ALBERT

Description: The landscape of chronic myeloid leukemia (CML) has radically changed since the introduction of tyrosine kinase inhibitor (TKI), imatinib (IM), now considered as standard therapy. Although excellent cytogenetic responses are obtained, minimal residual disease still persists in a proportion of patients (pts) when measured by serial molecular monitoring by quantitative real-time polymerase chain reaction (RQ-PCR) to measure BCR-ABL transcript levels (Baccarani M et al. Blood2006; 108:1809–20). We monitored BCR-ABL transcript levels by RQ-PCR in 176 chronic phase (CP) –complete cytogenetic response (CCyR) CML pts treated with IM. Median follow-up from start of therapy with IM was 35 (6–80) months. Pts were recruited from 33 centers in Argentina and 2 in Uruguay. Median follow up from the first assessment at our Institution was 18 (6–32) months. Seventy nine patients (45%) had received interferon as 1st line prior to IM and 97/176 (55%) pts received imatinib as 1st line. Eighty eight percent (155/176) pts had received IM 400mg/d and 12% (21/176) 600–800mg at study initiation. Fifty four percent presented with low Sokal score at diagnosis. Peripheral blood samples were tested by RQPCR every 6 months. Major molecular response (MMR) was defined as BCR-ABL/ABL ratio of

Description: Genetic modification of dendritic-cell (DC) function is an attractive approach to treat disease, either using mature DCs (mDCs) to immunize patients, or immature DCs (iDCs) to induce tolerance. Viral vectors are efficient at transducing DCs, and we have investigated the effect of transduction with a variety of viral vectors on the phenotype and function of DCs. Adenovirus (Ad), human immunodeficiency virus (HIV), equine anemia virus (EIAV), and Moloney murine leukemia virus (MMLV) all up-regulate costimulatory molecules and major histocompatibility complex (MHC) class II expression on DCs, as well as, in the case of Ad and lentiviral vectors, inducing production of Th1 and proinflammatory cytokines. Following transduction there is activation of double-stranded (ds) RNA-triggered pathways resulting in interferon (IFN) α/β production. In addition, the function of virally infected DCs is altered; iDCs have an increased, and mDCs a decreased, ability to stimulate a mixed lymphocyte reaction (MLR). Viral transduction of mDCs results in up-regulation of the indoleamine 2,3-dioxygenase (IDO) enzyme, which down-regulates T-cell responsiveness. Inhibition of IDO restores the ability of mDCs to stimulate an MLR, indicating that IDO is responsible for the modulation of mDC function. These data have important implications for the use of viral vectors in the transduction of DCs.

Description: We demonstrate that blockade of the MEK/ERK signaling module, using the small-molecule inhibitors PD184352 or PD325901 (PD), strikingly enhances arsenic trioxide (ATO)–induced cytotoxicity in human myeloma cell lines (HMCLs) and in tumor cells from patients with multiple myeloma (MM) through a caspase-dependent mechanism. In HMCLs retaining a functional p53, PD treatment greatly enhances the ATO-induced p53 accumulation and p73, a p53 paralog, cooperates with p53 in caspase activation and apoptosis induction. In HMCLs carrying a nonfunctional p53, cotreatment with PD strikingly elevates the (DR4 + DR5)/(DcR1 + DcR2) tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) receptors ratio and caspase-8 activation of ATO-treated cells. In MM cells, irrespective of p53 status, the combined PD/ATO treatment increases the level of the proapoptotic protein Bim (PD-mediated) and decreases antiapoptotic protein Mcl-1 (ATO-mediated). Moreover, Bim physically interacts with both DR4 and DR5 TRAIL receptors in PD/ATO-treated cells, and loss of Bim interferes with the activation of both extrinsic and intrinsic apoptotic pathways in response to PD/ATO. Finally, PD/ATO treatment induces tumor regression, prolongs survival, and is well tolerated in vivo in a human plasmacytoma xenograft model. These preclinical studies provide the framework for testing PD325901 and ATO combination therapy in clinical trials aimed to improve patient outcome in MM.

