ALBERT

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

Description: 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: Abstract 324 Introduction and classification: This is the largest adult T-ALL cohort treated according to immunologic subtypes. All patients were immunophenotyped in one central lab (Berlin). T-ALL (cyCD3+, CD7+) were subclassified into early T-ALL (sCD3-, CD1a-), thymic T-ALL (sCD3-/+, CD1a+) and mature T-ALL (sCD3+, CD1a-). T-ALL constitutes in 3 consecutive GMALL-studies 24% of ALL patients. Patients and methods: A total of 744 T-ALL pts (15 to 55 yrs) were accrued in 102 hospitals in the GMALL studies 05/93, 06/99 and 07/2003. In GMALL 05/93 239 adult T-ALL patients, were treated according to a multi-agent chemoprotocol. Stem cell transplantation (SCT) was not recommended in CR1. In GMALL studies 06/99 and 07/03 505 T-ALL pts received intensified chemotherapy; particularly with introduction of PEG-asparaginase in induction as well as HDMTX/PEG-Asp consolidation cycles. Based on study 05/93 results, SCT from sibling (Sib) as well as matched unrelated (MUD) donor in CR1 was recommended for all patients with early T-ALL, mature T-ALL and for high-risk (HR) pts with thymic T-ALL (defined as late CR, complex karyotype or MRD positivity (MRD+)). Results: T-ALL subtype distribution in the total cohort of 744 adult T-ALL was early-T 23% (N=170), thymic-T 56% (N=420), mature-T 21% (N=154), without any differences between the studies. GMALL Study 05/93: The overall CR rate was 86% (early-T 72%, thymic-T 93%, mature-T 84%. The lower CR rate in early T-ALL was mainly due to early death (19%). The overall CCR rate was 47% (early-T 45%, thymic-T 54%, mature-T 30%). The overall survival rate at 10 yrs for all pts was 47% (early-T 47%, thymic-T 55%, mature-T 25%). GMALL Study 06/99 and 07/03: Of the 505 patients, 87% achieved CR (early-T 84%, thymic-T 92%, mature-T 77%). PR/Failure was higher in early-T (13%) and mature-T (17%) compared to thymic-T (5%). Early death was 4% and equally distributed. 267 pts (64%) received chemotherapy only and the majority were 229 pts (86%) with thymic T-ALL, not considered for SCT in CR1. The CCR rate was 61%. The few early (n = 15) and mature (n = 23) T-ALL pts, which could not have a transplant in CR1, are a negative selection (e.g. early relapse, comorbidity, no donor) and their CCR rate was 33% and 22% respectively. This was due to a high relapse rate in early T-ALL (60%) and mature-T (74%) compared to 33% in thymic-T. Overall survival rate at 8 yrs for thymic T-ALL with chemotherapy was 68%, for the 77 adolescent pts (15 to 25 yrs) even 76%. Stem cell transplantation: 153 T-ALL pts in studies 06/99 and 07/03 received a SCT in first remission. SCT realisation rate in early T-ALL was 84%, in mature-T 68%. Overall CCR rate was 58% (early-T 47%, HR thymic-T 79%, mature-T 61%). Relapse rate after SCT was in early-T 33% and in mature-T 22%. The overall TRM rate was 18% despite more than half MUD SCT, without any TRM difference between the immunological subtypes. Overall survival rate after SCT in CR1 at 8 yrs was 53%, early-T 44%, thymic-T 67%, mature-T 59%. SCT modalit: 49% received alloSib, 55% alloMUD and 20% auto-SCT. Overall CCR rate after alloSib for the total cohort was 65% (early-T 60%, thymic-T 73% and mature-T 69%); for alloMUD total 55% (early-T 45%, thymic-T 77%, mature-T 61%) and for the small cohort of 20 pts with auto-SCT CCR was 35%. Conclusion: The strategy in three consecutive GMALL studies to stratify and treat adult T-ALL pts according to the immunologic T-subtypes was successful. Overall survival at 5 yrs could be improved to 56% from 44%. There was a particular improvement for mature T-ALL (49% vs. 30%) and early-T (40% vs. 33%). This was mainly due to a high realisation rate of SCT in early T-ALL and mature T-ALL and the substantial better results of SCT. Results of alloMUD SCT were comparable to alloSib SCT. The small cohort of HR thymic T-ALL pts also had a benefit from SCT. The excellent outcome of SR thymic T-ALL (∼ 50% of all T-ALL) with the OS of 68% and 76% in adolescents due to intensified chemo, partic. PEG-Asp, does not suggest SCT in CR1. Several molecular markers, such as ERG, BAALC, WT1, had in a retrospective analysis some prognostic relevance in this pt cohort. The new GMALL study generation will however focus in thymic T-ALL on early evaluation of MRD to decide for SCT (MRD+) or not (MRD-) whereas early/mature T-ALL remain allocated to high risk groups with SCT in CR1. Supported by Deutsche Krebshilfe 702657Ho2 and BMBF 01GI9971/8 Disclosures: No relevant conflicts of interest to declare.

