Autologous Recovery Research Articles

Survival of patients with documented autologous recovery after SCT for severe aplastic anemia: a study by the WPSAA of the EBMT

Graft rejection, with persistent pancytopenia, is well documented after allogeneic BMT (hematopoietic SCT (HSCT)) for severe aplastic anemia (SAA) and the prognosis is poor. The recovery of host-hematopoiesis, autologous recovery (AR), after allogeneic HSCT is a rare event and the incidence and long-term survival are unknown. We report a retrospective analysis of consecutive patients in the Aplastic Anaemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT-WPSAA) registry between 1973 and 2005. A total of 45 cases of AR, of 1205 patients transplanted for SAA in 57 centers are reported. We describe characteristics and long-term outcome of patients with AR, compared with SAA patients from participating transplant centers without AR (n=1024) and patients with graft rejection (n=136) without autologous recovery. The estimated cumulative incidence of AR was 4.2% (3.1-5.6) (confidence interval (CI) 95%) with an OS of 84% (95% CI 83-107%). The OS of the control group was 74% (81-90) at 10 years of follow up, whereas the patients with graft failure had an OS of 16% (CI 12-28%). This retrospective analysis establishes the incidence and long-term survival of patients experiencing AR after allogeneic HSCT for SAA.

Quality Oversight Needed for Intraoperative Autologous Blood Recovery and Readministration

Quality Oversight Needed for Intraoperative Autologous Blood Recovery and Readministration

Quality Oversight Needed for Intraoperative Autologous Blood Recovery and Readministration

Quality Oversight Needed for Intraoperative Autologous Blood Recovery and Readministration

Myeloablative irradiation in non-human primates

We used total body irradiation (TBI) as conditioning for cord blood transplantation studies in pigtailed macaques. In these studies, different doses of TBI were explored to obtain optimal myelosuppression with acceptable radiation-related side effects. Four macaques received TBI ranging from 800 to 1320 cGy, followed by standard post-transplant care. Hematopoietic recovery was monitored by CBC and donor contribution by variable number of tandem repeats analysis. Animals receiving 800 or 1020 cGy TBI tolerated the irradiation well with autologous recovery of neutrophils within 24 days. At a dose of 1200 cGy, neither autologous recovery nor extramedullary toxicity was observed. A fourth animal received 1320 cGy of TBI and suffered significant toxicity necessitating termination of the study. We conclude that previously considered myeloablative doses of TBI allowed for autologous recovery in the pigtailed macaque, and that a dose of 1200 cGy may be most appropriate, providing both myeloablation and acceptable non-hematopoietic toxicities.

Reduced-intensity conditioning using fludarabine with either antithymocyte globulin and BU, or low-dose TBI allowing allogeneic hematopoietic SCT

In a single-center study, we analyzed the outcomes of 66 patients with advanced hematological malignancies receiving two reduced-intensity conditioning regimens for allogeneic transplantation: fludarabine and low-dose TBI (flu/TBI, n=25), or fludarabine, antithymocyte globulin and BU (flu/ATG/BU, n=41). The selection criteria were based on the hypothesis that flu/TBI patients were expected to achieve autologous recovery in the event of non-engraftment. Sixty-three patients (95%) engrafted. Regimen-related mortality at day 100 and 1 year was 6 and 15%, respectively. With median follow-up of 50.4 months, survival did not differ by regimen. Multivariate analysis confirmed that the type of regimen did not affect relapse. In patients achieving full donor chimerism by day 30, those conditioned with flu/TBI showed greater overall survival (P=0.02). Engraftment failure occurred in two patients (3%), both of whom received flu/TBI. We conclude that conditioning with flu/TBI or flu/ATG/BU yields comparable survival and remission outcomes. By contrast to our hypothesis, patients receiving flu/TBI who subsequently failed engraftment did not achieve autologous recovery. Yet, rapid attainment of full donor chimerism after flu/TBI is associated with greater survival than after flu/ATG/BU. Further, larger prospective randomized studies are required to define the advantage of one regimen over the other.

Evidence for anti-tumour effect of allogeneic haematopoietic SCT in cases without sustained donor engraftment

Remissions of haematological malignancies have been reported after allo-SCT, despite donor cell rejection, suggesting that sustained allogeneic engraftment is not mandatory to obtain a lasting anti-tumour effect. To evaluate the potential benefit from transient post-allo-SCT alloreactivity, we took advantage of the Société Française de Greffe de Moëlle et Thérapie Cellulaire (SFGM-TC) registry to colligate 14 patients with an efficient and long-lasting allogeneic (GVL) effect after allo-SCT for haematological malignancies, despite transient or absent engraftment. None received a second allogeneic graft after autologous recovery. The median duration of remission after autologous reconstitution was 118 (12-252) months. Although we cannot exclude the possibility that some patients were cured before allo-SCT, this retrospective analysis does strongly suggest that an efficient GVL effect can be observed without sustained donor engraftment, and that the transient presence of donor T cells might be sufficient to induce a powerful GVL effect.

Single Donor 4/6 Matched Banked Unrelated Cord Blood Is An Effective Graft Source for Children Undergoing Myeloablative Transplantation for Malignant and Nonmalignant Disorders: Single Center Analysis of 318 Patients.

