Abstracts/Experimental Hematology 28 (2000) 31–131
`
`103
`
`There was a significant decrease in lin1 fraction of Sca-11 cells
`compared to both control mice and mice examined 2 to 3 days
`after cyclophosphamide. Generally, the purity of separation was
`lower and the apoptotic rate and susceptibility to Fas-mediated cell
`death was higher in mice recovering from cyclophosphamide dam-
`age. These findings demonstrated that not only numbers but also a
`quality of progenitors changed markedly during regeneration.
`
`226 Tuesday, July 11, 2000 (10:15–12:15)
`Session V-5: Stem and Progenitor Cell Transplantation:
`Experimental II
`SKIN-EXPLANT MODEL TO EVALUATE
`EFFECTIVENESS OF DEPLETION OF
`HLA-ALLOREACTIVE T-CELLS
`Markus Ege, Ute Schulz$, Ernst Holler$, Petra Gottlöber§,
`Hans Pillekamp§, Wilhelm Friedrich, intro by Bernhard Kubanek
`Dept of Pediatrics and §Dept. of Dermatology, University of Ulm,
`$Dept. of Haematology and Oncology, University of Regensburg,
`Germany
`
`Depletion of host specific alloreactive T-cells from donor lym-
`phocytes is an attractive tool for donor lymphocyte infusions after
`stem cell grafting. We intended to establish a preclinical method to
`evaluate effectiveness and safety of this approach. The skin ex-
`plant model is a powerful method to predict the risk of GvHD in
`the HLA-identical setting, and we adapted this model to the HLA-
`haploidentical situation. Unmanipulated donor T-cells are com-
`pared with donor T-cells following depletion of host specific T-cells.
`Prior to depletion donor MNC are incubated in the presence of ir-
`radiated recipient MNC and after a period of 5 days activated T-cells,
`positive for CD25, CD69, CD71 and HLA-DR, are magnetically
`removed (VarioMacs, Miltenyi). A repeat MLC of unmanipulated
`donor cells. Fresh skin biopsies are divided into pieces of 1 - 2 mm
`diameter and incubated with 106 cultured T-cells in 20% autolo-
`gous serum for 72 hours, fixed in formaldehyde and stained with
`haematoxylin-eosin. Skin alterations are graded according to Learner
`et al. In 3 separate experiments undepleted cells caused subepider-
`mal cleft formation corresponding to a grade III to IV GvHD reac-
`tion. In contrast, depleted cells showed only grade I alterations, as
`similarly observed in medium control. Our preliminary data sug-
`gest that this approach could represent a useful tool to determine
`effectiveness of magnetic depletion of alloreactive T-cells and be
`valuable to assess the safety of these cells prior to infusion.
`
`227 Sunday, July 9, 2000 (18:30–19:30)
`Poster Session I: Acute Leukemia: Basic Research
`INDUCTION OF APOPTOSIS AND GROWTH
`INHIBITION IN MYELOID MALIGNANCIES BY
`ARSENIC TRIOXIDE (As203)
`M. Rojewski, W. U. Knauf, E. Thiel, H. Schrezenmeier
`Medical Clinic III, University Hospital Benjamin Franklin, Free
`University of Berlin, Germany
`
`Clinical efficacy of As203 has been shown in patients with re-
`lapsed acute promyelocytic leukemia (APL). There is evidence
`As203 effects not only events specific for APL but also may target
`mechanism involved in the pathogenesis of other myeloid malig-
`nancies. We assessed susceptibility of induction of apoptosis by
`As203 (0,01-10 mM) and other agents (e.g. etoposide) in various
`myeloid and non-myeloid malignant cell lines. Apoptotic cells are
`measured by staining with annexin-V and 7-amino-actinomycin-D
`
`(7-AAD). The cell lines displayed different kinetics of response
`and different sensitivities of As203. Minimum concentration of
`As203 for induction of apoptosis after appropriate incubation was 1
`mM. High concentrations of As203 (5 mM) induced apoptosis after
`72 hours. With 5 mM As203: . 75% for NB-4, CEM and MV-4-11,
`50–75% for PBL-985, ML-2, MV-11, 20–50% for HL-60, HEL,
`Jurkat, K-562, PBL-985, U-937, KG-1, KG-1a. 1 mM As203 in-
`duced apoptosis in NB-4, HL-60, U-937, CEM, HL-60, KG-1a,
`PBL-985, ML-2, and MV-4-11 but not in HEL, K-562, KG-1, and
`Jurkat after up to 35 days of incubation. However, proliferation of
`HEL, K-562, and Jurkat non-apoptotic subpopulations was re-
`duced when treated with 1 mM As203. This anti-proliferative effect
`seems to be independent of apoptosis induction. MDR cell lines
`CEM-C1 and CEM-C2 were sensitive to 1 mM As203. 50% of cells
`were annexin-V positive after 3 day for CEM, 6 days for CEM-C1
`and 16 days for CEM-C2. MDR-cell lines HL-60-MX-1 and HL-
`60-MX-2 were resistant to 1 mM As203. These data reveal that
`As203 induced apoptosis is not restricted to cell lines with the
`translocation t(15;17). The molecular mechanisms involved in ap-
`optosis induction by As203 are under investigation. Preliminary re-
`sults indicate an upregulation of p53, Caspase 3 and an alteration
`of the Bcl-x expression in CEM after 4-6 hours of incubation with
`10 mM As203. In non-APL cell lines, apoptosis was induced in
`vitro by concentrations of As203 that are achievable also in vivo af-
`ter i.v. infusion of well-tolerated As203 doses. Thus, As203 might
`be a suitable therapeutic agent for myeloid malignancies other than
`APL and non-myeloid malignancies.
