O, OH, N) and electronically-excited atoms and molecules. A kinetic model of blood coagulation under influence of DBD plasma was constructed. The model assumes contact flux of positive ions from DBD plasma into the surface of the blood being treated. Once at the surface, these ions recombine, leading to formation of aqueous Hydrogen ions which catalyze the release of Calcium ions into the blood. The addition of Calcium ions to blood speeds up the coagulation process proportionally to the amount of ions added. The model demonstrates thrombin formation in the presence of DBD plasma peaking and occurring within significantly less time compared to thrombin formation without DBD plasma3. Such medically relevant demonstrations and mathematical explanations have allowed us to develop a portable device that may prove useful in situations where control of bleeding is crucial. In addition, because of the potential for simultaneous sterilization, this device may also help to decrease infections. This pioneering technology will find applicability in many clinical situations: sterilization of human tissue surfaces prior to surgery and sterilization of catheters, a well-known cause of morbidity in hospitals.

Description: Hematopoietic stem cells (HSCs) arise, self-renew, or give rise to all hematopoietic lineages through the effects of transcription factors activated by signaling cascades. Lyl-1 encodes a transcription factor containing a basic helix-hoop-helix (bHLH) motif closely related to scl/tal, which controls numerous decisions in embryonic and adult hematopoiesis. We report here that Lyl-1 null mice are viable and display normal blood cell counts, except for a reduced number of B cells resulting from a partial block after the pro-B stage. Nevertheless, the deletion of Lyl-1 results in a diminution in the frequency of immature progenitors (Lin–, CD34–, sca-1+, c-kit+ [LSK], and LSK-side population [LSK-SP]) and in S12 colony-forming unit (CFU-S12) and long-term culture-initiating cell (LTC-IC) content in embryonic day 14 fetal liver (E14 FL) and adult bone marrow (BM). More important, Lyl-1–/– E14 FL cells and BM are severely impaired in their competitive reconstituting abilities, especially with respect to B and T lineage reconstitution. Thus, ablation of Lyl-1 quantitatively and functionally affects HSCs, a cell population that transcribes Lyl-1 more actively than their differentiated progenies. Our results demonstrate for the first time that Lyl-1 functions are important for HSC properties and B-cell differentiation and that they are largely distinct from scl functions.

Description: The Stroke Prevention Trial in Sickle Cell Anemia (STOP) was a randomized trial to evaluate whether chronic transfusion could prevent initial stroke in children with sickle-cell anemia at high risk as determined by transcranial Doppler (TCD). The trial demonstrated a large benefit of transfusion and was halted early. After termination of the trial, patients participated in a posttrial follow-up study. More patients in the transfusion group (70%) elected transfusion for primary stroke prevention compared with those on standard care (45%). Six patients with persistently abnormal TCD results developed stroke. A minority with initially abnormal TCD results remained stroke-free without transfusion. Except for lower baseline and follow-up TCD velocities compared with those with stroke, no predictive features of this apparent lower-risk subgroup could be determined. TCD results at last testing in 108 patients that did not have stroke were: normal (44.4%), conditional (26.9%), abnormal (22.2%), and inadequate (6.5%). Patients on transfusion were more likely to have normal TCD results. Transfusion resulted in iron overload and alloimmunization, but no infection. The study provides new information on acceptance rates and long-term effects of transfusion. Persistent TCD elevation signals ongoing stroke risk. Reduction in TCD results over time without transfusion is observed in some patients and requires further study.

Description: Recent advances have increased the purity of hematopoietic stem cells (HSCs) isolated from young mouse bone marrow. However, little attention has been paid to the purity of HSCs from other contexts. Although Thy-1lowSca-1+Lineage-c-kit+ cells from young bone marrow are highly enriched for HSCs (1 in 5 cells gives long-term multilineage reconstitution after transplantation into irradiated mice), the same population from old, reconstituted, or cytokine-mobilized mice engrafts much less efficiently (1 in 78 to 1 in 185 cells gives long-term multilineage reconstitution). To test whether we could increase the purity of HSCs isolated from these contexts, we examined the SLAM family markers CD150 and CD48. All detectable HSCs from old, reconstituted, and cyclophosphamide/G-CSF-mobilized mice were CD150+CD48-, just as in normal young bone marrow. Thy-1lowSca-1+Lineage-c-kit+ cells from old, reconstituted, or mobilized mice included mainly CD48+ and/or CD150- cells that lacked reconstituting ability. CD150+CD48-Sca-1+Lineage-c-kit+ cells from old, reconstituted, or mobilized mice were much more highly enriched for HSCs, with 1 in 3 to 1 in 7 cells giving long-term multilineage reconstitution. SLAM family receptor expression is conserved among HSCs from diverse contexts, and HSCs from old, reconstituted, and mobilized mice engraft relatively efficiently after transplantation when contaminating cells are eliminated.

Description: A PRoliferation-Inducing TNF Ligand (APRIL) costimulates B-cell activation. When overexpressed in mice, APRIL induces B-cell neoplasia, reminiscent of human B-cell chronic lymphoid leukemia (B-CLL). We analyzed APRIL expression in situ in human non-Hodgkin lymphomas. APRIL up-regulation was only observed in high-grade B-cell lymphomas, diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma (BL). Up-regulation was seen in 46% and 20% of DLBCL and BL, respectively. In DLBCL, neutrophils, constitutively producing APRIL and infiltrating the tumor tissue, were the main cellular source of APRIL. Rare DLBCL cases showed a predominance of histiocytes or mesenchymal cells as APRIL source. APRIL secreted by neutrophils accumulated on tumor cells via proteoglycan binding. In addition to proteoglycans, DLBCL tumor cells expressed the APRIL signaling receptor, TACI and/or BCMA, indicating that these tumor cells are fully equipped to respond to APRIL. A retrospective clinical analysis revealed a significant correlation between high expression of APRIL in tumor lesions and decreased overall patient survival rate. Hence, APRIL produced by inflammatory cells infiltrating lymphoma lesions may increase tumor aggressiveness and affect disease outcome.

Description: We evaluated the ability of 2 human mAbs directed against TRAILR1 (HGS-ETR1) and TRAILR2 (HGS-ETR2) to kill human myeloma cells. HGS-ETR1 and HGS-ETR2 mAbs killed 15 and 9 human myeloma cell lines (HMCLs; n = 22), respectively. IL-6, the major survival and growth factor for these HMCLs, did not prevent their killing. Killing induced by either HGS-ETR1 or HGS-ETR2 was correlated with the cleavage of Mcl-1L, a major molecule for myeloma survival. Mcl-1L cleavage and anti-TRAILR HMCL killing were dependent on caspase activation. Kinetic studies showed that Mcl-1L cleavage occurred very early (less than 1 hour) and became drastic once caspase 3 was activated. Our data showed that both the extrinsic (caspase 8, Bid) and the intrinsic (caspase 9) pathways are activated by anti–TRAIL mAb. Finally, we showed that the HGS-ETR1 and, to a lesser extent, the HGS-ETR2 mAbs were able to induce the killing of primary myeloma cells. Of note, HGS-ETR1 mAb was able to induce the death of medullary and extramedullary myeloma cells collected from patients at relapse. Taken together, our data clearly encourage clinical trials of anti–TRAILR1 mAb in multiple myeloma, especially for patients whose disease is in relapse, at the time of drug resistance.

Description: The purpose of this study is to compare our standard chemotherapy regimen (CHVP [cyclophosphamide, doxorubicin, teniposide, and prednisone]) plus interferon with 4 courses of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) followed by high-dose therapy with autologous stem cell transplantation (ASCT) in treatment-naive patients with advanced follicular lymphoma. Four hundred one patients were included from July 1994 to March 2001: 209 received 12 cycles of CHVP plus interferon α for 18 months (CHVP-I arm) and 192 received 4 cycles of CHOP followed by high-dose therapy (HDT) with total body irradiation and ASCT (CHOP-HDT arm). Overall response rates were similar in both groups (79% and 78% after induction chemotherapy, respectively). One hundred thirty-one of the 150 patients eligible for HDT underwent transplantation (87%). Intent-to-treat analysis after a median follow-up of 7.5 years showed that there was no difference between the 2 arms for overall survival (P = .53) or event-free survival (P = .11). Patients with a complete response at the end of the induction therapy had a statistically longer event-free survival and overall survival (P = .02 and 〈 .001, respectively). After long-term follow-up, our study showed that there was no statistically significant benefit in favor of first-line high-dose therapy in patients with follicular lymphoma. High-dose therapy should be reserved for relapsing patients.

Description: Dendritic cells (DCs) are recognized as the most potent antigen-presenting cells of the immune system with the unique ability to initiate and maintain primary immune responses. In order to better characterize the functional and phenotypic features of DCs, a subtractive cDNA library to identify differentially expressed genes in monocyte-derived DCs (MDCs) was constructed. Using this approach, we found that the epithelial transcription factor ESE-3, which was previously shown to be exclusively expressed in cells of epithelial origin, is differentially expressed in MDCs. This was further confirmed by reverse transcriptase–polymerase chain reaction (RT-PCR) and Western blot analyses. The expression of ESE-3 is up-regulated upon maturation of MDCs and inhibited by treating the cells with IL-10 or IFN-γ. Knockdown experiments using siRNA suggest that ESE-3 plays an important role during MDC development. Our results might help to improve the phenotypic characterization of DCs and lead to a better understanding of the cellular mechanisms involved in antigen presentation and T-cell stimulation.

Description: To prospectively assess the applicability of reduced-intensity conditioning hematopoietic stem cell transplantation (RIC-HSCT), we wrote a protocol in which all untreated patients 50 years or older with acute myeloid leukemia (AML) and unfavorable cytogenetics would be evaluated during induction for a possible RIC-HSCT in first complete remission (CR1). Ninety-nine of 259 patients entered CR. Fifty-three of the 99 were seen by the Transplant Service with the remainder not seen because of illness, lack/unavailability of siblings, refusal, or, primarily, unclear reasons (21 patients). A donor was identified for 26 patients (21 sibling, 5 unrelated) with RIC-HSCT performed in 14 (13 sibling). Results in consulted patients suggested that 50% or fewer of the 85 patients who did not undergo transplantation were potential transplant candidates. We attempted to find one or more chemotherapy pair-mates for each patient who underwent transplantation based on cytogenetics, age, and a relapse-free survival (RFS) time that was more than or equal to the time from CR1 to RIC-HSCT in the patient who underwent transplantation. Thirty-two of the 39 matches favored (longer RFS) RIC-HSCT and 7, chemotherapy. The probability that the corresponding beta distribution was different than expected (ie, that RIC-HSCT was superior) was 0.99 (P = .004). Results were similar with respect to survival. While RIC-HSCT thus seems of interest, methods are needed to extend its applicability.

Description: Mantle cell lymphoma (MCL) is associated with poor clinical outcome among all the B-cell malignancies. MCL cells have a characteristic phenotype including IgM+, IgD+, CD5+, CD10−, CD19+, CD20+, Bcl2+, CD23−, CD24+, and have a (11:14) (q13:q32) translocation, involving the Bcl-1 locus and the IgH heavy chain joining region, resulting in up-regulation of Bcl-1/PRAD-1 with increased Cyclin D1 expression. In spite of our understanding of B cell development, we do not have effective therapeutic approach to treat MCL. This warrants an immediate understanding of the biology MCL cells, and key signaling pathways underlying the disease progression. Further, these key signaling pathways involved in MCL could be specifically targeted, which may have potential therapeutic value. Emerging evidences have revealed that sonic hedgehog (Shh) signaling promotes tumor cell proliferation, and protects them from apoptosis in different cancers such as pancreatic cancer, prostate cancer, gastric cancer, medulloblastoma, basal cell carcinoma, breast cancer, etc. In addition, targeting the Shh signaling has been shown as a potential therapeutic approach to treat some of these cancers. However, the role of Shh signaling has not been reported in any of B cell malignancies including MCL. Therefore, we studied the status of Shh signaling molecules in the proliferation and survival of MCL cells in vitro using JVM-2, Granta-519, Jeko-1 and Z138 cell lines and human primary MCL cells. Our results demonstrate that the molecules involved in the Shh signaling like patched and smoothened receptors, and the target transcription factors, GLI were over expressed in Granta-519, Jeko-1, Z-138 and JVM-2 MCL cell lines, and patients primary MCL cells compared to normal human B lymphocytes. Addition of exogenous Shh increased the proliferation of JVM-2 cells in vitro. There was an increased transcripts level of GLI1, BCL2 and Cyclin D1 (CCND1) in JVM-2 cells following the addition of Shh. Addition of Shh-signaling inhibitor Cyclopamine abrogated the increased proliferation and transcription of the above genes induced by Shh in JVM-2 cells (figure 1). Furthermore, the Gli2 anti-sense oligonucleotide decreased CCND1 and BCL2 transcript expression, as well as decreased the proliferation of JVM2 cells in vitro (figure 2). Taken together, these results suggest that Shh-Gli signaling may be one of the pathways that regulate the Bcl-2 and Cyclin D1 activation and thereby regulate the proliferation and apoptosis of MCL cells. Therefore, further dissecting of the Shh-Gli mediated regulation of Bcl2 signaling may have potential implications in advancing the treatment for MCL. Figure 1: Figure 1:. Figure 2: Figure 2:.

Description: Deletions and/or mutations of p53 are relatively rare and late events in the natural history of B-cell chronic lymphocytic leukemia (B-CLL). However, it is unknown whether p53 signaling is functional in B-CLL and if targeted nongenotoxic activation of the p53 pathway by using nutlin-3, a small molecule inhibitor of the p53/MDM2 interaction, is sufficient to kill B-CLL cells. In vitro treatment with nutlin-3 induced a significant cytotoxicity on primary CD19+ B-CLL cells, but not on normal CD19+ B lymphocytes, peripheral-blood mononuclear cells, or bone marrow hematopoietic progenitors. Among 29 B-CLL samples examined, only one was resistant to nutlin-3–mediated cytotoxicity. The induction of p53 by nutlin-3 in B-CLL samples was accompanied by alterations of the mitochondrial potential and activation of the caspase-dependent apoptotic pathway. Among several genes related to the p53 pathway, nutlin-3 up-regulated the steady-state mRNA levels of PCNA, CDKN1A/p21, GDF15, TNFRSF10B/TRAIL-R2, TP53I3/PIG3, and GADD45. This profile of gene activation showed a partial overlapping with that induced by the genotoxic drug fludarabine. Moreover, nutlin-3 synergized with both fludarabine and chlorambucil in inducing B-CLL apoptosis. Our data strongly suggest that nutlin-3 should be further investigated for clinical applications in the treatment of B-CLL.

Description: Investigators in the United Kingdom have shown that hereditary amyloidosis can be misdiagnosed as Ig light-chain (AL) amyloidosis because family history is an ineffective screen, and tissue staining used to type amyloid is unreliable. Misdiagnosis of AL can lead to inappropriate use of chemotherapy and failure to diagnose a hereditary disease. Over a 3-year period we sought to determine how often both possible sources of amyloidosis occurred in the same patient. We employed an algorithm based on established data and patterns of amyloidosis in order to focus the screening effort. Of 178 consecutive patients referred for amyloidosis, 54 were screened by polymerase chain reaction techniques with primers designed to detect transthyretin, apolipoprotein AI, apolipoprotein AII, fibrinogen Aα, and lysozyme variants. Three patients (6% of those screened and 2% of symptomatic patients) had both a monoclonal gammopathy and a hereditary variant. These results justify further study of screening for hereditary variants in patients with apparent AL, and highlight the need for practical techniques for identifying fibrils extracted from tissue.

Description: Essential thrombocythemia (ET) is heterogeneous with respect to natural history, X-chromosome inactivation patterns (XCIPs), and presence of the V617F mutation in Janus kinase 2 (JAK2). We studied 111 patients with ET; 39% were JAK2 mutant positive, and clone size (percentage mutant JAK2) was concordant with XCIP when constitutive T-cell patterns were taken into account. JAK2 mutant clones were present in both clonal and polyclonal cases as determined by XCIP, and the former had higher mutant JAK2 levels (median 26% versus 16%; P = .001). No change was observed in serial XCIP analysis of 14 polyclonal patients over a median follow-up of 61 months. Furthermore, 18 of 19 mutant-positive patients showed no significant change in mutant JAK2 level over a median follow-up of 47 months. These results suggest that, in many cases of ET, a small stable clone containing a JAK2 mutation can be maintained as a subpopulation for many years.

Description: The suppressive capacity of naturally occurring mouse CD4+CD25+ T cells on T-cell activation has been well documented. The present study is focused on the interaction of CD4+CD25+ T cells and B cells. By coculturing preactivated CD4+CD25+ T cells with B cells in the presence of polyclonal B-cell activators, we found that B-cell proliferation was significantly suppressed. The suppression of B-cell proliferation was due to increased cell death caused by the CD4+CD25+ T cells in a cell-contact–dependent manner. The induction of B-cell death is not mediated by Fas–Fas ligand pathway, but surprisingly, depends on the up-regulation of perforin and granzymes in the CD4+CD25+ T cells. Furthermore, activated CD4+CD25+ T cells preferentially killed antigen-presenting but not bystander B cells. Our results demonstrate that CD4+CD25+ T cells can act directly on B cells and suggest that the prevention of autoimmunity by CD4+CD25+ T cells can be explained, at least in part, by the direct regulation of B-cell function.