Background: Despite millions of donors in unrelated registries, many patients, in particular those belonging to ethnic minorities can not find a suitable donor in a timely fashion. In contrast, with an inventory of 250,000 banked cord blood unit, almost every patient will find a 4/6 cord blood unit matched by low resolution HLA-A and –B and high resolution –DRB1 typing. The current study evaluates the outcomes of umbilical cord blood transplants (UCBT) performed using a 4/6 matched donor treated at a single center.Methods: Between August 1993 and November 2007 a total of 318 consecutive pediatric patients (under 21 years old) underwent UCBT from a single 4/6 unit after myeloablative conditioning regimen. The patients were a median of 6.1 (range 0.05–20.33) years and weighed a median of 21.2 (range 3.27–118.4) kg at transplant. Overall, 36.5% (n=116) were girls and 39.2% (n=123) were CMV seropositive. A significant proportion (34.6%) of patient identified as belonging to ethnic and racial minorities. Sixty-five percent (n=205) of the patients had malignant diseases including acute lymphoblastic leukemia (n=87), acute myeloid leukemia (n=58), infant leukemia (n=14), and others (n=46). The nonmalignant patients (n=113) included inherited metabolic disorders (n=75), primary immunodeficiency diseases (n=16), bone marrow failure syndromes (n=11), and others (n=11). The cellular composition of the donor cord blood unit showed a median pre-cryopreservation total nucleated cell (TNC) dose of 6.2×107/kg (range 0.9–38.2), infused TNC of 4.8×107/kg (range 0.5–27.4), infused CD34 of 1.8×105/kg (range 0.02–104.8), and infused CFU of 3.6×104/kg (range 0.0–49.9). Kaplan-Meier estimates of survival were calculated using log-rank test. Descriptive statistics were used for other analyses.Results: The median time to engraftment (ANC>500/mm3 and platelets>50K/uL) were 25 and 83 days. By day 42, 87.4% had achieved ANC>500/mm3 and by day 180, 74.7% had achieved platelets>50K/uL. Acute grades III/IV GvHD developed in 15.5% while the incidence of extensive chronic GvHD at 2 years was 15.2% in evaluable patients. A total of 9.7% of patients had either primary graft failure (n=19) or autologous recovery (n=12). The probabilities of overall survival (OS) are presented in the table.N1-yr OS3-yr OS5-yr OSDiagnosis GroupMalignant20552%44%43%Nonmalignant11359%51%46%Patient CMV statusPositive12345%38%38%Negative19062%52%49%TNC cryopreservedx107/Kg<2.52931%24%24%2.5 – 4.999251%43%43%5.0 – 7.57956%50%48%> 7.511863%53%48%Conclusions: A 4/6 single donor unit is an effective graft source for patients younger than 21 yrs and should be seriously considered if the cryopreserved cell dose is more than 2.5×107TNC/kg. The use of 4/6 matched units will make transplant accessible to many more patients in particular those of ethnic and racial minorities who are unable to find a suitable adult unrelated donor.

Trans-Atlantic Consensus on the Medical Management of Radiation Accident Victims

Hematologists will play an important role in the management of radiation accident victims. In 2005, an EBMT (European group for Blood and Marrow Transplantation) International Consensus Meeting defined a unified basis for the medical management of radiation accident victims (Gorin NC et al, Ann Hematol. 2006; 85(10):671–9). The core of this consensus was the 2001 “METREPOL” (Medical Treatment Protocols for Radiation Accident) clinical grading of irradiated victims, based on data from 800 victims in 70 previous accidents. The “METREPOL approach” to triage concentrates on the medical examination of the patient in order to assess the “severity of effect” and the “indicators of repair” of the 4 most important organ systems: hematopoietic, neurovascular, gastrointestinal and skin. A report of the medical preparedness was recently published by the US Radiation Injury Treatment Network (RITN) (Weinstock et al, Blood 2008; 111(12):5440–5; Fliedner et al, Blood 2008; 111(12):5757–8) and supplemented by the EBMT. In addition, the US Department of Health and Human Services REMM (Radiation Event Medical Management; http://remm.nlm.gov) web-based system allows for the continual up-date of medical management and provides information for medical personnel. A workshop including experts from both sides of the Atlantic was organised to take forward a consensus on management of radiation victims, including appropriateness and indications for hematopoietic stem cell transplantation (HSCT). What is achievable following a radiation event will be dictated by the size and scope of the event. It is not expected that hematologists will be involved in the primary triage of victims. Instead, the primary aims for medical management by hematologists include:reducing short and long term morbidity using available supportive care and medical countermeasures,providing expertise to non-hematologists who may be called upon to manage victims with cytopenias,collecting clinical data and potentially biospecimens from radiation victims and 4) reassuring those who have not received significant doses of irradiation (i.e.”worried well”).Also, the clinical triage would determine whether an autologous recovery of hemopoiesis is possible or not. If the hemopoiesis is irreversibly damaged, then reconstitution of hemopoiesis through HSCT may be the only possibility. All other cases are likely to restore hemopoiesis on their own but may require bridging of the pancytopenic phases. The consensus for the use of cytokines was that: G-CSF is to be commenced in all patients with peripheral blood absolute neutrophil count (ANC) of <500/uL at any time-point. An estimate of dose based on the victim's location at the time of the event (i.e., geographic dose reconstruction) may be available after some incidents;if exposure was estimated as >300cGy or >200cGy with combined injury, then G-CSF is to be commenced independent of ANC. As the supplies of G-CSF may be limited after a mass event, G-CSF should NOT be given to patients who do not meet these criteria. Evidence for the use of pegylated G-CSF does not favour its use if G-CSF is available. Erythropoiesis stimulating agents are not to be used routinely. Victims who require transfusions should receive irradiated, leukodepleted products, if possible. It is expected that only a small fraction of victims would be candidates for allogeneic HSCT. Indications, timing and guidance for considering an allogeneic HSCT will be presented. There was also discussion on the importance of harmonizing European and US medical education curriculum on medical response to catastrophic radiological incidents where it will be very important that medical specialists in many countries will need to work together in the response.

