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fludarabine (Flu) Nonmyeloablative conditioning for allogeneic stem cell transplants (alloSCT)
Abstracts/Experimental Hematology 28 (2000) 31–131
with 4.1 (2.7–7.8) reticulocytes. All pts. had hyperbilirubinemia and increased LDH levels. Their haptoglobulin dropped to less than 50 mg%. In 3 recipients increased levels of isohemagglutinin IgG titers were observed. The pts. were treated successfully with multiple RBC transfusions, fluids, diuretics, plasma exchange, high dose immunoglobulins, steroids and erythropoietin. Since NST achieves a state of mixed chimerism without ablation of the recipients’ B cell response to RBC-mismatched antigens, we conclude that as it may be associated with an increased risk of delayed hemolysis. 133
at a concentration of 5⫻103 cellsⲐml in flask (10 mlⲐflask) for 2 weeks at 37⬚C in 5% CO2 in the presence of interleukin (IL) -6 (10ngⲐml), IL-11 (10ngⲐml), Flt-3 ligand (50ngⲐml) and thrombopoietin (10ngⲐml). We investigated the immunophenotype of exvivo expanded CB⫺ CD34⫹ cells at the onset of culture (T0) and 2 weeks later (2w) using three-colours flow cytometry and the following MoAbs: anti -CD34, -CD38, -CD33, -CDw90 and CD13 (Beckton-Dickinson). The table reports the percentages of CD34⫹ cell subsets:
Tuesday, July 11, 2000 (10:15–12:15) Session V-4: Stem Cell and Progenitor Cell Transplantation: Clinical
ENGRAFTMENT FOLLOWING LOW DOSE TOTAL BODY IRRADIATION (TBI) (200 CgY)ⲐFLUDARABINE (Flu) NONMYELOABLATIVE CONDITIONING FOR ALLOGENEIC STEM CELL TRANSPLANTS (alloSCT) A. Nagler, R. OR, M. Brune, G. Varadi, M. Shapira, S. Slavin BMT Department, Hadassah Jerusalem, Israel and Hematology Department, Sahlgrenska, Sweden We recently demonstrated that it is possible to achieve fast and stable engraftment with minimal organ toxicity using FluⲐBuⲐATG conditioning for alloSCT. We have now evaluated the possibility of achieving similar results with Flu and low dose TBI (200cGy). Eleven patients underwent alloSCT from fully matched sibling donors following the TBIⲐFlu conditioning protocol. Six had AML (resist.dis.Ⲑrelapse—5, CR-1), one ALL (relapse), one NHL (resist.dis.) and 3 had genetic disease. Eight were male and 3 female, of median age 38 (11–63) years. The patients were transplanted with G-CSF mobilized PBSC (1–2 phereses). The graft consisted of 13.2 (3.1–18.5) ⫻ 108Ⲑkg MNC, 9.3 (1.7–13.5) ⫻ 106Ⲑkg CD34⫹ cells and 2.8 (1.9–4.8) ⫻ 108Ⲑkg CD3⫹ T cells. In the 9 patients who engrafted the ANC never dropped below 0.5 ⫻ 109ⲐL and the plt. counts were never below 20 ⫻ 109ⲐL. Eight of the patients had transient chimerism but lost the graft and died one month post alloSCT from his basic disease (AML). Two patients who did not engraft (NHL-1, AML-1) had persistent disease, and died. None of the patients suffered from mucositis, liver, renal, pulmonary or other organ toxicity. Grade I-II transient acute GVHD of the skin was observed in 6 patients. Donor lymphocyte infusion was administered to 2 patients because of relapse and partial chimerism, respectively. The relapsing patient developed grade IV acute GVHD and died. Seven of the 11 patients (acute leukemia-4, genetic disease-3) are alive with no GVHD and no toxicity, 5 (4-10) months post alloSCT. We conclude that low dose TBI (200cGy) may be substituted for Bu and ATG as it induces stable engraftment with mixed or complete chimerism with minimal organ toxicity following alloSCT from matched sibling donors. 134
73
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Cord Blood Transplantation
IMMUNOPHENOTYPING OF EX VIVO EXPANDED CD34⫹ CELLS FROM CORD BLOOD L. Porretti*, L. Lazzari*, R. Lopa*, S. Lucchi*, G. Puglisi*, M. Scalamogna*, P. Rebulla*, G. Sirchia* (Intr. by D. Soligo) Centro Trasfusionale e di Immunologia dei Trapianti, IRCCS Ospedale Maggiore, Milan, Italy CD34⫹ cells purified from 2 cord blood (CB) units using immunomagnetic columns were cultured in stroma free liquid culture
