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: 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: Human cytomegalovirus (HCMV) infection remains a major and life threatening infectious complication after allogeneic stem cells transplantation (SCT). We performed a Dendritic cell (DC) vaccination trial by utilizing HCMV peptide loaded mature DC to boost HCMV specific T-cell responses which have been demonstrated to be protective against the development of HCMV disease. DCs were pulsed with nonamer peptides from the HCMV proteins pp65 and pp150, restricted by the HLA-class I elements A1,A2,A3,A11,A68 and B7. We enrolled 24 allogeneic SCT recipients, 6 patients received prophylactic vaccination in view of high risk for HCMV disease. 18 patients were vaccinated therapeutically after HCMV reactivation failed to respond to 4 weeks course of antiviral chemotherapy. Our primary objectives were safety and feasibility of DC vaccination after allogeneic SCT. As all patients with active HCMV infections already received antiviral chemotherapy at the time of DC vaccination, viral load was not a suitable efficacy parameter. Thus, evaluation of efficacy as a secondary objective was based on reconstitution of HCMV-specific CTL responses and long term control of HCMV infection. The study protocol was approved by the local ethical committee and all patients gave written informed consent. DC were generated under GMP conditions and displayed typical surface markers of mature DC (CD1a+/CD14−/CD83+). No local or systemic acute side effects occurred during the first week post vaccination. An observation period of 3 months was determined to evaluate long term side effects and control of HCMV infection. Six patients died during this observation period from other causes and one had no follow-up blood samples, so, 17 patients (5 received prophylactic and 12 received therapeutic vaccination) were evaluable. Only one patient developed Graft-Versus-Host-Disease (GVHD) grade III of the skin and gut. Due to a time lag of 2 months between vaccination and the onset of GVHD, a causative relationship seems to be unlikely. Four of the five patients receiving prophylactic vaccination never showed HCMV reactivation. 10 patients from the therapeutic group cleared their HCMV infection after a mean of 50 days post vaccination. Therefore, 15 of the 17 evaluable patients demonstrated control of HCMV infection after DC vaccination. Among these 15 patients, 10 had detectable specific T-cell response against the vaccine peptides after a mean of 23 days post vaccination. Only 2 from these 10 patients developed a further HCMV reactivation after high dose steroid therapy. Our results show that no relevant side effect was observed in this first DC vaccination trial among allogeneic SCT patients. Additionally, DC are able to induce an efficient peptide specific immune response among allogeneic SCT patients which is capable to protect against HCMV reactivation. In the future, further investigations should be performed to evaluate the feasibility of DC vaccination not only against other infectious complications but also against tumour associated antigens to induce specific T-cell response effectively targeting the particular tumour cell.

Description: Background: Follicular lymphoma (FL) is the second most common type of lymphoma in the United States. The clinical course is variable--some patients have an indolent course while others experience relatively aggressive disease, transformation to higher grade, and short survival. Despite a wide range of treatment options, no consensus exists concerning optimal therapy. Clinical predictors of outcome such as the FL International Prognostic Index (FLIPI) exist; however, biologic risk factors are needed to assist in providing accurate risk stratification. Recent data have suggested that the anti-tumor immune response may affect the clinical course and survival. In this study we sought to identify biologic indicators of prognosis in FL patients using a tissue microarray. Methods: Biopsies from 94 newly diagnosed FL patients who presented between 1985 and 2002 were reviewed for cytologic grade (CG)(Mann-Berard criteria, WHO classification) and presence of diffuse areas (DAs) prior to placement in a tissue microarray (two 1.5 mm cores/case). Cases with diffuse large B-cell components were not included. Immune response was assessed with immunostains for CD3 and CD25 as indices of T-cell infiltration and activation, respectively, and CD68 and CD163 to evaluate macrophage infiltration. Lymphoma cells were evaluated for bcl-2 expression, as well as CD10 and MUM1 expression to assess germinal center phenotype. Initial and subsequent patient management was individualized and varied considerably. Clinical data were obtained for FLIPI and overall survival (OS). Statistical analysis was performed using P ≤ 0.10 as significant for univariable and P ≤ 0.05 for multivariable analysis. Results: The patients consisted of 50 males (53.2%) and 44 