Facilitation of Thymic and Bone Marrow Engraftment by Hematopoietic Progenitors Expanded Ex-Vivo Using the Notch Ligand Delta1

Previously, we demonstrated that activation of Notch receptors by culture of murine lin− Sca-1+c-kit+ (LSK) hematopoietic progenitors with the Notch ligand Delta1ext-IgG, consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG, promoted early T cell differentiation and increased the number of progenitors capable of short-term lymphoid and myeloid reconstitution. Here we show accelerated thymic engraftment and T cell recovery after hematopoietic cell transplantation (HCT) in recipients of LSK cells cultured with Delta1ext-IgG compared to recipients of non-cultured LSK cells or control human IgG-cultured LSK cells. Furthermore, data suggest that addition of Delta1ext-IgG cultured LSK cells to non-cultured LSK cells have the potential to accelerated T cell recovery by the non-cultured LSK cells by facilitating thymic engraftment by the non-cultured LSK cells. 103 Ly5a LSK cells cultured with Delta1ext-IgG for 21 days generated a 108-fold increase in total number of cells compared to culture with control IgG. Lethally irradiated C57Bl/6 mice received 105 C57Bl/6 GFP+ LSK cells alone or along with 103 Ly5a LSK cells or 106 Lya5a Delta1ext-IgG-cultured cells or 106 Ly5a IgG cultured cells. We monitored egraftment by difference in CD45 as well as GFP. Mice were sacraficed and blood, bone marrow and thymus were harvested at 1,2,3 and 4 wks after HCT. At 3 wks after HCT, blood samples from recipients of Delta1ext-IgG-cultured cells contained at least 4-fold higher total number of CD3+ cells compared to the other control groups (p<0.05). Furthermore, recipients of Delta1ext-IgG-cultured cells contained at least 3-fold higher numbers of CD3+ cells derived from the GFP+ LSK cells compared to the other control groups (p<0.05). 1 wk after HCT, thymuses and BM from recipients of Delta1ext-IgG-cultured cells showed at lest a 3- and 7-fold increase, respectively, in the number of GFP+ cells compared to the other control groups (p<0.05and p<0.007). Previously, we have shown our system generates committed and non-committed T cell progenitors with enhanced T cell reconstitution potential. Here, we demonstrate that the addition of Delta1ext-IgG-cultured LSK cells to a non-cultured LSK graft has the potential to facilitate BM and thymic engraftment of the non-cultured LSK cells. By using the GFP+ LSK cells, we were able to demonstrate that the facilitation of early BM and thymic recovery was due to the increased engraftment/homing of the GFP+ cells rather than autologous recovery of the host. Thus this report describes a novel and clinically feasible ex-vivo culture system using Delta1 for the generation of T cell progenitors as a means to accelerate initial T-cell recovery as well as facilitating BM and thymic engraftment after HCT.

Prophylactic Rituximab after Reduced Intensity Conditioning Transplantation Results in Low Chronic Gvhd

