ALBERT

Description: In this study five monoclonal antibodies (MoAbs) to T-cell receptor (TCR) proteins (WT31, alpha F1, beta F1, TCR delta-1 and delta TCS-1) were used to identify discrete maturative stages in 40 cases of T-cell acute lymphoblastic leukemia (T-ALL). These MoAbs reacted exclusively with CD3+ T cells and did not label B-lineage and myeloid cells. In 17 of the 40 T-ALL cases studied the leukemic blasts lacked membrane and cytoplasmic TCR chains (group I). In 12 cases cells did not have membrane CD3/TCR but expressed cytoplasmic TCR proteins heterogenously: nine cases had cytoplasmic TCR beta chains (beta F1+, alpha F1-; group II), one case had cytoplasmic TCR alpha chains (alpha F1+, beta F1-; group III), and two cases were labeled by both alpha F1 and beta F1 MoAbs (group IV). The remaining 11 cases were mCD3+: nine were TCR alpha beta+ (group Va) and two exhibited TCR gamma delta (TCR delta-1+, delta TCS-1+; group Vb). The analysis of the TCR beta, -gamma, and - delta gene configurations in 23 of the 40 T-ALLs showed that: (1) the lack of TCR protein expression was due to the lack of TCR gene rearrangements only in one of nine cases; (2) five of five TCR beta+, TCR alpha- cases studied had germline TCR alpha genes (ie, no detectable TCR delta gene deletions); (3) seven of eight cases with TCR delta gene deletions expressed TCR alpha proteins, whereas in 12 of 20 of the T-ALLs with TCR beta gene rearrangements the synthesis of the corresponding protein occurred; only 2 of 16 cases with rearranged TCR delta genes expressed TCR delta chains. The T-ALL categories identified with anti-TCR MoAbs did not have additional characteristic phenotypic patterns and may correspond to the normal stages of T-cell development more precisely than those defined by other differentiation antigens.

Description: B-Chronic lymphocytic leukemia (B-CLL) patients whose malignant cells harbour unmutated immunoglobulin heavy chain variable region (IgVH) genes or express the zeta-associated protein tyrosine kinase ZAP-70 show a worse prognosis than do patients with mutated IgVH genes or ZAP-70−ve expression. The inability of malignant cells to activate the pro-apoptotic p53 pathway in response to ionizing radiation (IR) also correlates with a poor prognosis. We studied ZAP-70 expression and IgVH mutation status in 161 patients with B-CLL in order to determine the degree of concordance between these two prognostic criteria (M104/F57, wbc 2.44–576x109/l lymphocytes 0.56–287x109/l). We also studied the functional status of the p53 pathway and the apoptotic response to ionizing radiation in cells from a subset of patients from both prognostic categories. A human ZAP-70 antibody (clone 2F3-2) was conjugated to the Alexa Fluor 488 dye using a zenon mouse IgG labelling kit and used for a FACS based assay. FACS results were expressed as a ratio of B-cell mean cell fluorescence to T-cell mean cell fluorescence with a cut off at 〉 0.75 identifying a ZAP-70+ve sub-group. IgVH mutational status was studied by sequence analysis of FR1/JH polymerase chain reaction products. The ability of 5Gy ionizing radiation to augment levels of p53 and its transcriptional target p21CIP1 was quantified by western blot analysis. Cleavage of the caspase 3 target poly(ADP ribose) polymerase (PARP) was used as a measure of apoptosis induction. ZAP-70+ve expression was observed in 25% (41/161) of the samples with a median ratio of 0.85 (range 0.76–1.46) while the remaining 120 samples were ZAP-70−ve, with a median ratio of 0.56 (range 0.19–0.73). IgVH mutation status was analysed in 92 of these patients. Assignment of prognostic category by both criteria was concordant in 72/92 (78.2%) of the cases of which 54/92 (58.6%) were ZAP−ve/IgVH mutated (good prognosis) and 18/92 (19.5%) were ZAP+ve/IgVH unmutated (poor prognosis) patients. The remaining 21.7% were discordant, ie., either ZAP+ve/IgVH mutated (5.4%) or ZAP−ve/IgVH unmutated (16.3%). Isolates from 5/6 ZAP+ve/IgVH unmutated patients upregulated p53 in response to IR but nevertheless failed to initiate PARP cleavage, suggestive of a block in the apoptotic pathway distal to p53 induction. In 9 ZAP−ve/IgVH mutated isolates studied, 7 induced p53, p21 and PARP cleavage following IR. In conclusion, this large cohort of CLL patients demonstrated a good correlation between ZAP-70 expression and IgVH mutational status in identifying a poor prognosis sub-group. However, this prognostic category, as defined by both IgVH mutation status and ZAP-70 expression failed in some cases to predict the ability of B-CLL cells to induce an apoptotic response to DNA damage in vitro. Induction of the p53 pathway was not always sufficient to secure an apoptotic response, especially in the poor prognosis group. A combination of ZAP-70 and IgVH analysis with a functional assay for DNA damage-induced apoptosis will identify individuals in either prognostic category who are unlikely to respond to conventional cytotoxic drugs. Alternative therapeutic strategies independent of DNA damage-inducing agents may be of value in the treatment of these patients.