`
`228 Monday, July 10, 2000 (9:45–11:15)
`Session III-1: Acute Leukemia: Clinical and Basic
`Research
`IMPROVEMENT IN CYTOPENIAS OF PATIENTS WITH
`MYELODYSPLASTIC SYNDROMES (MDS) IN
`RESPONSE TO THALIDOMIDE
`A. Raza, L. Lisak*, L. Little*, C. Andrews*, P. Meyer*, M. Ekbal*,
`P. Venugopal
`Rush Cancer Institute, Chicago, Illinois
`
`Recent studies investigating the pathogenesis of MDS have
`yielded two important biological insights, both of which can be ex-
`ploited for therapeutic purposes. The first is that cytopenias may
`be the result of excessive cytokine-induced apoptosis of hemato-
`poietic cells, and second that the bone marrows (BM) of MDS pa-
`tients are highly vascular. Thalidomide was chosen as a potentially
`useful agent since it has both anti-cytokine and anti-angiogenic
`properties. The protocol provided for a starting dose of 100 mg po
`qhs of thalidomide, increased as tolerated to 400 mg. Patients be-
`longing to all FAB categories were eligible. Response meant an in-
`crease in Hb by 2.0 Gm/dL and/or a 50% reduction in packed red
`blood cell (PRBC) transfusions, increase in platelets by 30,000/ml
`or increase in absolute neutrophil count (ANC) by 500/ml. 83 pa-
`tients with a confirmed diagnosis of MDS have been accrued on
`the study and 31 have completed 12 weeks and are available for re-
`sponse evaluation. The median age was 68 years; there were 19
`males and 12 females. 18 had RA, 6 RARS, 6 had RAEB, and 1
`had CMMoL. 29 had primary MDS, while 2 had received prior
`therapy for breast cancer. Twenty-four patients were dependent on
`PRBC transfusions, while 6 were platelet dependent. The most fre-
`quent side effects were constipation, fatigue, and fluid retention
`while neuro-toxicity was avoided to a large extent because of pro-
`
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`104
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`Abstracts/Experimental Hematology 28 (2000) 31–131
`
`phylaxis with pyridoxine. 21 patients experienced rather signifi-
`cant partial response. The most striking responses were seen in the
`erythroid series with 8 patients achieving complete transfusion in-
`dependence and 13 increasing their hemoglobin by more than 2
`Gm/dL. Among the 13 platelet responders, there were 3 who in-
`creased their counts by .2000,000/ml and 4 by 100,000/ml. The
`best responses were seen in the RA/RARS patients, while 3/6
`RAEB patients showed some disease evolution. Interestingly, 2
`patients showed a response only after thalidomide was stopped.
`One of these in fact improved his hemoglobin from 7 to 16 Gm/dL,
`his platelets from 18,000/ml to 71,000/ml and normalized his ANC
`a full THREE months after stopping thalidomide, mimicking the
`effect of ATG and suggesting an immune-modulatory role of thali-
`domide in addition to its ant-cytokine and anti-angiogenic actions.
`In summary therefore, thalidomide is an exciting new addition to
`MDS therapeutic armament and needs further investigations.
`
`229 Sunday, July 9, 2000 (14:15–16:00)
`Session II-3: Signal Transduction
`OVEREXPRESSION OF THE H-RAS ONCOGENE IN
`PRIMARY PRIMITIVE HUMAN HEMATOPOIETIC
`CELLS ALTERS PROLIFERATION
`AND DIFFERENTIATION
`Craig Dorrell1,2*, Daniel S. Pereira3*, Robert G. Hawley4 and
`J. E. Dick1,2
`Department of Genetics, Hospital for Sick Children1 and
`Department of Molecular and Medical Genetics, University of
`Toronto2, Toronto, ON. ImClone Systems Inc., New York, NY3,
`Holland Laboratory, American Red Cross, Rockville, MD4
`
`The Ras pathway plays a central role in the control of cellular
`proliferation and differentiation. In order to examine the role of
`Ras signaling in hematopoietic regulation, we transduced CD341-
`enriched human cord blood cells with an MSCV-based retroviral
`vector encoding R12 activated H-Ras. The effects upon prolifera-
`tion and differentiation were then examined in vitro. Compared to
`cells transduced with a control vector, H-Ras expressing cells had
`an elevated frequency of CFU-M (76 6 16% vs. 22 6 13%) and
`increased monocyte/macrophage cell frequency in long-term sus-
`pension cultures (69 6 10% CD141 vs. 28 6 8% CD141). H-Ras
`expression also impaired CFC survival/expansion in suspension
`culture relative to controls. To determine the effects of decreasing
`the levels of H-Ras signaling, the farnesyltransferase inhibitor
`66177 (Schering-Plough) was used. At low doses, the inhibitor did
`not significantly reduce the frequency of monocyte/macrophage
`lineage cells, but led to the appearance of primitive/blastic monocytic
`colonies and cells with extended proliferative and self-renewal ca-
`pacities. Upon removal of the inhibitor, these cells rapidly differ-
`entiated into large, adherent macrophages. These results suggest
`that the level of Ras pathway signaling is an important determinant
`of myeloid cell fate, and may illustrate a manner in which Ras acti-
`vation in primitive hematopoietic cells contributes to leukemogenesis.
