ALBERT

Description: Weibel-Palade bodies (WPBs) are elongated secretory granules of endothelial cells that are packed with tubules composed of von Willebrand factor (VWF), a multimeric protein required for hemostasis. Disruption of tubular packing prevents the orderly secretion of VWF multimers and blocks the subsequent binding of platelets. The cigar-like shape and tubular cross section of WPBs are conserved in all vertebrates, but little is known about how VWF specifies this packing arrangement. Starting from recombinant 82 kDa VWF propeptide (domains D1D2) and 114 kDa disulfide-bonded D’D3 dimer, we now have assembled tubules reversibly in vitro with the same dimensions as VWF tubules in WPBs. Assembly was induced at pH 6.2, reversed at pH 7.4, and required Ca2+. Recombinant D’D3 dimers did not self-associate at pH 7.4 or pH 6.2, with or without Ca2+. Without Ca2+, VWF propeptide did not bind to D’D3 dimers. At pH 7.4, with Ca2+, VWF propeptide formed noncovalent 160 kDa dimers and, when mixed with D’D3 dimers, assembled a 280 kDa complex of two propeptides and one D’D3 dimer as shown by gel filtration chromatography and multi-angle light scattering. Lowering the pH to 6.2 caused the formation of 〉3 MDa aggregates with the same stoichiometry, which dissociated upon adding EDTA or raising the pH to 7.4. Quick-freeze deep-etch EM showed that the large aggregates are hollow right-handed tubular helices. The iterative helical real space reconstruction method was used to make 3D reconstructions of the tubules at 22 Å resolution from negative stain EM images (Figure, left). Tubules consist of a right-handed helix with axial rise of 26.2 Å and twist of 85.6 degrees per subunit, or 4.2 subunits per 11 nm turn. The dimensions (outside diameter 25 nm, inside diameter 12 nm) are similar to those of tubules in WPBs in thin sections of endothelial cells by transmission EM (Figure, right and its insert). Each subunit contains one D’D3 dimer flanked by two D1D2 propeptides (Figure, center). Each D’D3 dimer makes a total of six contacts with D1D2 domains. Each D1D2 propeptide makes three contacts with D’D3 and just one end-to-end homotypic contact. The spatial arrangement of these building blocks and inter-domain contacts in tubules suggest a model by which decreasing pH along the secretory pathway coordinates the formation of intersubunit disulfide bonds with the tubular packaging of VWF multimers. Within the WPB, Ca2+-dependent and pH-dependent binding of D1D2 to D’D3 domains stabilizes the packing of VWF multimers into tubules, which behave as constrained springs. Upon secretion, the increased pH weakens these constraints and permits the helical tubules to unfurl into flowing blood without tangling. Figure Figure

Description: Abstract 3544 Poster Board III-481 Objective To explore the impact and mechanism of keratinocyte growth factor (KGF) infusion on immune reconstitution post murine allogeneic umbilical cord blood cell transplantation (UCBT). Methods Mix cord blood was obtained at E19.5 from several litters of B6 females and was used as umbilical cord blood (UCB) graft. Thirty-two BALB/c mice were randomly assigned to 4 groups with 8 mice per group in the first cohort UCBT, mice were reconstituted with PBS or 1×106, 2×106, 3×106 UCB mononuclear cells (MNCs). Twenty-four BALB/c mice were randomly assigned to 3 groups with 8 mice per group in the second cohort UCBT, mice were injected with 1×106, 2×106 or 3×106 MNCs. All mice received platelet transfusion on +8d. Sixteen BALB/c mice were randomly assigned to 2 groups with 8 mice per group in the third cohort UCBT, mice were injected s.c. with KGF or PBS before TBI. All mice were injected with 2×106 MNCs and were supported with platelet transfusion on +8d. We studied survival, splenic lymphoid cell subsets, sjTREC assay after UCBT. Results The survival rates at +100d of mice resconstituted with allogeneic 1×106, 2×106, 3×106 UCB MNCs were 25%, 37.5% and 37.5% in the first cohort UCBT,respectively. The survival rates at +100d of mice injected with 1×106, 2×106, 3×106 MNCs and with platelet support were 87.5%, 100.0% and 100.0%, respectively. The splenic T, NKT, NK and B cell counts in PBS treated control mice on +35d were (9.57±0.74)×106, (0.64±0.06)×106, (1.43±0.10)×106 and (19.13±1.50)×106, respectively. While in KGF treated mice on +35d were (13.47±0.74)×106, (0.89±0.03)×106, (1.79±0.04)×106 and (20.50±0.91)×106, respectively. The level of sjTREC in control mice was 182.2±10.7copies per 105 cells Gwhile that of KGF treated mice was 224.2±9.6 copied per 105 cells and was higher than that of control mice (P=0.019). Conclusion Peripheral blood abtained from E19.5d fetus is rich in hematopoietic stem cells. We established a murine allogeneic UCBT model with platelet support on +8d. KGF treatment could enhance immune reconstitution after UCBT. Disclosures: No relevant conflicts of interest to declare.

