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Umbilical cord blood (UCB) has been successfully used for haemopoietic stem cell transplantation, although its use has been cautiously limited to paediatric patients because of the reduced volume produced. The clinical results have confirmed that either engraftment or survival significantly correlate with cell dose infused. We have standardized a culture method providing in a short time a significant amplification of both committed progenitors and primitive stem cells for clinical use. Eight-day culture of UCB cells with flt3L/SCF/PIXY 321 induced a 10-fold amplification of CD34+ cells and the expansion of multipotent (CFU-GEMM) and committed (CFU-GM, BFU-E) progenitors respectively of 5-, 7- and 9-fold over input cells. As to the early stem cell pool, the primitive CD34+Thy-1+ cell fraction increased 6-fold and the LTC-IC were amplified 17-fold. Furthermore, the in vitro proliferation was detected by the gradual loss of fluorescence of the CD34+ cells tracked at day 0 with the dye PKH26. After 8 d of amplification >6% of the CD34+ cells remained intensely fluorescent. This subpopulation represents a deeply quiescent cell fraction unresponsive to cytokines and very enriched of primitive stem cells. These cells are most likely to be responsible for long-term reconstitution after transplant.

We prospectively studied the chimaerism status in the bone marrow (BM) and peripheral blood (PB) of 23 patients receiving umbilical cord (UCB, 14 cases) or BM (nine cases) transplants from unrelated donors by PCR amplification of four individual-specific VNTR genetic loci. Haematological engraftment, with persistent full donor pattern. was observed in 10/14 (72%) patients receiving UCB and in 9/9 (100%) patients transplanted with marrow from an unrelated donor (MUD). In contrast, the remaining four patients converted to an autologous pattern. Three out of these four patients had an early autologous haematological reconstitution reaching a neutrophil level >0.5 x 10(9)/l at days 27, 33 and 37 after transplant, respectively. In all three of these patients, chimaerism analysis demonstrated an early appearance of donor cells (i.e. within 35 d after UCB transplant) showing a transient full donor (one case) or mixed chimaerism condition (two cases). Despite the early autologous haemopoietic reconstitution, one of the three patients died of GVHD at day 60, which was explained by the demonstration of low levels of donor lymphoid cells. In the MUD group all nine patients converted to a persistent full donor pattern with haematological reconstitution, accompanied in two of them by transient mixed chimaerism lasting to days 60 and 270 after transplant. Our data show that monitoring of chimaerism may predict graft failure with or without early autologous haemopoietic reconstitution in patients receiving unrelated UCB transplants. Furthermore, chimaerism analysis may identify, in patients with autologous reconstitution, those at risk of severe GVHD in whom immunosuppressive therapy should not be discontinued.

Umbilical cord blood (UCB) is a source of stem cells for allogeneic haematopoietic transplantation in paediatric and adult patients with haematological malignancies and other indications. Voluntary donation is the basis for the success of unrelated UCB transplantation programmes. In the last few years a growing number of private banks offer their services to expectant parents, to store UCB for future use. The debate concerning UCB donation and private preservation has been ongoing for several years. The aims of this single centre study were to explore knowledge about UCB stem cells and attitudes towards voluntary UCB donation or private UCB preservation among both blood donors and pregnant women.This study was conducted at the "Sapienza" University of Rome. Two types of anonymous questionnaires were prepared: one type was administered to 1,000 blood donors while the other type was distributed to 300 pregnant women.Most blood donors as well as the majority of pregnant women had some general knowledge about UCB (89% and 93%, respectively) and were aware of the possibility of donating it (82% and 95%). However, the level of knowledge regarding current therapeutic use resulted generally low, only 91 (10%) among informed blood donors and 69 (31%) among informed pregnant women gave a correct answer. The survey revealed a preference for voluntary donation both among blood donors (76%) and among pregnant woman (55%). Indeed, a minority of blood donors (6.5%) and of pregnant women (9%) would opt to store UCB for private use.The study raises the following considerations: (i) the large support for UCB donation expressed by blood donors and by pregnant women suggests that UCB preservation does not represent an obstacle to the expansion of UCB donation and to development of unrelated transplantation programmes; (ii) information about UCB donation and preservation should be carefully given by professionals and institutions.