Description: Abstract 2177 Poster Board II-154 Resistance toward Imatinib and other Bcr-Abl tyrosine kinase inhibitors remains an increasing clinical problem in the treatment of advanced stages of chronic myeloid leukemia. Thus novel therapeutic strategies are needed to address the emerging problem of Imatinib resistance. Previous preclinical studies reported that the MEK inhibitors PD184352 or PD0325901 (Pfizer), strikingly enhances ATO-mediated apoptosis in Acute Myelogenous Leukemia and in Multiple myeloma. The aim of this study was to investigate whether the combined treatment with PD184352 (PD) and ATO has cytotoxic effects on murine Ba/F3 cells expressing wild-type (wt) or various imatinib-resistant mutant forms of Bcr-Abl, including T315I. We first analyzed the pharmacologic interactions between PD and ATO using a fixed-ratio experimental design in Bcr-Abl Ba/F3p210wt, Ba/F3p210T315I and Ba/F3p210Y253F cell lines and found that the combined treatment with PD plus ATO resulted in the synergistic induction of apoptosis in all cell lines tested (Chou-Talalay method): the averaged Combination Index values calculated from the ED50 (50% effective dose), ED75 and ED90, in PD plus ATO treated cells were 0.72± 0.19, 0.61± 0.04 and 0.69± 0.09 in BCR-ABL Ba/F3p210wt, Ba/F3p210T315I, and Ba/F3p210Y253F respectively. Synergistic interaction between ATO and PD0325901, a derivative of PD184352, was demonstrated in all tested cell lines. In order to investigate the molecular effectors involved in PD/ATO-induced apoptosis we first evaluated its effects on Bcr-Abl protein expression and CrkL phosphorylation, a well-known downstream target of Bcr-Abl. Immunoblotting analyses demonstrated that treatment for 24 to 48 hours of Ba/F3p210wt and Ba/F3p210T315I cells with PD (1μM) or ATO (1μM) alone or in combination had no effects on levels of total Bcr-Abl or phospho—CrkL thereby indicating that the combination PD/ATO does not act via Bcr-Abl oncogenic signaling. Therefore, we studied whether p53 and the p53-related gene p73 are molecular targets of the combined treatment in Ba/F3p210wt and Ba/F3p210T315I cell lines. We found that monotreatment with neither PD nor ATO 1 μM (or their combination) was able to induce p53 accumulation, whereas the combination PD/ATO promoted the accumulation of the proapoptotic and antiproliferative TA-p73α protein and reduced the levels of the antiapoptotic and proproliferative dominant-negative ΔN-p73α in both cell lines. Consistent with these results, we found that PD greatly enhanced the ATO-induced Puma expression, mitochondrial depolarization, caspase-3 activation, and apoptosis in Ba/F3p210wt and Ba/F3p210T315I cells and functional knock-out of p73 gene expression by small interfering (si)RNAs significantly reduced (P〈 .05 Dunnett test) the PD/ATO induced mitochondrial depolarization. To determine whether the PD plus ATO efficacy observed in vitro for BaF3 cells expressing mutant forms of Bcr-Abl was recapitulated in vivo, we studied PD/ATO combination in a mouse model of Imatinib-resistant, Bcr-Abl—dependent disease. Severe combined immunodeficient mice were injected intravenously with Ba/F3 cells expressing Bcr-Abl-T315I isoform. Mice with Bcr-Abl-T315I—induced leukemia were treated with the MEK inhibitor PD0325901 (10 mg/kg; orally) plus ATO (3.75 mg/kg; intraperitoneally) or Imatinib (50 mg/kg, twice daily; intraperitoneally) or vehicle for three weeks. Untreated or Imatinib-treated mice harboring the T315I isoform developed aggressive disease, with massive liver and splenic infiltration, typically resulting in death in 32 days. However, mice harboring the T315I isoform showed significantly prolonged survival when treated with PD/ATO (43 days, P=.001, Kaplan-Meier method and compared using the log-rank test). Moreover, histopathological analysis of 20 days Imatinib-treated mice revealed infiltration of the liver and spleen. In contrast, histopathological analysis of organs from PD/ATO-treated mice demonstrated normal tissue architecture. Consistent with these results immumoblottig analysis of the lysates from livers and spleens revealed a marked expression of Bcr-Abl protein in mice treated with Imatinib or vehicle. Our preclinical in vitro and in vivo studies suggest that a strategy combining ATO with disruption of MEK pathway could represent an effective therapeutic strategy for the treatment of Imatinib-resistant Bcr-Abl-positive leukemias, including those harboring the T315I mutation. Disclosures: No relevant conflicts of interest to declare.