Description: Overcoming disease persistence in chronic myeloid leukemia (CML) patients is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL positive cells in presence of imatinib mesylate (IM, Gleevec™) and nilotinib (AMN107, NI), a novel more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84-cell clones (R-CM) was found to substantially protect IM-naïve LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony stimulating factor (GM-CSF) which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the JAK-2/STAT-5 signaling pathway in GM-CSF-receptor alpha receptor (CD116) expressing cells, including primary CD34+/CD116+ GM-progenitors (GMP). In a cell based resistance induction assay, GM-CSF enabled the evolution of IM- and NI-resistant colonies. When analysing GM-CSF expression in over 120 patient samples of first diagnosis CML patients and resistant patient, elevated mRNA and protein levels of GM-CSF were detected exclusively in IM-resistant patient samples, suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly, inhibition of JAK-2 with AG490 abrogated GM-CSF-mediated STAT-5 phosphorylation and NI resistance in vitro. Together, this suggests that adaptive autocrine secretion of GM-CSF and cytokines in general may be a novel IM and NI resistance mechanism, which may also contribute to CML-persistence. GM-CSF protects CML-progenitors via a BCR/ABL-independent activation of the anti-apoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF-induced IM and NI progenitor cell resistance providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML.

Description: CAM307 is a randomized Phase III trial comparing the efficacy and safety of alemtuzumab (CAM) with chlorambucil (CHLO). The trial enrolled 297 previously untreated patients (pts) requiring therapy according to NCI-WG criteria. Pts were randomized 1:1 to CAM (n=149) vs CHLO (n=148) using standard dosing regimens. Fluorescence in situ hybridization (FISH) on interphase nuclei of lymphocytes isolated from blood was analyzed for cytogenetic abnormalities prior to the start of therapy. FISH analysis was performed using 13 DNA probes to detect chromosomal aberrations in 17p13.1 (P53), 13q14 (RB1, D13S319 and D13S25), 11q22.3-11q23 (ATM and MLL), 6q27 (subtelomere), 6q21 (chromosome 6q21/alphasatellite 6 cocktail probe), trisomy 8q24 (c-myc), trisomy 12 (CEP12) and translocations involving the locus of immunoglobulin heavy chain gene (IGH, 14q32.33). Samples were analyzed in 282 pts (95%); chromosomal aberrations were detected in 231 pts (82%) while 51 pts (18%) exhibited a normal interphase FISH pattern. The most frequent abnormalities were deletions (del) at loci 13q (49%), sole del 13q (24%), 11q (19%), 17p (7 %), 6q (4 %), and trisomies 12 (14%) and 8q (5%). Translocations IGH, 14q32.33 were detected in 10 pts (4%). An exploratory analysis was performed to correlate time to event variables (assessed by an independent response review panel) with cytogenetics. Overall 165 pts (59%) revealed combination abnormalities. The most frequently observed chromosomal associations were: del 13q + del 14q (N=20, 12%), del 11q + del 13q (N=17, 10%), del 11q + del 13q + del 14q (N=11, 7%), del 11q + del 14q (N=7, 4%), trisomy 12 + del 13q (N=5, 3%), del 13q + del 17p (N=4, 2%), del 11q + trisomy 12 (N=3, 2%) and del 17p + del 6q (N=3, 2%). Coexistence of del 17p and del 11q was not observed. Although del 13q was observed with all chromosome abnormalities, nearly half of the cases del 13q14.3 (D13S25 and D13S319) coincided with an ATM deletion (11q22.3). FISH analysis has allowed the detection of uncommon abnormalities: tetraploidy (n=1), hyperdiploidy (n=1), trisomy 18 (n=1) and c-myc oncogene amplification (〉15 copies per nuclei) (n=2). The latter is a well known abnormality in solid tumors but rarely seen in leukemia. In addition, del of the IGH variable region was detected in 70 pts. The biological and clinical significance of this abnormality is to be investigated. Conclusions: Overall, 82% of treatment naïve BCLL pts revealed cytogenetic aberrations and 59% were combination abnormalities. CAM307 demonstrates a significant improvement in PFS in pts treated with CAM vs CHLO who present with del 13q as the sole abnormality; no difference in pts with del 11q. However, a trend towards improved PFS was observed in pts with trisomy 12 and del 17p, which did not reach significance due to small sample size. Further investigation of CAM therapy in high risk cytogenetic subgroups is warranted.