`
`230 Monday, July 10, 2000 (16:00–17:00)
`Poster Session II: Stem and Progenitor Cell
`Transplantation: Clinical Research
`ALLOGENEIC BONE MARROW TRANSPLANTATION
`FOR ADVANCED SOLID TUMORS
`D. Blaise, J. O. Bay, P. Viens, N. Vey, C. Faucher, J. Fleury,
`J. J. Grob, A. M. Stoppa, B. Choufi, R. Plagne and D. Maraninchi
`Institut Paoli Calmettes, Marseille & Centre J Perrin, Clermont
`Ferrand, France
`
`Pts with metastatic solid tumors, refractory to chemotherapy,
`present, a poor prognosis. Allogeneic BMT delivers the best dis-
`ease control in leukemia. Thus we proposed this strategy to 12 pts
`(age: 37(21–45); M/F 5 3/9) with refractory solid tumors (Breast:
`3, Kidney: 3; Melanoma: 3; Ovarian: 2; Sarcoma: 1). All received
`an allo BMT from a identical sibling. The first 6 pts were prepared
`with Buscy200 regimen and a GVHD prophylaxis associating
`CSA and MTX. The 6 others received Busulfan (8 mg/kg) 1 Flu-
`darabine 1 ATG (Mini) and CSA as GVHD prophylaxis. Only 3
`pts received post graft GCSF. One patient (ovarian) died on day 5
`from disease progression. All others had full hematological recov-
`ery. Among 9 studied pts, 5 had mixed donor chimerism (90%
`(90–99.7)) and 4 full DC. 4 pts presented grade 2 AGVHD. 3 pts
`received DLI prior day 100. Only 1 pt (refractory ovarian carci-
`noma) experienced an objective response: this was correlated with
`GVHD evolution : 20 mths after transplant, after a new progres-
`sion, she is receiving DLI. 3 pts are alive at 8, 10 and 20 mths with
`measurable disease. 9 pts decreased at 3 mths (1-10) (progression:
`8; TRM: 1 (Buscy regimen)). These data indicates that in this situ-
`ation allo BMT is feasible notably using mini regimen. However
`the speed of disease evolution limits the possibility to obtain an
`allo effect inviting to treat these pts sooner. Based on these data, a
`multicenter trial is presently ongoing in France.
`
`231 Tuesday, July 11, 2000 (8:15–9:45)
`Plenary Session VI: Presidential Symposium
`CHRONIC GVHD IS INCREASED AFTER ALLO BLOOD
`CELL TRANSPLANTATION BUT IS ASSOCIATED WITH
`A REDUCTION OF RELAPSE RATE: RESULTS OF A
`RANDOMIZED STUDY
`D. Blaise, M. Kuentz, J. H. Bourhis, N. Milpied, L. Sutton,
`J. P. Jouet, M. Attal, P. Bordigoni, J. Y. Cahn, J. M. Boiron,
`M. Michallet for the SFGM
`We recently reported the results of a randomized study compar-
`ing allo BMT (N 5 53) and BCT (N 5 48) (JCO, Vol. 18, N.3,
`Feb 2000) for early stage leukemia: this study establishes that BCT
`is associated with better hematological recovery, lower costs, no
`more AGVHD and identical outcome. In the present analysis, with
`a longer follow-up (27 mths (13-41)), we focus on cGVHD.
`cGVHD is more frequent after BCT (28/48 (58%) vs. 16/53 (30%)
`(p , 0.02)) and more severe (extensive cGVHD 5 61% after BCT
`vs. 25% (p , 0.05)). In addition there is a trend for late cGVHD in
`BCT (43% started after 6 months vs. 19% in BMT). No causal fac-
`tor has been found so far. cGVHD has a major effect on relapse
`control : only 1 pt relapsed of the 39 with cGVHD vs. 7 of 55 with-
`out (2 year relapse KM estimate by : 4% (1-18) vs. 18% (9-34) : p ,
`0.03). This effect persisted when the analysis was restricted to
`BCT population (patients with c GVHD : 0 relapse out of 24 pa-
`tients; without cGVHD : 3 of 20 (2 year KM relapse estimate 5
`22% (7-51)) : p , 0.04). However this did not equate so far into a
`
`DR. REDDY’S LABS., INC. EX. 1021 PAGE 2
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