ALBERT

Beschreibung: Although the analysis of chimerism induction has become an important diagnostic tool for providing better clinical management of patients undergoing allogeneic HSCT, the techniques have not been fully standardized. Moreover, it is currently unknown whether the onset of graft-versus-host disease (GVHD) is related to the status of mixed chimerism (MC) or complete donor-type chimerism (CDC). First, to validate our chimerism analysis system, we performed experiments with artificially mixed cell samples from healthy volunteers to examine the reliability of short tandem repeat (STR) determination by quantitative polymerase chain reaction (PCR). We confirmed a linear correlation between the proportion of mixed cells and the calculated ratio, with a correlation coefficient of 0.99, which enables the detection of target cells at 3% (median standard deviation, 1.6%). Next, using this validated system, we prospectively evaluated the kinetics of chimerism in CD3+, CD19+, and peripheral blood mononuclear cells (PBMC), for correlation with the occurrence of GVHD in 19 patients with various hematological diseases (median age, 53y) who had received allogeneic HSCT from an HLA-identical sibling donor between July 2003 and February 2004. The preparative regimen was fludarabine/busulfan (BU) (n=12) or cladribine/BU (n=7). GVHD prophylaxis consisted of cyclosporin alone (n=8), cyclosporin plus short-term methotrexate (n=7), or tacrolimus (n=4). Chimerism analysis was repeated weekly after transplantation. We evaluated 405 consecutive blood samples from these 19 recipients, 12 of whom developed acute GVHD. Six of these 12 patients showed MC, i.e. 91% (83–94%; MC) donor cells in the CD3+ fraction at the onset of GVHD, but all except one subsequently achieved CDC within a median of 15 (7–33) days without additional DLI. The remaining patient showed persistent MC and relapsed 158 days after transplantation. We found that the presence of MC in the CD3+ fraction is rather common at the onset of acute GVHD, but GVHD subsequently eradicates residual host hematopoietic cells. Alternatively, GVHD is part of a clinical manifestation of an immune reaction that is related to the induction of CDC.

Beschreibung: Recent introduction of molecular targeted drugs such as bortezomib and IMIDs in the clinical settings has achieved the improved treatment outcome of multiple myeloma (MM). However, MM is still an incurable disease and these drugs also possess serious adverse reactions, therefore, safe and more effective therapy should be established. Artesunate (ART) is a semi-synthetic derivative of artemisinin and is widely used for the treatment of malaria as a salvage therapy. Recent in vitro studies showed that ART also has an anti-tumor activity against several cancer cell lines. We thus investigated whether ART could possess anti-myeloma activity and demonstrated that apoptosis of myeloma cells is strongly induced by ART. Interestingly, we also found that this activity is mediated through heat shock protein (Hsp) 27-dependent pathway. Several MM cell lines (IM9, OPM2, RPMI8226, U266) were treated with various concentrations of ART for 48 hours, and MTT assays were performed to assess the anti-myeloma activity. IM9, OPM2, RPMI8226 cells showed the striking reduction of viability (up to 40% at 1μM and up to 90% at 10μM) in a dose- and time-dependent manner, whereas ART has less inhibitory effect on U266 cell line. ART induced G1 arrest of IM9 cells, and apoptosis was confirmed by decreased mitochondrial membrane potential, and flow cytometric analysis using AnnexinV/PI staining. Colony assay showed that ART has no growth inhibitory effect on normal CD34-positive bone marrow cells even at a concentration of 10μM. Immunoblot analyses demonstrated the activation of caspases-3 and -9, and the decreased expression of pro-apoptotic protein Bid. Interestingly, heat shock protein (Hsp) 27 was downregulated in myeloma cell lines (IM9, RPMI8226) which are sensitive to the ART treatment. In contrast, downregulation of Hsp 27 was not observed in U266 cells which are resistant to ART. Other anti-apoptotic Hsps (Hsp70, 90), as well as Bcl-2 family proteins (Bcl-2, Bax, Bad, Bcl-xL), Akt, MDM2, p53 and MAPKs (SAPK/JNK, p38, ERK1/2) were not affected by ART. Quantitative RT-PCR analysis showed that ART did not influence the mRNA expression of Hsp27, suggesting that ART could downregulate the Hsp27 protein at a posttranscriptional or posttranslational levels. Overexpression of Hsp27 cDNA by transfection method in ART-sensitive myeloma cell lines demonstrated that these cell lines acquired resistance to ART and apoptosis was not induced at a concentration which ART is effective to the parental cells. Our preliminary data using knockdown procedure of Hsp27 mRNA by RNAi-expressing lentivirus showed the growth suppression of myeloma cells, which apparently suggested that Hsp27 could confer a critical function in the proliferation of myeloma cells and that downregulation of Hsp27 could result in induction of apoptosis in MM. Hsp27 has an important role in anti-apoptotic effect against various stresses to the cells. Our data implies a novel mechanism that downregulation of Hsp27 by ART could induce misfolding of several client proteins indispensable for the cell growth, which results in the induction of apoptosis in MM cells. In conclusion, ART could become a candidate of safe and effective therapeutic drug for MM, and Hsp 27 might be a potential molecular target for the treatment of MM.