To assess the effectiveness of the search for an unrelated donor on the outcome of patients with high-risk acute lymphoblastic leukemia, we analyzed prospectively 136 patients who underwent a search for cord blood (CB) and an unrelated volunteer donor (UD) at the same time. The probability of finding a donor was 58.2%, 70.3%, and 75.7% at 3, 6, and 12 months, respectively. The median time to find a donor was 1.8 months for CB and 3.5 months for UD. Of the 99 patients with a donor, 38.4% failed to undergo the transplant because of a relapse observed at a median of 4 months from the start of the search. In univariate analysis, absence of relapse during the search (p < 0.0001) and transplant (p = 0.004) showed a positive impact on long-term survival. In multivariate analysis, relapse during the search remained the key factor affecting survival (p < 0.0001). Since an extension of the search beyond 3 months enables only a slight increase in the probability of finding a donor compared to the increased risk of relapse, the time of the search should not exceed the 3-month time point. The simultaneous search for CB and UD increases the likelihood of performing a timely transplant.

The aim of the study was to analyze and compare the functional properties and the gene expression profile of regulatory T cells (Tregs) isolated from cord blood (CB) units (n = 23) and from the peripheral blood (PB) of adult normal donors (n = 13). Tregs were purified from mononuclear cells and expanded for 6 days with anti-CD3, anti-CD28, and IL-2. CB and PB Tregs presented similar immunophenotypic features. However, Tregs isolated from CB presented a much higher expansion capacity; this was confirmed by the genomic characterization that showed in CB-derived Tregs significant enrichments of genes involved in cell proliferation, chromatin modification, and regulation of gene expression. All samples were positive for the FoxP3 gene and protein after expansion. CB and PB expanded Tregs exerted a comparable and potent suppressive function on the proliferative reaction of autologous T cells stimulated by allogeneic dendritic cells and presented a high in vitro IL-10 production capacity. Gene profile analysis also revealed for PB Tregs significant enrichments of genes involved in the adaptive immune response. These data offer further insights into the understanding of the biology of CB transplantation indicating a possible role played by CB Tregs in the suppression of the allogeneic T cell response.

The aim of this single center study was to assess the impact of pre-transplant factors on long-term follow-up in young patients affected by high-risk acute lymphoblastic leukemia (ALL) who underwent an unrelated cord blood transplant (CBT). The conditioning regimens, graft-versus-host disease (GVHD) prophylaxis and supportive policies were uniform for all patients.We analyzed the results of CBT performed in 30 patients, aged18 years, affected by high risk ALL. As conditioning regimen, all patients received 12 Gy fractionated total body irradiation, etoposide, cyclophosphamide and horse anti-lymphocyte globulin. GVHD prophylaxis consisted of 6-methylprednisolone and cyclosporine A. RESULTS The cumulative incidence of engraftment was 93% (95% CI:0.85-0.93). The cumulative incidence of grade III-IV acute and chronic GVHD was 7% (95% CI:0.01-0.19) and 33% (95% CI: 0.17-0.64), respectively. The 9-year cumulative incidence of transplant-related mortality and relapse was 34% (95% CI:0.13-0.45) and 31% (95% CI:0.16-0.61), respectively. The 9-year overall survival, leukemia-free survival and event-free survival were 42% (95% CI:0.52-0.93), 47% (95% CI:0.25-0.61) and 46% (95% CI:0.33-0.61), respectively. A number of CFU-GM1 x 10(4)/Kg of recipient body weight was the only factor that negatively affected all outcome parameters both in univariate and multivariate analyses.The infused cell dose expressed as in vitro progenitor cell growth represents the most important pre-transplant factor affecting the long-term outcome after an unrelated CBT in young patients with high risk ALL. The number of CFU-GM should thus be considered in the selection process of cord blood units for transplant.