Description: Background. There are known prognostic factors in Acute myeloid leukemia (AML) patients, being the cytogenetic analysis the strongest as single predictor of disease relapse or poor therapy response. Recently, alterations in FLT3 gene (Internal tandem duplications-ITD and D835/836 mutations) are frequently detected by PCR in 30–35% of AML patients (pts) and would be associated with aggressive disease. This study reports the molecular characterization of 82 AML pts from Argentina and Uruguay, mostly of Spanish-Italian origin, studied between 1996 to 2005. Design and Methods. This study was based on 82 pts: 71 adults, median age 36 yrs, (range 25–80) and 11 children (median age: 11 yrs, range. 3–17 yrs). Cytogenetic risk was established in 77 pts by kariotyping, PCR and FISH: 49% (n=38) with low risk, 38% (n=29) with standard risk, and 13% (n=10)with high risk. The FAB distribution (n=75) was: M0=2,7% (n=2), M1= 6,7% (n=5); M2=17,3% (n=13); M3=46,7% (n=35); M4=16% (n=12); M5=6,7% (n=5); M6=4% (n=3). Clinical endpoints and follow up were available for 45 pts and 56 pts, respectively. A total of 42 pts achieved complete remission (CR), 12 pts had relapse of disease, 10 pts underwent early death without completing induction (ED pts), and 7 pts died after treatment. Prognostic factors considered were: Age 〉 55 years, WBC average, WBC 〉 100 x 106/L, % Blasts in bone marrow, Secondary etiology (therapy related/MDS). JM and TKD domain coding sequences were amplified by PCR for characterization of ITD and D835/836 mutations, respectively. Results and Interpretation. FLT3 mutations could be demonstrated in 23% (19/82 pts): ITD =16% (13/82), D835/836 =7% (6/82). The median follow-up time was 36 months (range 1 – 96 m). A total of 48% (n=27) of pts. were still alive without relapse at the end of this study. Higher incidence of Flt3 mutations [ITD+ and D835/836+] were found in: 41,7% (n=5) of pts with no achievement of CR (n=12) Vs 7,1% (n=3) pts with CR (n=42) (p=0.01), and in 38% (n=5) of the death patients group (n=13) Vs. 7,4% (n=2) pts still alive without relapse (n=27) (p=0,027).The WBC average was significantly higher in the ITD+ group (69,38x106/L) Vs ITD(−) group (9,27x106/L) (p=0.001). ITD mutation was more frequent in pts with WBC 〉100x106/L (83,3%) Vs WBC 100.106/L. Both type of FLT3 mutations (ITD and D835/836) were associated with early death in the cohort. This colaborative study showed that FLT3 mutational status had to be considered as important tool in prognosis of AML pts, however further follow up with larger number of pts is required to fully address its association with poor clinical outcome.