Description: Design/methods: Rituximab added to 8 cycles of CVP (R-CVP) chemotherapy improves time to progression and duration of response in previously untreated patients with stage III/IV CD20 positive follicular NHL compared with CVP alone (Marcus et al Blood2005;105:1417–23). A protocol pre-planned analysis of this study with a median follow-up of 53 months has now been performed. Results: A total of 321 patients (median age 53 years) were recruited. Eighty-three percent of patients in both arms had intermediate to high-risk disease according to the Follicular Lymphoma International Prognostic Index (FLIPI, score 2–5). Median time to progression or death (TTP) in the R-CVP arm was 34 months compared with 15 months in the CVP arm, p

Description: SGN-30 is a monoclonal antibody directed against the CD30 antigen expressed on some hematologic malignancies. Based on encouraging phase I data, a multicenter phase II study was conducted treating patients with refractory or recurrent CD30-positive ALCL with an ECOG performance status of ≤ 2. Thirty-nine patients (24M, 15F) with ALCL were enrolled, with a median age of 57 (range 23–82) and a median of 3 prior therapies (range 2–5). Nine patients had previously received a stem cell transplant. Eighty-five percent of tumors were negative for ALK, a poor prognostic factor. SGN-30 was administred at 6 mg/kg/wk (90 minute infusion, premedications were not required) for 6 consecutive weeks. After 24 patients were enrolled, the dose was escalated to 12 mg/kg/wk in subsequent patients. (Patients with stable disease or objective response were eligible to receive additional cycles of SGN-30. Five patients received ≥ 2 cycles of SGN-30.) Response assessments, as determined by CT scans, were performed 2 weeks after the last infusion. Best response is shown below: CR PR SD PD Pending Eval ORR *Both CRs have ongoing durations of 〉365 days; both patients received additional cycles of SGN-30. **PRs had durations of 27, 53, 139 and 167 days; two additional patients have ongoing durations of 86+ and 25+ days. ***Three SDs have ongoing durations of 96+, 365+, and 365+ days. Two additional patients had SD for 71 and 174 days. 2* 6** 5*** 24 2 21% Three drug-related toxicities ≥ Grade 3 were reported (each was considered possibly related to SGN-30): 1) lymphopenia, 2) catheter related infection and 3) urticaria. No other significant hematologic or biochemical toxicities have been observed. There was one definitely related serious adverse event (Grade 2) in a patient who experienced a transient exacerbation of his cutaneous lesions after 2 doses of SGN-30 but achieved a partial response after continuing on study. This phase II study represents one of the largest prospectively designed trials in relapsed/refractory ALCL and demonstrates good tolerability and clinically meaningful antitumor activity of SGN-30, especially in ALK negative patients who have a particularly poor prognosis.

Description: Adaptive immunity is triggered at the immune synapse, where peptide-major histocompatibility complexes and costimulatory molecules expressed by dendritic cells (DCs) are physically presented to T cells. Here we describe transmission of the inflammatory monoamine serotonin (5-hydroxytryptamine [5-HT]) between these cells. DCs take up 5-HT from the microenvironment and from activated T cells (that synthesize 5-HT) and this uptake is inhibited by the antidepressant, fluoxetine. Expression of 5-HT transporters (SERTs) is regulated by DC maturation, exposure to microbial stimuli, and physical interactions with T cells. Significantly, 5-HT sequestered by DCs is stored within LAMP-1+ vesicles and subsequently released via Ca2+-dependent exocytosis, which was confirmed by amperometric recordings. In turn, extracellular 5-HT can reduce T-cell levels of cAMP, a modulator of T-cell activation. Thus, through the uptake of 5-HT at sites of inflammation, and from activated T cells, DCs may shuttle 5-HT to naive T cells and thereby modulate T-cell proliferation and differentiation. These data constitute the first direct measurement of triggered exocytosis by DCs and reveal a new and rapid type of signaling that may be optimized by the intimate synaptic environment between DCs and T cells. Moreover, these results highlight an important role for 5-HT signaling in immune function and the potential consequences of commonly used drugs that target 5-HT uptake and release.

Description: The λ interferons (IFN-λs), also known as IL-28 and IL-29, are coexpressed with IFN-β after Toll-like–receptor (TLR) stimulation in human monocyte–derived dendritic cells (DCs). IFN-λ shares with type I IFNs an intracellular signaling pathway that drives the expression of a common set of genes. However, IFN-λ signaling is initiated through a membrane receptor system distinct from that of type I IFNs. Because IFNs produced by DCs in response to TLR stimulation are critical in the differentiation and maturation of DCs, we sought to investigate whether IFN-λ exhibits specific effects on DC differentiation. In this work, we show that DCs acquire IFN-λ responsiveness through the expression of the specific IFN-λ receptor chain during their differentiation from monocytes. IFN-λ–treated DCs express high levels of major histocompatibility complex class I (MHC class I) and MHC class II but low levels of costimulatory molecules. However, they express CCR7 and acquire the ability to migrate to lymph nodes when intravenously injected into SCID/Bg mice. In mixed lymphocyte reaction (MLR) cultures, IFN-λ–treated DCs specifically induced IL-2–dependent proliferation of a CD4+CD25+Foxp3+ T-cell subset with contact-dependent suppressive activity on T-cell proliferation initiated by fully mature DCs. IFN-λs are thus able to generate tolerogenic DCs, an activity that could thwart IFN-β functions.

Description: We ascertained the prevalence of self-reported late occurrence of diabetes, hypertension, and cardiovascular (CV) disease in 1089 hematopoietic cell transplantation (HCT) survivors who underwent HCT between 1974 and 1998, survived at least 2 years, and were not currently taking immunosuppressant agents and compared them with 383 sibling controls. All subjects completed a 255-item health questionnaire. The mean age at survey completion was 39.3 years for survivors and 38.6 years for siblings; mean follow-up was 8.6 years. Adjusting for age, sex, race, and body mass index (BMI), survivors of allogeneic HCT were 3.65 times (95% confidence interval [CI], 1.82-7.32) more likely to report diabetes than siblings and 2.06 times (95% CI, 1.39-3.04) more likely to report hypertension compared with siblings but did not report other CV outcomes with any greater frequency. Recipients of autologous HCTs were no more likely than siblings to report any of the outcomes studied. Allogeneic HCT survivors were also more likely to develop hypertension (odds ratio [OR] = 2.31; 95% CI, 1.45-3.67) than autologous recipients. Total body irradiation (TBI) exposure was associated with an increased risk of diabetes (OR = 3.42; 95% CI, 1.55-7.52). Thus, HCT survivors have a higher age- and BMI-adjusted risk of diabetes and hypertension, potentially leading to a higher than expected risk of CV events with age.

Description: We report a novel chromosomal translocation t(4;12)(q11;q13) in a 37-year-old male with acute promyelocytic leukemia (APL)-like morphologic changes but lacking RARα rearrangements. His blast cells had some morphologic features evocative APL such as heavy azurophilic granules, bundles of Auer rods except regular round or oval nuclei. Immunophenotype of blast cells showed positivity for CD13 and CD33, and negativity for CD34 and HLA-DR which were compatible with the diagnosis of APL. Disseminated intravascular coagulation (DIC) was present. Chromosome study of BM cells showed that 29 out of 35 metaphases had a consistent karyotype of 46, XY, t (4;12)(q11;q13) which was confirmed by chromosome painting with whole chromosome paint probes 4 and 12. However, fluorescence in site hybridization, reverse transcriptase-polymerase chain reaction (RT-PCR) and multiplex RT-PCR did not demonstrate any evidence for RARα rearrangements including PML-RARα, PLZF-RARα, NuMA-RARα, NPM-RARαand STAT5b-RARα fusion genes. Sequential treatment with arsenic trioxide and chemotherapy had no effect, he died of bleeding due to DIC. In view of the fact as mentioned above, this case should be rated as transitional M2–M3.

Description: To explore the clinical and experimental features of acute leukemia(AL) with trisomy 4, a retrospective study on the clinical and laboratory data in 21 cases of AL with trisomy 4 was performed. Chromosomes were prepared using direct method and/or short-term(24h) cultures of bone marrow cells. Karyotypic analysis was carried out using R- banding techique. 14 cases were studied by interphase FISH using a chromosome 4-specific α-satellite DNA probe labeled by spectrum Green to ascertain the presence of a clone with trisomy 4. 5 cases with t(8;21) revealed by karyotypic analysis were detected by dual-color FISH using t(8;21) translocaton probe to confirm the AML1/ETO rearrangement. The patients with AL and trisomy 4 had a median age of 50 years and a median survival of 10 months. They all had de novo AL expect two cases with secondary AL. M2 was the most frequent FAB subtype in this series (9/21 cases). The initial leukocyte count more than 10×109/L was seen in 16 cases. An enlargement of liver, spleen and/or lymph nodes in varying degrees was found in 15 cases. Among 15 cases received immunophenotypic analysis, 11 cases showed CD34 positivity and 6 cases co-expressed myeloid and lymphocytic antigens. Karyotypic analysis disclosed clonal trisomy 4 in 18 cases and one cell with +4 in 3 cases. Isolated trisomy 4 was found in 7 cases, while 14 cases had other abnormalities besides trisomy 4 including numerical abnormality (+6) in one case, and structural abnormalities in 13 cases among which t(8;21) was found in 8 cases. Interphase FISH confirmed that all 13 cases including 3 cases having one cell with +4 on karyotypic analysis had clonal trisomy 4. Dual-color FISH confirmed that all 5 cases with t(8;21) had AML1/ETO rearrangment. So we concluded that AL patients with trisomy 4 had unique clinical features and a poor prognosis.

Description: Flow cytometric approaches can resolve cell position in the cell cycle based on DNA content, and these analyses are widely used in the study of cell growth, cell cycle regulation, and oncology. These applications require dyes that bind to DNA in a stoichiometric manner and which, with the exception of some UV-excited dyes like Hoechst 33342, require fixation, permeabilization and RNAse treatment for reproducible results. The Vybrant® DyeCycle™ stains are DNA-selective, cell membrane-permeant dyes that show greatly-enhanced fluorescence when bound to DNA. DyeCycle violet stain can be excited by a 405 nm laser, DyeCycle green stain by a 488 nm laser, and DyeCycle orange stain by either 488 nm or 532 nm lasers. (Figure 1) No fixation, permeabilization or addition of RNAse is required for stoichiometric binding of the dye to DNA. These dyes show similar performance on live Jurkat cells to Hoechst 33342 and DRAQ5: G0/G1 peak CV generally less than 6% and G2/G1 ratio greater than 1.8. The DyeCycle stains have been tested on a variety of cells: Jurkat, CHO, NIH 3T3, HL60, HEK cells, and peripheral blood leukocytes. Staining was optimized by cell type using time, temperature, cell concentration and dye concentration. The dyes can be used on cells in the presence of media components, including serum and divalent cations. Cells with damaged membranes exhibit different DyeCycle stain uptake patterns from live cells and can be excluded from analysis with spectrally-resolved viability dyes, such as SYTOX® blue and SYTOX red dyes. The DyeCycle stains have been used with antibody staining against surface antigens to evaluate the cycle profile of subpopulations. Generally, antibody conjugates with red-emitting fluorophores, such as allophycocyanin and phycoerythrin tandem fluorophores, were used because the required concentrations of the DyeCycle stains produced significant emission across fluorescein and phycoerythrin detection channels. The DyeCycle stains have also been combined with viability and apoptotic markers in testing of cells induced to apoptosis with camptothecin. DyeCycle orange stain, in particular, was found to define a sub-G0 population in late apoptotic cells. Finally, the DyeCycle stains cause some retardation of cell division, but do not demonstrate the toxicity observed with DRAQ5. The adherent cell lines, HEK and NIH 3T3, were stained with DyeCycle violet stain and DyeCycle orange stain, respectively before being were sorted under sterile conditions based on G0/G1 and G2/M population fluorescence. Sorted populations demonstrated the appropriate fluorescence when verified by reanalysis, and all sorted populations attached normally and expanded over 1 to 3 days post-sort. DyeCycle stains allow resolution of cell cycle information in viable cells against the dynamic background of cell activity using common lasers, as well as the ability to sort cells based on position in the cell cycle. Figure 1. ModFit analysis of live jurkat cells labeled with 5 μM Vybrant DyeCycle violet stain or 10 μM of either Vybrant DyeCycle green or orange stains. Figure 1. ModFit analysis of live jurkat cells labeled with 5 μM Vybrant DyeCycle violet stain or 10 μM of either Vybrant DyeCycle green or orange stains.

Description: Late-onset erythropoietic protoporphyria (EPP) is a rare complication of myelodysplastic syndrome (MDS) but has not been described in association with a myeloproliferative disorder (MPD). EPP is normally an inherited disorder characterized by photosensitivity that starts in early childhood and results from overproduction of protoporphyrin secondary to ferrochelatase (FECH) deficiency. Severe liver disease occurs in 1% to 2% of patients. Here we report that severe photosensitivity and cholestatic liver disease in a patient with a myeloproliferative disorder was caused by excess protoporphyrin production from a clone of hematopoietic cells in which one FECH allele had been deleted. Our observations suggest that the usual explanation for the association of late-onset EPP with MPD and MDS is acquired somatic mutation of one FECH allele in bone marrow and show for the first time that the consequent overproduction of protoporphyrin may be severe enough to cause acute liver damage.

Description: The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin insensitive and was recently shown to regulate the prosurvival kinase AKT by phosphorylation on Ser473. We investigated the molecular effects of mTOR inhibition by the rapamycin derivatives (RDs) temsirolimus (CCI-779) and everolimus (RAD001) in acute myeloid leukemia (AML) cells. Unexpectedly, RDs not only inhibited the mTOR complex 1 (mTORC1) containing mTOR and raptor with decreased p70S6K, 4EPB1 phosphorylation, and GLUT1 mRNA, but also blocked AKT activation via inhibition of mTORC2 formation. This resulted in suppression of phosphorylation of the direct AKT substrate FKHR and decreased transcription of D-cyclins in AML cells. Similar observations were made in samples from patients with hematologic malignancies who received RDs in clinical studies. Our study provides the first evidence that rapamycin derivatives inhibit AKT signaling in primary AML cells both in vitro and in vivo, and supports the therapeutic potential of mTOR inhibition strategies in leukemias.

Description: In the adult, platelets are derived from unipotential megakaryocyte colony-forming cells (Meg-CFCs) that arise from bipotential megakaryocyte/erythroid progenitors (MEPs). To better define the developmental origin of the megakaryocyte lineage, several aspects of megakaryopoiesis, including progenitors, maturing megakaryocytes, and circulating platelets, were examined in the murine embryo. We found that a majority of hemangioblast precursors during early gastrulation contains megakaryocyte potential. Combining progenitor assays with immunohistochemical analysis, we identified 2 waves of MEPs in the yolk sac associated with the primitive and definitive erythroid lineages. Primitive MEPs emerge at E7.25 along with megakaryocyte and primitive erythroid progenitors, indicating that primitive hematopoiesis is bilineage in nature. Subsequently, definitive MEPs expand in the yolk sac with Meg-CFCs and definitive erythroid progenitors. The first GP1bβ-positive cells in the conceptus were identified in the yolk sac at E9.5, while large, highly reticulated platelets were detected in the embryonic bloodstream beginning at E10.5. At this time, the number of megakaryocyte progenitors begins to decline in the yolk sac and expand in the fetal liver. We conclude that the megakaryocyte lineage initially originates from hemangioblast precursors during early gastrulation and is closely associated both with primitive and with definitive erythroid lineages in the yolk sac prior to the transition of hematopoiesis to intraembryonic sites.

Description: Practice guidelines for use of an erythropoietic agent (EpA) have been previously developed for patients with all cancers, but these have not specifically addressed non-myeloid hematological malignancies. Given issues of benefit, cost, access to treatment and practice variation for these patients, the Cancer Care Ontario Program in Evidence - Based Care (CCO PEBC) conducted a systematic review and has developed a practice guideline. Entries to MEDLINE, CANCERLIT, EMBASE, the Cochrane Library databases, and abstracts of the American Societies of Clinical Oncology (ASCO) and Hematology (ASH) were searched. A hierarchy of outcomes was developed that included survival, quality of life (QoL), transfusion requirements and improvements in hemoglobin concentration/hematocrit. To validate conclusions and recommendations, the practice guideline was sent to Ontario practitioners in July 2006; responses are now being collated. Eighteen reports (14 articles, 4 abstracts) of randomized trials met eligibility criteria. None of the 3 trials reporting survival outcomes detected a benefit with use of an EpA. Of 7 trials evaluating QoL, 6 reported superior outcomes in patients receiving an EpA. None of those trials sufficiently reported methodologic parameters required by proposed guidelines for analyzing, interpreting and reporting QoL measures. Use of an EpA significantly reduced the proportion of patients transfused in 5 trials evaluating this outcome; reductions ranged from 15% to 40%. The absolute risk reduction ranged from 15% to 24%; the number needed to treat to prevent a transfusion ranged from 4 to 6. Use of an EpA was not associated with a reduction in the mean/median number of units transfused. In 10 studies, either a statistically significant increase in the hemoglobin concentration/hematocrit or in the proportion of patients with an increase in the hemoglobin concentration/hematocrit was seen. For this practice guideline, we interpreted the evidence as providing insufficient data to justify use of an EpA in order to improve survival or QoL. However, there are compelling data showing that use of an EpA reduces the risk of requiring a transfusion. Initiatives such as the Commission of Inquiry on the Blood System in Canada (“the Krever Commission”), state that alternatives to transfusion be offered to patients because of associated known and potentially unknown adverse consequences of blood products. Thus, the use of an EpA is recommended as an alternative to transfusion. However, the decision to use an EpA to reduce transfusion requirements should primarily consider individual patient values and the likelihood that a patient will require a transfusion so that patients are not exposed to unnecessary treatment and the health care system to additional costs.