Description: B-cell chronic lymphocytic leukaemia (CLL) is a heterogeneous disease with a variable clinical course. The disease is characterised by the proliferation in the bone marrow and lymph node of a clonal population of CD5+ve cells that accumulates in the peripheral blood. Therefore, the characteristics of the proliferative compartment are important in determining the kinetics of disease progression in CLL and the sensitivity of the malignant cells to cytotoxic drugs. However, laboratory studies on drug sensitivity of CLL have been performed exclusively on resting circulating peripheral blood cells since it is not feasible to obtain cells from the proliferating pool in sufficient numbers for in vitro analysis. CLL cells can be stimulated to proliferate in vitro using CpG oligonucleotides (ODN) and other factors. The aim of the present study was to generate and validate an in vitro model using malignant cells from the peripheral blood of patients with CLL. The expression pattern of proteins eg., survivin in this model should mimic that in proliferating CLL cells in the bone marrow and lymph nodes. Survivin is a member of the family of inhibitor of apoptosis (IAP) proteins with an additional role in cell cycle progression. Survivin has been shown to be expressed in proliferating bone marrow and lymphoid cells. Cells from patients with CLL were activated for 72h with a combination of ODN (1μM), IL-2 (100u/ml) and CD40L (0.5μg/ml) (ODN*). Activated cells retained their characteristic CLL immunophenotype as determined by the continued expression of CD5, CD19, CD23 and CD25 (n=5). Cell proliferation was confirmed by increased incorporation of 3H-thymidine into DNA in activated cells (n=12). Novel findings in the ODN* activated CLL cells were significant increases in expression of CD38 (n=7, p=0.0001) and of T-cell zeta associated protein (ZAP-70) tyrosine kinase (n=14, p=0.0005). The increased expression of both these proteins in circulating peripheral blood CLL cells has been associated with poor prognosis. All six ODN* activated CLL isolates analysed by western blotting showed increased survivin expression with no constitutive expression in the controls. Drug sensitivity was studied in cells from eight patients using the MTT assay. Activated cells showed significantly greater resistance to chlorambucil (median IC50=164.4±28.18μM) compared to control cells (median IC50=93.63±14.96μM, p=0.044). Figure 1 shows representative IC50 curves. The increased resistance of the activated cells to chlorambucil may be a consequence of the upregulation of survivin. In summary, the in vitro model replicates several key features of authentic proliferating CLL cells found in bone marrow and lymph nodes. It also shows increased resistance to the conventional drug chlorambucil. This model may be of value in evaluating novel drugs and drug combinations which may be more effective in killing the proliferating population that maintain the malignant cell population in CLL. Figure Figure