From July 1995 to December 2001, 42 patients with leukemia aged 1-42 years underwent cord blood transplant (CBT) from unrelated, < or = 2 antigen HLA mismatched donors. In all, 26 patients were in < or = 2nd complete remission and 16 in more advanced phase. Conditioning regimens, graft-versus-host disease (GVHD) prophylaxis and supportive policy were uniform for all patients. The cumulative incidence of engraftment was 90% (95% CI: 0.78-0.91). The cumulative incidence of III-IV grade acute- and chronic-GVHD was 9% (95% CI: 0.04-0.24) and 35% (95% CI: 0.21-0.60), respectively. The 4-year cumulative incidence of transplant-related mortality (TRM) and relapse was 28% (95% CI: 0.17-0.47) and 25% (95% CI: 0.14-0.45), respectively. The 4-year overall survival (OS), leukemia-free survival (LFS) and event-free survival (EFS) were 45% (95% CI: 0.27-0.63), 47% (95% CI: 0.30-0.64) and 46% (95% CI: 0.30-0.62), respectively. In multivariate analysis, the most important factor affecting outcomes was the CFU-GM dose, associated with CMV serology (P=0.003 and 0.04, respectively) in influencing OS and with patient sex (P=0.008 and 0.03, respectively) in influencing LFS. Finally, CFU-GM dose was the only factor that affected EFS significantly (P=0.02). In conclusion, the infused cell dose expressed as in vitro progenitor cell growth is highly predictive of outcomes after an unrelated CBT and should be considered the main parameter in selecting cord blood units for transplant.

Anti-P1 is frequently found by transfusion services because a large percentage of P2 individuals have this antibody in the serum. 1 It is usually a cold-reactive antibody without clinical siLmificance3 Some authors a suggest that routine examination of donor red cells for P1 antigen is not necessary, because of its apparent clinical insitmiflcance. We report a severe delayed hemolytic transfusion reaction caused by anti-P1 alloantibody.

Abstract 4195 Directed family cord blood (DCB) storage provides hemopoietic stem cell source for transplantation (HSCT) for families with an existing or a potentially future recipient with HSCT curable disease (D). The National CB Banks Network in Italy (ITCBN) has a leading role in providing public DCB service for high-risk families, in compliance to GITMO directives for eligibility criteria (HSCT curable D: malignant MD, non MD, inherited ID). To provide best cost-effective practices recommendations it is important to report on DCB procedures and HSCT rate (HSCT-R) among public Banks. By 12.12.2008 almost 1800 DCB units were stored in 18 Italian Banks and 104 (9%) issued for HSCT. The present survey aims at summarizing the over 15 yrs DCB experience among 5 ITCBN Banks active since 1997 (range 1990-1997), and including 670 DCB units. Results Preliminary analysis reports a 94% overall compliance to eligibility criteria directives, and overall HSCT- R for an alive sibling of 12% (63/522); the 63 HSCT were 97% matched, for curing ID in 84% and with 72 % overall survival outcome. Different policies among Banks were compared (Bank vs others: 1) eligibility criteria distribution : Bank PV06 DCB for MD Conclusion. Advice for public long-term DCB storage should depend on HLA compatibility, potential recipient, disease progression, and likelihood/ timing of using DCB; Italian CB Banks are actively cohoperating to find best public banking practices to ensure a CDB storage system that is ethical, cost effective and responsive to patient needs. Disclosures: No relevant conflicts of interest to declare.