Description: The treatment of ARL is complicated by the numerous immuno-chemotherapy, antiretroviral, and prophylactic options available to lymphoma specialists. The optimal management in the era of combination antiretroviral therapy (cART) is unclear. We administered a survey instrument to determine physician preferences and perceptions in the management of ARL and to assess the variability in treatment in Canada. The survey was developed with items grouped into key domains of ARL management (physician demographics, attitudes, and treatment preferences) and piloted for content validity and clarity. The final questionnaire was administered to lymphoma physicians with valid contact information in the provinces of Ontario (ON; n=155) and British Columbia (BC; n=48). The Dillman Tailored Design Method was followed for multi-modality (internet and standard mail) survey administration. Of 196 physicians, 131 either responded by completing the questionnaire (n=117; 60% response rate) or declining to participate (n=14; 7%). Most responders were male (63%), white (70%), practicing in an academic setting (63%), and belonged to a median age group range of 41–50 years. The majority (98%) had a positive attitude towards the treatment of ARL, as measured by a previously validated 2-item attitude scale. However, barriers to adequate care were still identified; 84% of physicians agreed that uncontrolled human immunodeficiency virus infection represented a major barrier to ARL care and 54% agreed that a patient’s concurrent intravenous drug abuse impaired care. Most physicians recommended the concomitant use of cART in the care of their patients with ARL (n=72 of 109 responses; 66%). Similarly, a majority of respondents recommended CHOP-like regimens (cyclophosphamide, doxorubicin, vincristine, and prednisone; n=92 of 108 responses; 85%) to form the backbone of chemotherapy. The addition of the rituximab was preferred by 41% physicians but not by 40% others, with remaining respondents unsure of the agent’s role. In logistic regression analysis, use of rituximab was predicted only by location of practice (province), after adjusting for other potential predictors including physician age, race, gender, practice environment (academic vs. community), years of experience, ARL patient volume, and modality of survey response. Physicians from BC were much more likely to administer rituximab than ON practitioners (OR 44.5; 95% CI: 7.76–255.0, p

Description: Background Patients with beta Thalassemia Major (TM) require life-long blood transfusions, which often cause iron overload that may increase patients’ morbidity and mortality. Iron Chelation Treatment (ICT), aimed to reduce iron overload, is based on 8–12 hour infusions of Deferoxamine (DFO) for 5–7 days/week, and/or Deferiprone (L1) orally administered. Current ICT can be related to low satisfaction, low compliance, and potentially negative consequences on clinical effectiveness, patients’ wellbeing and on healthcare costs. Aims: To investigate the Health-Related Quality-of-Life (HRQoL) of TM patients and their satisfaction with ICT. Methods: The Italian-THAlassemia-Cost-&-Outcomes-Assessment (ITHACA) was a naturalistic multicentre study conducted to evaluate costs, HRQoL, compliance and treatment satisfaction in TM patients undergoing ICT for at least 3 years, enrolled at Italian Thalassemia Care Centers. HRQoL was measured in 〉14 years old patients with 2 generic instruments: EQ-5D; Short Form-36 (SF-36). To measure satisfaction 〉12 years old patients received a 28-item instrument consisting of 4 domains: ‘perceived effectiveness’, ‘acceptance’, ‘burden’, and ‘side effects’. Each domain scored from from 1 (very dissatisfied) to 5 (very satisfied). Results Based on 126 patients: median age 29.4 years (12.3–48.5), 49.6% male. At enrolment 48.0% were using DFO, 33.6% L1, 18.4% were treated with DFO+L1. 86.5% of patients had at least one TM-related complication, 13.5% changed treatment regimen at least once in a median of 11.6 months before enrolment. With EQ-5D profile patients reported moderate problems with ‘mobility’ (9.1%), ‘self care’ (0.8%), ‘usual activities’ (23.5%), moderate or severe ‘pain/discomfort’ (60.5%) and ‘anxiety/depression’ (39.5%). Mean EQ-5D-Visual Analogue Scale was 73.0 (30–100). The SF-36 Physical Component Summary mean(SD) score was 47.7(8.4), while the mean score estimated in the Italian general population comparable for age and sex is 53.3; the Mental Component Summary mean(SD) score in TM patients was 45.1(8.8), while the Italian general population mean score was 47.7. Mean satisfaction scores were 4.29 (perceived effectiveness), 3.37 (acceptance), 3.87 (burden), and 3.57 (side effects). Simple linear regression analysis showed that satisfaction with burden (r2=12.6%, p

Description: Background: Current guidelines offer numerous options for initiating therapy in patients with untreated, advanced stage follicular lymphoma (FL). Selecting among these options that include watchful waiting, single-agent and combination chemotherapy, monoclonal antibodies, and radioimmunotherapy, remains challenging. Recent data suggest that chemotherapy combined with a monoclonal antibody may alter patterns of relapse and overall survival for pts with FL (Fisher, Blood 2004). While rituximab (R) chemotherapy combinations have become commonly used for untreated pts with FL, to date, the optimal first-line therapy remains undefined. To address this issue, we updated a systematic literature review and performed a meta-analysis of first-line therapy for untreated FL that examined the effect of various chemotherapy regimens combined with R on response rates and survival in patients with untreated FL. Methods: The comprehensive systematic review included searches the Cochrane Central Register of Controlled Trials (Cochrane Library Issue 1, 2003), MEDLINE (1/1996–6/2006), EMBASE (1/1980–7/2006), American Society of Hematology Annual Meeting abstracts (2002–2005), and American Society of Clinical Oncology Annual Meeting abstracts (1995–2006). Each database was searched using combinations of the term follicular lymphoma and the terms for medications and treatment regimens. Inclusion criteria for studies were as follows: 1) Inclusion of patients with untreated stage III/IV FL grades 1, 2, or 3; 2) Intervention with chemotherapy and/or immunotherapy, radioimmunotherapy, or watchful waiting; 3) Reporting in English of the following treatment outcome measures specifically for patients with FL: CR/CR-unconfirmed, overall response rate (OR), and at least one form of survival data. Abstracts subsequently published as papers were excluded. Extracted data included pre-treatment disease status, treatment regimen, median follow-up time, progression free survival, overall survival, CR and OR. The following treatment strategies from peer-review publications were analyzed: single agent R, R-CVP, R-CHOP, and fludarabine-combinations with R (R-Fcom). In meta-analyses of selected studies, we utilized the Mantel-Haenszel (fixed effects model) and DerSimonain and Laird (random effects) methods to calculate the risk difference comparing treatment regimens’ CR/CRu to the spontaneous CR in patients undergoing watchful waiting (4.6%; Ardeshna et al. Lancet, 2003). Results: In total, over 3135 abstracts were reviewed to identify 11 studies meeting the inclusion criteria for this analysis. These studies included data from 3144 patients. Only one study presenting CR data for R-CVP (36%, 95% confidence interval: 28%–44%) met inclusion criteria. The meta-analyses estimated the CR rate associated with single-agent R to be 30% (95% CI: 20%–40%), R-CHOP to be 62% (30%–94%), and R-Fcom to be 85% (76%–94%) (random effects; see Figure). Conclusions: R-CHOP and R-fludarabine combinations appear to produce the highest CR rates for untreated pts with FL. Meta-analysis can aid clinicians in therapeutic decision making as they weight the risks and benefits of various regimens for newly diagnosed pts. Figure Figure

Description: More than 21 million prescriptions for warfarin are written yearly in the US. Yet, in spite of its importance, vitamin K epoxide reductase (VKOR), the target of warfarin, has resisted purification since its identification in 1972. We report the first successful purification and reconstitution of activity of a recombinant human vitamin K epoxide reductase. A series of detergents were screened to determine that best for solubilization of VKOR from microsomes. Detergents tested that were effective in solubilization of VKOR also led to loss of measurable activity. This loss of activity supports our previous prediction that VKOR is embedded in and requires a membrane environment for enzymatic activity. The short-chain phospholipid, DHPC (1,2-Dihexanoyl-sn-Glycero-3-Phosphocholine) was the detergent of choice to efficiently extract VKOR from the microsomes, even though this reagent completely inhibited enzyme activity. Partial reconstitution was achieved on-column by washing with 0.4 % dioleoylphosphatidylcholine/0.4% deoxycholate. Complete recovery of activity was achieved by removing the deoxycholate through dialysis in the presence of the reducing reagent, THP (Tris(hydroxypropyl)phosphine). During dialysis, the solution became cloudy indicating the formation of membrane-like structure. Purified recombinant VKOR is ~21 kDa (~18.5 kDa + tag); fully active; and over 93% pure. The concentration of warfarin for 50% inhibition is the same for purified protein and microsomes. It has been reported and assumed that VKOR is a multi-subunit enzyme. Our results, however, suggest that a single peptide can accomplish the reaction. The trace amounts of contaminating proteins were identified by mass spectrometry; however, none are apparently relevant to the VKOR reaction. Moreover, the turn-over number of purified VKOR (0.25 sec-1 is approximately two-fold higher than microsomes and about 10 fold higher than the turnover number of gamma-glutamyl carboxylase for CO2 addition. In addition to the vitamin K epoxide to vitamin K reaction, our results also indicate that VKOR can efficiently convert vitamin K to vitamin K epoxide. Our results suggest that ancillary proteins (other than a thioredoxin-like enzyme) are not necessary for full VKOR activity. This purification will allow further characterization of VKOR in relation to other components of the vitamin K cycle and should facilitate its structural determination.

Description: Objectives: Emergency room (ER) evaluation may differ when physicians see patients in the context of clinical trials compared to routine care. We aimed to determine whether patterns in the use of a systemic pretest probability (PTP) stratification tool and D-dimer testing change over time and whether this influences their clinical utility. Methods: Charts were reviewed of 974 cases that had D-dimer testing for VTE exclusion in two time periods; 474 consecutive patients evaluated between 07/02 and 10/02 and 500 between 07/04 and 10/04. The former cohort was managed by ER physicians that had just participated in a clinical trial on VTE diagnosis including PTP assessment and D-dimer testing, whereas the latter group had received no formal training after 2002. In both cohorts, pretest scoring as low, intermediate or high risk according to Wells criteria for DVT and PE was performed in every case since this was requested from the laboratory. D-dimer testing (Vidas® D-Dimer, Biomerieux) was performed only in the low and moderate risk group after reception of the PTP assessment form. Physicians were also asked to check out a form detailing individual Wells criteria leading to the overall assessment but were not mandated to do so in order to obtain the D-dimer result from the laboratory. Results: Pretest probability was evaluated as low, moderate or high in 66,7%, 31,9% and 1,3% vs. 78,4%, 21,2% and 0% (all comparisons are 2002 vs. 2004). There was a significant increase in proportion of undetailed evaluations of PTP (32,7% vs. 61%). Detailed forms in both cohorts had similar risk distribution (low/moderate risk 51,7/46,4% vs. 59,2/40,8%) whereas undetailed forms were usually quoted as low risk (97,4% vs. 90.5%). Number of D-dimer tests performed per month was stable over the three year period of observation and in the two evaluated cohorts (119/mth vs. 125/mth). D-dimer results were negative in 299/474 cases (63,1%) in 2002 and 359/500 (71,8%) in 2004 (p=0,003), although this difference was less apparent when analysed according to the individual PTP risk groups (low/moderate risk 77,3/51,9% vs. 71,2/48,3%). Incidence of VTE events decreased over time from 5,3 to 1,6% (p=0,002). Incidence in the low/moderate risk groups was 1,6%/10,6% vs. 0,3%/6,6%. Only one false negative result (popliteal vein DVT) was observed in the two cohorts (NPP = 99,9%). Conclusion: Our results show a decreasing incidence in VTE over time in an ER population screened by D-dimer testing and PTP even though the number of tests performed were stable over time. This was accompanied by a decreasing number of cases considered to have an intermediate PTP. These findings suggest a change of practice over time resulting in an increasing use of D-dimer testing for very low risk patients and a decrease in their use for intermediate risk patients. A decrease in the proper use of the PTP tool over time might result in overestimation of the physician perceived risk and therefore lead to an increase in imaging resource utilisation. Broader studies including imaging prescription trends over time will be needed to confirm this hypothesis.

Description: 44 patients with relapsed or refractory CD20+ NHL were enrolled on a phase I trial of dose-escalated 90YZ followed by high-dose BEAM and autotransplant in which the 90YZ dose was patient-specific based on dosimetry. 90YZ doses were calculated to deliver cohort-defined radiation doses (100–1700 cGy) to critical organs (liver, lung or kidney) with 3–6 patients per cohort. On D-22, rituximab (R) 250 mg/m2 was infused followed by 111In Zevalin®. Imaging was performed immediately and at 4, 24, 72 and 144 hours; dosimetry was performed on D-15. On D-14, R was followed immediately by 90YZ at the cohort-prescribed dose. On D-6 through D-1, patients received high-dose BEAM. On D0, a minimum of 2.0 × 106 CD 34+ cells/kg was infused and G-CSF 5 μg/kg SQ daily begun. The median age was 54 (range: 25–73) years. NHL histologic subtypes were as follows: 16% mantle cell, 57% diffuse aggressive, 11% low-grade and 16% transformed. 43% had recieved 3 or more chemotherapy regimens, and 70% had received R either alone or in combination with chemotherapy. The toxicity profile was similar to that associated with high-dose BEAM. The most common grade III/IV toxicities were infection, fever, stomatitis, nausea, vomiting, diarrhea. One patient experienced transient veno-occlusive disease at the 700 cGy dose level. Two dose-limiting toxicities occurred at the 1700 cGy dose level: one patient with grade 4 stomatitis died of pneumonia and sepsis on D+10, and one patient experienced septic emboli to the lung on D+13. Engraftment occurred at a median of 10 days (range: 8–18 ) to granulocytes ≥ 500/μL, and 21 days (range: 11–40 days) to platelets ≥20,000/μL. One heavily pretreated patient developed MDS on D+291 and expired of sepsis on D+483. With a median follow-up of 21 months, the three year overall and progression-free survivals are 52% and 37%, respectively. 90YZ Zevalin ® Dosing for Cohorts 5–9 (median;range) Cohort (cGy) Total Dose (mCi) mCi/kg 900(n=3) 28(27–37) 0.3(0.3–0.4) 1100(n=5) 51(29–65) 0.6(0.5–0.8) 1300(n=5) 61(39–98) 0.6(0.5–1.1) 1500(n=6) 73(51–99) 0.9(0.5–1.4) 1700(n=3) 98(82–104) 1.2(1.1–1.2) For dosimetry-based trials, 1500 cGy to the critical organ (nearly always liver) is the recommended dose. Although patient-specific doses calculated to deliver a cohort-prescribed absorbed radiation dose to the critical organ were highly variable, the two dose-limiting toxicities ocurred at nearly identical doses (1.14, 1.20 mCi/kg) when calculated according to weight. Whereas eight other patients safely received at least twice the conventional 0.4 mCi/kg dose, 0.8 mCi/kg is the recommended dose for phase II studies, if dosing is to be based on weight and not dosimetry. Outcomes are encouraging given the high-risk patient population. A phase III trial will be required to establish whether or not the addition of 90YZ to high-dose BEAM improves outcomes in NHL patients undergoing autotransplant.

Description: The 60-month follow-up data for patients randomized to imatinib in IRIS demonstrated significant clinical improvements in survival rates and QALYs gained (17,09 years;13,58 QALYs) in patients newly diagnosed with chronic myeloid leukemia (CML) and treated with imatinib in comparison to patients under interferon-alpha (INF-α) (9,10 years;6,31 QALYS) as first line therapy. Although reimbursed as second line therapy for chronic phase CML patients who did not respond to INF-α, imatinib was not considered for public reimbursement as first line treatment in Brazil based solely on drug costs. An economic evaluation of imatinib as first line treatment versus INF-α was performed under the Brazilian Public Heathcare System perspective, according to the long-term follow-up data from IRIS, literature recommendations and prior health technology assessment from the National Institute for Clinical Excellence (NICE) to consider long term follow-up survival and adverse events costs. This study was aimed to evaluate the cost-effectiveness of imatinib compared with IFN-α for first-line treatment of chronic myeloid leukemia under the Brazilian public healthcare system perspective. For the economic model, a base case of 100 patients for each treatment option was constructed focused on drug costs and adverse events. Drug costs were estimated based on the Brazilian public healthcare reimbursement payment (APAC-SUS) for chronic phase CML treatment. Febrile neutropenia (grade III and IV), depression, nausea and abnormal liver-function results were considered as adverse events. Clinical guidelines and protocols from two public hematology Brazilian centers, Fundação Pró-Sangue FM-USP and Instituto Nacional do Cancer, were used to estimate adverse events treatment costs. Adverse events frequency for all grades was based on data published by NICE and the Agency for Health Care Research and Quality. Due to the high crossover rate from INF-α to Imatinib group observed in the IRIS study (90% from INF-α to imatinib within a year of study entry) the estimated life time survival for INF-α treatment group was based on the European Study Group on Interferon in Chronic Myeloid Leukemia. Utilities values from the IRIS study were used for both groups. Annual discount rates were of 6% for costs and 1.5% for QALYs. The annual average costs for the treatment of adverse effects in the INF-α were 1.83 higher than the imatinib group. Adverse events lifetime costs for INF-α were 24% higher than imatinib, even tough imatinib granted a projected 6.3 years survival advantage over INF-α. The resulting incremental cost-effectiveness ratio (ICER) of imatinib, compared with IFNα, considering adverse events was US$ 18,637 per QALY gained. Assuming a conservative cost-effectiveness threshold of less than US$ 25,500, which is three times the GDP per capita in Brazil (US$ 8,500 in 2005), the ICER for imatinib compared with INF-α falls within the range considered by the World Heath Organization as a cost-effective fist line treatment for patients newly diagnosed with CML.

Description: The developing fetal immune system provides a unique opportunity to manipulate normal immunologic development for therapeutic prenatal and anticipated postnatal interventions. In previous studies we have shown that allogeneic in utero hematopoietic cell transplantation (IUHCT) results in donor specific tolerance that can subsequently facilitate non-myeloablative postnatal cellular or organ transplants. It follows that in utero injection of transduced hematopoietic stem cells (HSC) could potentially induce tolerance to a transgene encoded protein. We hypothesized that expression of a transduced antigenic protein by HSC and their progeny would alter thymic T cell development resulting in deletion of antigen specific T-cells. To test this hypothesis, we used the mammary tumor virus (MTV) superantigen system to evaluate the effect of IUHCT of transduced HSC on T cell development. In this system, expression of different MTV oncogenes by different I-E+ strains of mice results in deletion of T cells expressing the relevant Vβ T cell receptor. Specifically, mice which are Mtv7+ delete T cells expressing the Vβ6 T-cell receptor. In this study, CD150+CD48− enriched Balb/c (I-E+ Mtv7−) HSC were transduced with an HIV-based lentivirus expressing MTV7 under an MND promoter. 1.5E+05 transduced cells were injected intravascularly via the vitelline vein into E14 Balb/c fetuses. Non-injected age matched naive Balb/c mice served as the control group. The peripheral blood (PB) and thymuses of injected fetuses and control mice were harvested at day of life (DOL) 10, 20 and 60 and analyzed by flow cytometry for T lymphocyte Vβ6 expression. Additionally, the T cell composition of the thymus was assessed at DOL10 for CD4 and CD8 single positive (SP) and CD4/CD8 double positive (DP) cells. Thymic flow cytometric analysis at DOL10 revealed that greater than 98% of the T cells were CD4CD8 DP, a stage that has not yet undergone negative selection. No significant difference was noted in the percentage of thymic Vβ6+ DP T-cells at this time point or at DOL20 and DOL60. In contrast, there was a significant decrease in the percentage of Vβ6+ peripheral blood SP cells in those mice injected with MTV7 transduced HSC relative to control mice at DOL10, DOL20 and DOL60 (p

Description: After the report of two cases of leukaemia caused by insertional mutagenesis of a retroviral vector in children with SCID, it became clear that safety issues of therapeutic gene transfer must be addressed more thoroughly. We analysed whether gene transfer into mature T cells and haematopoietic stem cells bear the same risk of generating T cell leukaemia through activation of specific T cell oncogenes, such as LMO2, TCL1 and ΔTrkA. To address this issue, we used the Rag-1 mouse model, which allows long term analysis of transplanted T cells and haematopoietic stem cells. We were able to transduce mature T cells and haematopoietic stem cells of C57BL/6 (Ly5.1) donor mice with oncoretroviral vectors expressing LMO2, TCL1 and ΔTrkA. Transduction efficacies of up to 70% were achieved for mature T cells and approximately 90% for haematopoietic stem cells. After transplantation into Rag-1-deficient recipients, stem cell transplanted animals developed T cell lymphomas/leukemia for all investigated oncogenes after characteristic incubation times, mostly of a CD8+CD4+ double positive phenotype. T cell lymphomas were characterised by gross thymic mass, splenomegaly and heavily enlarged lymph nodes, although none of the control- vector- transduced mice developed lymphoma/leukaemia. LM PCR analysis revealed mono- or oligoclonality of the tumours. T cell transplanted animals showed no signs of leukaemia development so far. However, after several attempts, one immortalized T cell progenitor clone could be generated after transduction with LMO2. Our results so far indicate that mature T cells are less susceptible to transformation by known T cell proto-oncogenes, but the studies are still ongoing.