Background: B cells are implicated in the pathophysiology of chronic GVHD. We hypothesize that prophylactic anti-B cell therapy delivered two months after reduced intensity conditioning (RIC) transplantation would prevent or reduce chronic GVHD incidence from the historical 50%. Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) were the target diseases as they are B cell malignancies with clear allogeneic GVL benefit.Methods: CLL and MCL patients were conditioned with total lymphoid irradiation (TLI) 80 cGy in 10 fractions, d-11 to d-1 and anti-thymocyte globulin (ATG) 1.5mg/kg/day, d-11 to d-7 (total 7.5mg/kg). PBPC were infused on day 0. Primary GVHD prophylaxis was cyclosporine (CSA) on d-3 with taper by 6 months, and MMF from day 0 until d28 for related donors, d100 for unrelated donors. Rituximab (375 mg/m2/week ×4) was infused on days 56, 63, 70, and 77 post-transplant.Results: 36 patients accrued to the study (median age 57, range 31–66 yrs), with 34 patients completing the 4 rituximab infusions. All 22 CLL patients were high risk (fludarabinerefractory, unmutated VH-IgG, or P53 deletion). The 14 MCL patients included 4 patients in PR and 10 patients in CR status at transplant. Median follow-up is 20 months. Twenty patients had sibling donors; 16, unrelated donors. Median CD34 cell dose was 7.5 CD34/kg. All patients had donor cell engraftment except for one patient who had graft failure with stable autologous recovery. Full donor chimerism (PB CD3>95%) was achieved in 14 out of 31 patients (45%) by day 90. However, all but 5 patients had achieved full donor chimerism at 1 year. The incidence of grade 2–4 acute GVHD was 6%. The incidence of chronic GVHD was 18%. Day 100 NRM was 0% and 1-year NRM was 3%. Ten relapses have occurred (5 CLL, 5 MCL). Estimated FFP and OS at 2 years for CLL patients is 82%(CI +/−16%) and 73% (CI +/−33%), respectively; for MCL patients, 64% and 68%, respectively Full donor chimerism was associated with persistent disease remission. Twelve of 19 VHIg mutated CLL patients have achieved minimal residual disease (MRD) by quantitative allele-specific oligonucleotide-IgH PCR (ASO-Q-PCR). DLI was given to 5 CLL patients and 1 MCL who had relapsed and had not achieved full donor chimerism.. There were no infusional toxicities with rituximab. Transient rituximab related neutropenia occurred in 10 patients d100–150. Post-transplant infections included influenza B, RSV, fungal sinusitis, pseudomonas, klebsiella infection, VZV reactivation, and one PTLD before day 56 rituximab. All recovered Of 22 patients at risk for CMV reactivation, 10 reactivated ( range 4 to 56 days post-HCT). Therefore, rituximab did not contribute to the CMV reactivation or increase infection incidence.Conclusion: Prophylactic rituximab infusion post RIC transplantation is well tolerated, provides safe donor B cell depletion without detrimental effect on engraftment or infection incidence, and is associated with a low incidence of chronic GVHD while maintaining GVL. A randomized trial of rituximab prophylaxis after allogeneic HCT is warranted.

Allogeneic bone marrow transplantation for severe aplastic anaemia--the London experience.

Using the Seattle protocol with minor modifications, 23 patients with severe aplastic anaemia received allogeneic bone marrow transplants from HLA/mixed leucocyte culture matched sibs in three London centres between 1973 and 1977. Ten patients (43.5%) are alive 6 months to 5 years after transplantation, and are well with full haemopoietic reconstitution, two with autologous bone marrow recovery following the graft procedure. A failure of the marrow graft to take, or take followed by rejection occurred in 12 patients (52%). Failure of marrow recovery was associated with a high early mortality from bacterial or fungal infection. The only survivors amongst those who rejected the first graft were four patients in whom a subsequent graft from the same donor was successful, and two in whom autologous recovery occurred. Graft versus host disease (GVHD) occurred in seven patients, and was fatal in one case. The most frequent complication after successful engraftment was varicella-zoster infection which occurred in five patients and was fatal in one patient. The overall results compare favourably with those from other transplant centres, but the high rate of graft rejection and low incidence of GVHD differ from other series. The results should encourage further referral of patients with severe AA for bone marrow transplantation.

Unrelated Cord Blood Cell Transplantation for Acquired Severe Aplastic Anemia: The Report from the Japan Cord Blood Bank Network.

Treatment approaches for patients with severe aplastic anemia (SAA), who failed immunosuppressive therapy and lack a bone marrow donor remains a great challenge. Unrelated cord blood transplantation (UCBT) has not been recommended for SAA because of historical poor outcome with high rate of engraftment failure. To evaluate the current feasibility of UCBT in SAA, we retrospectively analyzed the outcomes of 31 patients (median age 28 years old; ranged 0.9–72.3 years old) with SAA, who received UCBT as the first graft between 1998 and 2006 in Japan. Median disease duration before UCBT was 337 day (31–5063 days). By serology, HLA loci were matched in 4 recipient-donor pairs and mismatched (1–2 loci) in 27 patients. A minimum cell count of 2 × 107 nucleated cells/kg body weight was infused in all patients. Engraftment was observed in 17 of 24 evaluable patients. The median times to achieve a neutrophil count ≥ 0.5 × 109/l and a platelet count ≥ 50 × 109/l were 19 days (range 12–35 days) and 59 days (range 39–145 days), respectively. The results of chimerism analysis were available in 9 of them and all of them showed complete donor chimerism (>99%) except one with autologous recovery. Late rejection was seen in one patient. Acute GVHD (≥ grade II) was observed in 5 of 18 evaluable patients (grade II; n=4, grade III; n=1) (cumulative incidence =17.1%) and chronic GVHD was observed in 4 of 14 evaluable patients (extensive: n=1, limited: n=3) (cumulative incidence =19.7%). Currently, 13 patients are alive, having survived for median 22.5 months (ranged 3 to 77 months) after UCBT (overall survival at 2 years=40%). Causes of death of 18 patients were following: graft failure (n=7), bacterial/fungal infections (n=3), hepatic veno-occlusive disease (n=3), and others (n=5). The conditioning regimen appeared to be the most important factor for the outcome and low dose total body irradiation (2–4 Gy) + fludarabine (90–250/ mg/m2) and cyclophosphamide (50–100 mg/kg or 2250/mg/m2) (n=5) gave the best outcome with 80% of survival. The GVHD prophylaxis with single agent (cyclosporine or tacrolimus) related with a better engraftment rate than 2 or more agents (84.4% vs 47.3%, p=0.02). These results suggest that UCBT can be a salvage treatment for patients without a bone marrow donor and warrant further evaluation in prospective studies. Optimization of conditioning regimen will improve the engraftment and outcome of UCBT.