females (46.8%), with a median age at diagnosis of 58 (range 24–89) years. The FLIPI distribution was 34 low risk (0–1), 26 intermediate risk (2), and 34 high risk (〉2). There were 31 deaths. The median survival was 174.1 months and the median follow-up among surviving patients was 68.8 months (range 19.5–196.3). Cox proportional hazards analysis for risk of death showed no association with CG, DAs, or expression of bcl-2, CD3 (intrafollicular), CD25, CD68, or CD163. A high FLIPI score and lack of CD10 expression were both of borderline significance for higher risk of death (HR 2.06, 95% CI 0.86–4.92; P = 0.10 and HR 2.8, 95% CI 0.81–9.65; P = 0.10, respectively). MUM1 was expressed in 23 of 87 evaluable cases and was also associated with higher risk of death (HR 2.14, 95% CI 0.97–4.71; P = 0.059). Multivariable analysis showed that only MUM1 expression was associated with a higher risk of death (HR 2.30, 95% CI 1.04–5.12; P = 0.04). Conclusions: These data demonstrate that expression of MUM1 in FL cells, consistent with a late germinal center/post germinal center phenotype, identifies a group of FL patients with poor prognosis. We found no correlation between survival and host immune response, using individual analysis of immunohistochemical markers of T-cell infiltration/activation (CD3 and CD25) or macrophage infiltration (CD68 and CD163). More detailed analysis of molecules involved in the transition of centrocyte to post-germinal center B-cells in FL is warranted.

Description: The prothrombinase complex, composed of the enzyme factor Xa, the cofactor factor Va, and the substrate prothrombin associated on a cell surface in the presence of divalent metal ions, catalyzes the activation of prothrombin to thrombin 300,000-fold more effectively than the enzyme, factor Xa, alone. We have demonstrated that amino acids E323, Y324 and E330, V331 are binding sites for factor Xa on the factor Va heavy chain and are required for coordinating the spatial arrangement of enzyme and substrate directing prothrombin cleavage at two spatially distinct sites. We have also demonstrated that amino acid region 332–336 contains residues that are involved in cofactor function. Peptide studies have identified amino acid residues 334DY335 as major participants in factor Va cofactor activity. We have employed site-directed mutagenesis to study the effect of these amino acids on the catalytic efficiency of prothrombinase. Recombinant factor V molecules with the mutations D334K and Y335F, designated factor VKF, and D334A and Y335A, designated factor VAA were produced, transiently transfected, expressed in COS7L cells, and purified. Kinetic studies demonstrate that while factor VaKF has a KD for factor Xa similar to the KD observed for wild type factor Va, the kcat of prothrombinase assembled with factor VaKF has approximately a 1.5-fold decreased value compared to kcat of prothrombinase assembled with the wild type cofactor molecule. On the contrary, prothrombinase assembled with factor VaAA was found to have a nearly 10-fold decrease kcat, compared to prothrombinase assembled with wild type factor Va. This data suggest that not all amino acid substitutions are well tolerated at positions 334–335. Analysis of the sequence 323–340 using the recently published completed model of coagulation factor Va (pdb entry 1Y61) revealed that amino acids 334–335 are located at the end of a beta-sheet. To ascertain the importance of these mutants and their contribution to cofactor activity we have combined the mutations of amino acids 334–335 with mutations at amino acids 323–324 (E323F, Y324F) and 330–331 (E330M, V331I). We thus created quadruple mutants resulting in recombinant factor VFF/KF, factor VFF/AA, factor VMI/KF and factor VMI/AA. These molecules were transiently expressed in COS-7L cells and studied for their ability to be incorporated into prothrombinase. Free energies associated with the catalytic efficiencies of prothrombinase assembled with each mutant were also calculated (ΔΔGint). The ΔΔGint of interaction for the double mutants, factor VaFF/KF and factor VaMI/KF, had positive values indicating that the side chains of amino acids 330EV331, 323EY324 and 334DY335 located in and around the factor Xa binding site interact in a synergistic manner resulting in the destabilization of the transition state complex and a decelerated rate of catalysis. Conversely, combining the factor Xa binding site mutants with recombinant factor VaAA result in ΔΔGint values of approximately zero. In conclusion, the data demonstrate that replacement of amino acids 334–335 by two hydrophilic residues results in decreased cofactor function. In contrast, replacement of these amino acids by two small hydrophobic residues do not appear to be well tolerated by the cofactor resulting in severely impaired cofactor activity. Altogether, these data demonstrate the importance of amino acid residues D334 and Y335 for the rearrangement of enzyme and substrate required for efficient catalysis.