Description: AMG 531 is a novel thrombopoeisis-stimulating peptibody that increases platelet production by targeting the thrombopoietin receptor. The trial described here is an ongoing, open-label, extension study of patients with immune thrombocytopenic purpura (ITP) assessing the safety and efficacy of long-term AMG 531 treatment of ITP, as well as patient-reported health-related quality of life (HRQOL). Eligible patients have completed a previous AMG 531 study in ITP. Patients previously treated with AMG 531 receive a starting dose that is the same as the final dose given in the previous study; placebo-treated patients begin the extension study with an AMG 531 dose of 1 μg/kg. The dose is skipped, decreased, maintained, or increased based on platelet count. Analysis was performed on the 36 patients (25 female, 11 male; 29 white, 6 Hispanic, 1 black) for whom complete data are currently available. The mean age is 50.0 ± 13.0 (SD) years. Most patients (83%) had a splenectomy prior to this study. HRQOL is being assessed with an ITP disease-specific measure, the ITP Patient Assessment Questionnaire (ITP-PAQ). This report describes the first use of the ITP-PAQ to measure treatment effect. The ITP-PAQ has 10 scales: 4 Physical Health (PH)—Symptoms, Fatigue, Bother, and Activity; 2 Emotional Health (EH)—Psychological and Fear; 3 Quality of Life (QOL)—Overall, Social, and Work; and 1 Women’s Reproductive Health (WRH). Each scale is scored 0 (worst)—100 (best). A planned interim analysis was conducted to evaluate changes in HRQOL from week 1 (pretreatment) to week 24. Results indicated improvement in the areas of PH-Symptoms, with a 7.9-unit increase (p=0.007), PH-Fatigue, with a 7.8-unit increase (p=0.006), PH-Bother, with a 9.4-unit increase (p=0.025), PH-Activity, with a 7.4-unit increase (p=0.032), and QOL-Work, with a 7.9-unit increase (p=0.014). An analysis of minimal clinically important difference will be conducted when the sample size is large enough to determine if statistically significant values are clinically meaningful. A comparison of HRQOL in patients with a durable platelet response (doubling of the baseline count and ≥50×109/L at 6 or more weeks during weeks 17–24 in the absence of rescue medication) versus those without a durable response showed a trend for greater improvement among the responders in all 10 scales, with significant differences for PH-Symptoms (p=0.02), PH-Bother (p=0.02), PH-Activity (p=0.01), EH-Psychological (p=0.01), and QOL-Overall (p=0.03). Individualized dosing of AMG 531 provides a unique therapeutic option for managing platelet count in ITP while improving HRQOL.

Description: Myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (sAML) are diseases of the hematopoietic stem cell that are potentially curable by allogeneic stem cells transplantation (ASCT). MDS and sAML are most frequent in elderly patients with significant co-morbidity. Here we report our results with reduced intensity conditioning regimen and preemptive donor lymphocyte transfusions that have been successful in patients with advanced and refractory AML. Sixty seven patients were treated for MDS and 90 patients for sAML. The FLAMSA regimen consisted of a 4 day course of chemotherapy (Ara-C 2g/m2, Fludarabin 30 mg/m2, and amsacrin 100 mg/m2) followed by 3 days rest and reduced intensity conditioning with 4 Gy total body irradiation (TBI), cyclophosphamide (CY) +/− antithymocyte globulin (ATG). Overall survival at 10 years was 50% for MDS and 26% for sAML (p=0.014, log rank). In the MDS group most patients had preleukemic forms (IPSS 3 and 4). Survival at 5 years was 58% with FLAMSA, 44% with myeloablative conditioning with TBI and 67% with Busulfan (BUS). Non-relapse mortality at 5 years was 41% with FLAMSA, 20% for BUS and 50% for TBI. The relapse or progression rate was 0% for FLAMSA, 24 % for BUS and TBI respectively. In sAML the 5 year survival was 17% for FLAMSA, 57 % for BUS and 30% for TBI. Non-relapse mortality was 60% for FLAMSA, 36% for BUS and 66% for TBI, whereas the relapse rate was 62% for FLAMSA, 12.5 % for BUS and 37% for TBI (p=0.04). In multivariate analysis adjusted for disease, conditioning treatment and source of stem cells – marrow vs. mobilized blood – differences were not significant and the only significant prognostic factor was age less than 30 years (p=0.003). Therefore patients of the age of 60 years and older with AML were treated with FLAMSA containing BUS (8mg/kg in two days) instead of 4 Gy TBI. The overall survival at 2 years was 64%, 61% with FLAMSA-TBI and 70% with FLAMSA-BUS. The non-relapse mortality was 9% for FLAMSA-BUS and 37% for FLAMSA-TBI; the relapse rate was 25% at 2 years. At day 120 patients received donor lymphocytes (DLT) in 3 escalating doses from 1 × 106/kg, 5 × 106/kg on day 150 and 1 × 107/kg on day 180, if they had no GVHD, infection or relapse. Preemptive DLT appear to prevent relapse when compared to a historical control and contemporary patients not given DLT. We conclude from these data that MDS/sAML may have a different biology in young patients, and that in the elderly we can optimize the treatment with FLAMSA-BUS as a dose reduced regimen and preemptive immunotherapy for maintenance of remission.

Description: The presentation of leukemic antigens can be improved in AML and MDS by in vitro conversion of leukemic cells in leukemia-derived DC (DCleu), thereby forming a platform for the generation of leukemia-specific cytotoxic lymphocytes (CTL). In preliminary analyses with 140 AML and 60 MDS-cases we could already define optimal serum-free culture conditions to generate DC/DCleu.(Kufner 2005 I–III). Now we want to predict or correlate the clinical response to a DC/CTL-based immunotherapy by detailed analyses of the ex vivo generated/activated DC/DCleu and T-cells: 1)By a combination of 3 different DC-generating methods (‘MCM-mimic’, Lee 2003; ‘Ca-Ionophore’, Houtenbos 2003; ‘Picibanil’, Sato 2003) we can generate DC/DCleu in every case of AML/MDS, independently from FAB-type or karyotype. DC/DCleu are quantified according to their surface DC/blast-marker profiles. On average 42–45%/39–66% DC in AML/MDS could be generated with 48–54%/39–51% mature (CD83+) and 31–34%/23–31% migratory (CCR7+) DC. 45–65% of DC were ‘DCleu’; on average 47% of blasts are convertible to DCleu.. 2) In AML-patients who had presented with a relapse after SCT we could correlate a better ex vivo convertibility of blasts to DCleu with the patients’ in vivo response to a GM-CSF/Donor-lymphocyte Infusion (DLI)-therapy of their relapse after SCT (33% vs 7% to DCleu convertible blasts in ‘non-responders’). 3) A ‘Mixed lymphocyte culture’ (MLC) of autologous AML-patients’ or allogeneic donor-T-cells showed an on average higher proliferation and stimulation of DC-primed compared to MNC-primed T-cells: Upregulation of CD80/CD86-CD28;CD40-CD154;CD137L-CD137; moreover DC-priming yielded higher proportions of CD4+ cells, CD3+CD45RO+ memory cells CCR4+ T-cells (+59%, +52%, +91%) compared to MNC-primed T-cells (+35%, +13%, +44%) and a higher leukaemia-cytolytic activity (average 62%) compared to MNC-stimulated CTL (average 26%). 4) A detailed analysis of data showed great individual variations depending on the quality and composition of DC and T-cells: a) non-DC-primed autologous or allogeneic T-cells an lead to an increase of naive blasts after 3h incubation with blasts b) in cases with an ineffective DC-mediated ex vivo lysis of naïve blasts lower proportions of mature DC (29% vs 63%), DCleu (41% vs 68%) or a reduced T-cell proliferation or even loss of CD4/CD8/memory T-cells were seen. In summary our data show 1. that DC/DCleu can be generated in every single AML/MDS-case. 2. Grade of ex-vivo generability of DC/DCleu correlates with the in vivo response to a GM-CSF/DLI-relapse therapy. 3. Composition and quality of DC and autologous or donor T-cells after DC-priming provides informations about the activability and quality of CTLs in individual patients. We conclude, that ex vivo analysis of the DC/anti-leukemic T-cell-activability is necessary to develop and select promising anti-leukemia-directed T-cells for the immunotherapy of AML and MDS.

Description: The involvement of mediators of the innate immune response in immunological processes after allogeneic haematopoietic stem cell transplantation (HSCT) has been an important finding. Toll-like receptor (TLR) signalling plays a key role in the innate response to pathogens. The MyD88 adapter-like (Mal) protein is involved in TLR 2, 4 and 9 signalling. A polymorphism leading to a non functional variant (Ser → Leu 180) has been reported to be protective in cerebral malaria due to a reduced inflammatory response (O’Neill LA, ASH 2005). A dominant negative form of Mal inhibits NFκB activation by TLR-4 and LPS (Fitzgerald KA, Nature 2001). As bacterial products like LPS are believed to contribute to initiation and maintenance of graft versus host disease (GvHD), we hypothesised that one or two non functional Mal variants (Ser → Leu 180) present in donor or recipient cells might reduce the incidence of GvHD and improve survival after allogeneic HSCT. 163 donor recipient sibling pairs transplanted with full or reduced intensity conditioning without T depletion were identified from the Eurobank database. DNA was amplified using allele specific PCR primers: allele 1 (Ser)5′ CACCATCCCCCTGCTGTC; allele 2 (Leu) 5′ CACCATCCCCCTGCTGTT; reverse 5′ TGACGAAAGCCATCAG. The allele frequency was: allele 1 (Ser) 0.84; allele 2 (Leu) 0.16. There were no significant differences between the cohorts in age, gender mismatch, immunosuppression, conditioning or transplant center. The overall incidence of acute and chronic GvHD was reduced (83 vs 75% and 63 vs 52% respectively), when both donor and recipient possessed allele 2 (p=0.247 and p=0.155). A similar trend could be seen when the donor alone had at least one uncommon allele. The 5 year projected survival was not different for patient/donor pairs for any given combination of alleles. To further test the functional relevance of the Mal polymorphism, PBMCs from 33 Donor/Recipient pairs were stimulated with LPS or PHA and TNFα levels were analysed after 24h and 72h respectively. After LPS stimulation the median TNFα levels were lower when one or two uncommon alleles were present (1871 vs 1774 vs 988 pg/ml; p=0.9 and p=0.178). PHA stimulated cells also showed lower median TNFα levels, approaching statistical significance for the combined group carrying at least one uncommon allele (6656 vs 5173 pg/ml, p=0.085) and being statistically significant when two uncommon alleles were present (6656 vs 1495 pg/ml, p=0.031). We conclude that the presence of a non-functional Mal variant especially in donor cells may reduce the incidence of acute and chronic GvHD, although this did not reach statistical significance in this group of patients. There was no impact on survival. A larger cohort is currently being investigated.

Description: Background: Maintaining T cell function and survival after ex vivo manipulation remains a major challenge in adoptive immunotherapy. We previously demonstrated that murine T cells activated and expanded ex-vivo with anti-CD3 and anti-CD28 antibody-coated magnetic beads (CD3/CD28 beads) exhibit reduced GVHD-inducing potential compared to naïve (unmanipulated) T cells in a murine allogeneic BMT model. Blood diagnostics are commonly used to help identify patterns of biomarkers in multiple disease states. Objective: Determine if plasma profiles of 59 different analytes are altered in murine allogeneic BMT recipients who receive ex-vivo activated, suicide gene transduced and selected (Td) T cells compared with animals that received naïve suicide gene expressing T cells. Methods: Murine T cells were Td with a chimeric CD34-thymidine kinase (CD34-TK) fusion suicide gene. High efficiency (70%) gene transfer of CD34-TK to C57BL/6 (B6) murine T cells was accomplished 24 h after CD3/CD28 bead activation and gene-modified cells were purified to 〉 96% by CD34 immunomagnetic selection 2 days post-infection. Naïve B6 CD34-TK-expressing T cells were purified from the spleens of CD34-TK transgenic mice the day of BMT. To induce GVHD, lethally irradiated BALB/c allogeneic recipients were given T cell depleted (TCD) B6 BM supplemented with or without B6 CD34-TK purified naïve or Td T cells. Animals were bled 1 week after BMT and EDTA plasma samples were prepared and frozen. Quantitative measurements of 59 analytes were obtained using a rodent multi-analyte profile test (MAP test; Charles River Lab) on the plasma of 2 mice that received naïve T cells and died from GVHD on days 16 and 21 after BMT, 2 mice that received Td T cells and died from GVHD on day 34 after BMT, and 2 mice that received TCD BM only and survived 〉 100 days. Results: As before, we found that ex vivo activation of the donor T cells before BMT significantly prolonged the survival of mice transplanted with the Td T cells compared with mice receiving naïve T cells (p = 0.0028). Eighteen analytes, including IFN-γ, IL-2, IL-3, IL-4, IL-7, IL-11, and TNF-α were below the detection limits of the rodent MAP test for all samples analyzed. Twenty-six analytes, including VEGF, SCF, MIP-2, MIP-1α, MIP-3β, MCP-5, IL-1β, IL-18 and GM-CSF were detectable but not significantly different than the BM only controls for either the naïve or Td T cell groups. Nine analytes, including eotaxin, MIP-1γ, MCP-3, MCP-1, and IL-10 were similarly increased 2- to 3-fold in both the Td and naïve T cell groups compared to the BM only control. Interestingly, recipients of naive T cells exhibited increased plasma levels of growth hormone (≥14-fold), MIP-1β (4.6-fold), IP-10 (2.3-fold), and lymphotactin (2-fold), as well as decreased levels of leptin (≥14-fold), compared to both the BM only and Td T cell groups. This extreme modulation of growth hormone and leptin levels is particularly interesting given the fact that leptin influences local growth hormone secretion from lymphocytes and that both of these analytes have multiple biologic effects on T cells. Finally, only a single analyte, monocyte derived chemokine (MDC), was increased (4-fold) in mice that received Td T cells compared to the naïve T cell recipients. Conclusion: These results indicate that several plasma analytes with important immunological functions are altered when donor T cells are manipulated ex-vivo. These alterations may account for the decreased GVHD-inducing potential of ex vivo manipulated T cells after allogeneic BMT.

Description: Allogeneic bone marrow transplantation (BMT) from an HLA-identical sibling is a curative form of therapy for patients with acquired severe aplastic anemia. Survival has significantly improved over the past 3 decades. The actuarial risk of rejection has been reduced to about 7%. Improved results with survival in excess of 90% have been reported. Current preparative therapies are associated with early and late sequelae such as acute and chronic graft-versus-host disease (aGvHD or chGvHD, respectively) and secondary tumors. Two patients (6 years and 11 years old) with SAA, who had an HLA-identical sibling donor could not proceed with myeloablative therapy at the time of transplant due to delay in results of chromosome stability and fragility in one patient and abnormal pulmonary function in the second. Both received reduced intensity preparative regimen with Fludarabine (30 mg/m2×4 doses) Cyclophosphamide (5 mg/kg×4 doses) and rabbit ATG (1.5 mg/kg×4 doses) followed by an unmanipulated allogeneic BMT from their HLA-identical sibling donors. Graft versus host disease prophylaxis consisted of Cyclosporine from day -1 and Methotrexate 15 mg/m2 on day +1 and 10 mg/m2 on days +3, +6, +11 after transplant. Myeloid engraftment occurred on day +15 and day +28. The time to a platelet count 〉20,000 unsupported was +11 days and +29 days. No transplant-related toxicities, including mucositis or alopecia, were recorded. There were no signs for aGvHD or chGvHD. The patients continue with full donor chimerism 27 months and 130 days post transplant, respectively. This data suggests that a non-myeloablative, immunosuppressive regimen is sufficient to provide a stable engraftment in the patients with SAA. This approach may be associated with decreased transplant-related short- and long-term toxicities. A larger study is needed to fully evaluate the outcome and the toxicity associated with this conditioning.