CB-unit 1 Phenotype of cells CD34⫹ CD34⫹/38⫺/33⫺ CD34⫹/38⫺/33⫹ CD34⫹/38⫹/33⫹ CD34⫹/38⫺/w90⫹ CD34⫹/33⫹/13⫹
CB-unit 2
T0
2w
T0
2w
71 1.2 21.5 69.2 2.7 45
10 0.4 8.3 90.8 0.14 97
85 0.1 ⬍0.1 64.5 0.1 43
19 6 66 25.4 1 95
The expansions of CB-units 1 and 2 were: nucleated cells 2.63 and 2.4 log; CD34⫹ cells 1.78 and 1.76 log. These culture conditions determined a marked myelo-monocytic lineage commitment. However, CD38 was not expressed on 8.7 and 72% of CD34⫹ cells at 2w. These data support that CD8 may not be the hallmark of uncommitment during CD stem cell ex vivo expansion. 135
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Cord Blood Transplantation
THE INFLUENCE OF MODE OF DELIVERY ON HEMATOPOIETIC STEM CELLS IN UMBILICAL CORD BLOOD COLLECTIONS L. Porretti*, G. Puglisi*, R. Lopa*, L. Leechi*, P. Rebulla*, M. Scalamogna*, G. Sirchia* (Intr. by D. Soligo) Centro Trasfusionale e di Immunologia dei Trapianti IRCCS Ospedale Maggiore, Milan, Italy We evaluated the influence of mode of delivery on the hematopoietic stem cell (HSCs) content (colony-forming cells, CFC, and the number of CD34⫹ cells) of 120 consecutive umbilical cord blood (UCB) units collected in our bank iin 1999. Ninety-eight units were collected after vaginal delivery (VD) and 22 after cesarean section (CS). Table 1 reports mean and SD of the HSCs content of VD and CS units and the results of the statistical analysis:
Table 1 VD-UCB CS-UCB t-test
CFC/l
CD34⫹ cells/l
CFC/CD34⫹ cells
41.4 (21.2) 42.2 (22.4) p ⫽ 0.403
68.3 (38) 95.2 (38) p ⫽ 0.003
0.67 (0.34) 0.45 (0.19) p ⫽ 0.005
CS-UCB showed a significant higher number of CD34⫹ cell counts not associated with a corresponding increment of CFC counts. In order to expand this observation we evaluated in another series of 22 VD-UCB and 12 CS-UCB, the percentages of apoptotic (Annexin-V⫹Ⲑ7-aminoactinomicin⫺) and necrotic (Annexin-V⫹Ⲑ7aminoactinomicin⫹) CD34⫹ cells by flow-cytometry. Table 2 re-
with 4.1 (2.7–7.8) reticulocytes. All pts. had hyperbilirubinemia and increased LDH levels. Their haptoglobulin dropped to less than 50 mg%. In 3 recipients increased levels of isohemagglutinin IgG titers were observed. The pts. were treated successfully with multiple RBC transfusions, fluids, diuretics, plasma exchange, high dose immunoglobulins, steroids and erythropoietin. Since NST achieves a state of mixed chimerism without ablation of the recipients’ B cell response to RBC-mismatched antigens, we conclude that as it may be associated with an increased risk of delayed hemolysis. 133
at a concentration of 5⫻103 cellsⲐml in flask (10 mlⲐflask) for 2 weeks at 37⬚C in 5% CO2 in the presence of interleukin (IL) -6 (10ngⲐml), IL-11 (10ngⲐml), Flt-3 ligand (50ngⲐml) and thrombopoietin (10ngⲐml). We investigated the immunophenotype of exvivo expanded CB⫺ CD34⫹ cells at the onset of culture (T0) and 2 weeks later (2w) using three-colours flow cytometry and the following MoAbs: anti -CD34, -CD38, -CD33, -CDw90 and CD13 (Beckton-Dickinson). The table reports the percentages of CD34⫹ cell subsets:
Tuesday, July 11, 2000 (10:15–12:15) Session V-4: Stem Cell and Progenitor Cell Transplantation: Clinical
ENGRAFTMENT FOLLOWING LOW DOSE TOTAL BODY IRRADIATION (TBI) (200 CgY)ⲐFLUDARABINE (Flu) NONMYELOABLATIVE CONDITIONING FOR ALLOGENEIC STEM CELL TRANSPLANTS (alloSCT) A. Nagler, R. OR, M. Brune, G. Varadi, M. Shapira, S. Slavin BMT Department, Hadassah Jerusalem, Israel and Hematology Department, Sahlgrenska, Sweden We recently demonstrated that it is possible to achieve fast and stable engraftment with minimal organ toxicity using FluⲐBuⲐATG conditioning for alloSCT. We have now evaluated the possibility of achieving similar results with Flu and low dose TBI (200cGy). Eleven patients underwent alloSCT from fully matched sibling donors following the TBIⲐFlu conditioning protocol. Six had AML (resist.dis.