Description: We have used confocal laser microscopy and a novel “voxel”-based imaging software to study the dynamics of platelet aggregation and thrombus formation when anticoagulated blood was perfused over collagen-coated surfaces at shear rates simulating arterial flow. The objective was to evaluate the three-dimensional growth of platelet thrombi over time (“4-D” imaging). Blood from healthy donors, anticoagulated with either PPACK (80 μM) or, depending on the type of experiment, with trisodium citrate (11 mM), was incubated with mepacrine (10 μM) to render platelets fluorescent. Blood was aspirated with a syringe pump through a rectangular perfusion chamber (flow path height of 80 μm) at a flow rate of 160 or 480 μl per min to provide initial shear rates of 500 or 1,500 sec−1, respectively. Prior to perfusion, glass coverslips were coated with fibrillar type I collagen (Roche Diagnostics, Mannheim, Germany) prepared in 0.5 M acedic acid, pH 2.8, and blocked with 2 % BSA. The chamber was mounted on a Zeiss Axiovert 100M/LSM 510 invert laser scanning confocal microscope (Carl Zeiss, Oberkochem, Germany). Upon perfusion, a series of stacks, i.e. 30 confocal optical sections, from the bottom to the apex of the forming platelet aggregate or thrombus, were obtained every 25 sec with a 488-nm laser and a scanning time of 〈 500 msec on an area of 26,450 μm2. Images corresponding to an area of 0.202 μm2 were analyzed by a “voxel”-based procedure, whereby a voxel is defined by a volume of 0.202 μm3 (0.45 μm x 0.45 μm x 1 μm). For calibration, fluorescent beads (Invitrogen, Carlsbad, CA, USA) were used, and the volume coresponding to a 1.0 μm thick stack was calculated pursuant to the voxel technique. A threshold was applied to distinguish adherent platelets from the background. Using these procedures, a uniform profile of thrombus formation and volume was observed (n=7). With citrate anticoagulated blood at an initial shear rate of 500 sec−1, thrombus growth begun after a lag phase of 220 sec, and, after 420 sec, thrombus volume reached a maximum (mean ± SD, 5x104 ± 4.9x103 μm3). Thrombus progression occurred in a two-step way with an apical growth (height extension) at the interval of 220 and 300 sec, and a further growth in the plane section at the interval of 300 and 420 sec after perfusion. Prolonged perfusion resulted in markedly abnormal flow pattern due to thrombus growth and increased shear rates. Again at an initial shear rate of 500 sec−1, platelet aggregate formation and thrombus progression were completely suppressed in the presence of anti-αIIbβ3 antibody (abciximab, 4 μg/ml). Interestingly, the polymorphism (HPA-1, PlA) of the β subunit of αIIbβ3 had a dramatic effect on thrombus growth. Thus, when comparing blood from homozygous carriers of HPA-1b (n=8) and HPA-1a (n=8), thrombus formation and progression occurred more rapidly with HPA-1b than with HPA-1a platelets, resulting in significantly larger thrombi from HPA-1b than from HPA-1a individuals (p=0.001). In conclusion, the voxel-based determination of thrombus formation and progression in vitro provides an appropriate technique to assess volumina of thrombi. Moreover, this technique can detect phenotypic differences related to an αIIbβ3 polymorphism which is postulated to modulate platelet thrombogenicity.