Description: Haploidentical transplantation (H-SCT) has been an alternative allogenetic transplant option for the high risk lukemia patients who do not have an HLA-identical related donor. The high dose of stem cell infusion and intensive GVHD prophylaxis treatment were applied in H-SCT to overcome the haploidentical major histo compatibility barrier and to achieve engraftment. We speculated that the dynamics of donor chimerism are different from conventional myeloablative allogeneic bone marrow transplantation(allo-BMT). Methods Quantitative analysis of chimerism were performed by multiplex PCR amplification of STR markers (STR-PCR) in unfractionated nucleated cells from bone marrow or whole blood of 30 high risk leukemia patients who received H-SCT(14 females,16 males; median age 25 years). Eighteen of 30 patients received G-CSF primed bone marrow transplantation and another 12 patients received G-CSF primed bone marrow and peripheral blood stem cell transplantation. The conditioning regimen consisted of modified BU/CY or modified total body irradiation(TBI) plus CY. One patient received cyclosporine (CSA), short-term methotrexate(MTX) and mycophenolate mofetil (MMF) as the regimen of prophylaxis of GVHD. Twenty-four patients received CSA, MTX and MMF plus ATG/ALG for GVHD prophylaxis. The combination of CSA, MTX, MMF, ATG/ALG and CD25 monoclonal antibody for preventing GVHD in 5 patients. Results: All of the patients except one showed initial donor engraftment and just one patient presented primary graft failure. The early chimerism and its kinetics after transplantation showed that the donor chimerism became dominant (DC 84.1% 〉60%) by day +3, which preceded the detection of hematologic engraftment by a median of 9 days. On day +7, donor cells of majority patients had achieved stable engraftment. The median chimerism was 92.4%;The probability of achieving full donor chimerism (FDC) in PB was 56.3% on day +14, 61.5% on day +28;Comparing with the group of standard allo-BMT, median quantity of CD34+ cells and CD3+ T lymphocyte cells of H-SCT group were 2 and 3 times higher. Furthermore GVHD prophylaxis was more intensive with ATG/ALG and MMF in H-SCT group. The median percentage of donor chimerism of H-SCT was significantly higher than that of BMT on the day of +7d, +14d and +1month. Meanwhile there were difference in the rates of FDC among H-SCT and BMT(61.5% VS 20%, p90%), and another 10 patients achieved stable FDC(DC ≥95%). Conclusion: In the early stage after transplantation, the engraftment of donor cells of H-SCT was more rapidly and completely than that of conventional BMT owing to the high quantity of donor CD34+ cells, T lymphocyte cells and the more intensive immunosuppressive therapy.

Description: Overcoming imatinib mesylate (IM) resistance and disease persistence in patients with chronic myeloid leukemia (CML) is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL+ cells in the presence of IM and nilotinib (NI; AMN107), a novel, more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84 cell clones (R-CM) was found to substantially protect IM-naive LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony-stimulating factor (GM-CSF), which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the janus kinases 2 (JAK-2)/signal transducer and activator of transcription 5 (STAT-5) signaling pathway in GM-CSF receptor α receptor (CD116)–expressing cells, including primary CD34+/CD116+ GM progenitors (GMPs). Elevated mRNA and protein levels of GM-CSF were detected in IM-resistant patient samples, suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly, inhibition of JAK-2 with AG490 abrogated GM-CSF–mediated STAT-5 phosphorylation and NI resistance in vitro. Together, adaptive autocrine secretion of GM-CSF mediates BCR/ABL-independent IM and NI resistance via activation of the antiapoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF–induced IM and NI progenitor cell resistance, providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML.