Beschreibung: Abstract 2349 Introduction: Incidence of hematological malignancies is higher in elderly population. However, standard chemotherapy has not been established and the potential role of RICBT has remained unclear. This study reports the results of RICBT for elderly patients with hematological malignancies, retrospectively. Objective: To investigate the feasibility of RICBT. Primary endpoints were engraftment and overall survival (OS). secondary endpoint was transplant-related mortality (TRM). Patients and Methods: Between Feb.2009 and Jun.2010, 29 patients (median age 70 years, range 58–76) received RICBT for hematological malignancies. Primary diseases were divided into 2 groups; advanced (intermediate and high risk; n=21) or standard (n=8). All cases in 70's were included in the high risk. Median follow up time was 238 days (range 8–464).Conditioning regimen and GVHD prophylaxis consisted of fludarabine, busulfan or cyclophosphamide, and TBI 2Gy with tacrolimus± MTX for 70 years (n=11) and 500/μL) and platelet engraftment (〉20,000/μL) were observed in 89.6% (95% CI; 79–100) at day 60 (median; 18.0 days, range; 13–28), and 62.1% at day 100 (median; 36.5 days, range; 17–60), respectively. Neutrophile engraftment was 100% in TBI regimen (median; 18 days, range; 13–28), 75% in ATG regimen (95% CI; 51–100, median; 19 days, range; 16–27)(p=0.04). Platelet engraftment was 77% in TBI regimen, 42% in ATG regimen, respectively (p=0.06). Primary graft failure occurred in 3% of all cases. Cumulative incidence of acute GVHD (II-IV) was 58% (95% CI; 39–78) at day 100 (median; 27.5 days, range; 13–82), 81% (95% CI; 62–100, median; 28 days, range; 17–82) in TBI regimen and 12.5% (95% CI; 0–35, median; 13 days, range; 13) in ATG regimen, respectively (p=0.01). The 1-year estimated OS was 51% (95% CI: 25–77) in all cases, 51% (95% CI: 13–90) in TBI, and 48% (95% CI: 18–77) in ATG, respectively (p=0.08). According to the age, OS was 80% (95% CI: 45–80) in 50's, 53% (95% CI: 20–86) in 60's, and 38% (95% CI: 0–77) in 70's (P=0.0765). Causes of TRM included infections (n=5 including 3 cases in 70's), TMA (n=2). Incidence of TRM at day 100 was 20% (95% CI; 4–35, median; 47 days, range; 8–79), 7% in TBI regimen (95% CI; 0–21, median; 79days), 41% in ATG regimen (95% CI; 9–72, median; 30 days, range; 9–72). Incidence of TRM, according to the age, was 40%,0%, and 52% in 50's,60's, 70's, respectively. Discussion and Conclusion: Because of the high incidence of high risk disease and TRM, despite low incidence of GVHD, RICBT is associated with a low OS in patients over 70 years compared with those who are under 70 years. Eligibility of RICBT needs to be investigated, especially in 70's patients, and further studies are warranted to clarify the safety in elderly patients. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: CD47 belongs to the immunoglobulin superfamily, and is expressed as a 50 kDa cell surface antigen in a wide variety of tissues. CD47 is associated with integrins of β1, β2, and β3, and serves as both a receptor for thrombospondin (TSP) and a ligand for the transmembrane signal regulatory protein SIRP-α . CD47 has a number of different functions, including platelet activation, cell motility, leukocyte adhesion, migration and phagocytosis. Recently, it was reported that the ligation of CD47 rapidly induces cell death in T-cells and chronic lymphocytic leukemic B cells (B-CLL) in a caspase-independent manner (Nat Med, 1999;5:1277–1284). These findings indicate that CD47 ligation may have potential as a new therapeutic approach for lymphoid malignancies. B-CLL is the most common hematological malignancy in Western countries. Although new therapeutic agents such as fludarabine and 2-chlorodeoxyadenosine have been introduced in the clinic, B-CLL is not considered curable, and thus there is an immediate need for new, effective drugs. In an attempt