Description: Protein 4.1R (4.1R), a vital component of the red cell membrane cytoskeleton, stabilizes the spectrin-actin lattice and attaches it to the embedded membrane proteins. The inclusion of exon 16, which encodes peptides critical for spectrin/actin binding, occurs via an intricate interplay between the auxiliary cis-elements and transacting factors. An intronic splicing enhancer, UGCAUG, is present in triplicate and is situated between two polypyrimidinetract-binding (PTB) sites, TCTT, in the intron downstream of exon 16. In addition, PTB binding sites are also present in triplicate in the upstream intron of exon 16. In this study, we characterized the splicing factors that orchestrate the erythroid differentiation stage-specific switch in exon 16 splicing through these cis-elements using two cell systems: mouse erythroleukemia cells (MELC) that can be induced to erythroid differentiation and G1E-ER cells that undergo synchronous erythroid maturation after induced GATA-1 expression. We identified two RBM9 isoforms (RBM9-1A and RBM9-1F) with distinct amino-termini that interact with the intronic splicing enhancer UGCAUG. The expression of RBM9-1A is erythroid-specific while RBM9-1F can be detected in a wide variety of cell types. Real-time PCR and Western blot analyses showed that RBM9-1A expression is significantly increased while RBM9-1F is reduced during induced erythroid differentiation in both MELC and G1E-ER4 cells. The up-regulation of RBM9-1A correlated with exon 16 inclusion in differentiated cells. Furthermore, the inhibition of RBM9 expression by isoform specific-shRNA reversed 1A enhancing activity, but not that of 1F on exon 16 inclusion in differentiated cells. Thus, exon 16 splicing is mediated by a cell type-specific RBM9 isoform and its up-regulation in late erythroid differentiation is vital for exon 16 splicing. However, over-expression of PTB completely diminished the enhancing effect of RBM9-1A on exon 16 splicing in both differentiated MELC and G1E-ER4 cells, suggesting that PTB plays a role in exon 16 splicing. We analyzed PTB expression and its effect on the exon 16 splicing switch during erythroid differentiation. PTB, a repressive regulator of alternative splicing, binds to the exon 16 upstream and downstream intronic silencers. Its over-expression reduced exon 16 inclusion in both endogenous 4.1R and transfected exon 16 minigenes. Moreover, PTB expression was down-regulated and coincided with increased exon 16 splicing during erythroid differentiation suggesting that regulated expression of repressor PTB mediates exon 16 splicing. Our results further suggest that the differentiation-specific exon 16 splicing switch is achieved by varying the amount of either ubiquitously expressed or cell-type specific activators and inhibitors, and hence the relative efficiency of spliceosome recruitment in the exon inclusion pathway.

Description: Three patients with Ph1 (+), BCR/ABL+ chronic myelogenous leukemia were allografted, two with unrelated compatible placental blood cells and one from an HLA compatible sibling. The three patients engrafted successfully, achieved mixed chimerism and all cleared the BCR/ABL fusion transcript. Despíte the fact that the three patients lost the partial chimerism, they have remained in complete molecular remissions 7 months, fourteen months and five years after the allografts. It is possible that the iatrogenic induction of a transient chimerism and in turn of a transient graft versus leukemia effect, might have a role in the induction of the sustained molecular remission of these three individuals with CML despite the fact that the three patients lost the graft; however other possible explanations can be offered. A longer follow up of the patients is mandatory to further clarify these observations.

Description: Background: In allogeneic BMT patients, the presence of allo-reactive donor CD4+ T cells in the graft were reported to be the primary cause of GvHD. Moreover, donor T-cells are required to promote the stem cell engraftment and to decrease the disease relapse. A number of studies also reported that a subset of CD4+CD25+ T cells usually generated de novo from the thymus that expressed FoxP3 regulate the T cells allo-reactivity in vivo. Thus, to establish a therapeutically useful adoptive T-cells immunotherapy, we depleted the CD4+ T cells from the graft and transplanted along with T cell depleted (TCD) BM cells in clinically relevant parent to F1 experimental allogeneic BMT model. Our hypothesis is that CD4-depleted graft will not cause GvHD, preserve the thymic function, homeostatically produce donor BM-derived CD4+ T cells along with FoxP3+CD4+CD25+ regulatory T cells with beneficial anti-opportunistic infection and anti-tumor effects. Methods: We used a parent (C57BL/6) to (C57BL/6 X BALB/c)CB6F1 allogeneic BMT model with a combination of TCD BM and splenocytes as the hematopoietic graft. CD4+ or CD8+ cells were selectively depleted from the splenocytes of C57BL/6 donor mice using MACS column. 1×106 CD4-depleted splenocytes or a mixture of 2×106 CD8-depleted and 1×106 CD4-depleted splenocytes and/or grafts containing 10×106 unfractionated splenocytes along with 5×106 TCD BM cells harvested from the congeneic C57BL/6 donor mice, were adoptively transferred to lethally irradiated (11Gy) CB6F1 mice. GvHD was monitored twice weekly by weight loss and other clinical signs. After 50 days post transplant recipients mice were bled or sacrificed and lymphocytes isolated from blood and different organs were analyzed by multicolor FACS. Results: Within 50 days of transplant the recipients of CD4-depleted splenocytes had 100% survival without GvHD whereas recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes suffered from severe GvHD (%weight loss below 20%) with 50% survival. Surprisingly, very significantly expansion of total CD4+ T cells (37% ± 7% of lymphocytes, CD4:CD8 ratio 6:1) occurred in the blood of recipients of CD4-depleted splenocytes. In contrast the recipients of mixture of CD4- and CD8-depleted splenocytes DLI or whole splenocytes had only few CD4+ T cells (~2% ± 2% of lymphocytes, CD4:CD8 ratio 1:2). Over 90% of the CD4+ T cells in the blood of recipients of CD4-depleted splenocytes were from the donor BM and included significantly higher number of CD25+CD4+ T cells compared with the recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes. Similarly, significantly increased numbers of FoxP3+CD25+CD4+ regularity T cells were also found in the spleen and thymus of recipients of CD4-depleted splenocytes compared with the recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes (p

Description: The role of the IFN-g in the development of GVHD remains enigmatic. Whereas it has been shown that GVHD can occur in the absence of IFN-g, there are partially contradicting results how IFN-g can modulate GVHD. Thus, it has been suggested that blocking IFN-g might ameliorate gut GVHD in rodent BMT models and its direct cytopathic effects in human ex vivo skin explant models. However, there is also data supporting the notion that IFN-g is important for limiting GVHD by induction of activation induced cell death (AICD) in donor T cells. Furthermore, it has been demonstrated in preclinical BMT models that IFN-g may accelerate or mitigate GVHD depending on the conditioning intensity. We have recently demonstrated that mitigation of GVHD by treatment with the Histone deacetylase Inhibitor (HDACi) suberonylanilide hydroxamic acid (SAHA) is associated with early downregulation of STAT1 phosphorylation in the spleen and host epithelial GVHD target organs. To further understand the role of STAT1 in the development of GVHD we studied STAT1 and p-STAT1 (Tyr701) expression by immunohistochemistry in the GVHD target organs liver, small bowel and colon following induction of GVHD and correlated these findings with the presence of lamina propria (LP) lymphocytes, typical features of GVHD-induced tissue damage (crypt cell apoptosis, crypt regeneration) and expression of tissue cytokines/chemokines. GVHD was induced in the fully MHC mismatched BALB/c to B6 strain combination following lethal irradiation with 975 rad and animals were sacrificed on days +1, +3 and +6. As detected by western blots p-STAT1 expression became detectable on day +1 in the spleen and on days +3 in the liver, small bowel and colon. Compared to untreated controls immunohistochemical p-STAT1 staining became apparent on day +3 post-BMT in the small bowel and colon of syngeneic controls and GVHD animals. In syngeneic controls p-STAT1 expression decreased again on day +6. In contrast, a further significant increase in p-STAT1 staining was observed in animals with GVHD in the colon and small bowel on day +6. In the colon this significant increase in crypt cell p-STAT1 staining was associated with the presence of LP infiltrating lymphocytes and coincided with the maximal features of tissue damage (luminal sloughing, crypt destruction and crypt apoptosis). In line with these results IFN-g protein expression became detectable in colon tissue lysates on day +6 supporting the role of IFN-g producing infiltrating donor T cells in causing STAT1 activation and tissue damage. We conclude that in comparison to untreated controls STAT1 activation can be observed in the colon and small bowel starting on day +3 in animals with GVHD and syngeneic controls. Whereas pSTAT1 staining peaks at day +3 in syngeneic controls and declines thereafter, maximal STAT1 activation occurs in GVHD animals on day +6, coincides with detectable IFN-g expression and is accompanied by LP infiltration and features of severe GVHD-related tissue damage. To fully understand the role of IFN-g in the development of gut GVHD further studies are warranted to delineate the role of STAT1 dependent and independent signaling pathways in the development of GVHD.

Description: UCB is an attractive source for unrelated HSCT of benign indications; however, cell dosage is a critical factor for UCB HSCT. The red cell depletion (RD) and post-thaw wash techniques that are widely used incur significant nucleated cell loss; therefore, two strategies to minimize cell loss are to deplete plasma, but not the red blood cells (PD) during processing and forego post-thaw wash. A retrospective audited analysis was performed on 61 patients with benign disorders who were transplanted with 68 PD UCB units (8 double cords) with 29 thalassemias, 8 AA, 5 WAS, 5 SCID, 2 osteoporosis, 2 sickle cell disease, 2 Hemophagocytic Lymphohistiocytosis, 2 Hurler Syndrome, 1 CGD, 1 Fanconi’s Anemia, 1 Leroy I-Cell Disease, 1 Lymphohistiocytosis, 1 OIMD, and 1 Alpha Mannosidosis. Transplant characteristics: patient median age 4.25 years old (range 0.3–39); median weight 17 kg (range 5–76); male 56%; median # HLA ABDR matches of 5.0 (12–6/6; 19–5/6; 23–4/6; 5–3/6, 1–2/6); median pre-freeze TNC dose 8.1 x 107/kg; median post-thaw TNC dose as reported by TC 7.7 x 107/kg; median pre-freeze CD34 dose 3.1 x 105/kg; transplants outside of U.S.− 32 (52%); non-myeloablative − 9; 44% post-thaw washed (W), 56% infused without post-thaw wash (NW). The Kaplan-Meier estimates of 3-month ANC500 and 6-month platelet 20K and 50K engraftment are 87±5%, 83±6%, and 84±6% respectively. The median time to engraftment for ANC 500, platelet 20K, and 50K are 20 days (range 11–64), 46 days (range 13–153), and 61 days (range 21–171) respectively. No major adverse event was observed in either the W or the NW group, and the median time to engraftment for ANC 500, platelet 20K and 50K for W vs. NW were 21 vs. 19 days, 55 vs. 44.5 days, and 76 vs. 59 days respectively. The incidence of reported grade II–IV acute GVHD was 29%, and 10% had grade III–IV acute GVHD. 33% developed limited chronic GVHD, and 15% developed extensive chronic GVHD. With a median follow-up of 219 days (range 4–1402 days), the Kaplan-Meier estimates of 1-year TRM, OS and disease-free survival were 20±6%, 78±6% and 72±6% respectively. These results demonstrate that HSCT using unrelated PD UCB can be performed safely with outstanding results in patients with benign disorders, and post-thaw washing may delay engraftment of HSCT using PD UCB.

Description: Reduced-intensity conditioning regimens (RIC) had become a classical strategy of allogeneic hematopoietic stem cell transplantation (HSCT) and many patients are now transplanted with unrelated donor. The aim of this restrospective study was to evaluate the impact of HLA mismatches between donor (D) and recipient (R) at the allelic level on survival after RIC. We analyzed 103 patients registered in France from Jan 1999 to Dec 2003 with a median age of 46 years (18–67). All patients had hematologic malignancies: AL (n=35), MM (n=18), CLL (n=5), NHL (n=11), HD (n=9), CML (n=12), MDS (n=9), and MPS (n=4). 39% of the patients were in an advanced phase of the disease at time of HSCT. Anti-thymocytes globulins (ATG) were part of the conditioning regimen for 77% of patients. The main source of stem cells was PBSC (n=65). Seventy-one D/R pairs (69%) were 10/10 HLA match at the allelic level. Mismatches concerned 5, 6, 15, 2 and 7 D/R pairs for HLA-A, -B, -C, -DRB1 and -DQB1, respectively. The results showed that 96% of patients engrafted. Acute GVHD grade II to IV and grade III/IV occurred in 46% and 19% of patients, respectively. The risk of developing cGvHD was 45% at 2 years. Overall survival (OS) was 42% at five years. Among the 47 patients alive, the median disease free survival (DFS) was 28 months. Among non-HLA parameters studied, the only factor associated with a good OS was the diagnosis of lymphoid disease (HD or NHL or CLL) (p=0.003). Recipient age

Description: Curative strategies for hematologic malignancies include alloHSCT, which has become a treatment option for older pts, as well as those with more extensive prior therapy and comorbidities. This has stimulated research on the development of less toxic but comparably effective approaches to transplantation at the level of cytoreduction, alternate graft sources, graft manipulation, and GvHD prophylaxis. To this end, between 7/2001 and 12/2005, we administered TCD HSCTs, derived from HLA-M or HLA-MM URDs, to 36 adult pts as treatment for a variety of hematologic malignancies on a clinical trial. The conditioning regimen was designed to reduce regimen related toxicity while preserving adequate immunosuppression to allow for engraftment. The TCD grafts allowed transplantation across HLA disparate pt-donor pairs without the use of additional renal and hepatotoxic GvHD prophylaxis. The conditioning regimen consisted of hyperfractionated total body irradiation (HFTBI) (1375 cGy), thiotepa (5mg/kg) x 2d, fludarabine (25mg/m2) x 5d and antithymocyte globulin (ATG) x 2d. HLA typing was performed by DNA SSOP analyses for A,B,C, DRB1, and DQB1, and donors were matched at ≥8 of 10 alleles. Pts received TCD-PBSC (n=29) or TCD-BM (n=7) from HLA-M (21 pairs) or HLA-MM (15 pairs) URDs. PBSCs were TCD by Isolex 300i CD34+ selection followed by sheep E-rosette depletion, and BMs were depleted with soybean agglutination and sheep E-rosette depletion. The median age was 40.6 (range 18–63)yrs; 10 pts ≥ 50 yrs. Diseases included AML and ALL CR1 (only standard or high risk), AML and ALL CR2, ALL ≥ CR3, acute biphenotypic leukemia, CML in CP, MDS, T-PLL. The median followup is 22 (range 6–55) mos. All evaluable pts engrafted neutrophils, 31 of 35 evaluable pts engrafted platelets. Four pts died of complications prior to platelet engraftment, including one pt with late graft failure. The 100d non-relapse mortality was 20% with most (〉50%) deaths due to infection. The incidence of acute (a) grade II–III and chronic (c) GvHD was low for the entire group of 36 pts at 11% and 28%, respectively, when compared to that of unmodified transplantation. The incidence of aGvHD was 14% and 7%, and cGvHD (majority - limited) was 26% and 30% for HLA-M and HLA-MM transplant pairs, respectively. Estimated 3 yr DFS is 60% for both HLA-M and HLA-MM transplants, and 83% for standard risk and 41% for high risk disease pts. Only one pt has relapsed. These results indicate that a transplantation strategy using HFTBI, thiotepa, fludarabine and ATG followed by TCD PBSC or BM, but without posttransplant immunomodulating agents, is well-tolerated in an older patient group (median age 40 yrs) even with HLA-MM URDs. Although this approach appears to provide an antileukemic effect for acute leukemia pts transplanted in remission of acute leukemia, this will need to be confirmed with longer followup.

Description: Background. Conventional matching of patients and their unrelated donor (UD) hematopoietic stem cells (HSC) by 4-digit molecular HLA typing is associated with lengthy donor searches and elevated social costs. 80% of UD transplants are performed across DPB1 mismatches which, if involving disparity in host versus graft (HvG) direction for an immunogenic T cell epitope, have been shown to be associated with poor clinical outcome of transplantation for hematopoietic malignancies and beta-thalassemia. In this study we have developed an innovative approach of DPB1 epitope- rather than allele-specific matching, by only two PCR reactions (epitope-specific typing; EST). Moreover, we have determined allelic DPB1 frequencies in Italy, confronted them with the ones previously reported for other ethnic groups, and calculated the probability of finding non-permissive DPB1 mismatches in unrelated HSC donor searches. Methods. High resolution genomic DPB1 typing and EST were performed in parallel on blood samples taken from 112 healthy unrelated Italian blood donors. Results. EST of DPB1 alleles encoding the immunogenic T cell epitope yielded 100% concordant results with high resolution DPB1 typing in all 112 samples studied, and is therefore suitable to univocally determine the presence or absence of non-permissive DPB1 disparities. The overall frequency of DPB1 alleles encoding the shared T cell epitope in the Italian population was 23.15%. Importantly, we show that based on DPB1 allelic polymorphism in the four ethnic groups representative of the world-wide UD registries, over 75% of UD matched for the other HLA loci will not present a DPB1 epitope disparity in HvG direction, demonstrating that prospective UD-recipient DPB1 matching by EST does not significantly limit the number of suitable donors, and has a negligible impact on the time and cost of the search. Conclusions. EST is a challenging alternative to conventional tissue typing which, if applied more broadly to HLA loci other than DPB1, could fundamentally change current approaches to UD searches.