Description: The prognosis for patients with chronic myeloid leukemia (CML) in blast crisis (BC) remains dismal even with the availability of the BCR-ABL tyrosine kinase inhibitor imatinib, since it only offers short-term benefit in most cases. Allogeneic hematopoietic stem cell transplantation (HSCT) seems to be a viable option for BC-CML patients who attained remission. We treated 10 patients (9 males, 1 female) with ablative allogeneic HSCT, who achieved second chronic phase (CP) by the use of imatinib after onset of BC between October 2003 and August 2006. Median patient age was 32 years (range, 17–46). Imatinib was given orally at daily doses ranging from 600 to 800mg according to patients tolerance for at least 2 months (range, 2–5) prior to HSCT Among them, 4 patients received HSCT from human leukocyte antigen mismatched haplo-identical family donors, the others underwent a transplant from HLA matched related (n=5) or unrelated (n=1) donors. At the time of transplantation, 5 patients were in complete hematologic response with 3 patients achieved a cytogenetic response, 5 patients were in partial hematologic response. After a median follow-up of 26 months (range, 10–44), 6 (60%) out of the 10 patients were alive with mean Karnofsky score reaching 80. Among them, 5 patients achieved a molecular remission. 1 patient died in relapse 4 months after transplantation, the others died of severe acute graft-versus-host disease and associated infections. No unusual organ toxicities and engraftment difficulties were observed. Extensive chronic GVHD developed in 3 of 6 patients who could be evaluated. Patients transplanted with haplo-identical donors had a high treatment-related modality, 3 out of 4 patients died. These results suggest that allogeneic HSCT may represent a feasible treatment for patients with CML in second CP attained by imatinib after onset of BC especially when a suitable donor is available.

Description: Abstract 4325 Objective Retrospective analysis the therapeutic and side effect of the rabbit antithymocyte (ATG) versus swine ALG within the preparative regimen of allogeneic hematopoietic transplantation (allo-HSCT) for graft versus host disease (GVHD) prophylaxis. Methods Totally 102 patients who had admitted to our hospital and been treated by allo-HSCT with the preparative regimen including ATG/ALG were followed up from June 2002 to June 2008. They were divided into ATG group and ALG group. The allergic reaction, effect of GVHD prophylaxis, transplantation related mortality (TRM), disease free survival (DFS) and relapse rate (RR) of these groups were retrospectively analyzed. Cumulative rate were analyzed by the Kaplan-Meier method and the factors associated with the III?‘‡W AGVHD were analyzed with the COX regression model. Results ALG group had more allergic reaction than ATG, but ATG group had more bacteremia and cytomegalovirus (CMV) antigenaemia. The haematopoiesis reconstitution was comparable in two groups. The III?‘‡W AGVHD, two-year TRM,DFS and RR were (40% vs 21%,p=0.028),(54% vs 29%,p=0.039),(41% vs 53%,p=0.174),(10% vs 24%,p=0.306),respectively in ATG/ALG groups. In multivariate analysis,10mg/kg ATG as a protective variable to III?‘‡W AGVHD occurrence(RR=0.53 ;95%CI, 0.38?‘0.71),The CD3+ cell counts of administration was associated with an increased risk for III?‘‡W GVHD(RR=4.43 ;95%CI, 3.87?‘4.95). Conclusion 10mg/kg ATG significantly decreased the risks for III?‘‡W AGVHD and extensive chronic GVHD(ecGVHD); The lethal infections became the most important cause of death in the ATG group, but the increased risk for infection did not neutralize the reduction of TRM induced by the decrease of severe GVHD. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract□F Objective To analyze the complete remission (CR) rate, disease free survival (DFS) and overall survival (OS) of de novo acute myeloid leukemia (AML) patients induced with HAD (Homoharringtonine-HHT, cytosine arabinoside-AraC, daunorubicin-DNR) regimen containing intermediate dose AraC (ID-AraC) and to explore the impact of cytogenetic abnormalities on the prognosis. Methods 87 AML patients were treated with HAD regimen containing ID-AraC as induction therapy. HAD regimen was as follow: HHT 2mg/m2.d, Day 1–7; Ara-C 100mg/m2, Day 1–4, 1–1.5g/m2/q12h, Day 5–7; DNR 40 mg/m2.d, Day 1–3. CR rate, DFS and OS were calculated.83 patients who had karyotype results were divided into 3 groups according to SWOG criteria respectively. Differences in CR rate, DFS and OS among different groups were evaluated. Results The CR rate of the 87 cases was 80/87 (92%). Median DFS of the 80 CR patients was NR (not reach). DFS rates at 1 and 3 years were 76.3% and 63.4% respectively. The median OS of the 87 patients was 16 (range from 2 to 67) months. OS rates at 1 and 3 years were 86.0% and 58.7% respectively. According to SWOG criteria, CR rate, median DFS and OS were 100%, NR for the favorable group„dG88.9%, NR and 16 months for the intermediate group„dG83.3%, 4.5 months and 7.5 months for the adverse group. The differences among the three groups were statistically significant excepting for CR rate between adverse and intermediate groups. Conclusions HAD regimen containing ID-AraC as induction chemotherapy regimen is very effective in de novo AML of adult patients, can achieve higher CR rate and longer survival time than standard dose HAD regimen. Most of the patients were able to endure therapy. Cytogenetics is the important prognostic factor for AML patients. The differences among the three groups were statistically significant.