Abstract 4085 Poster Board III-1020 Introduction Umbilical cord blood (CB) stem cells are now broadly used in the unrelated stem cell transplant setting and comparative studies with different stem cell sources have shown that CB transplant is characterized by a lower risk of graft-versus-host disease (GVHD). The immaturity of CB T cells has been generally regarded as the main contributing factor accounting for this phenomenon; the possible role played by CB regulatory T cells (Tregs) for the suppression of the allogeneic T-cell response is now under investigation, but very scare data are so far available. Aim of this study was to analyze and compare the functional properties and the gene expression profile of Tregs expanded from CB units with those expanded from the peripheral blood (PB) of adult normal donors. Methods Tregs were purified from mononuclear cells obtained from 23 CB units and from the PB of 13 adult normal donors using the CD4+CD25+ regulatory T-cell isolation kit (Miltenyi Biotec) and expanded for 6 days in 96-well U-Bottom plates coated with the anti-CD3 (5 ug/ml) and anti-CD28 (5 ug/ml) MoAbs in the presence of IL-2 (100 U/ml). Immunophenotypic analyses were performed before and after expansion. To assess their suppressive functions, expanded Tregs were seeded with autologous effector T cells stimulated with allogeneic dendritic cells (DC) pulsed with apoptotic leukemic blasts, then incubated with [3H]-thymidine and counted in a beta-counter. Suppressor activity was measured as [3H]-thymidine incorporation in the presence or absence of Tregs. The IL-10 production capacity of expanded Tregs was tested using an ELISA assay. The two-sided student t test was used to evaluate the significance of differences between groups. Gene expression profile experiments were performed using the HGU133 Plus 2.0 arrays (Affymetrix); statistical analyses were carried out using the dChip software; a t test was used to evaluate the presence of specifically expressed classes of genes. Functional annotation analysis was performed using the DAVID software. Results CB and PB Tregs presented similar immunophenotypic appearances before and after expansion. Im particular, after expansion they presented a comparable expression of surface CD4, CD25, CD62L, CCR5 and CD45RO, and of cytoplasmic CTLA-4 and Foxp3, while they both were negative for the CD45RA antigen, thus indicating the loss of their naïve features. On the contrary, Tregs obtained from CB (n=23) presented a much higher expansion capacity compared to those obtained from PB (n=13): mean fold increase (range), CB 10.3 (1.6-24), PB 3.9 (1.5-10), p 0.003. CB expanded Tregs (n=6) exerted a potent suppressive function on the proliferative reaction of T cells stimulated by allogeneic DC, that resulted inferior even though not significantly compared to that exerted by PB expanded Tregs (n=5): mean fold reduction (range), CB 7.8 (2.5-15.1), PB 14.3 (1.5-23.7), p 0.14. Tregs expanded from CB (n=4) and PB (n=1) presented a high and comparable in vitro IL-10 production capacity: mean pg/ml (range), CB 326.5 (226-426), PB 382. Gene expression profile analysis showed a higher number of upregulated genes in Tregs expanded from CB (n=2) compared to Tregs expanded from PB (n=3); among them, a significant enrichment of genes involved in cell proliferation, cell cycle checkpoints, signal transduction, cell differentiation, apoptosis, TGF-β receptor pathway and the GrNH pathway was observed. This suggests that CB Tregs retain a more undifferentiated program and are characterized by the high expression of genes which might provide an advantage in cell expansion. Finally, when looking at the Foxp3 gene expression levels, no difference was observed between the two populations. Conclusions These results demonstrate that Tregs contained in CB retain an expansion potential superior to that of Tregs isolated from the PB of normal donors, as confirmed by functional analyses and gene profile. Tregs expanded from CB and PB seem to exert a potent and comparable suppressive function of the proliferative effect in mixed lymphocyte reaction assays. The maintaining of the modulatory properties after expansion is confirmed by the expression of the Foxp3 gene and protein, and by the production of IL-10. These data offer further insights into the understanding of the biology of CB transplantation indicating a possible role played by CB Tregs in the suppression of the allogeneic T-cell response. Disclosures: No relevant conflicts of interest to declare.

20 CML patients with hematological (5 pts) or only cytogenetic (15 pts) relapse occurring after allogeneic BMT have been treated with alpha-2b-interferon (IFN) at a starting dose of 5 times 106 IU/m2, subcutaneously, three times a week. All 5 patients with hematological relapse achieved hematological remission without reduction of bone marrow Ph1-positive cells. With a median follow-up of 43 months (range 6–48) from the hematological relapse, 3 patients are alive and 2 patients died from non-lymphoid blast crisis. 7 out of 15 patients with only cytogenetic relapse remain in hematological remission at a median of 37 months (range 3–45) from cytogenetic relapse, with 2 patients achieving complete cytogenetic remission confirmed at the molecular level by disappearance of the bcr rearranged band. With a median follow-up of 21 months (range 6–46), 8 patients progressed from cytogenetic to hematological relapse: 4 patients died from blast crisis and the other 4 patients are currently alive in chronic phase. For the 15 patients, the actuarial survival from BMT is 71% at 5 years.