Description: Abstract 3214 BACKGROUND Type 1 Gaucher disease (GD1) is a chronic, multisystem disease that varies considerably among individuals with regard to organ involvement, presentation, severity, and progression rate. Because hematologic signs and symptoms are common (e.g., anemia, thrombocytopenia, splenomegaly, and hepatomegaly), treatment is often guided by a hematologist. OBJECTIVE To describe the safety and tolerability of velaglucerase alfa after 15 months of treatment in an ongoing, open-label extension study (HGT-GCB-044 [ClinicalTrials.gov identifier, NCT00635427]) in GD1 patients who received either velaglucerase alfa or imiglucerase in a preceding 9-month, double-blind, randomized, Phase III trial (HGT-GCB-039 [NCT00553631]). METHODS In HGT-GCB-039, treatment-naïve GD1 patients aged ≥2 years were randomized to velaglucerase alfa or imiglucerase as a continuous 60-minute intravenous infusion (60 U/kg body weight every other week [EOW]; 9 months). HGT-GCB-039 completers could enroll in extension study HGT-GCB-044, receiving velaglucerase alfa (60 U/kg EOW; ongoing). Safety and tolerability were assessed after 15 months of participation in HGT-GCB-044 (i.e., after a total of 2 years of enzyme replacement therapy). RESULTS Of 35 patients enrolled in HGT-GCB-039, 34 received study drug and 32 completed the trial (1 velaglucerase alfa patient was lost to follow-up after a serious adverse event [AE] of life-threatening convulsion, which was considered unrelated to treatment; 1 imiglucerase patient withdrew consent because of AEs). This analysis included only the 32 patients completing HGT-GCB-039 who enrolled in the HGT-GCB-044 extension phase: 16 had received velaglucerase alfa (median age, 38 years [range, 8–60 years]; 50% [n=8] male; 56% [n=9] splenectomized) and 16 had received imiglucerase (median age, 27 years [range, 4–59 years]; 44% [n=7] male; 63% [n=10] splenectomized). These patients had received velaglucerase alfa for 24 months (continuous velaglucerase alfa group) or imiglucerase for 9 months + velaglucerase alfa for 15 months (imiglucerase-switch group) at the time of the current analysis. No differences were observed between the safety profiles of the continuous velaglucerase alfa and imiglucerase-switch treatment arms (Table). AEs most commonly reported (≥20% of patients) during the extension phase were nasopharyngitis and arthralgia in the continuous velaglucerase alfa group and headache and upper respiratory tract infection in the imiglucerase-switch group. Most AEs were mild or moderate in severity and, apart from the patient who was lost to follow-up in HGT-GCB-039 after a convulsion, there were no other life-threatening AEs. None of the serious AEs were considered drug related. The proportion of patients with infusion-related AEs remained low in HGT-GCB-044. Anti-imiglucerase antibodies developed in 4 patients receiving imiglucerase in trial HGT-GCB-039, 1 of whom discontinued following multiple infusion-related AEs and did not enter HGT-GCB-044. This patient also had anti-velaglucerase alfa antibodies, which was attributed to assay cross-reactivity, as he had never been exposed to velaglucerase alfa. No patients developed anti-velaglucerase alfa antibodies during the first 9 or subsequent 15 months of drug exposure. CONCLUSIONS Velaglucerase alfa was generally well tolerated in both the continuous velaglucerase alfa and imiglucerase-switch arms of these controlled clinical trials. Although 4 patients had anti-imiglucerase antibodies in trial HGT-GCB-039, no patient has developed anti-velaglucerase alfa antibodies over the course of the trials to date. Disclosures: Mehta: Shire HGT: Consultancy. Giraldo:Shire HGT: Consultancy. Kisinovsky:Shire Argentina: Consultancy. Kishnani:Shire HGT: Honoraria; Genzyme: Honoraria; Pfizer: Honoraria. Barton:Shire HGT: Employment. Wang:Shire HGT: Employment. Crombez:Shire HGT: Employment. Bhirangi:Shire HGT: Employment. Zimran:Shire HGT: Consultancy; Protalix: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Genzyme: Research Funding; Actelion: Honoraria; Pfizer: Honoraria.

Description: In this study five monoclonal antibodies (MoAbs) to T-cell receptor (TCR) proteins (WT31, alpha F1, beta F1, TCR delta-1 and delta TCS-1) were used to identify discrete maturative stages in 40 cases of T-cell acute lymphoblastic leukemia (T-ALL). These MoAbs reacted exclusively with CD3+ T cells and did not label B-lineage and myeloid cells. In 17 of the 40 T-ALL cases studied the leukemic blasts lacked membrane and cytoplasmic TCR chains (group I). In 12 cases cells did not have membrane CD3/TCR but expressed cytoplasmic TCR proteins heterogenously: nine cases had cytoplasmic TCR beta chains (beta F1+, alpha F1-; group II), one case had cytoplasmic TCR alpha chains (alpha F1+, beta F1-; group III), and two cases were labeled by both alpha F1 and beta F1 MoAbs (group IV). The remaining 11 cases were mCD3+: nine were TCR alpha beta+ (group Va) and two exhibited TCR gamma delta (TCR delta-1+, delta TCS-1+; group Vb). The analysis of the TCR beta, -gamma, and - delta gene configurations in 23 of the 40 T-ALLs showed that: (1) the lack of TCR protein expression was due to the lack of TCR gene rearrangements only in one of nine cases; (2) five of five TCR beta+, TCR alpha- cases studied had germline TCR alpha genes (ie, no detectable TCR delta gene deletions); (3) seven of eight cases with TCR delta gene deletions expressed TCR alpha proteins, whereas in 12 of 20 of the T-ALLs with TCR beta gene rearrangements the synthesis of the corresponding protein occurred; only 2 of 16 cases with rearranged TCR delta genes expressed TCR delta chains. The T-ALL categories identified with anti-TCR MoAbs did not have additional characteristic phenotypic patterns and may correspond to the normal stages of T-cell development more precisely than those defined by other differentiation antigens.