Description: Physiological concentrations of NaCl inhibit the hydrolysis of von Willebrand Factor (VWF) by ADAMTS-13. This effect is linked to the specific binding of chloride ions to VWF. Urea-induced unfolding was measured in the presence of NaCl, CH3COONa and NaClO4 at pH 8.0, 25°C, for multimeric VWF, the recombinant A1-A2-A3 VWF domains and the A1 domain. Chloride stabilizes the folded conformation of the A1-A2-A3 and A1 domains more efficiently than acetate but less strongly than perchlorate. Spectrofluorometric studies showed that chloride binds to both the A1 and A1-A2 domain, but not to the isolated A2 domain. Binding of Cl− to both wild type (WT) and the natural mutant (2B VWD) p.R1306W A1-A2-A3 domains of VWF has a large heat capacity change equal to −1.0 and −0.4 Kcal mol-1 K-1 for WT, and p.R1306W A1-A2-A3 domains, respectively. This result implies that a burial of a vast apolar surface area is caused by conformational transitions linked to chloride binding. At any temperature, chloride affinity was higher for WT than for the mutant p.R1306W form. Chloride ions inhibit hydrolysis by ADAMTS-13 of the A1-A2-A3 and A1-A2 domains in the presence of either urea or high shear stress (40 dyn/cm2), while this effect was either absent or negligible in experiments using A2 and A2-A3 domains. Steady-state kinetic experiments showed that chloride ions inhibit allosterically the cleavage by ADAMTS-13 of the WT A1-A2-A3, whereas this effect was significantly reduced in p.R1306W form, as a consequence of the reduced chloride affinity (Figure 1). On the whole, these findings showed the existence of a conformational linkage between the A1 and A2 domains in the VWF molecule. The p.R1306W natural mutant, that has an higher affinity for GpIb, bears a mutation which stabilizes a conformation of the A1 domain in a GpIb-bound like state and reduces at the same time the A1 domain affinity for chloride. These findings suggest that in some type 2B VWD forms chloride ions bind with lower affinity and the rate of hydrolysis by ADAMTS-13 increases. This can contribute, along with the enhanced binding of high molecular weight VWF multimers to platelet GpIb molecules, to the depletion of these VWF forms and to the hemorrhagic diathesis usually observed in these patients. Figure 1 Linkage graph showing the kcat/Km values of hydrolysis of WT A1-A2-A3 (•), p. R1306 W(○) WT A1-A2 (□) isolated A2 domain (•), and A2-A3 domains (▿). The continuous lines were drawn to eq 4 with the best - fit parameter values k+ = 7.26 ± 0.2 × 104 M−1 sec−1 kCI = 1.23 ± 0.3 × 104M−1 sec −1, Kd = 35 ± 5 mM (WT A1-A2-A3); k+ = 1.36 ± 0.1 × 105 M−1 sec −1 kCI = 3 ± 0.5 × 104 M−1 sec−1 , kd = 158 ± 20 mM (p R1306W), k+ 7.47 ± 0.3 × 104 M−1 sec−1 kCI = 1.38 ± 0.3 × 104 M−1 sec−1, Kd = 36.2 ± 8 mM(A1-A2). No significative NaCl effect was observed for both isolated A2 and A2 -A3 domains Figure 1. Linkage graph showing the kcat/Km values of hydrolysis of WT A1-A2-A3 (•), p. R1306 W(○) WT A1-A2 (□) isolated A2 domain (•), and A2-A3 domains (▿). The continuous lines were drawn to eq 4 with the best - fit parameter values k+ = 7.26 ± 0.2 × 104 M−1 sec−1 kCI = 1.23 ± 0.3 × 104M−1 sec −1, Kd = 35 ± 5 mM (WT A1-A2-A3); k+ = 1.36 ± 0.1 × 105 M−1 sec −1 kCI = 3 ± 0.5 × 104 M−1 sec−1 , kd = 158 ± 20 mM (p R1306W), k+ 7.47 ± 0.3 × 104 M−1 sec−1 kCI = 1.38 ± 0.3 × 104 M−1 sec−1, Kd = 36.2 ± 8 mM(A1-A2). No significative NaCl effect was observed for both isolated A2 and A2 -A3 domains