CD4+CD25+ T-regulatory (Treg) cells have been shown to regulate self and allograft tolerance and to suppress allogeneic immune response. Studies in mouse models of bone marrow (BM) transplantation have shown that the infusion of culture-expanded Tregs can be effective in preventing and suppressing graft-versus-host disease (GVHD), while still allowing a graft-versus-leukemia effect (GVL). Cord blood (CB) represents the most recent source of hematopietic stem cell transplantation (HSCT) used in the unrelated transplant setting. Comparative studies have shown that unrelated CB transplant is an acceptable alternative to unrelated BM transplantation, characterized by a lower risk of transplant-related mortality due to GVHD. CB has been reported to contain Tregs, but minimal data are available on these cells. Aim of this study was to compare the suppressive functions of Tregs obtained from CB units with those obtained from the peripheral blood (PB) of patients who have undergone an allogeneic BM transplantation and of normal donors. Treg cells were purified from mononuclear cells obtained from CB units or PB using the CD4+CD25+ regulatory T-cell isolation kit (Miltenyi Biotec) and expanded for 6 days in 96-well U-Bottom plates coated with anti-CD3 and anti-CD28 MoAbs plus IL-2. To assess the suppressive functions of Treg cells, expanded CD4+CD25+ Tregs from CB units or PB were seeded with naïve autologous effector CD4+CD25- T cells stimulated with allogeneic dendritic cells (DC) pulsed with apoptotic leukemic blasts, then incubated with [3H]-thymidine and counted in a beta-counter. Suppressor activity was measured as [3H]-thymidine incorporation in the presence or absence of Tregs. The immunophenotypic analysis of Tregs from CB units - in terms of expression of surface CD4, CD25, CD62L, cytoplasmic CTLA-4 and Foxp3 - before and after expansion, did not show any specific peculiarity. CB units (n = 6) contained a proportion of Tregs comparable to those contained in the PB of allotransplanted patients (n = 8) or in the PB of normal donors (n = 8) (mean % ± SD, CB 0.28% ± 0.41; PB patients 0.4% ± 0.4; PB donors CB 0.45% ± 0.29). On the contrary, Tregs from CB units and from the PB of allotransplanted patients showed a higher expansion capacity when compared to Tregs from the PB of normal donors (mean fold increase ± SD, CB units 11.5 ± 9.0; PB patients 9.6 ± 5.0; PB donors CB 5.2 ± 2.7). Preliminary data also show that Tregs expanded from CB units (n = 2) exert a higher suppressive function on the proliferative reaction of T cells stimulated by allogeneic DC if compared to Tregs expanded from the PB of allotransplanted patients (n = 2) (mean fold reduction ± SD, CB 5.25 ± 3.7; PB patients 3.8 ± 3.3). These results demonstrate that Treg cells contained in CB units present an expansion capacity superior to that of Tregs from PB of normal donors; moreover, these expanded cells seem to exert a potent suppressive function of the proliferative effect in mixed lymphocyte reaction culture assays. These data offer further insights in the understanding of the biology of CB transplantation and may indicate a possible clinical use of expanded Tregs for the prevention and management of GVHD in the setting of CB transplantation.

In the last 3 years, 14 children with high-risk leukemia (11 ALL, 2 AML and 1 CML) underwent cord blood transplantation from unrelated HLA-mismatched donors at a median of 99 days from the start of search. Eight patients were transplanted in second CR, one in accelerated phase, three at relapse and two patients in first CR. Conditioning regimen (fractionated TBI, etoposide, CY and anti-lymphocyte serum) and prophylaxis of GVHD (CsA and 6-methylprednisolone) were identical for all patients. Neutrophils >0.5x10(9)/l were reached at a median of 33 days from transplant, but in four cases we observed an autologous hematopoietic reconstitution (three spontaneous, one after autologous BM rescue). Acute and chronic GVHD were observed in 10/14 and 3/8 evaluable cases, respectively. Three patients died of transplant-related toxicity and three patients relapsed. The probabilities of event-free, disease-free and overall survival were 50, 53 and 64%, respectively. Cord blood transplant from HLA-mismatched unrelated donor is a valid option for the treatment of children with high-risk leukemia. With our eligibility criteria, conditioning regimen and prophylaxis of graft-versus-host disease, the main obstacles to successful transplant were represented by graft failure and fatal acute GVHD.