Brain Sparing Conditioning Regimen and Umbilical Cord Blood Transplantation for Inherited High Risk Neurologic Metabolic Diseases.

In addition to the well established role of hematopoietic stem cell transplantation (HSCT) in the treatment of malignancies, HSCT is utilized for a number of neurologic metabolic diseases (typically, enzyme deficiencies) due to the ability of donor graft to provide an ongoing source of enzyme that can be taken up by recipient's cells. Many of these diseases damage the white matter of the brain, and after onset of symptoms are characteristically progressive and lethal. Major limitations to the success of HSCT as therapy for these diseases are graft failure and toxicity to the brain from conditioning, resulting in disease progression. Thus, we reasoned that it would be advantageous to develop a less intensive conditioning regimen that minimally contributed to central nervous system toxicity while providing sufficient immunosuppressive to permit engraftment from unrelated cord blood donors. We are testing a reduced intensity regimen including Campath 1H (1.5 mg/kg), clofarabine (200 mg/m2), melphalan (140 mg/m2), and low dose total body irradiation (200 cGy) for this patient population. To gain insight into the persistence of Campath 1H, we measured serum levels within 30 hours prior to the cord blood infusion. We report outcomes in three adults and three children:DiagnosisAge (years)UCB Graft; HLA matchNC dose (×108/kgCD34+ dose (×106/kg)Campath 1H serum level*Donor Engraftment1MLD44dUCB; 4/6, 5/60.440.68Undectectable0%2ALD9dUCB; 5/6, 5/60.901.43699100%3ML1dUCB; 5/6, 5/61.433.041,575100%4TS1sUCB; 6/60.971.332,250100%5MLD42dUCB; 5/6, 6/60.760.942,72860%6MLD42dUCB; 5/6, 5/60.220.24Not done100%Legend: MLD, metachromatic leukodystrophy; ALD, adrenoleukodystrophy; ML, mucolipidosis type II; TS, Tay-Sachs disease; NC, nucleated cell; dUCB, double unit umbilical cord blood; sUCB, single unit umbilical cord blood; ND, not detected; Eng, engraftment; HLA matching is reported for antigen level HLA-A, B and allele level DRB1. *Campath 1H levels (nanograms/mL) are reported as an average of two measurements of samples diluted 10x. Cumulative doses for both UCB units are shown. The most recent donor chimerism is reported. To decrease the risk of graft rejection and prevent graft versus host disease (GvHD) patients received cyclosporine and mycophenolate mofetil. Patient 2 developed grade II skin and gastrointestinal acute GvHD, treated successfully with systemic and topical steroids. All patients are alive; none experienced progressive deterioration of neurologic function in the peri-transplant period. Our data suggest that this conditioning regimen is minimally toxic to the brain. The two subjects with the lowest Campath 1H concentrations had autologous recovery (patient 1) and GvHD (patient 2), suggesting that in vivo T cell depletion with Campath 1H may be beneficial. These results suggest that novel modifications in the transplant process may provide opportunities to decrease neurologic toxicity and maintain optimal neurologic function in this high risk population.

Cord Blood Transplantation after Non Myeloablative Conditioning (Minneapolis Protocol): Impact on Transplant Outcomes in 112 Adults with Hematologic Malignancy. A French Multicentric Study of the Société Française de Greffe de Moelle et de Thérapie Cellulaire (SFGM-TC) and Eurocord.