Description: We previously demonstrated that natural killer (NK) cells generated after haploidentical stem-cell transplantation (SCT) are blocked at an immature state characterized by phenotypic features and impaired functioning, a blockage that may affect transplantation outcome (Nguyen et al. Blood 2005). Hypothesizing that the absence of mature donor T cells in the graft may affect NK cell differentiation and function, we examined NK cells from 21 patients who received haploidentical SCT from relatives for advanced malignant hematopoietic disease and underwent either partial (pTCD) (CD3+ in the graft 〉1x105/Kg, mean: 6.9x105/Kg; n=11) or extensive (e-TCD) (CD3+ in the graft

Description: Background: Allogeneic hematopoietic stem cell transplantation (SCT) has been established as a curative therapy for β-thalassemia major which is prevalent among Thais. Donors are usually patients’ siblings but the chance to find an optimal HLA-identical is just about 30%. Thus clinical trial using alternative donors such as partially-mismatched related donors or matched unrelated donors could be a good choice. Methods: To study the efficacy and results, we retrospectively review our experience with β-thalassemia pediatric patients undergoing transplantation from July 1999 to July 2006. Results: There were 43 patients categorized into HLA-identical siblings group (n=26, 13 male and 13 female) and alternative donors group (n=17, 14 male and 3 female). Median age and weight were 7.2 years (1.2–14.8 years), 19.8 kg (9.8–34 kg) and 7.7 years (1.4–14.8 years), 21.5 kg (11–33 kg), respectively. Amongst 26 matched siblings there were 17 bone marrow (BM), 4 peripheral blood stem cell (PBSC), and 5 cord blood (CB) donors. Of the alternative group stem cells derived from 12 matched unrelated, 2 two-antigen mismatched sibling CB, 2 one-antigen mismatched unrelated CB, and 1 one-allele mismatched unrelated BM donors. Myeloablative conditioning regimen consisted of busulfan (oral or intravenous) and cyclophosphamide for matched siblings SCT. Anti-thymocyte globulin was added for alternative donors SCT. Fludarabine was also used in addition for unrelated donors SCT. Graft-vs-host disease (GvHD) prophylaxis consisted of cyclosporine plus methotrexate except for unrelated SCT that tacrolimus plus methotrexate were used instead. Median numbers of infused CD34+ cells of the matched siblings group were 11.1x106/kg (3.7–35.4) in BMT (n=17), 9.3x106/kg (7.1–12.8) in PBSCT (n=4), and 0.94x105/kg (0.2–5.3) in CBT (n=5), compared to 5.5x106/kg (1.5–18.1) in BMT (n=10), 11.1x106/kg (11–17.2) in PBSCT (n=3), and 2.1x105/kg (1.5–3) in CBT (n=4) of the alternative group. Median times to neutrophil and platelet engraftments in each groups were 15 and 33 days, compared to 17 and 41 days, respectively. 39 successful donor engraftments were achieved from 38 HLA-matched (25 related and 13 unrelated) donors and from 1 mismatched unrelated CB unit. The remaining 1 of the matched related group and 3 of the alternative group did not engrafted, all but one case subsequently resumed autologous recovery. Acute GvHD occurred in 4 matched-sibling and 5 unrelated SCT recipients. After immunosuppressive therapy the lesions were completely resolved in all but two patients, one who suffered from severe grade IV intestinal GvHD and another who later developed extensive chronic GvHD which required treatment longer than 4 years. There were 4 mortalities only in the alternative group: 3 patients died post-engraftment due to severe fatal GvHD (n=1), cerebral aspergillosis (n=1), severe veno-occlusive disease with liver failure (n=1; Pesaro class 3); and one died from neutropenic sepsis 3-week after 4/6 matched related CBT. Median follow-up time for surviving patients was 53.5 months (2–84 months). Overall and disease-free survival in HLA-matched SCT (n=38) recipients were 94.7% (n=36) and 92.1% (n=35), compared to in HLA-mismatched (n=5) 60% (n=3) and 20% (n=1), respectively. Conclusions: SCT for β-thalassemia major using HLA-matched donors, either related or unrelated, had superior results than HLA-mismatched. In situation of unavailable matched sibling donors, the search for an appropriate complete HLA-matched unrelated volunteer donor using high resolution DNA typing is essential for favorable outcomes.

Description: The Raf/MEK/ERK signaling module plays a pivotal role in the regulation of cell proliferation, survival, and differentiation. Our group, among others, has recently demonstrated that this pathway is frequently dysregulated in hematological malignancies and may constitute an attractive therapeutic target, particularly in AML. Here we investigated the effects of PD0325901, a novel MEK inhibitor, on phospho-protein expression, gene expression profiles, cell proliferation, and apoptosis in cell line models of AML, ALL, multiple myeloma (MM), ex vivo-cultured primary AML blasts, and oncogene-transformed hematopoietic cells. AML cell lines (OCI-AML2, OCI-AML3, HL-60) were strikingly sensitive to PD0325901 (IC50: 5–19 nM), NB4 (APL) and U266 (MM) showed intermediate sensitivity (IC50: 822 and 724 nM), while all the lymphoid cell lines tested and the myeloid cell lines U937 and KG1 were resistant (IC50 〉 1000 nM). Cell growth inhibition was due to inhibition of cell cycle progression and induction of apoptosis. A statistically significant reduction in the proportion of S-phase cells (p=0.01) and increase in the percentage of apoptotic cells (p=0.019) was also observed in 18 primary AML samples in response to 100 nM PD0325901. Analysis of the correlation between sensitivity/resistance to PD0325901 and Ras/Raf mutation status is currently ongoing. PD0325901 effects were also examined in a panel of IL-3-dependent murine myeloid FDC-P1 cell lines transformed to grow in response to 11 different oncogenes in the absence of IL-3. Fms-, Ras-, Raf-1-, B-Raf-, MEK1-, IGF-1R-, and STAT5a-transformed FDC-P1 cells were very sensitive to PD0325901 (IC50: ~ 1 nM), while A-Raf-, BCR-ABL-, EGFR- or Src-transformed cells were 10 to 100 fold less sensitive (IC50: 10 to 100 nM); the parental, IL-3 dependent FDC-P1 cell line had an IC50 〉 1000 nM. Analysis of the phosphorylation levels of 18 different target proteins after treatment with 10 nM PD0325901 showed a 5- to 8-fold reduction in ERK-1/2, observed only in sensitive cell lines, and a 2-fold reduction in JNK and STAT3 phosphorylation. PD0325901 (10 nM) treatment also profoundly altered the gene expression profile of the sensitive cell line OCI-AML3: 96 genes were modulated after 24 h (37 up- and 59 down-regulated), most of which involved in cell cycle regulation. Changes in cyclin D1 and D3, cyclin E, and cdc 25A were also validated at the protein level. Overall, PD0325901 shows potent growth-inhibitory and pro-apoptotic activity, indicating that MEK may be an appropriate therapeutic target in an array of different hematological malignancies. Further preclinical/clinical development of this compound is warranted, particularly in myeloid leukemias.

Description: Survivin has recently been shown to critically regulate erythroid versus megakaryocytic differentiation in mouse hematopoietic stem cells. Its role in human hematopoiesis has not been fully elucidated yet. To answer the question whether dysregulation of expression of survivin and its protein stabilisator HSP90 contributes to the ineffective erythropoiesis in myelodysplastic syndrome (MDS), we have generated an in vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Cell harvest was at days 0, 4, 7 and 11. Expression analysis of survivin splicing variants (wt, 2a, d3ex) and of HSP90 was done by real time RT-PCR (qPCR) for each lineage at each time point. Furthermore, epigenetic analysis of key regulatory genes by methylation specific PCR and qPCR to detect DNMT1 (maintenance methylation) and DNMT3a and 3b (de novo methylation) expression was performed for each of the experimental conditions. RNA expression of all survivin isoforms and of HSP90 was continously upregulated during normal erythropoiesis. During late megakaryopoiesis of normal hematopoietic stem cells a significant downregulation was seen with elevated expression values for survivin wt and survivin 2b during early thrombopoiesis. In contrast, during MDS erythropoiesis a significant downregulation of expression of all survivin isoforms and HSP90 during late erythropoiesis was observed. Lower expression for survivin wt, survivin 2b and HSP90 was seen during early MDS megakaryopoiesis. Promotor methylation analyisis revealed MDS specific aberrant methylation for survivin, particularly during MDS high risk erythropoiesis (p=0.02). However, expression of DNMT1 was only elevated during erythropoiesis and in low risk MDS. A significant inverse correlation between RNA expression of DNMT enzymes and survivin splicing variants could not be detected. In conclusion our data support the essential role for survivin in regulation of erythropoietic versus megakaryopoietic differentiation of hematopoietic precursor cells. The dysregulation of survivin expression during MDS erythropoiesis may therefore account for the ineffective erythropoiesis known in MDS. Furthermore, downregulation of survivin (protein) stabilisator HSP90 might additionally contribute to this effect. Although an inverse correlation with overexpression of DNMTs and downregulation of survivin splicing variants was not seen, the aberrant survivin promoter methylation during MDS erythropoiesis indicates that epigenetic dysregulation results in low survivin expression.

Description: Activating mutations of the FLT3-receptor tyrosine kinase such as the short internal tandem duplication (ITD) represent the single most common genetic aberration in patients with acute myeloid leukemia (AML). FLT3-ITD mutations are present in about 25% of AML patients and are associated with high blast counts and a higher rate of relapse after conventional chemotherapy. In search for a valid model system for FLT3-ITD AML we greatly improved retroviral gene transfer of the mutated FLT3 receptor into human G-CSF mobilized CD34+ peripheral blood progenitor cells (PBPCs). In the past, retroviral vector production for FLT3 had been hampered by low virus titers related to transgene toxicity in the virus producing cells. Virus vector production in the presence of the FLT3 inhibitor GTP14562 strongly increased the virus vector titer. Subsequent ultracentrifugation and resuspension of the concentrated virus vector in fresh medium enabled us to consistently transduce CD34+ PBSCs to levels of greater than 50%. For individually tested CD34+ PBPC from 5 donors, co-cultivation of FLT3-ITD overexpressing PBPC on the irradiated mouse stromal cell line M210-B4 resulted in a 6 fold increased formation of early cobble stones and a dramatic proliferation advantage for suspended cells compared to naïve, GFP-transduced or FLT3-wildtype transduced PBPCs. The enhanced proliferation of the FLT3-ITD transduced cells was associated with decreased CD34 expression compared to control-transduced cells. Addition of the CXCR4 chemokine inhibitor AMD3100 to the co-culture decreased the cobble stone formation and growth advantage of FLT3-ITD transduced PBPCs. In contrast, cultivating the same FLT3-ITD overexpressing PBPCs in liquid culture (X-VIVO10, 1%HSA, TPO, SCF, FLT3 and IL-3) resulted in a moderate or no growth advantage or in an even reduced cell viability (dependent on the donor PBPCs used), which indirectly highlights the influence of stromal cells on FLT3-ITD overexpressing PBPCs. And in contrast to previously published data from murine cells, addition of the CXCR4 ligand stromal cell derived factor-1 (SDF-1) to liquid cultures did not confer an additional growth advantage for FLT3-ITD positive cells. Using an improved transduction protocol we demonstrate that the robust FLT3-ITD mediated growth advantage for human PBPCs is dependent on co-cultivation on a stromal cell layer and we show that the enhanced proliferation can be partly inhibited by the CXCR4 inhibitor AMD3100. Studies to delineate factors that confer FLT3-ITD specific cell growth on stromal cells are under way.

Description: Experience with unrelated allogeneic UCB stem cell transplantation in the non-myeloablative setting is limited because of concerns of graft failure due to limited cell content. Dual UCB graft infusion has been explored to overcome cell dose limitations, but influence on engraftment is unclear. This single institution phase I trial examined RIC followed by single UCB transplantation for patients unable to tolerate fully myeloablative regimens and lacking a matched adult donor. Conditioning consisted of TBI 200 cGy, fludarabine 175 mg/m², cyclophosphamide 2 gm/m², ATG 60 mg/kg. Trial design specified selection of UCB grafts to be matched at 4/6 HLA loci or better with one unit infused if cryopreserved nucleated cell dose exceeded 2.5 x 107/kg recipient weight. If no adequate single UCB unit to meet these criteria was available, study patients received 2 UCB units containing a combined cell dose exceeding 1.5 x 107/kg. Grafts were analyzed via flow cytometry post thaw for stem cell and lymphocyte expression of surface antigens, among them CD4, CD8, CD7, CD34, CD38, CD45RA, CD45RO. 23 patients have been enrolled, the majority diagnosed with AML (n=17). Median age was 47 (range 25–68) years. Single UCB units meeting stated criteria were identified for all but one study patient, who was excluded from graft analysis. The time to engraftment was measured from the date of transplantation to the date of 3 successive days ANC≥500/μL attained. In addition, we assessed time to peripheral blood donor lymphocyte chimerism ≥ 60%. Patients were censored at the date of last follow-up or the date of death. 94% (95%CI: 77-.99%) of patients achieved ANC≥500/μL by T+36 with median of 26.5 ( range 11–55) days. 68% (95%CI: 48–85%) of patients (n=15) achieved 〉60% donor derived chimerism by median of 22 (range 14–35) days. Median cryopreserved and infused total nucleated cell dose was 2.85 x 107 and 2.5 x 107 cells/kg, respectively. Median infused CD34 cell dose was 1.71 (range 0.21–5.39) x 105 cells/kg. By univariate analysis the dose of infused UCB graft T-cells including CD4 and CD8 T-cells co-expressing CD45RO, and CD34 stem cells co-expressing CD7 and CD38 correlated with neutrophil recovery, p=0.046, 0.008, and 0.033, respectively. Median overall survival at this early interim analysis has not been reached; with 86% and 65% survival at 3 and 24 months, respectively. Event free survival is 78% and 44.5% at 3 and 24 months, respectively. In summary, in this heavily pretreated group of advanced age patients, RIC and single unit unrelated allogeneic UCB stem cell transplantation is safe. Identification of a single UCB graft of HLA match 4/6 meeting cell dose requirement 2.5x107/kg is identified in the vast majority of full size adult patients. Engraftment and survival rates are higher than reported in single UCB transplants following administration of myeloablative regimens and similar to recent reports for RIC with double UCB graft infusion. The potential benefit of infusion of 1 vs. 2 UCB units after RIC in adult patients remains to be fully evaluated.

Description: T-cell depletion of allogeneic hematopoietic stem cell graft (HSCT) represents the most powerful approach to prevent graft-versus-host disease (GvHD), thus allowing to overcome HLA barriers in patients with high risk malignancies, lacking a conventional donor. We hypothesized that early add-back of suicide-gene transduced donor lymphocytes (TK cells) to leukemic patients undergoing haploidentical HSCT (haplo-HSCT) could provide early immune-reconstitution and selective control of GvHD. In a phase II clinical trial (protocol MMTK007), 17 of 29 enrolled pts, (median age 52), received add-backs of 10^7/kg TK cells 42 days after haplo-HSCT. TK cells engraftment, observed in 14 patients, was necessary and sufficient for a rapid and effective immunereconstitution (IR), with a median of 144 (101–336) CD3+, 59 (28–149) CD4+ and 86 (52–279) CD8+ cells/mcl at day 100 after HSCT. Accordingly, engraftment of TK cells was tightly correlated with clinical outcome: while 3/3 pts who failed TK cells engraftment died of infections, only 1/14 TK engrafted patients died from infections (last event at day +166). As shown in Table I, the immune repertoire of treated patients improved significantly at 6 months post transplant and normalized completely in 12 months. High numbers of T cell precursors specific for CMV and EBV were detected at immune reconstitution (median of 86 and 69 gIFN specific spots/10^5 PBMC respectively) and predicted subsequent freedom from viral reactivation (p=0.002). Six pts developed acute (GvHD), (grade I to IV) that was always completely abrogated by the suicide system. Overall survival of TK cells treated patients is 50% at three years. These results indicate that TK-DLI abolish late mortality after CD34+ haplo-SCT in adults. A phase III multicentric study will start in 2007 to validate prospectively the advantage of TK-DLI in haplo-SCT.

Description: Pre-clinical data in monkeys receiving non-myeloablative conditioning followed by MHC-mismatched kidney and bone marrow transplantation show that transient chimerism is sufficient to permit achievement of long-term tolerance to a simultaneous donor renal allograft. Our group has recently reported successful induction of tolerance to donor kidneys in patients with advanced multiple myeloma and renal failure through combined bone marrow and kidney transplantation in the HLA-identical setting. On the basis of these results, five end-stage renal failure patients without malignant disease received simultaneous kidney and bone marrow transplantation from haploidentical HLA mismatched related donor after non-myeloablative conditioning with MEDI-507 (anti-CD2 humanized mAb; MedImmune), cyclophosphamide, thymic irradiation and peritransplant cyclosporine. Transplantation of kidney and bone marrow were both performed on Day 0. All patients developed initial mixed chimerism but lost their chimerism by Day 21. We have analyzed three patients who successfully discontinued immunosuppression on Days +240, +422 and +244. At a follow-up of 47, 38, and 17 months, all three patients are off immunosuppression without allograft rejection. T-cell counts exceeded 100 cells/μL by Day +128, +21, +21, respectively, and a high early prevalence of CD4+CD25high cells was detected. Post-transplant in vitro alloreactivity assays (bulk MLR/CML) showed the development of long-lasting donor-specific unresponsiveness in all three patients. In patients in whom renal tubular epithelial cells (RTEC) were cultured from the donor kidney, no killing of donor RTEC was detected post-transplant. We also assessed alloresponses in chemorefractory lymphoma patients receiving haploidentical bone marrow transplantation with a similar conditioning regimen. In contrast to the recipients of combined kidney/bone marrow transplants, these patients showed sustained global hyporesponsiveness in CML and MLR. However, loss of donor chimerism was associated with the eventual appearance of measurable anti-donor CML and/or MLR responses. In contrast, donor-specific and host-specific unresponsiveness with strong anti-3rd party responses developed in a sustained mixed chimera who received haploidentical stem cell transplantation with a modification of this conditioning protocol (i.e. different dose of MEDI-507, Isolex-selected CD34+ cells from G-CSF mobilized PBMC and the addition of fludarabine). In summary, we have obtained proof of principle that durable multilineage mixed chimerism with donor- and host-specific tolerance can be achieved without GVHD in humans receiving haploidentical HCT. In recipients of combined kidney/bone marrow transplants but not in recipients of bone marrow alone, patients who lose chimerism develop donor-specific unresponsiveness. These studies point to a role for the renal allograft in maintaining long-term tolerance following loss of initial donor chimerism.

Description: Backround: Arsenic trioxide (ATO) has been shown to be synergistic with melphalan both in vitro and in vivo. We conducted a phase I/II trial to determine the safety and efficacy of a combination of arsenic trioxide, melphalan and ascorbic acid (AA) as preparative regimen in patients undergoing high-dose therapy (HDT) and autologous hematopoietic progenitor cell transplantation for multiple myeloma (MM). We also assessed the impact ATO levels on melphalan pharmacokinetics (PK), engraftment and toxicity. Methods: Forty-eight patients with secretory myeloma (23 females, 25 males; median age: 54, range: 3570) were treated between 4/04 and 8/05. All patient received melphalan 100 mg/m2 IV on days -4 and -3 and AA 1000 mg/day IV on days -9 to -3. Patients were randomized to 3 arms; no ATO (arm 1), ATO 0.15 mg/kg IV on days -9 to -3 (arm 2) and ATO 0.25 mg/kg IV on days -9 to -3 (arm 3). Twelve patients had disease progression or relapse after a prior autograft. Median CD34 cells dose infused was 4.5 x 106/kg (range 2.3–10.9). Results: Patients in all 3 arms were evenly matched. With a median F/U of 14.0 months (range 6–25) post autograft, no dose-limiting toxicity or non-relapse mortality was seen. Toxicity was limited to grade I or II nausea, vomiting and diarrhea. Median ATO levels on day 0 in arms 1, 2 and 3 were 0.2, 26.3 and 46.2 ng/ml, respectively. Melphalan PK was not altered by ATO pretreatment. Median time to neutrophil engraftment (ANC 〉500/ dl) was 9 days. There were no engraftment failures or delays in the ATO arms. CR rate for the entire group was 23%, and total response rate (CR + PR) was 75%. 1-year Progression-free survival (PFS) and overall survival (OS) were 75% and 95%, respectively. There was no significant difference in CR, RR, PFS or OS between the 3 arms (p = 0.9, 0.9, 0.4 and 0.6, respectively). A prior autologous transplant (p = 0.02) and abnormal cytogenetics at transplant (p = 0.04) were associated with a significantly shorter remission. Conclusions: ATO + melphalan + ascorbic acid is a safe, effective and well tolerated preparative regimen for patients with multiple myeloma undergoing an autotransplant. A longer follow up is needed to assess the impact of ATO on progression-free and overall survival.