Description: The evolutionarily conserved Notch receptors play important roles in cell fate decisions. Ligation of Notch-1 causes differentiation of T/NK cell precursors from HPCs and is critical for development of T cells in the thymic microenvironment. Five known Notch ligands exist in mammals (Delta1, Delta3, Delta4, Jagged1, and Jagged2), and Delta4 in particular has a greater capacity to support T cell development than Delta1. Notch ligation through Delta1 has also been shown to potentiate CD56+ NK cell differentiation from human HPCs in the presence of IL-15, although the phenotype and functionality of these cells has not been extensively described. We compared the ability of the 5 mammalian Notch ligands to induce the development of functional NK cells from human CD34+ HPCs derived from umbilical cord blood (CB). CD34+ cells isolated from CB were cultured in RPMI + 10% FBS on a murine stromal cell line, OP-9, expressing one of the five mammalian Notch receptors (Jagged1, Jagged2, Delta1, Delta3, or Delta4) or OP-9 cells transfected with vector alone, in the presence of IL-7, Flt3 ligand (FL), and IL-15. After three weeks of culture, development of CD56+CD3− cells was greatly accelerated by the ligands Jagged2 (53.4 +/− 5.5% CD56+CD3− cells), Delta-1 (38.6 +/− 5.7%), and Delta-4 (65.0 +/− 3.9%) versus culture in the absence of ligand (17.6 +/−10.3%, p = 〈 0.02) or in the presence of Jagged1 or Delta3. By 5 weeks, the percentage of NK cells seen in cultures containing Jagged2, Delta1, or Delta4 reached 80–90%. These NK cells expressed CD117 but only partially expressed CD94, with positivity ranging from 12.1 to 34.1% of NK cells derived from these 3 ligands after 5 weeks in culture; similarly, few CD16+ NK cells were seen in these cultures (0 to 12.1% of NK cells after 5 weeks). KIR expression in more than 1% of NK cells was not identified under any culture condition. In preliminary experiments, the addition of IL-2 or IL-21, both of which have been shown to induce KIR expression in non-Notch mediated models of NK cell development, did not significantly alter the percentages of NK cells expressing CD94, CD16, or KIR. Because the ligands Jagged2 and Delta4 induced the highest percentages of NK cells in culture, we examined the cytotoxic activity of these cells. NK cells derived from Jagged2 or Delta4 ligation expressed perforin and displayed in vitro cytotoxic activity against the human leukemia cell lines K562 (34.1% or 40.8% target cell lysis, respectively, at an E:T of 10:1) and HL-60 (14.1% or 31.6%, respectively). These cells also produced IFN-gamma, with Delta4 cultures producing higher levels of IFN-gamma versus Jagged2 cultures (1112 vs. 163.9 pg/ml, respectively). These data demonstrate that the Notch ligands vary in their ability to induce differentiation of NK cells from human CD34+ HPCs. Jagged2 and Delta4 in particular have greater capacity to generate functional NK cells which have cytolytic activity and can secrete IFN-gamma, while at the same time lacking a majority of inhibitory NK receptors (KIR and the NKG family of receptors which dimerize with CD94). The generation of cytolytic KIR-negative NK cells is of interest for cellular therapy against malignancies that are susceptible to NK cell killing.