Description: Karyotypic instability is strongly associated with multiple myeloma (MM). According to the chromosome number pattern, two major groups are recognized: hyperdiploid (H) tumors, associated with recurrent trisomies involving non-random chromosomes (3, 5, 7, 9, 11, 15, 19 and 21); and non hyperdiploid (NH) tumors associated with hypodiploid, pseudodiploid or near-tetraploid karyotypes. MM patients are approximately equally distributed between the two categories; notably, the most recurrent IGH translocations and chromosome 13 deletion appear to be prevalently associated with NH-MM, whereas recent evidences have suggested that H-MM correlates with a favorable prognosis. To molecularly characterize these two genetic categories, we performed a gene expression profiling analysis on 66 newly-diagnosed MM, characterized by FISH analyses for IGH translocations, 13q14 deletions and additional copies of chromosomes 1, 11 and 19. The ploidy status was investigated by combining two recently proposed FISH approaches (Wuilleme S. et al., 2004; Chng W.J. et al., 2005). The gene expression profiles of highly purified MM plasma cells have been generated by means of high-density oligonucleotide arrays (Affymetrix GeneChip U133A) and subsequently analyzed using unsupervised and supervised approaches (two-dimensional hierarchical clustering and SAM, respectively). The differential expression of 229 genes distinguished the 28 H-MM from the 38 NH-MM cases. The 208 upregulated genes in H-MM mapped mainly on the chromosomes involved in hyperdiploidy, while a significant percentage (29%) of the 21 genes upregulated in NH-MM were localized on 16q. The identified transcripts have been further validated on a publicly available gene expression dataset of an independent cohort of 64 MM patients (Carrasco et al., 2005). Notably, the global classification rate for the 64 cases resulted of 81%, confirming the validity of the identified transcriptional fingerprint. A functional analysis revealed a significant fraction of genes involved in protein biosynthesis (38%), transcriptional machinery and oxidative phosphorylation. Furthermore, an integrative genomic approach using a model-free statistical method (LAP, locally adaptive statistical procedure) supported these findings, allowing the identification in H-MM of globally upregulated regions on the chromosomes 3, 5, 9, 15 and 19, along with the downregulation of a region on 16q arm. Remarkably, two sub-groups are clearly distinguishable within H-MM group, one associated with chromosome 11 gain and the other showing 1q gain and chromosome 13 deletion. A supervised analysis of the H-11 vs H-13/1 patients identified 57 differentially expressed genes. Eleven of the 18 genes up-regulated in the H-11 group mapped to chromosome 11, whereas 21 of the 39 genes up-regulated in the H-13/1 group mapped to the 1q region. Notably, CCND2 resulted the most significantly upregulated gene in H-13/1 group. Our data reinforce the importance of combining cytogenetics and gene expression approaches for a better definition of the genetic alterations in MM and provide a molecular and genomic framework for dissection of disease pathogenesis and clinical management.