Description: Increased understanding of the local injection site infiltrate in response to tumor vaccines may facilitate more effective anti-cancer vaccination strategies. A pilot vaccination strategy was developed to determine if K562/GM-CSF immunotherapy could enhance T cell reactivity and clinical responses in CML patients undergoing therapy with imatinib mesylate. K562/GM-CSF is a tumor vaccine derived from a CML cell line that expresses several defined CML associated antigens and has been genetically modified to secrete GM-CSF. We undertook a correlative project comparing the cellular infiltrates from pre- and post-vaccination skin biopsies in this immunotherapy trial. GM-CSF producing tumor vaccines are effective in recruiting antigen presenting cells (APCs), however alone they are insufficient to initiate APC maturation. Because topical imiquimod (a Toll-like receptor 7 agonist) is known to enhance the in vivo maturation of the recruited APCs, the vaccines (1 x 10*8 cells distributed over 10 injection sites) were given with or without topical 5% imiquimod cream. Imiquimod was applied 4 hrs post-vaccination and then 3d and 5d post-vaccination to injection sites, with at least 1 site left without imiquimod treatment. A series of 4 vaccines were administered in 3 wk intervals. Six mm punch biopsies were taken at baseline, and 3d following the 1st and 4th vaccination. Biopsies were performed at the imiquimod site with the largest area of induration, as well as a site not exposed to imiquimod. Immunohistochemistry of CD3, CD4, CD8, CD1a (Langerhans cell (LC)), factor XIIIa (dermal dendritic cell), and CD68 (monocyte/macrophage) and Geimsa staining was performed. Staining is reported as number of live cells per mm2 in the epidermis and dermis for CD1a+ cells and dermis for the remaining stains. Fifteen subjects agreed to the procedures as part of the clinical trial. Mean area of induration of imiquimod sites was increased significantly compared to the non-imiquimod sites after both the first (p=0.005) and fourth vaccinations (p=0.068). Geimsa staining revealed significant increases in proportion of neutrophils, eosinophils, and mononuclear cells to total number of staining cells after the 1st vaccination in the sites treated with imiquimod compared to the pre-vaccination biopsies while the increases at the sites without imiquimod treatment did not reach statistical significance. We observed increases in CD3+, CD4+, and CD8+ cells at post-vaccination sites. Interestingly, the total number of CD1a+ LCs in the area measured did not appear to be affected by the administration of imiquimod and was constant after vaccinations. However, distribution of CD1a+ LCs shifted from the epidermis to the dermis after vaccination. In addition we observed recruitment of factor XIIIa+ dermal dendritic cells and CD68+ macrophages to the vaccination site that was increased by imiquimod. Epidermal APCs known as LCs migrate to the dermis, yet maintain homeostasis of the total LCs following vaccination independent of Imiquimod application. A correlation across subjects between these histologic features and clinical response to vaccination is on-going.

Description: Because cell lines can be adapted to proteasome inhibition in vitro (Pfeifer G. et al., Science 283: 978–981, 1999.), secondary resistance of malignant cells towards Bortezomib (Velcade®) in vivo is a likely scenario. We have succeeded to adapt the human AML cell line HL-60 to Bortezomib in vitro, so that the adapted subline (HL-60a) shows normal viability and growth rate at 40 nM Velcade®, while cell death is induced above 10nM in the parental line. We hypothesized that alternative proteolytic pathways might allow the continued proliferation and survival of Bortezomib-adapted cells, and assessed the activity-profiles of proteasomal subunits, ubiquitin-specific proteases and lysosomal cysteine proteases in a functional proteomics approach, using recently developed synthetic affinity probes. These tools for the first time allow semiquantitative visualization of the individual members of these protease families, based on their activity, in contrast to western blot which lacks activity information or to the turnover of fluorogenic substrates, which is not truly protease-specific. After 72h of culture in Bortezomib-free medium (wash out phase), HL-60a cells contained significantly reduced levels of active proteasomal β1,β2, and β5 subunits, compared to the parental line, as confirmed by a reduced turnover of the β5-selective fluorogenic substrate Suc-LLVY-AMC. A panel of 7 different ubiquitin-specific proteases (USP) was visualized in both types of cells, using the affinity probe HAUbVS. Of these, a 97 kD USP that we have identified as USP14 by mass spectrometry-based sequencing, was significantly upregulated in HL-60a cells. By contrast, the activity of lysosomal cysteine proteases remained unchanged in HL-60a cells. Because the cytosolic protease tripeptidyl peptidase II (TPPII) can partially substitute for proteasome activity in lactacystine-treated cell lines, we assessed TPPII-activity using a fluorogenic substrate. We observed a significant upregulation of TPPII-activity in HL-60a cells, compared to non-adapted controls. Inhibition of TPPII, however, was not sufficient to restore Bortezomib-sensitivity in HL-60a cells. Interestingly, the combination of Bortezomib with the HIV protease inhibitor Ritonavir induced synergistic cytotoxicity both in HL-60 and HL-60a cells. Thus, Bortezomib-resistance is accompabied by upregulation of protease activities in alternative pathways. Combining different protease inhibitors like Bortezomib and Ritonavir might be a promising option to overcome primary or secondary Bortezomib resistance.