Description: Hemorrhagic cystitis (HC) is a serious complication after hematopoietic stem cell transplantation (HSCT). The pathogenesis of HC in adults is not fully understood and may be influenced by BK virus infection, type of transplant, conditioning regimen, stem cell source, and graft-versus-host disease. Little is known about the development of HC in children after HCST, especially about the association with BK virus infection. Therefore, we retrospectively analyzed the incidence, risk factors and BK virus association of HC in 165 consecutive children (median age, 12 years) who underwent peripheral blood stem cell (n=97; T-cell depleted: n=48) or bone marrow transplantation (n=68) between 1/2000 and 12/2006 in a single center. Fifty nine patients received autologous HSCT and 106 patients underwent allogeneic HSCT. Nineteen of the 165 patients (11.5%) developed HC after a median of 33 days (range, 1–98 days). All 19 patients with HC underwent allogeneic HSCT and showed BK viruria after transplantation. An acute graft-versus-host disease was significantly more frequent in children with HC (P 〈 .001). Significant risk factors in univariate binary logistic analyses were age 〉 12 years (OR, 3.275; P 〈 .031), use of busulfan (OR, 3.514; P 〈 .013), use of busulfan and cyclophosphamide in combination (OR, 4.935; P 〈 .002), and an unrelated donor (OR, 3.309; P 〈 .043). Independent risk factors in multivariate binary logistic analyses were age 〉 12 years and the combination of busulfan and cyclophosphamide. We suggest that cyclophosphamide is toxic to the urinary bladder and busulfan enhances this effect. Furthermore, we analyzed the BK virus load in urine by real-time polymerase chain reaction. We found in patients without HC a significantly increased number of BK virus copies in urine in children older than 12 years (P 〈 .009) and in children who received antithymocyte globulin (P 〈 .001). In addition, BK virus load in urine was significantly increased in children who suffered from HC. Thirteen of 14 children with HC had a BK virus load in urine 〉107 copies/mL (P 〈 .001). We observed in individual BK virus profiles an increase of BK virus copies in urine before the onset of HC. We conclude that HC in children is a disease of multiple etiologies. Allogeneic HSCT, the combination of busulfan and cyclophosphamide, age 〉 12 years, and an unrelated donor are risk factors for the development of HC in childhood. Increased BK virus load in urine of more than 107 copies/mL may lead to HC. Therefore, it is useful to quantify BK virus in urine in those children with above mentioned risk factors to initiate early treatment or to prevent the development of HC.

Description: In 1999 the German Multicenter Study Group for Adult ALL (GMALL) activated a pilot study (GMALL 06/99). One major aim was to develop a new, shortened and intensified induction regimen based on the following new principles compared to previous GMALL trials: 1) Dexamethasone (DEXA) instead of prednisone to improve antileukemic activity and prophylaxis of CNS relapse 2) prephase with cyclophosphamide (CYCLO) 3) G-CSF parallel to chemo 4) intensified daunorubicin with two 2day cycles (DNR) vs 4 wkly applications 5)1 dose PEG-L-Asparaginase (ASP) instead of 14 d conventional ASP Induction I was followed by GMALL induction phase II as previously reported and a uniform consolidation I. Remission control took place on d24 and d44. Thereafter treatment was risk adapted. Induction I consisted of DEXA, CYCLO and G-CSF. In addition pts received PEG-ASP 1000 U/m2 (d13), vincristin 2 mg (d4,11,18) and DNR 45 mg/m2 (d4+5,11+12). The regimen was modified by 3 amendments which separated the study to 4 pilot phases. The major modifications referred to reduction of DEXA/CYCLO and earlier application of G-CSF. Table 1: Major modifications of induction phase I Drug Pilot 1 Pilot 2 Pilot 3 DEXA 40 mg/m2 (d1–3) 10 mg/m2 (d4–17) 10 mg/m2 (d 1–6,11–16) 10 mg/m2 (d 1–5,11–14) CYCLO 200 mg/m2 (d1–3) none none G-CSF from d13 from d4 from d4 Overall 843 pts were included between 4/99 and 10/03. The median age was 36 (15–65) yrs. Subtypes distribution was c-/pre B 65%, pro B 8%, early T 8%, thymic 14%, mature T 6%. 23% had Ph/BCR-ABL+ ALL. The overall CR rate was 83%, with 12% failure/PR and 7% early death (ED). Significant differences were detected for the pilot phases (p=.0008). The high mortality in pilot I was mainly due to infections. With lower doses of DEXA the rate of ED (p=.0002) and severe infections decreased significantly whereas the failure rate increased slightly. The earlier application of G-CSF contributed to a significant decrease of grade III/IV granulocytopenias and probably also mucositis. Table 2: Results and major toxicities (grade III/IV) of induction therapy Pilot 1 Pilot 2 Pilot3 P Evaluable 103 100 605 CR 76% 83% 82% .0008 PR/Failure 9% 9% 14% ED 16% 8% 5% Survival (3y) 45% 47% 47% 〉.05 Granulopenia 84% 72% 69% .008 Median duration 17d 15d 12d