Description: Abstract 3044 Poster Board II-1020 ADAMTS-13 cleaves high molecular weight von Willebrand factor (VWF) realeased by the endothelium in order to prevent massive intravascular platelets adhesion and aggregation as pathologically observed in thrombotic thrombocytopenic purpura. ADAMTS-13 is present at low levels in plasma. We therefore surmised that platelets are able to specifically bind the metalloprotease on their surface, hereby concentrating the enzyme where it is most required. Immunofluorescent studies were then performed to see whether or not ADAMTS-13 bound to the platelet plasma membrane using as primary antibody a murine anti-human ADAMTS-13 monoclonal antibody (13E2). A positive membrane fluorescent signal was detected using as a source of platelets either a normal donor or a patient with type III Von Willebrand disease, demonstrating that ADAMTS-13 is located on the platelet surface independently from VWF. This results were confirmed by flow cytometry analysis. In all 10 normal individuals tested 13E2 binding to not-permeabilized platelets was detected with a FITC antimurine secondary antibody. With this as background, 96 wells polystyrene NUNC plates were coated either with albumin or fibrinogen or VWF or recombinant ADAMTS-13 (each at 10 μg/ml) and then blocked with 5% albumin overnight. Washed platelets (100,000/μl), incubated with divalent cations, were then let adhere to the wells for 1 hr at 37°C. After extensive washing adherent platelets were lysed, p-nitrophenilphosphate was added and the reaction was stopped with NaOH 2M. Detection was done by assessing optical density at 405 nm. Binding of washed platelets preincubated with 2mM CaCl2 and/or 2mM MgCl2 to wells covered with immobilized recombinant ADAMTS-13 was significantly higher than binding to wells coated with albumin (p

Description: Accelerated osteoclastogenesis is the major event promoting the skeletal impairment in multiple myeloma (MM). Osteoclasts (OC) are directly activated by myeloma cells (MC), although these cells themselves may apparently undergo to OC-like morphologic transformation and produce bone erosion in vitro. Since OCs exert their function and promote osteoclastogenesis through activation of several adhesion molecules, including avb3, we investigated the role of this integrin expressed by MCs in their OC-like activity in vitro. Bone marrow MCs were purified from eight patients with severe skeletal disease (group A) and from two patients without bone lesions (group B). U266 and RPMI-8226 MC lines were the controls. Semi-nested PCR assessed the CDR3 immunoglobulin (Ig) gene rearrangement, whereas OC markers including TRAcP, cathepsin-k, calcitonin-receptor, carbonic anhydrase and vATPase were evaluated by RT-PCR. The cytoskeletal rearrangement of F-actin was analyzed by immunofluorescence. av and b3 expression on MCs was evaluated by flow-cytometry, whereas bone erosion on calcium phosphate discs and number of pits was measured by dedicated software. The effect of avb3 stimulation on the activation of osteoclastogenic function was investigated by exploring the phosphorilation of transcriptional kinases and downstream molecules, as ERK1/2 and cFos, respectively. The primary role of avb3 in OC-like functional transdifferentiation was explored in MCs by siRNA silencing for both chains. Ontogenetic derivation from the B-cell lineage was confirmed by the monoclonal CDR3 rearrangement, CD138/CD38 and Pax-5 expression. Cells from patients of group A expressed OC markers, in contrast with those of group B or U266 and RPMI-8226. Formation of the F-actin ring confirmed the differentiation of MCs toward the OC-like phenotype. Cells from group A expressed av and b3 (80±7% and 75±9%) similarly to U266 and RPMI-8226 (〉90% in both instances), whereas a minimal expression was demonstrated in group B (av:6±2%; b3:8±3%). avb3+ cells produced a high number of erosive pits, at variance from avb3− cells (35±8 vs. 4±1 pits/cm2 ). The highest phosphorilation of ERK1/2 and expression of cFos was revealed in patients of group A as compared to B (840±110 OD and 905±210 OD vs. 270±35 OD and 315±80 OD, p

Description: Foxp3+ regulatory T cells (Tregs) play a central role in maintaining immune tolerance. A reduction in the function of Tregs is a key feature of autoimmune diseases, whereas their expansion in malignant diseases leads to the suppression of host antitumor responses. We analyzed the absolute number of CD4+ and CD8+ Tregs in the peripheral blood of 52 patients with myelodysplastic syndrome (MDS) and show a significant correlation between increased number of CD4+ Tregs and MDS subgroups with 5% or more bone marrow blasts (P 〈 .001), high International Prognostic Scoring System (IPSS) score (P 〈 .001), and disease progression (P 〈 .001), whereas no correlation between CD8+ Tregs and prognostic variables was observed. The CD4+ Tregs showed a polyclonal spectratype, and the percentage of the naive subset was significantly higher in the high-risk patients compared with low-risk or healthy age-matched donors (P = .032). Our data suggest that CD4+ Treg expansion is a feature of high-risk MDS and progression to aggressive subtypes of the disease.