Description: A proportion of haematological malignancies over express the multidrug resistance (MDR) efflux proteins - P-glycoprotein (P-gp), multidrug resistance protein (MRP1) and lung resistance proteins (LRP) that confer resistance to a number of structurally unrelated cytotoxic drugs. The clinical significance of the observed over expression of P-gp in B-CLL regardless of stage or treatment of the disease is unclear, although a protective effect on cell survival has been reported. Recent evidence suggest that a sub-group of B-CLL exist with an aggressive clinical course that is often resistant to treatment. B-CLL cells from this subgroup of patients have unmutated immunoglobulin heavy chain variable region (IgVH) and express zeta-associated protein (ZAP-70). We analysed the association between the expression of MDR proteins and the prognosis indicator ZAP-70 in cells from 46 B-CLL patients (30M/16F, 33 stage A, 7 B, 6 C) for (i) ZAP-70 expression using an antibody (clone 2F3.2)-Alexa Fluor 488 conjugate by flow cytometry and expressed as a ratio of B to T-cell mean cell fluorescence, (ii) the expression of P-gp, MRP1 and LRP protein by flow cytometry using specific antibodies and expressed as MCF ratio to isotype controls, (iii) flow cytometric analysis of functional activity of P-gp expressed as MCF ratio in the presence and absence of verapamil, a known inhibitor of P-gp, (iv) IC50 (μg/ml) for cytotoxic drugs 2-chlorodeoxyadenosine (CdA), chlorambucil (Chl) and fludarabine (FdR) using the cytotoxicity MTT assay and (v) IgVH mutational status by sequence analysis of FR1/JH polymerase chain reaction products in 29/46 (63%) of patients. 13/46 (28%) patients were ZAP-70+ve and the remaining 33/46 (72%) were ZAP-70−ve. P-gp expression (median 1.59 in both groups p=0.85) and P-gp functional activity (median 2.01 vs 1.84 p=0.77) were not statistically different between ZAP-70+ve and ZAP-70−ve groups. There was no significant difference in the expressions of MRP1 (median 2.77 vs 3.48 p=0.31) or LRP (median 4.87 vs 5.18 p=0.56) between ZAP-70+ve and ZAP-70−ve patients. The IC50 levels for CdA, Chl and FdR between ZAP+ve (median 0.04, 6.4 & 0.54mg/ml respectively) and ZAP−ve groups (median 0.065, 12.15 & 0.72 respectively) were not statistically significant (p=0.67, 0.45, 0.36 respectively). 9 ZAP-70+ve and 15 ZAP-70−ve patients were sequentially investigated over a median period of 3 years for expression of P-gp, MRP1, LRP and P-gp function. There were no significant changes in P-gp expression and P-gp function between presentation and follow samples over this period. However, a significant increase in expression of MRP1 (median at presentation 1.65 and follow up 5.23 p= 0.0001) and LRP (median at presentation 3.92 and follow up 5.84 p= 0.0002 respectively) were observed for the ZAP-70−ve group. IgVH mutation analysis was concordant with ZAP-70 expression in 6 patients with poor prognosis (ZAP-70+ve/IgVH unmutated) and in 16 with good prognosis (ZAP-70−ve/IgVH mutated). Statistical analysis of MDR protein expression and IC50 in this smaller cohort of patients provided data similar to that in the main group. MDR proteins, their activity and in vitro drug sensitivity appear not to significantly contribute to the poor prognosis subset of B-CLL identified by their ZAP-70+ve/IgVH unmutated status. The significance of increased MRP1 and LRP expression in the good prognosis ZAP−ve group is unclear.

Description: The Weather Research and Forecasting model (WRF) double-moment 6-class microphysics scheme (WDM6) implements a double-moment bulk microphysical parameterization of clouds and precipitation and is applicable in mesoscale and general circulation models. WDM6 extends the WRF single-moment 6-class microphysics scheme (WSM6) by incorporating the number concentrations for cloud and rainwater along with a prognostic variable of cloud condensation nuclei (CCN) number concentration. Moreover, it predicts the mixing ratios of six water species (water vapor, cloud droplets, cloud ice, snow, rain, and graupel), similar to WSM6. This paper describes improving the computational performance of WDM6 by exploiting its inherent fine-grained parallelism using the NVIDIA graphics processing unit (GPU). Compared to the single-threaded CPU, a single GPU implementation of WDM6 obtains a speedup of 150× with the input/output (I/O) transfer and 206× without the I/O transfer. Using four GPUs, the speedup reaches 347× and 715×, respectively.