Description: For some patients (pts) with hematologic malignancies (HM) undergoing PBPC transplantation, the approved regimen of 3 daily doses of palifermin 60 μg/kg prior to myeloablative conditioning therapy may be inconvenient. This study was initially developed to demonstrate the non-inferiority of various collapsed pre-conditioning dosing regimens (180 μg/kg) in reducing the incidence of severe OM (WHO grade 3 or 4) induced by fTBI and HD chemotherapy in pts with HM undergoing PBPC transplantation. The study was stopped early because of slower than expected accrual. Methods: Pts were 18 to 74 years old with HM, a Karnofsky performance score ≥70%, and eligible for fTBI (12 Gy) followed by optional VP-16 (60 mg/kg) and cyclophosphamide (100 mg/kg), and autologous PBPC support. Pts were randomized (1:1:1:1) to receive palifermin 60 μg/kg administered once daily for 3 days prior to the start of fTBI (Arm A) versus palifermin 180 μg/kg administered once only on day -1 (Arm B); day -2 (Arm C); or day -3 (Arm D) prior to the start of fTBI and stratified by VP-16 use and by the number of days of TBI fractions. All pts received palifermin 60 μg/kg for 3 days post transplant (days 0, 1, 2). Results are summarized as point estimates. Efficacy was analyzed for all evaluable patients as randomized; sensitivity analyses were done for all evaluable patients as treated. Results: Of the 47 pts randomized, 1 pt was excluded from analyses due to delayed informed consent. The primary efficacy endpoint, incidence of severe OM, is summarized in Table 1. The overall incidence of severe OM for all pts was 61% (28/46), with a mean duration (SD) of 4.3 days (4.4). Severe OM incidences were 82% (9/11) in Arm A and 54% (19/35) in the combined collapsed dose groups (Arms B+C+D). Severe OM rates were 60% (6/10), 31% (4/13), and 75% (9/12) in Arms B, C and D, respectively. A similar distribution of the incidence of severe OM was seen in the sensitivity analysis (Table 1). All arms had a similar incidence of adverse events. Conclusion: The overall incidence and duration of severe OM observed in this trial was consistent with the efficacy data of palifermin demonstrated in the pivotal randomized phase 3 trial (Spielberger et al, NEJM 351:10–17, 2004). The collapsed dosing regimen (180 μg/kg) was well tolerated, and the efficacy of palifermin in the collapsed dose arms is similar to Arm A, possibly allowing for increased patient and caregiver convenience. The small sample sizes limit the ability to compare the individual collapsed dose arms; however, the timing of palifermin collapsed dose administration may have an impact on the efficacy of OM reduction. Incidence of Severe OM Analysis Subseta Arm A Arm B Arm C Arm D aPrimary: Analyzed according to randomized treatment assignment. Sensitivity: Analyzed according to actual treamtent received Primary 9/11 (82%) 6/10 (60%) 4/13 (31%) 9/12 (75%) Sensitivity 7/10 (70%) 8/11 (73%) 5/13 (38%) 8/12 (67%)

Description: Aim: To assess the role of autologous transplantation (AT) compared with chemotherapy (CT) for low risk relapsed childhood acute lymphoblastic leukaemia (ALL). Patients and Methods: Since 1997 at a single Institution, 30 pediatric consecutive patients (pts), lacking a compatible related donor, underwent immunologically purified peripheral stem cell AT, for B cell precursor ALL in second remission (CR2) after late (〉30 ms after diagnosis) or extra-medullary (BM) relapse, belonging to S1–S2 BFM risk group. Since 2000 the positivity of minimal residual disease (MRD) at transplant was considered an exclusion criterium. For each AT patient all possible controls were selected among 236 S1 or S2 pts in CR2 treated with CT in all BFM Centers, matched for: site of relapse, CR1 duration, relapse period and waiting time to transplant. Outcome data are expressed according to the KM estimator for the AT group; a weighted version of the KM estimator is used for the CT group in order to account for the variable proportion of matching; a p-value for the comparison at 4 years (ys) is provided by a permutation test. The role of gender and age at relapse are assessed in a multivariate analysis by a Cox model. The impact of MRD is evaluated. Results: 103 CT controls were selected with a median matching ratio of 4 controls (1–8) for each AT patient. Eight pts in the AT group presented with subsequent relapse at a median of 17 ms (11–48) and 50 in the CT group at a median of 22 ms (5–59) after first relapse; 6 of 8 and 18 of 50 relapsed pts in the two groups underwent allogeneic transplant in CR3 and 4 and 15 of them, respectively, are alive. All events were relapses and no pts died of treatment related complications in CR2. The probability of DFS at 4 ys was 71.3% (SE 8.8) for the AT pts and 44.3% (SE 6.2) for the CT group (p-value: 0.0165) and the probability of survival was 85.8% (SE 6.6) and 65.7% (ES 6.5) (p-value: 0.0385) with a median follow-up of 4.9 ys. The advantage of AT was consistent within the subgroups of late BM and extra-BM relapsed pts. Age and gender did not significantly affect DFS (HR male vs female: 1.82, p-value: 0.08; age 〉10 ys vs

Description: The outcome of patients (pts) with PTCL receiving conventional therapy is dismal. Because of this, there is an increasing interest to investigate intensive treatments in these patients. The aim of this study was to analyze the toxicity, response and outcome of a phase II trial that includes high-dose chemotherapy (CT) plus ASCT as first-line treatment for pts with PTCL. Forty-one pts (30M/11F; median age: 47 yrs.) diagnosed with PTCL (excluding cutaneous and anaplastic ALK+), in stages II-IV and 0.1). Four-year OS was 57% (95%CI: 31–83%) and 71% (95%CI: 37–100%), respectively (p〉0.1). In summary, in this series of patients with PTCL a relatively high CR rate was obtained with high-dose CHOP/ESHAP followed by ASCT. Toxicity was manageable. The contribution of ASCT to pts outcome is debatable because of the absence of significant differences in OS and EFS of patients in CR transplanted vs. those not transplanted. Novel strategies aimed at increasing the CR rate in these patients warrant investigation.

Description: Systemic light-chain amyloidosis (AL) is a protein deposition disorder and a monoclonal plasma cell disease. Treatment of AL has focused on the reduction or elimination of the clonal plasma cells that make amyloid-forming light chains. High-dose melphalan and stem cell transplant (MEL SCT) is an effective therapy for selected AL patients. At 3 months post-SCT, response of the clonal plasma cell disease can be reliably scored; the achievement of a complete response (CR= immunofixation negative) is usually associated with extended survival while no response (NR= 〈 50% reduction) is not. Many factors likely contribute to this distribution of responses. In order to identify factors specific to clonal plasma cells, gene expression profiles (GEP; Affymetrix U133 PLUS 2.0) were obtained using FACS-sorted CD138+/DAPI- plasma cells (〉95% pure) from untreated AL patients before SCT. Supervised analyses were then performed based on responses at 3 months post-SCT, comparing the CR (n=4) and NR (n=5) groups. The basic analysis was a t-test, filtering genes for differential expression at p

Description: NM-HCT from related donors who share at least one HLA haplotype with the recipient is a treatment option for patients with hematologic malignancies who do not have suitable HLA-matched related or unrelated donors. We have shown that addition of cyclophosphamide pre-transplant (29 mg/kg) and post-transplant (50 mg/kg on day+3 or days+3 and +4) to a standard nonmyeloablative conditioning regimen of fludarabine and TBI combined with tacrolimus and MMF prophylaxis of GvHD is an effective means of achieving complete engraftment of donor T-cells and granulocytes with low non-relapse mortality and an incidence and severity of acute and chronic GvHD which does not differ from studies of NM-HCT using HLA-matched donors. In a study of 89 patients (FHCRC,N=30; JH,N=59) with advanced myeloid and lymphoid malignancies, patients with relapsed HL had significantly better survival than patients with other diagnoses. Fifteen patients with HL were studied (median follow-up of 17 mo). Patients were heavily pre-treated with a median number of prior cytotoxic therapy regimens of 5 (range: 3–10); 14 patients had failed prior autologous HCT with a median time of 18 mo from auto-HCT to NM-HCT. Donors for 9 patients were HLA-mismatched at ≥4/10 loci. All evaluable patients were complete donor CD3 and CD33 chimeras by day +28 (one patient died on day +28 and was not evaluable). Clinically significant acute GvHD occurred in 9/14 (64%) patients and Grades III/IV GvHD in 3/14 (21%) patients. Extensive chronic GvHD occurred in 5/14 (36%) patients. Two patients (13%) died of non-relapse causes at days +236 and +633 secondary to chronic GvHD. Median failure-free survival (FFS) was 21 mo compared to 6 mo for patients with other lymphoid malignancies (N=23) or myeloid malignancies [(N=51), see Figure]. The hazard of mortality was less among patients with HL compared to those with other lymphoid malignancies [hazard ratio (HR)=0.36 (p=0.05)] yet patients with myeloid malignancies had a similar hazard of mortality compared to those with lymphoid malignancies other than HL [HR=0.88 (p=0.66)]. Difference in FFS between HL and other lymphoid malignancies was not statistically significant [HR=0.54 (p=0.16)], nor was the difference between myeloid and other lymphoid malignancies [HR=1.26 (p=0.44)]. More patients will need to be studied to better demonstrate a graft-versus-lymphoma effect of haploidentical transplantation in relapsed HL. NM-HCT from haploidentical donors may be a good option for such patients who have a limited window of opportunity to proceed to transplant if responsive to salvage chemotherapy. Figure Figure Figure Figure

Description: Background. We previously reported data from 103 pts receiving PBSC from HLA-matched unrelated donors after conditioning with 2 Gy total body irradiation (TBI) plus 90 mg/m2 fludarabine. Postgrafting immunosuppression included MMF (administered from day 0 until day +40 with taper through day +96), and CSP (given from day -3 to day +100, with taper through day 180) (historical group). The incidences of grade III–IV acute and chronic extensive GVHD were 14% and 48%, respectively. Several studies have suggested that CSP could prevent activation-induced cell death of T-cells, and thus potentially delay the eradication of donor-versus-host alloreactive T-cells, preventing tolerance induction. Conversely, antimetabolite inhibitors, such as MMF, could delete alloreactive T-cells by induction of activation-induced cell death and apoptosis, and thus might favor tolerance induction Pts and Methods. Here, we investigated whether postgrafting immunosuppression with prolonged MMF (given at 15 mg/kg orally thrice a day from day 0 to day +30, then at 15 mg/kg orally twice a day until day 150, and then tapered at day 150 and discontinued at day 180) and truncated CSP (abruptly discontinued on day 80), would promote tolerance induction and reduce the incidence of GVHD (current protocol, n=71) after unrelated HCT with nonmyeloablative conditioning. Results. Sustained donor engraftment was achieved in 68 pts (96%) in the current protocol, versus in 98 pts (95%) in the historical group. Grades II, III and IV acute GVHD were seen in 36 (50.7%), 11 (15.5%) and 7 (9.9%) pts, respectively, in the current protocol, versus 39 (37.9%), 11 (10.7%) and 4 (3.9%) pts, respectively, in the historical group. The incidences of grade II–IV and grade III–IV acute GVHD were 75% and 23% in the current protocol, versus 52% (P=.05) and 14% (P=.14) in the historical group. The 1-yr incidence of chronic GVHD was 46% in the current protocol, versus 48% in the historical group. Finally, the 1-yr probabilities of relapse, nonrelapse mortality and progression-free survival were 24%, 36% and 40%, respectively, in the current protocol, versus 26% (P=.9), 18% (P=.005) and 56% (P=.04) in the historical group. The increased incidence of nonrelapse mortality in the current protocol was not due only to the increased incidence of grade II–IV acute GVHD, since nonrelapse mortality remained significantly higher in the current protocol than in the historical group after adjusting for occurrence of grade II and grade III–IV acute GVHD (P=.04). Evaluation of pretransplant comorbidities is ongoing. Conclusions. Postgrafting immunosuppression with prolonged MMF and truncated CSP failed to decrease the incidence of GVHD after nonmyeloablative conditioning with URD. Ongoing efforts are directed at reducing the risk of acute GVHD after nonmyeloablative conditioning for unrelated donors by replacing tacrolimus for CSP, and by adding sirolimus to MMF and CSP.

Description: Introduction and aim of study. Cyclosporin A (CsA) is commonly used as prophylaxis against Graft versus Host disease (GVHD) after allogeneic haematopoietic stem cell transplantation (SCT). Therapeutic drug monitoring is necessary because of its unpredictable absorption and narrow therapeutic window. Usually dose adjustments are based on trough levels, although these correlate poorly with CsA systemic exposure in adults. The aim of this study was to investigate intravenous and oral CsA pharmacokinetics in children after SCT and to develop a limited sampling strategy in order to determine CsA systemic exposure. Methods. Pharmacokinetics was investigated in children, aged 1.8 to 16.1 yrs, 2 to 43 days after stem cell infusion (median: 25 days), after intravenous or oral administration of CsA in, respectively, 9 and 8 children. Parameter estimation was performed using nonlinear mixed effect modeling as implemented in the NONMEM program. Results and conclusions. Pharmacokinetics were described adequately with a two-compartment model with lag time (population estimates: Cl=10.5 l/h; Vc=15.5 L; Vp=60.6L; t ½ absorption=1.01 h, Tlag=0.6 h). Combination of Cthrough and a Bayesian fitting procedure with the pharmacokinetic model can adequately estimate the systemic exposure to CsA (r2=0.90, Figure 1). The estimation of actual AUC improves when more concentration time-points are used. No relation between body weight and clearance was found (Figure 2). Based on these data, CsA should not be dosed per kilogram body weight. A regimen with a fixed dose for all, adjusted on through levels in combination with the described pharmacokinetic model, may be more appropriate. (open circles: oral administration; closed circles: intravenous administration) Figure Figure Figure Figure

Description: The use of in vivo Alemtuzumab in reduced intensity conditioning (RIC) stem cell transplantation for AML has been reported to be associated with low non-relapse mortality (NRM) and favourable survival outcomes. However, Alemtuzumab depletes the alloreactive donor T cells and recipient antigen presenting cells that mediate graft versus leukaemia (GvL) and graft versus host disease (GvHD). We report the analysis of 90 patients from the British Society for Blood and Marrow Transplantation (BSBMT) registry comparing T-cell replete and Alemtuzumab-containing protocols in HLA-identical sibling RIC transplants for AML. Patient characteristics were: median age at diagnosis-50 years; 46%-male, 54%-female; diagnostic karyotypes according to MRC AML criteria-13% good risk, 76% standard risk, 11% poor risk; 67%-CR1, 24%-CR2, 9-refractory/relapsed disease/PR. Conditioning protocols were: fludarabine/melphalan (66%), fludarabine/busulphan (17%), fludarabine/cyclophosphamide (11%), others (6%). 51 patients (57%) received in vivo Alemtuzumab and 37 patients (41%) did not receive any T-depleting antibodies. 2 patients (2%) received ALG/ATG and were excluded from subsequent analyses comparing the effect of Alemtuzumab with T-replete transplants. The median CD34 cell dose was 4.41x106/kg (0.77–15.8). The actuarial overall survival (OS) and progression-free survival (PFS) at 5 years for all patients were 53% and 47% respectively. The NRM and relapse risk (RR) at 5 years were 16% and 49% respectively. The majority of the relapses were within 2 years of transplant. Acute GvHD was either absent or Grade I in 75 patients (84%). Grade II-IV acute GvHD developed in 15 patients (16%). Extensive chronic GvHD occurred in 18/65 surviving ≥100 days (28%). A complete hematological remission(CR) at transplant predicted for OS at 3 years with 54% survival for CR1, 57% survival for CR≥2 and 0% survival for PR/relapse/refractory disease (p

Description: Allogeneic SCT remains an effective strategy for inducing durable remission in CML. RIC regimens are less myelosuppressive, but adequately immunosuppressive, allowing for engraftment with acceptable treatment-related mortality (TRM) in older pts who otherwise would not be candidates for SCT. The long-term antitumor effect of this approach is not well-established. This is relevant in CML, since many pts present for SCT with advanced disease after failing tyrosine kinase inhibitors (TKI). Patients, Methods: We evaluated outcomes of 64 CML pts (40 M/24 F) with median age 52 yrs (range 18–72) treated from June 1996 to April 2005 with FAI (fludarabine 120 mg/m2, Ara-C 8 gm/m2, idarubicin 36 mg/m2), FM140 (fludarabine 120 mg/m2, melphalan 140 mg/m2 +/− Ara-C 2 gm/m2) or FM180 (fludarabine 120 mg/m2, melphalan 180 mg/m2) and unmanipulated stem cells. Disease stage at time of study entry was first chronic (n=13), second chronic (n=17), accelerated (n=29), or blast phase (n=5), with median time from diagnosis to SCT of 2.6 yrs (range 0.5–20.3). Stem cell source was bone marrow (n=38) or peripheral blood (n=26), and donor type was matched related (n=30), 1 Ag mismatched related (n=4), or matched unrelated (n=30). Graft vs. host disease (GVHD) prophylaxis consisted of tacrolimus and mini-dose methotrexate in all but 6 pts (CSA-based). Anti-thymocyte-globulin was added to all pts other than matched related. Maintenance therapy with TKIs following SCT was not used. Multivariate analysis was done using Cox proportional hazards regression. Results: 22 pts were alive at a median follow up of 7 yrs from SCT (range 0.8–9.8). OS and PFS were 48% and 30%, respectively, at 2 yrs, and 33% and 30%, respectively, at 5 yrs. The cumulative incidence of acute GVHD grades II–IV and III–IV were 31% and 14%, respectively, and chronic GVHD was 32% (22% for extensive). TRM at 100 days, 1-, 2-, and 5- yrs were 2%, 14%, 20%, and 33%, respectively. There was no association between pt age, donor source, preparative regimen, or time to SCT and TRM. Disease recurrence accounted for 12 of 42 deaths. There were 3 cases of graft rejection, with 1 death from graft rejection. Only disease stage at time of SCT was significantly predictive in multivariate analysis for both OS and PFS. Pts with advanced disease had worse OS (HR 2.36, 95%CI 1.25–4.46, p=0.008, see figure) and PFS (HR 1.91, 95%CI 1.05–3.49, p=0.035) than pts in chronic phase. In multivariate for PFS, pts who developed grade I or II acute GVHD were less likely to progress compared to pts who did not develop any GVHD: grade I (HR 0.324, 95%CI 0.13–0.84, p=0.027) and grade II (HR 0.286, 95%CI 0.11–0.78, p=0.014). Conclusion: RIC SCT provides adequate disease control in chronic phase CML pts. The development of some GVHD is protective in this setting. TRM rates are acceptable but continue to increase over time. Alternative treatment strategies need to be explored in pts with accelerated or blast phase disease. Results may be improved with addition of TKI therapy post SCT. Survival by Disease Group Survival by Disease Group