Description: Several conflicting options regarding management of adult ALL are currently discussed. One major issue is the indication for SCT depending on risk factors and age, and the other is the recommendation to use unmodified pediatric protocols for “young” adults. Decision making on SCT is generally based on conventional risk factors – mainly disease characteristics – available at diagnosis, and decision making for “pediatric” chemotherapy on age. It is essential to develop more sophisticated criteria – also to reduce the risk of selection in clinical trials. In order to enhance prognostic models and to better address individual patient characteristics and the course of disease we reanalysed conventional prognostic factors together with new patient specific factors in a large cohort of adult ALL patients. A total of 1657 well characterised pts (15–55 yrs) included in the risk stratified protocols of the German Multicenter Study Group (GMALL) 06/99 and 07/03 was analysed. Treatment and risk stratification have been described (Brüggemann, Blood2006: 107;1116). Age remained a highly significant factor for CR, survival (OS) and disease free survival (DFS). OS ranged from 58% for 15–25 yrs, 52% for 26–35 yrs, 43% for 36–45 yrs to 32% for 45–55 yrs (p=.0001). Poorer outcome with increasing age was mainly due to early death (ED) and death in CR. CR, OS (45% c/pre-B, 45% pro-B, 38% early T, 47% mature T and 64% thymic T;p

Description: Abstract 1361 Poster Board I-385 The expression of Nur77/NR4A1, one member of the Nur Orphan Nuclear Receptor family, is tightly regulated in many cell types, including myeloid and lymphoid lineages, and can change rapidly in response to a variety of external stimuli. Best known for its role in regulating apoptosis by either transcribing lethal genes or by initiating mitochondrial production of cytochrome c, Nur77 may also play other non-apoptotic roles. In an effort to understand the fate and function of cells that upregulate Nur77, we developed a transgenic Nur77-eGFP reporter mouse. Our analysis of cells in the peripheral blood of these mice reveals two populations of circulating cells that express GFP (Nur77) ‘constitutively’: T lymphocytes and a subset of monocytes. Our preliminary data suggest that the GFP+ T cells, which, interestingly are enriched for regulatory T cells, are recent thymic emigrants that have upregulated Nur77 as a consequence of TCR stimulation during maturation. Our observations also indicate that the GFP+ monocytes define a subpopulation of ‘patrolling’ cells that may ultimately differentiate into splenic dendritic cells. Understanding the identity of these circulating GFP+ cells should provide us with insights into the regulation and function of Nur77 itself. The analysis may also clarify the significance of phenotypic differences among blood cell subtypes by providing additional information about cell origin and activity. We thank the Arnold and Mabel Beckman Foundation (HH), HHMI (TB), and NSF (#MCB-0744570 (JP and NC)) for their support for this work. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 344 Hemorrhagic cystitis is a serious complication after hematopoietic stem cell transplantation (HSCT). The pathogenesis of hemorrhagic cystitis is not fully understood and is influenced by BK virus infection, type of transplant, conditioning regimen, stem cell source, and graft-versus-host disease. Nothing is known about human genetic factors and the development of BK virus-associated hemorrhagic cystitis in patients after HSCT. Toll-like receptors (TLR) are essential components of the innate immune system. TLR have been discovered as the most important class of pattern recognition receptors, involved in the host defense against bacteria, viruses, fungi, and protozoa. C3H/HeJ mice that lack functional TLR4 receptors did not develop cystitis after intravesical instillation of E. coli. Individuals with the Asp299Gly polymorphism of the TLR4 gene showed a diminished inflammatory responsiveness to endotoxin. Because of the requirement of TLR4 in inflammatory response we hypothesized that TLR4 Asp299Gly reduces the risk of BK virus-associated hemorrhagic cystitis after hematopoietic stem cell transplantation (HSCT). 