to establish a novel therapeutic agent for B-CLL, we first generated a murine monoclonal antibody against an extracellular domain of human CD47 (designated MABL). Soluble MABL (10μg/ml) with goat anti-mouse IgG (GAM) induced apoptosis of CD47-positive CCRF-CEM and MOLT-4 cells. However, because MABL caused hemoagglutination, we created a disulfide-stabilized dimer of a single-chain antibody fragment of MABL (S-S diabody) to get rid of this adverse effect. The S-S diabody did not cause hemoagglutination. Treatment with the S-S diabody (0–0.1 μg/ml for 0–24 hours) alone was fully active in inducing apoptosis of primary samples from patients with B-CLL and CD47-positive lymphoid cells (MOLT-4 and JOK-1) in a dose- and time-dependent manner. In addition, administration of the S-S diabody (30 mg/kg for 5 days) significantly prolonged the survival of SCID mice inoculated with JOK-1 cells compared with that of the control mice (median survival 21 days vs. more than 30 days, p

Beschreibung: Abstract 4495 Incidence of myeloid malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is higher in elderly population. However, standard chemotherapy has not been established and the potential role of RICBT has remained unclear. This study reports the results of RICBT for elderly patients with myeloid malignancies. To investigate the feasibility of RICBT. Primary endpoints were engraftment and overall survival (OS). 2nd endpoints were transplant-related mortality (TRM) and relapse rate. Between Mar.2009 and Jul.2011, 21 patients (median age 70 years, range 65–74) received RICBT for myeloid malignancies (de novo AML; n=7, MDS related; n=14). Primary diseases were divided into 2 groups; advanced (high risk; n=11) or standard (CR1 & 2; n=10). Median follow up 9.5 months (0.3–29).Conditioning regimen was fludarabine 200mg/m2, busulfan, and TBI 2Gy(n=9) or ATG 7.5mg/kg (n=12).Immunosuppresants were tacrolimus± MTX (n=13) or cyclosporine ± MTX (n=8). Median total nucleated cells (TNC): 2.6 × 10^7 cells (2.0–4.8); Median CD34+: 0.4 × 10^5 cells (0.2–2.9); HLA match: 5/6 (n=1), 4/6 (n=20). Time to event curves were plotted by using the actuarial method of Kaplan-Meier, and differences between curves were analyzed by log-rank tests. Neutrophile (〉500/μ L) and platelet recovery (〉20,000/μ L) were observed in 86% (95% CI; 82–87) at day 60 (median; 17 day, range; 12–27), 62% (95% CI; 41–83) at day 100 (median; 32 day, range; 41–83), respectively. Neutrophile engraftment was 100% in TBI regimen (median; 16 days, range; 13–20), 79% in ATG regimen (median; 19 days, range; 12–27) (P=0.09). Platelet engraftment was 86% (95% CI; 60–100) in TBI regimen, 42% (95% CI; 14–70) in ATG regimen, respectively (p=0.02). Cumulative incidence of acute GVHD (II-IV) was 33% (95% CI; 13–53) at day 100 (median; 24days, range; 10–82), 73% (95% CI; 17–82, median; 25 days, range; 17–82) in TBI regimen and 14% (95% CI; 0–33, median; 11.5 days, range; 10–13) in ATG regimen, respectively (P=0.02). The 1-year OS was 64% (95% CI; 46–86) in all cases, 71% (95% CI; 38–100) in TBI regimen, 63% (95% CI; 37–89) in ATG regimen, respectively (P=0.55,Figure1). According to the age, OS was 81% (95% CI; 57–100) in 60’s, 45% (95% CI; 14–76) in 70’s, respectively (P=0.04). Causes of TRM included infections (n=2) and late graft failure (n=1), all cases in 70’s and received ATG. Relapse rate was 19% (95% CI; 2–36) in all cases, 43% (95% CI; 6–80) in TBI regimen, and 7% (95% CI; 3–68) in ATG regimen at 500 days, respectively (P=0.06). RICBT with ATG regimen is associated with a high TRM in over 70 year-old high risk patients. However, ATG regimen may conduct low incidence of acute GVHD and relapse rate. Eligibility of RICBT needs to be investigated, especially in over 70 patients, and further studies are warranted to clarify the safety in elderly patients. Disclosures: No relevant conflicts of interest to declare.