Ⲑrelapse—5, CR-1), one ALL (relapse), one NHL (resist.dis.) and 3 had genetic disease. Eight were male and 3 female, of median age 38 (11–63) years. The patients were transplanted with G-CSF mobilized PBSC (1–2 phereses). The graft consisted of 13.2 (3.1–18.5) ⫻ 108Ⲑkg MNC, 9.3 (1.7–13.5) ⫻ 106Ⲑkg CD34⫹ cells and 2.8 (1.9–4.8) ⫻ 108Ⲑkg CD3⫹ T cells. In the 9 patients who engrafted the ANC never dropped below 0.5 ⫻ 109ⲐL and the plt. counts were never below 20 ⫻ 109ⲐL. Eight of the patients had transient chimerism but lost the graft and died one month post alloSCT from his basic disease (AML). Two patients who did not engraft (NHL-1, AML-1) had persistent disease, and died. None of the patients suffered from mucositis, liver, renal, pulmonary or other organ toxicity. Grade I-II transient acute GVHD of the skin was observed in 6 patients. Donor lymphocyte infusion was administered to 2 patients because of relapse and partial chimerism, respectively. The relapsing patient developed grade IV acute GVHD and died. Seven of the 11 patients (acute leukemia-4, genetic disease-3) are alive with no GVHD and no toxicity, 5 (4-10) months post alloSCT. We conclude that low dose TBI (200cGy) may be substituted for Bu and ATG as it induces stable engraftment with mixed or complete chimerism with minimal organ toxicity following alloSCT from matched sibling donors. 134
73
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Cord Blood Transplantation
IMMUNOPHENOTYPING OF EX VIVO EXPANDED CD34⫹ CELLS FROM CORD BLOOD L. Porretti*, L. Lazzari*, R. Lopa*, S. Lucchi*, G. Puglisi*, M. Scalamogna*, P. Rebulla*, G. Sirchia* (Intr. by D. Soligo) Centro Trasfusionale e di Immunologia dei Trapianti, IRCCS Ospedale Maggiore, Milan, Italy CD34⫹ cells purified from 2 cord blood (CB) units using immunomagnetic columns were cultured in stroma free liquid culture
CB-unit 1 Phenotype of cells CD34⫹ CD34⫹/38⫺/33⫺ CD34⫹/38⫺/33⫹ CD34⫹/38⫹/33⫹ CD34⫹/38⫺/w90⫹ CD34⫹/33⫹/13⫹
CB-unit 2
T0
2w
T0
2w
71 1.2 21.5 69.2 2.7 45
10 0.4 8.3 90.8 0.14 97
85 0.1 ⬍0.1 64.5 0.1 43
19 6 66 25.4 1 95
The expansions of CB-units 1 and 2 were: nucleated cells 2.63 and 2.4 log; CD34⫹ cells 1.78 and 1.76 log. These culture conditions determined a marked myelo-monocytic lineage commitment. However, CD38 was not expressed on 8.7 and 72% of CD34⫹ cells at 2w. These data support that CD8 may not be the hallmark of uncommitment during CD stem cell ex vivo expansion. 135
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Cord Blood Transplantation
THE INFLUENCE OF MODE OF DELIVERY ON HEMATOPOIETIC STEM CELLS IN UMBILICAL CORD BLOOD COLLECTIONS L. Porretti*, G. Puglisi*, R. Lopa*, L. Leechi*, P. Rebulla*, M. Scalamogna*, G. Sirchia* (Intr. by D. Soligo) Centro Trasfusionale e di Immunologia dei Trapianti IRCCS Ospedale Maggiore, Milan, Italy We evaluated the influence of mode of delivery on the hematopoietic stem cell (HSCs) content (colony-forming cells, CFC, and the number of CD34⫹ cells) of 120 consecutive umbilical cord blood (UCB) units collected in our bank iin 1999. Ninety-eight units were collected after vaginal delivery (VD) and 22 after cesarean section (CS). Table 1 reports mean and SD of the HSCs content of VD and CS units and the results of the statistical analysis:
Table 1 VD-UCB CS-UCB t-test
CFC/l
CD34⫹ cells/l
CFC/CD34⫹ cells
41.4 (21.2) 42.2 (22.4) p ⫽ 0.403
68.3 (38) 95.2 (38) p ⫽ 0.003
0.67 (0.34) 0.45 (0.19) p ⫽ 0.005
CS-UCB showed a significant higher number of CD34⫹ cell counts not associated with a corresponding increment of CFC counts. In order to expand this observation we evaluated in another series of 22 VD-UCB and 12 CS-UCB, the percentages of apoptotic (Annexin-V⫹Ⲑ7-aminoactinomicin⫺) and necrotic (Annexin-V⫹Ⲑ7aminoactinomicin⫹) CD34⫹ cells by flow-cytometry. Table 2 re-