166 children (median age, 13 years) with acute lymphoblastic leukemia (n=72), acute myeloid leukemia (n=38), myelodysplastic syndrome (n=21), chronic myeloid leukemia (n=12), genetic disease (n=12) or severe aplastic anemia (n=11) who underwent allogeneic bone marrow (n=105) or peripheral blood stem cell transplantation (n=61; T-cell depleted: n=31) in a single center and their respective donors were genotyped of TLR4 for rs4986790 (A896G, Asp299Gly) using TaqMan real-time polymerase chain reaction. The donor was HLA-matched unrelated in 57% of transplants or HLA-identical related in 33% of transplants. Conditioning regimen was myeloablative in all cases and based on total body irradiation in 48% of transplants or busulfan in 47% of transplants. Two forms of post-transplant immunosuppression predominated, cyclosporine A and methotrexate in 64% of transplants and cyclosporine A alone in 25% of transplants. The Asp299Gly variant was present in 21 of the 166 patients (12.6%) and in 24 of the 166 donors (14.4%). Interestingly, we found a significantly reduced incidence of BK virus-associated hemorrhagic cystitis in patients with the Asp299Gly variant (0% versus 22.8%, p=0.009). In addition, we observed a significantly reduced incidence of BK virus-associated hemorrhagic cystitis in patients who were transplanted from a donor with this specific variant (4.2% versus 22.5%; p=0.05). In ten of the donor-patient pairs the variant was present in both individuals and no hemorrhagic cystitis was observed. The occurrence of the TLR4 Asp299Gly variant, in either recipients or donors, had no significant impact on acute and chronic GVHD, relapse rate, bacterial and fungal infectious complications, transplant related mortality, and overall survival. In conclusion, the TLR4 Asp299Gly variant in the recipient and/or the donor confers strong protection against BK virus-associated hemorrhagic cystitis after HSCT in children. This study provides the first evidence that the innate immune system through TLR4 signaling pathway seems to play an important role in the pathogenesis of BK virus-associated hemorrhagic cystitis after HSCT. Disclosures: No relevant conflicts of interest to declare.

Description: Invasive fungal infections (IFI), in particular infections due to Aspergillus spp and Candida spp, still pose considerable problems in patients undergoing allogeneic stem cell transplantation (SCT). Despite the availability of new antifungal agents, morbidity and mortality of IFI are still unacceptable high. Although neutropenia is known as the single most important risk factor for IFI, there is a growing body of evidence that T cells play a major role in the defense against fungi. Therefore, adoptive immunotherapy with T cells against Candida spp. might be an interesting therapeutic option in patients undergoing allogeneic SCT. After overnight incubation of 1×108 peripheral blood mononuclear cells from 4 healthy individuals with cellular extracts of C.albicans, activated T cells were selected using the IFN-γ secretion-assay (Miltenyi Biotec, Bergisch Gladbach, Germany). After 14 days of culture, T cell clones were generated by limiting dilution and incubated for another 14 days. The median number of cells obtained was 2.6×107 (range, 0.85–5.75×107). Flow cytometry revealed a highly homogenous population of CD3+CD4+ cells (97.2% ± 2.6; n=6), of which an average of 8.6% (range, 4.8–58.2%) produced IFN-gamma on re-stimulation with C.albicans antigens, as assessed by intracellular cytokine staining assay. 20.5% (range, 5.8–72.4%) of the generated cells produced TNF-alpha, whereas no significant number of cells produced TH2 cytokines such as IL-4 and IL-10, indicating that the generated T cell clones were TH1 cells. The percentage of IFN-gamma producing T cells was significant upon stimulation with C.albicans and C.tropicalis, whereas less than 1% of cells produced IFN-gamma upon stimulation with antigens of other yeasts such as C.glabrata, Debaryomyces hansenii and Kluyveromyces lactis and molds such as A.fumigatus, Penicillium chrysogenum and Alternaria alternata. Compared to CD4+ T cells of the original fraction, the isolated and expanded anti-Candida T cells showed reduced alloreactivity, as assessed by means of CSFE. In addition, a strong proliferation of the generated anti-Candida T cells was seen after re-stimulation with C.albicans antigens. The potency of the generated T-cells to damage C.albicans was evaluated using the XTT assay. Compared to polymorphonucelar cells (PMNs), APCs and T-cells alone or to the combination of PMNs with T cells or APCs, respectively, the combination of PMNs, APCs and T-cells showed highest fungal damage (n=4). In conclusion, our data suggest that the isolation and expansion of anti-Candida T cells is possible and feasible. The generated T cells show low alloreactivity in vitro and increase the antimycotic potential of phagocytes. Thus, antimycotic T cells might become an important tool in the prophylaxis and therapy of IFI in patients after allogeneic SCT.