Description: Background: Retrospective studies have reported a correlation of higher ELISA optical density (OD) values for H-PF4 antibodies with occurrence of thrombotic events in patients (pts) with HIT; often with variable prophylactic direct thrombin inhibitor (DTI) use. Objectives: To compare positive OD (≥0.4) values with thrombotic risk and clinical outcome in pts with clinically-suspected or confirmed HIT. Design: Retrospective cohort study. Setting: IRB-approved, CAMC HIT Registry. Patients: 182 HIT pts, age ≥18 yrs with OD values ≥0.40 (EIA, GTI) enrolled from 3/11/05 to 12/27/07. Age 64.5±13.4 yrs; M/F (107/75); caucasian (96.2%); CV surgery (n=120), medical (n=45), gen/vasc surgery (n=17); heparin use: therapeutic (n=160), prophylaxis (n=17), catheter flushes (n=5). Measurements: mean OD±SD; objectively confirmed thrombosis (venous, arterial); all-cause mortality; length of stay (LOS); DTI use; composite outcome (new thrombosis, death, amputation); thrombotic composite outcome (new thrombosis, death from new thrombosis, amputation). Results: At HIT diagnosis, prevalent thrombosis occurred in 82 pts and isolated HIT occurred in 100 pts. Thrombotic presentations (n) included: venous alone (DVT-27, PE-9, DVT + PE-5), arterial alone (single event-28, multiple events-5), and venous plus arterial (8). Prevalent thromboses were symptomatic in 65 pts (79.3%). Fourteen pts developed a new thrombosis; 5 of them had isolated HIT. A total of 87 pts (47.8%) had “ever” thrombosis. The OD of the 182 pts was 1.12±0.76 with a cumulative thrombotic rate of 52.4%, 75.6% and 90.3% in OD ranges of 0.40–0.89, 0.4–1.49 and 0.4–2.29, respectively. No significant difference in rates of prevalent thrombosis was observed in any of 3 categorizations of OD values (% thrombosis): 0.40–0.99 vs. ≥1.00 (42.3% vs. 41.7%; P=0.457), 0.40–1.09 vs. ≥1.10 (47.9% vs. 40.0%; P=0.307) and 0.40–1.19 vs. ≥ 1.20 (47.5% vs. 40.0%; P=0.337). Among pts with “ever” vs. “never” thrombosis, there was no significant difference in OD values (1.14±0.84 vs. 1.10±0.69; P=0.692). Among pts with a new thrombosis, the OD was 1.45±1.36 (min 0.43, max 5.53). The OD was numerically higher in pts with isolated HIT who developed a new thrombosis vs. pts with “never” thrombosis (2.23±2.05 vs. 1.10±0.69; P=0.285). Males developed a higher rate of thrombosis than females (56.1% vs. 36.0%; P=0.008). Surgical pts comprised a higher proportion of “ever” thrombosis than medical pts (54.7% vs. 26.7%); P=0.001). All-cause mortality was significantly higher in the “ever” vs. “never” thrombosis groups (16.1% vs. 5.3%; P=0.017); highest among pts with isolated HIT (80.0%) and prevalent thrombosis who developed a new thrombosis (55.6%). HIT as cause or possible cause of mortality in pts with “never” thrombosis (1.1%) was significantly lower than those with “ever” thrombosis (10.3%, P=0.007) and those with new thrombosis (57.1%, P