The aim of this retrospective multicentric study was to assess the results of 112 consecutive umbilical cord blood transplantations (UCBT) following nonmyeloablative conditioning (NMA) performed in 20 French centres, between October 2003 and March 2007.Characteristics of the patients : Underlying disease: acute leukaemia: myeloid 60, lymphoblastic 17, lymphoma: 15, myelodysplasia: 5, myeloma: 4, Hodgkin: 4, chronic leukaemia: myeloid 3 and lymphoid 3 and 1 solid tumor. Median age at transplantation: 44 y (16–69), median weight: 62 kg (42–125), male: 49 (43%), CMV seropositivity: 64% ; 32 pts had received previous autologous and 3 allogeneic transplantation. The time between diagnosis and transplant was 19 months (3–174). Disease status at transplantation was early (31%), intermediate (37%) and advanced (32%). Median follow-up was 7 months (2–38). The original Minneapolis conditioning regimen was used in 106 (96%) pts and modified in 6 (4 or 6 Gy TBI: 4 pts; ATG: 2). Characteristics of the grafts: A single unit was infused in 77 pts (69%), two in 35 (31%). HLA compatibility was 6/6 in 6 pts, 5/6 in 36, 4/6 in 60, ≤ 3/6 in 6 ; 43 pts were ABO matched. Infused nucleated cells (NC) was 3.1×107/kg (1–9): 2.9 × 107/kg in single units and 3.7 × 107/kg in double units. Results: Neutrophils recovery was 85±4% at a median of 19 days (0–48) ; 14% pts experienced autologous recovery; 14% had mixed and 72% full donor chimerism at D+100. Univariate analysis indicated the low weight, previous transplantation, double units and HLA compatibility as significant factors for neutrophil recovery; however multivariate analysis did not find any significant factor. Acute GVHD was observed in 34±5% of pts: 21, 12 and 5 pts had grade II, III or IV aGVHD respectively and chronic GVHD in 16%. Non relapse mortality was 12±3% at 6 months ; relapse: 22±5% ; overall survival: 72±5%. Causes of death were relapse in 17 pts, GVHD in 2 pts, venocclusive disease and multiorgan failure in 5, infections in 4 and other toxicity in 3. DFS at 6 and 24 months were 68±5% and 65±5%, respectively. By univariate analysis, risk factors for DFS were age (>44y), weight, previous transplant, HLA disparity (0+1 vs 2+3), and NC dose (<3.1× 107/kg). Multivariate analysis identified 3 independent risk factors: HLA disparity, cell dose and age were still significant. This French experience of UCBT after NMA confirms the good results of the Minneapolis group (Brunstein et al. Blood 2007). Few events were observed between 6 and 24 mo and DFS remains high, however a longer follow up is needed. A prospective study of UCBT after NMA conditioning in AML is ongoing in France to identify the risk factors for DFS in a more homogeneous group of pts.

Correction of chronic granulomatous disease after second unrelated‐donor umbilical cord blood transplantation

Allogeneic hematopoietic stem cell transplantation (HSCT) is curative for chronic granulomatous disease (CGD), but many patients lack a suitably matched related donor. We report successful outcomes after mismatched, unrelated-donor umbilical cord blood transplantation (uUCBT) in two boys with X-linked CGD. Both patients experienced autologous recovery after first transplants, required second transplants to achieve durable donor engraftment, and are alive 27 and 15 months post-transplant. Both had invasive fungal disease and received granulocyte transfusions. In conclusion, uUCBT is effective in children with CGD, but immunosuppression in the conditioning regimen may need to be increased to decrease the risk of graft rejection.

Post-radiation pulmonary epithelial regeneration from transplanted adult stem cells

7022 Background: Pneumonitis and pulmonary damage are common toxicities of chest radiation therapy (RT), which may be reduced by transplantation of stem cells that differentiate into pulmonary epithelium. Multipotent Adult Progenitor Cells (MAPC) are adult bone marrow (BM) derived cells, which after IV injection in NOD-SCID mice, differentiate into epithelial and hematopoietic tissues (Nature 418:41). Here we tested the ability of mouse (m) MAPC to regenerate epithelia after RT, in Fanconi Anemia C (FAC) mouse model, which inherently has DNA repair defects and is over sensitive to DNA-cross linking agents. Methods: One million, enhanced-green fluorescent protein (eGFP)- expressing, wild-type mMAPC were injected IV in FAC mice, 4–24 hours after 7.5 Gy (followed by mitomycin C {MMC} 0.1mg/kg/week X 4 in a subset) or 11 Gy RT. Non-eGFP expressing BM was co-transplanted after myeloablative (11 Gy) RT. BM, lungs, intestine and liver of the recipients were analyzed 3–10 months post-transplant for eGFP+ cells and epithelial (CK+) or hematopoietic (CD45+) differentiation using flowcytometry (BM) &amp; immunohistochemistry. Control FAC mice were identically treated but without MAPC. Results: MAPC engraftment (&gt;1% eGFP+ cells) was absent in all control mice. Following 7.5 Gy RT, autologous BM recovery occurred in all (N=10) mice and 1 mouse had 2.7% donor-derived hematopoietic (eGFP+/CD45+) cells after MMC. In both recipients of 11Gy RT, analyzed 9–10 months post-transplant, 5–7% of all CD45+ BM cells were donor MAPC-derived. No donor MAPC-derived epithelial cells were seen in the intestine or liver. However, lungs of most recipients had clusters of donor MAPC-derived epithelial cells, estimated to comprise 1–8% of all pulmonary epithelial cells, consistent with homing of MAPC in lungs following IV injection and their epithelial differentiation in the post-radiation, regenerative, pulmonary micro- environment. The highest degree of MAPC-derived pulmonary regeneration was seen following 11Gy RT that was persistent at 10 months post- transplant. Conclusions: Following radiation, IV injected mMAPC differentiate into hematopoietic and pulmonary epithelial cells that persist long-term. MAPC transplantation has the potential for reducing radiation-induced pulmonary toxicity. No significant financial relationships to disclose.