Description: Allogeneic Hematopoietic Cell Transplantation (HCT) can cure hematologic malignancies through beneficial graft-v-leukemia (GVL) allo-immune responses, but its full potential is limited by graft-versus-host disease (GVHD). Recent studies demonstrate allogeneic antibodies develop against mHA encoded on the Y-chromosome, called H-Y antigens develop after sex-mismatch HCT in association with chronic GVHD and persistent disease remission. We hypothesize novel mHA can be serologically identified as targets of allogeneic antibody responses that develop after transplant and were absent pretransplant. Over 70,000 nonsynonymous single nucleotide polymorphisms (nsSNPs) encode polymorphic amino acid residues in human proteins that are potential mHA, and their analysis requires a novel high-throughput approach. ProtoArray™ (Invitrogen) displays five thousand full-length human proteins expressed as N-terminal GST-fusion proteins in a baculovirus system that are affinity purified under native conditions maintaining their cellular enzymatic activities/native conformations. These human antigens are printed in duplicate on nitrocellulose-coated glass slides. In order to determine if targets of allogeneic antibodies can be detected using microarray technology we tested three cGVHD patients for de novo Ab development after HCT using ProtoArrays™. Plasma from three male patients with AML was collected 1) prior to myeloablative HCT 2) from each HLA-identical sibling donor, and 3) one year after HCT. All three had developed extensive chronic GVHD. Plasma was diluted 1:150 and incubated on two microarrays providing replicate results. After washing, each microarray was incubated with anti-human IgG conjugated to Alexa Fluor 647 dye and detected. Negative controls include buffer, BSA, and GST, and their maximum fluorescent signals with these samples were 5,631 units with mean SD of 546. In contrast, influenza A protein is a positive control with fluorescent signal ranging 30,000–45,000 units. Correlation coefficients (R2 values) between duplicate slides ranged from 0.85 to 0.91. Data analysis was performed using Invitrogen’s Prospector Analyzer Software. Fluorescent intensity is measured for each “spot” and individual antigen results are reported as both flourescent signal intensity and a Z-score which is a measure of the intensity of a given signal relative to all of the other human protein targets reported in units of standard deviation. In order to identify human protein targets of allogeneic antibodies, each target antigen’s pretransplant Z-score was subtracted from his respective one year post-transplant Z-score yielding the “ΔZ” for that antigen. Using a conservative ΔZ of 0.1, these three patients developed new antibody responses for 67, 66, and 74 human proteins. Ninety-two percent of these proteins have known nsSNPs. No single protein was recognized by “new” antibodies in all three proteins, however polymorphic proteins Growth Arrest Specific-7 (GAS7), laminin A/C, and ribosomal protein S19 (RPS19) were recognized by two of the three patients after HCT. Results from plasma samples collected 3 and 6 months after HCT demonstrate the progression of alloimmune immune response with few new antibody responses at 3 months. Conclusion: Protein microarrays are an innovative, powerful tool for high-throughput global assessment of B cell alloimmunity after HCT. Microarray technology provides sufficient reproducibility for candidate mHA discovery. These novel mHA require validation by large clinically characterized patient samples.

Description: Constitutive expression of the chimeric NPM/ALK fusion protein encoded by the t(2;5)(p23;q35) is a key oncogenic event in the majority of pediatric anaplastic large cell lymphomas (ALCL). To determine the pathogenetic mechanisms involved in NPM/ALK-mediated lymphomagenesis we employed a mass spectrometry (MS)-based proteomics approach to identify changes in protein expression caused by the overexpression of NPM/ALK. We identified many proteins which were downstream targets of the FRAP/mTOR pathway including ribosomal S6 kinase (1.6-fold), translational initiation factor eIF (4.8-fold), ribosomal protein L11 (4.8-fold), eukaryotic translation initiation factor 3 (3.2-fold), translation initiation factor IF-2 homolog (4.3-fold) and translation initiation factor eIF-2alpha kinase (3.4-fold). The FRAP/mTOR pathway plays a key role in the regulation of cell growth and proliferation and positively regulates translation and ribosome biogenesis and is selectively inhibited by rapamycin. To determine the feasibility of targeting the FRAP/mTOR pathway by rapamycin for treatment of pediatric ALCLs, we determined the prevalence of expression of key proteins in the FRAP/mTOR pathway in pediatric ALCLs and correlated its expression with that of the ALK protein. In addition we determined the in vitro effect of rapamycin on the viability of cell lines derived from t(2;5)-positive ALCLs. We used formalin-fixed paraffin-embedded tissues of ALK-positive ALCLs (n=18) obtained from the Children’s Oncology Group clinical trials (CCG5941 and ANHL0131) and determined the expression of phospho-mTOR, phospho-70S6Kinase and phospho-S6 ribosomal protein using immunohistochemistry. The effect of rapamycin on the viability of cell lines derived from t(2;5)-positive ALCLs was determined by MTT assay and cell cycle analysis. Western blot analysis was performed to determine the effect of rapamycin on cell cycle proteins and apoptosis. Immunohistochemical studies demonstrated diffuse strong nuclear expression of phospho-mTOR in 17/18 cases, and nuclear and cytoplasmic phospho-70S6kinase expression in 15/18 cases. In addition, cytoplasmic expression of phospho-S6 ribosomal protein was observed in 18/18 (100%) of cases. Importantly, the reactive lymphocytes demonstrated negligible expression of all three proteins. Furthermore, rapamycin potently decreased the viability of SUDHL-1 cells (30% reduction by 10nM at 48 hours) and resulted in G1 cell cycle arrest without induction of caspase-3 activity. Western blot analysis demonstrated a reduction in the level of phospho-p70S6Kinase as well as 4EBP-1 levels. Our studies demonstrate overexpression of many proteins in the FRAP/mTOR pathway in NPM/ALK-positive ALCLs. Our data indicate that the majority of pediatric ALCLs express proteins in the FRAP/mTOR pathway and are constitutively activated. Furthermore, our in vitro data support the use of rapamycin as a therapeutic agent in ALK-positive ALCLs.

Description: A monoclonal B-cell Lymphocytosis (MBL) is detected in the peripheral blood of around 3% of otherwise healthy adults, the majority of these have a CLL immunophenotype. We have previously demonstrated that the cells in MBL are indistinguishable from good risk CLL, sharing the same immunophenotypic profile, genetic aberrations and IgVH gene usage. MBL exerts an increasing burden on haematology clinics with over 100 new patients diagnosed per annum in our regional haemato-oncology laboratory. This is largely as a result of the increased tendency to investigate patients with a mild lymphocyosis although

Description: Natural killer (NK) cells are among the first lymphocytes to recover following allogeneic hematopoietic stem cell transplantation (HSCT). Their levels may reflect both homeostatic and inflammatory processes. In this study we analyzed the early reconstitution of NK cells in fifty-one patients with hematologic malignancies after a reduced-intensity regimen consisting of fludarabine and cyclophosphamide and its relationship to the development of acute graft-versus-host disease (aGVHD) and cytomegalovirus (CMV) reactivation. NK cells were severely depleted by the fludarabine-cyclophosphamide conditioning regimen. On the day of the transplant, the NK levels had been reduced in all patients to 0 cells/microliter. Subsequent NK cell recovery was rapid. By two weeks post-transplant NK cells had recovered to median levels comparable those in the original hosts (median= 119 cells/microliter; range, 0–816) and by four weeks increased to a median of 161 cell/microliter (range, 6–701; p〈 0.0001) as compared to pre-transplant levels. By the second month post-transplant the median levels decreased and were maintained at pre-transplant levels. CMV antigenemia developed in 23 of the 51 patients at a median of 30 days post transplant (range, 20–213 days). The median NK cell reconstitution at 2 weeks in the patients who subsequently developed CMV antigenemia was 158 cells/microliter (range, 2–816) and was higher than that among patients without CMV antigenemia (median 71 NK cells/microliter, range 10–228;Wilcoxon rank sum test p = 0.05). Grade I to III aGVHD developed in 31 out of 51 patients (median time = 27 days; range, 11–92 days). The NK cell levels in the patients who developed aGVHD were significantly higher at two weeks post-transplant (median NK = 193 cells/microliter; range, 0–816) than those in the patients who did not (median NK = 66 cells/microliter; range 2–215) (p = 0.005 by Wilcoxon rank sum test). Furthermore, based on a Kaplan-Meier analysis of time to aGVHD as a function of the NK levels at two weeks post-transplant, patients who had NK cell counts greater than 195 cells/microliter at two weeks post transplant had a significantly higher probability of developing aGVHD (p = 0.0036 by log-rank test) Figure 1. These results suggest that the early NK cell reconstitution may serve as a biomarker of transplant outcomes, especially in predicting the development of acute GVHD. Figure Figure

Description: B cells are implicated in the pathophysiology of chronic GVHD, as evidenced by the association of Ab production against minor histocompatibility Ags with chronic GVHD, and rituximab efficacy in chronic GVHD. We hypothesize that anti-B cell therapy beginning 56 days after nonmyeloablative total lymphoid irradiation (TLI) and anti-thymocyte globulin (ATG) transplantation reduces chronic GVHD. Cognizant of possible GVL loss with B cell depletion, we treated patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) because any loss of GVL may be offset by rituximab’s direct anti-B cell tumor efficacy. Patients were conditioned with TLI (80 cGy in 10 fractions, d-11 to d-1) and ATG (1.5mg/kg/day, d-11 to d-7, total 7.5mg/kg). PBSC were infused on day 0. Primary GVHD prophylaxis was MMF from day 0 until d28 for MRD, d100 for URD and cyclosporine (CSA) tapered off by 6 months. To diminish donor B cell alloimmunity, rituximab (375 mg/m2) was infused on days 56, 63, 70, and 77 post-transplant. Fourteen patients have received HCT, and 11 (median age 57, range 46–65 yrs) have received four rituximab infusions (5 fludarabine-refractory, unmutated immunoglobulin heavy-chain variable region gene (IgVH) CLL; 6 MCL). Median follow-up is 290 days (108–381 days). No infusion related toxicities occurred. Transient leukopenia in all patients and frank neutropenia in one patient was noted as early as 30 days post-rituximab. There was one graft loss at day 50 in a patient with MCL progression. Only 3/11 patients had peripheral blood CD3 donor chimerism 〉95% before rituximab infusion day 56, but 6/7 patients achieved full donor chimerism post-rituximab day 180, showing rituximab has no detrimental effect on engraftment. Peripheral blood CD19+ B cells were undetectable in all patients days 90–270, and 2/2 evaluable patients at one year now have CD27+ CD19+ donor B cell reconstitution. Infections post-transplant include early CMV reactivation (5), influenza B (1), aspergillus sinusitis (1), ecthyma gangrenosum (1), VZV (1). All infections resolved. Non-relapse mortality at 100 days and currently is 0%. Four of the 5 unmutated IgVH CLL patients with diffuse lymphadenopathy and marrow involvement by 4-color flow and IgVH quantitation prior to HCT have achieved molecular CR by IgVH PCR. One CLL patient progressed 9 months after HCT and has received DLI. Four MCL patients were transplanted in PR; one achieved CR, 3 progressed before rituximab infusion and 2 subsequently died. The third progressed MCL patient responded to salvage chemotherapy with development of chronic GVHD and achieved CR. No patients developed acute GVHD. One patient developed chronic GI GVHD that resolved with six weeks of steroids. Rituximab infused 56–77 days after TLI-ATG is well tolerated and able to modulate donor B cell reconstitution without detrimental effect on engraftment or infection incidence. No acute GVHD occurred. This novel approach of rituximab infusion two months after TLI-ATG HCT provides safe in vivo peripheral donor B cell depletion with low chronic GVHD incidence thus far. Importantly, GVL is maintained with four CLL and two MCL patients achieving molecular CR after HCT. CLL/MCL Med Age Med f/u MRD/URD GVHD CD3〉95% Donor Chimerism Disease status pre-HCT»last f/u 5/6 57 yrs 290 days 9/2 aGVHD- none cGVHD- one,d257 D56 pre-rituximab: 3/11 pts〉95%; D180: 6/7 pts〉95% CLL: 4 PR»CR; 1PR»PD--DLI; MCL: 1PR»CR; 3PR»PD-2 deaths; 2CR»CR

Description: Relapse of malignancy is frequent in patients transplanted with advanced diseases. There is a relationship between higher Bu exposure and lower risk of relapse, but also higher transplant-related mortality, and data indicated the maximum tolerated daily Bu AUC is 〈 6000 microMol/min when Bu is used in combination with cyclophosphamide (Cy). To reduce transplant-related mortality, Flu has been substituted for Cy in many transplant centers. We hypothesized that pharmacokinetics-targeting of IV Bu, which results in a predictable systemic exposure, would produce values of tolerable Bu AUC that are higher for BuFlu than for BuCy. Our standard HCT regimen since July 2004 consists of once daily IV BuFlu where fludarabine 40 mg/m2 is given intravenously daily for four days and each infusion is followed immediately by an initial IV Bu dose of 130 mg/m2. Pharmacokinetic analysis is performed after the first infusion of busulfan; the goal is to adjust the busulfan doses for days 3 and 4 to achieve an average exposure targeted to an AUC of 4500–5600 microMol/min. Outcomes for the first sixty-nine patients were analyzed. Thirty-nine of 63 (62%) evaluable patients had their Bu doses adjusted, 30 increased [median adjustment 50% (8 – 175%)], and 9 decreased [median 30% (11 – 60%)], while 24 (38%) pts had an AUC within the desired range without adjustment. Most of the patients (98%) had hematological malignancies and 73% had high CIBMTR risk, median age was 48 (22–68) years, donors were HLA-identical siblings (33), matched (23) or mismatched unrelated donors (13). With a median patient follow-up of 392 (248 – 707) days, the non-relapse mortality was 4% at 100 days and 17% at one year. The K-M estimates of survival and event-free survival are 88%±4% and 83%±5% at 100 days, and 61%±6% and 51%±6% at 1 year, respectively. Subsequently, as part of a prospective, targeted AUC-dose finding trial, 20 patients received an initial Bu dose of 170 mg/m2 with subsequent doses targeted to achieve a 4-day average AUC of 5400–6600 microMol/min. Pharmacokinetic analysis was performed after the first and fourth infusion of busulfan. All patients had hematologic malignancies, 65% had high CIBMTR risk, median age was 48 (22 – 61) years, donors were HLA-A, B, C, DRB1, DQB1 matched siblings (11) or matched unrelated donors (9). Thirteen (65%) patients had their doses adjusted, six had it increased [median 37.1% (35.6 – 61.9 %)] and seven decreased [median 41.6% (18.3 – 55.2%)], while seven patients had an AUC within the desired range without adjustment. With a median follow-up of 111 (range 21–312) days, the 100-day K-M estimates of survival are 88%±8% and event-free survival 83%±9%. The complete observation of 100-day safety, relapse and survival will be presented at the meeting and data will determine further AUC escalation.

Description: Maribavir (MBV) is an oral antiviral drug with a unique mechanism of action against cytomegalovirus (CMV). Maribavir is currently in clinical development for use in recipients of stem cell or solid organ transplants, who are vulnerable to renal impairment caused by a variety of drugs or post-transplant complications. Characterization of the pharmacokinetic (PK) profile of maribavir in patients with renal impairment was needed to determine if dose adjustment is indicated. The effect of renal function on the PK of a single 400 mg dose of maribavir was evaluated in adults with normal renal function (creatinine clearance 〉80 mL/min) and adults with renal impairment classified as mild, moderate, or severe, as measured by creatinine clearance 50–80, 30–

Description: The presence of Hepatitis-B virus(HBV) surface antibody(HbsAb) is a strong indicator for the existence of HBV-immunity. Immunosuppressed allografted recipients are at risk of developing HBV hepatitis due to the potential loss of their immunity against HBV. The primary aim of the present study was to evaluate the incidence of HbsAb eradication post allo-transplantation, the potential risk factors and the impact of adoptive immunity transfer in the HbsAb loss. Additionally, to estimate the cumulative incidence of HBV infection in patients (pts) with HbsAb disappearance. Eighty-two immunized recipients aged 27(14–54) years and their donors were retrospectively studied. Fifty-six pts were naturally immunized while 26 were vaccinated. The median follow-up period was 36(6–132) months. Seventy-two were transplanted from siblings (4 with 1 Ag mismatch). Seven donors were matched unrelated volunteers, 2 were twins and 1 was haploidentical. Eighty-one pts received myeloablative conditioning. Marrow (BM) was infused in 23 pts, peripheral stem cells (PBSC) in 58 and BM plus PBSC in 1, with a median number of CD34:4,45x106/Kg, CD3:2,77x108/Kg, CD4:1,16x108/Kg, CD8: 1,06x108/Kg. Antithymocyte globulin (ATG) was administered to 15 and steroids to 54 pts. Thirty pts developed acute and 71 chronic graft versus host disease (cGVHD). Forty-six donors were non-immunized, 19 vaccinated and 13 naturally immunized. Data on immunity origin were missing for 4 donors. HbsAb disappearance was observed in 39/82 pts, 24(6–60) months post transplant with a probability of loss 90% at 5 years. Multivariate analysis revealed as significant risk factors for HbsAb loss the BM graft, ATG administration, age (