CD154 Blockade and Donor-Specific Transfusions in DLA-Identical Marrow Transplantation in Dogs Conditioned with 1-Gy Total Body Irradiation

Stable mixed donor/host chimerism has been reliably established in dogs given a sublethal dose (2 Gy) of total body irradiation (TBI) before and immunosuppression with mycophenolate mofetil (MMF) or rapamycin combined with cyclosporine (CSP) after marrow transplantation from dog leukocyte antigen (DLA)-identical littermates (hematopoietic cell transplantation [HCT]). When TBI was reduced to 1 Gy, only transient engraftment was observed. Here we investigated whether stable engraftment after 1-Gy TBI could be accomplished by reducing host-versus-donor immune responsiveness through preceding CD154 blockade and infusion of donor peripheral blood mononuclear cells (PBMCs). We found that the anti-human CD154 antibody, 5c8, cross-reacted with canine lymphocytes and blocked alloimmune responses in vitro. Based on pharmacokinetic studies, 6 dogs received a single intravenous injection of 5 mg/kg anti-CD154 antibody (on day −5), followed 1 day later by donor PBMCs. On day 0, the dogs were given 1 Gy of TBI and underwent DLA-identical marrow grafts. Postgraft immunosuppression consisted of MMF and CSP. All 6 dogs demonstrated initial engraftment; 3 dogs sustained the engraftment for >26 weeks, whereas 3 dogs rejected their grafts, after 9, 22, and 24 weeks, and survived with autologous recovery. Graft survival was significantly improved over that in 11 historical controls conditioned with 1-Gy TBI and given either MMF or rapamycin with CSP after HCT, all of which rejected their grafts between 3 and 12 weeks ( P = .03). Preceding donor PBMC infusion and CD154 blockade improved survival of DLA-identical marrow grafts after 1-Gy TBI.

Hematopoietic Stem Cell Transplantation (SCT) for Hematologic Malignancy from Matched Related Donors (MRD) with a Myeloablative Once Daily IV Busulfan (Bu)/Fludarabine (Flu) Based Regimen (FLUBUP) with Thymoglobulin: Outcomes in 200 Patients with over One Year Follow-Up.

Earlier reports indicated that a myelablative protocol based on once daily IV Bu with Flu plus rabbit antithymocyte globulin appeared effective and relatively well tolerated. Since 1999 this has been our standard regimen for all pts with hematologic malignancy, including those who might otherwise be candidates for non-myeloablative regimens. Two hundred MRD SCT patients (pts) were treated between 05/99 and 07/05. Chemotherapy comprised fludarabine 50mg/m2 on days -6 to -2 and IV busulfan (Busulfex, PDL Pharma) 3.2 mg/kg daily days -5 to -2 inclusive. Forty-six acute leukemia pts had additional TBI 200cGy x 2 on day (D) -1 or 0. Prophylaxis for GVHD was cyclosporine A, methotrexate with folinic acid and Thymoglobulin (Genzyme) 4.5 mg/kg in divided doses over 3 consecutive days pretransplant finishing D0. Cell source was mobilized blood cells (BCT) in 172 and marrow (BMT) in 28. Median age was 46 (range 18–65) and follow-up of survivors was 13–87 months (median 42). As engraftment and GVHD did not differ significantly data for BMT and BCT were combined. Two pts had graft failure (1 with autologous recovery) and two died before D28. The remaining 196 evaluable pts engrafted granulocytes at median of 16 days (range 2–42). Platelets engrafted in 194 pts at a median of 18 days (0–101), 2 died before engraftment on days 29 & 72. Incidence of acute GVHD grade II-IV, acute GVHD grade III-IV and chronic GVHD was 14±2%, 3±1% and 54±4% respectively. Five pts died before D100 of GVHD (1), candidiasis (1), influenza B (1), veno-occlusive disease (1) and myocardial infarction (1), after D100 15 deaths were related to GVHD (11), pneumonia/ARDS (2), graft failure (1) and pulmonary embolus (1). One case of PTLD resolved with rituxan, there was no serious neurological toxicity. For low-risk (acute leukemia CR1/CR2, CML CP1) pts 3 year TRM was 4±3% for those ≤45 years old (n = 54) and 6±4% for those >45 (n = 31). Corresponding figures for all others (high risk) were 6±4% (n = 40) and 27±7% (18±5% at one year, n = 75) (p = 0.04). [table 1] We conclude that FLUBUP appears to be well-tolerated with little mortality attributable to the regimen itself. In partular, concerns about excessive immune suppression (partularly causing PTLD) and neurotoxicity have not been substantiated. Chronic GVHD is the major cause of non-relapse death. Control of early morbidity and mortality from GVHD may allow intensification of the regimen, for example by adding TBI. There is also evidence that the safety can be improved by monitoring Bu levels. The effect of age on TRM is only apparent in high-risk pts, the challenge is to reduce mortality from chronic GVHD in these individuals without sacrificing graft-vs.-malignancy.3 year outcomes (%) for groups with >10 ptsDisease/stagensurvivalTRMAML CR1/2 (no TBI)3266±89±5AML CR1/2 (TBI)*1694±60AML advanced (no TBI)1822±1017±9MDS1579±1124±12ALL CR1/21573±118±7CML CP122100%0CLL/Richter's (as primary indication)1468±1316±10Multiple Myeloma1354±148±7NHL (low grade/mantle)2175±1017±9All pts20066±312±3* p = 0.07

Continuous Erythroblastosis Is Observed during Early Recovery Period after Allogeneic Umbilical Cord Blood Transplantation.

Objectives: The repopulation characteristics on human hematopoietic cells are crucial for clinical performance after allogeneic hematopoietic stem cell transplantation. We observed and compared the changes of blood cells during the early recovery period after allogeneic HSCT.Method: The study included patients from 19 to 62 years of age who were admitted to our hospital during Dec. 2000 to Mar. 2005 for the allogeneic HSCT, and in whom no serious complications were observed on transplantation. A total of 25 patients were eligible (10 patients of U-CBT with stable engraftment, 2 patients of U-CBT with no engraftment and autologous recovery thereafter, 8 patients of BMT and 5 patients of PBSCT). All the patients received G-CSF. From day 0 of transplantation, the number of blood cells was calculated mechanically and microscopically, and their morphology was microscopically determined with the stained smear-slide. Five smear slide glasses were made by hand on each of the two days, and the cells were stained with May-Giemsa solution.Result: Continuous erythroblastosis was observed in all the engrafted U-CBT cases, which was firstly observed between the same days as when blood WBC was increased in number, and five days later (on day 15 on average). Neither correlation of the ABO incompatibilities was observed to erythroblastosis, nor prominent hemolysis was observed. However, in BMT cases and in PBSCT cases, continuous erythroblastosis was observed in a few cases. Neither continuous erythroblastosis was observed in U-CBT cases that showed no engraftment of the transplanted graft.Discussion: These observations indicate that continuous erythroblastosis during early recovery period is specific for U-CBT, and may reflect the difference in the characters of hematopoietic stem cells.

Stem Cell Transplantation from HLA-Identical Siblings Versus Alternative Donors in β-Thalassemia Diseases.

Background: Allogeneic hematopoietic stem cell transplantation (SCT) has been established as a curative therapy for β-thalassemia major which is prevalent among Thais. Donors are usually patients' siblings but the chance to find an optimal HLA-identical is just about 30%. Thus clinical trial using alternative donors such as partially-mismatched related donors or matched unrelated donors could be a good choice.Methods: To study the efficacy and results, we retrospectively review our experience with β-thalassemia pediatric patients undergoing transplantation from July 1999 to July 2006.Results: There were 43 patients categorized into HLA-identical siblings group (n=26, 13 male and 13 female) and alternative donors group (n=17, 14 male and 3 female). Median age and weight were 7.2 years (1.2–14.8 years), 19.8 kg (9.8–34 kg) and 7.7 years (1.4–14.8 years), 21.5 kg (11–33 kg), respectively. Amongst 26 matched siblings there were 17 bone marrow (BM), 4 peripheral blood stem cell (PBSC), and 5 cord blood (CB) donors. Of the alternative group stem cells derived from 12 matched unrelated, 2 two-antigen mismatched sibling CB, 2 one-antigen mismatched unrelated CB, and 1 one-allele mismatched unrelated BM donors. Myeloablative conditioning regimen consisted of busulfan (oral or intravenous) and cyclophosphamide for matched siblings SCT. Anti-thymocyte globulin was added for alternative donors SCT. Fludarabine was also used in addition for unrelated donors SCT. Graft-vs-host disease (GvHD) prophylaxis consisted of cyclosporine plus methotrexate except for unrelated SCT that tacrolimus plus methotrexate were used instead. Median numbers of infused CD34+ cells of the matched siblings group were 11.1x106/kg (3.7–35.4) in BMT (n=17), 9.3x106/kg (7.1–12.8) in PBSCT (n=4), and 0.94x105/kg (0.2–5.3) in CBT (n=5), compared to 5.5x106/kg (1.5–18.1) in BMT (n=10), 11.1x106/kg (11–17.2) in PBSCT (n=3), and 2.1x105/kg (1.5–3) in CBT (n=4) of the alternative group. Median times to neutrophil and platelet engraftments in each groups were 15 and 33 days, compared to 17 and 41 days, respectively. 39 successful donor engraftments were achieved from 38 HLA-matched (25 related and 13 unrelated) donors and from 1 mismatched unrelated CB unit. The remaining 1 of the matched related group and 3 of the alternative group did not engrafted, all but one case subsequently resumed autologous recovery. Acute GvHD occurred in 4 matched-sibling and 5 unrelated SCT recipients. After immunosuppressive therapy the lesions were completely resolved in all but two patients, one who suffered from severe grade IV intestinal GvHD and another who later developed extensive chronic GvHD which required treatment longer than 4 years. There were 4 mortalities only in the alternative group: 3 patients died post-engraftment due to severe fatal GvHD (n=1), cerebral aspergillosis (n=1), severe veno-occlusive disease with liver failure (n=1; Pesaro class 3); and one died from neutropenic sepsis 3-week after 4/6 matched related CBT. Median follow-up time for surviving patients was 53.5 months (2–84 months). Overall and disease-free survival in HLA-matched SCT (n=38) recipients were 94.7% (n=36) and 92.1% (n=35), compared to in HLA-mismatched (n=5) 60% (n=3) and 20% (n=1), respectively.Conclusions: SCT for β-thalassemia major using HLA-matched donors, either related or unrelated, had superior results than HLA-mismatched. In situation of unavailable matched sibling donors, the search for an appropriate complete HLA-matched unrelated volunteer donor using high resolution DNA typing is essential for favorable outcomes.