Double Umbilical Cord Blood Research Articles

Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for Pediatric Patients with Treatment Related Myelodysplastic Syndrome or Acute Myelogenous Leukemia (tMDS/AML)

AbstractAbstract 2363The development of tMDS/tAML is a complication that can occur following chemotherapy (CT) or radiation (RT) therapy. Therapeutic approaches for such patients (pts) have included both CT and/or HSCT, with overall successful outcome in 30–35% of pts. From October 1994 to September 2009, 22 pts with tMDS/tAML received an allogeneic HSCT at our institution. The median age at presentation of primary disease was 15 years (range 3 months–26 years). The median age at time of HSCT was 21 years (range 8–29 years). There were 11 males and 11 females. Therapy related MDS/AML developed following RT and/or CT in pts with cancer (n=19) or a non-malignant disease (n=3). The initial malignancies consisted of Osteosarcoma (OS) n=5, Neuroblastoma (NB) n=3, Ewing Sarcoma n=3, Rhabdomyosarcoma n=2, Desmoplastic Round Cell Tumor n=1, Malignant Fibrous Histiocytoma n=1, Hodgkin Lymphoma n=1, Non-Hodgkin Lymphoma n=1, and AML n=1.Five pts proceeded to HSCT without induction chemotherapy, at which time four pts had refractory anemia (RA) and one pt had AML. The other 17 pts were treated with chemotherapy prior to transplant; 14 received high dose cytarabine at 3g/m2/dose every 12 hours × 4 doses with 13 pts achieving morphologic remission or secondary RA. Conditioning regimens were selected according to previous therapies: three pts had a total body irradiation (TBI) containing regimen; 11 pts received busulfan (Bu) (0.8mg/kg/dose every 6 hours × 10 doses) melphalan (Mel) (70mg/m2/dose × 2 doses) fludarabine (Flu) (25mg/m2/dose × 5 doses) for cytoreduction; and eight pts received myeloablative regimens containing Bu and/or Mel and/or thiotepa with doses modified for organ toxicity. Sixteen pts received T-cell depleted (TCD) bone marrow (n=2) or peripheral blood stem cell (PBSC) (n=14) grafts (including 11 pts following Bu-Mel-Flu); four received unmodified bone marrow (n=3) or PBSC (n=1); and two received a double umbilical cord blood transplant (DUCBT). Donors included HLA-matched (n=9), or mismatched related donors (n=3), HLA-matched (n=4), or mismatched (n=4) unrelated donors or DUCBT (n=2). Disease status at time of HSCT was: morphologic and cytogenetic CR (n=12); RA with positive cytogenetics (n=6); and refractory disease (n=4).The median follow up is 5.9 years (2.2-15.7 years). The five-year overall survival (OS) and disease free survival (DFS) rates for the entire group are 56.6% with 12 pts alive without evidence of either primary disease or tMDS/tAML. The OS and DFS rate for the 11 pts who received the Bu-Mel-Flu cytoreduction with TCD grafts was 54.5%. Ten pts died. Cause of death was relapse of MDS/AML (n=5) or of primary disease (n=2), GVHD (n=2), and infection (n=1). Four pts developed grade II-IV acute graft versus host disease (GVHD). Two pts recovered and two pts died from infectious complications related to GVHD. One patient developed localized chronic GVHD which resolved. Variables associated with a favorable outcome included: disease status with DFS of 63% for pts in RA or CR at HSCT compared to 0% for pts with >5% residual blasts in the bone marrow (p=0.04); age with DFS of 70% for pts <21 years old compared to 40% for pts ≥21 years old (p=0.51); and normalization of cytogenetic abnormalities with DFS of 50% for pts with normal cytogenetics compared to 33% for pts with persistent abnormalities (p=0.49).Our results suggest that the strategy of induction with high dose cytarabine-based chemotherapy followed by allogeneic stem cell transplantation improves the overall outcomes for pts with tMDS/tAML to those obtained in pts transplanted for primary MDS/AML. In addition, the use of T-cell depleted transplant may decrease the toxicity of transplantation in heavily pre-treated pts without increase in relapse rate. Disclosures:No relevant conflicts of interest to declare.

Immune Reconstitution After Double Umbilical Cord Blood Transplantation in Adults without In Vivo T Cell Depletion of the Graft.

AbstractAbstract 1246Infusion of two partially matched units can overcome the barrier of cell dose in umbilical cord blood transplantation (UCBT). Besides engraftment, restoration of adaptive immunity remains a major problem resulting in higher transplant-related mortality in UCBT compared to marrow or peripheral blood transplantation. According to reported data, prolonged immunodeficiency in adult recipients of single-unit UCBT is characterized by long-term impairment of thymopoiesis, whereas the impact of double UCBT on immune reconstitution has not been elucidated. We prospectively monitored cellular and humoral immunity in consecutive adult recipients of double UCBT to assess the time frame and pattern of immune reconstitution. From 08/2006 until 07/2010, 27 patients underwent double UCBT at our center. Twenty-two, who fulfilled defined criteria [conditioning without antithymocyte globulin (ATG), sustained donor engraftment, no relapse before day 100], were included in study analysis. Patients received double UCBT for high-risk acute leukemia or lymphoma (AML: 15, ALL: 5, type 2 dendritic cell leukemia: 1, hepatosplenic γδ T-cell lymphoma: 1), at a median age of 37 years (range, 16–59). The conditioning regimen was ablative in 16, and reduced-intensity in 6 cases. The combination of mycophenolate mofetil to calcineurin inhibitor (cyclosporine: 18, or FK506: 4) was used for prevention of acute graft-versus-host disease (GvHD). The majority of units (36/44) were 4/6 matched to recipient at HLA-A, -B, and -DRB1 loci, the remaining being 5/6 matched. Median total nucleated and CD34+ cell doses from both units were 4.36 × 10^7/kg (range, 2.65–6.86) and 1.56 × 10^5/kg (range, 0.6–5.13) at infusion, respectively. Intravenous immune globulin (IVIG) was routinely administered every 2–4 weeks during the first 6 months from transplant. Absolute counts of B, NK, and T lymphocytes as well as serum immunoglobulin levels (prior to infusion of IVIG), were routinely assessed at 1, 2, 3, 6, 9, 12, 15, 18, 24 months, and annually thereafter. Neutrophil engraftment (absolute count >500/μl) was achieved at a median of 19 days (range, 6–52). Twenty patients developed acute GvHD (grade II: 17, grade III-IV: 3). Chronic GvHD occurred in 4 patients, and was extensive in 2. Cytomegalovirus (CMV) infection was detected in 6 out of 16 seropositive recipients (at a median of 58, range 25–116, days after UCBT), and in none of the 6 seronegative. In addition, 5 patients developed HHV-6 infection, and 1 EBV-related lymphoproliferation. Relapse occurred in 4 patients. There were 5 deaths of infection, 3 of GvHD, and 2 of other causes. The absolute counts of T and B cells were extremely low early post transplant, while NK cells recovered from the first month and reached normal values by the second month (median count, 147/μl). The number of CD8+ T cells started to increase from the third month, and exceeded lower normal limit after 6 months (median count, 234/μl). CD4+ T cells remained substantially low (<100/μl) for the first 3 months, increased moderately afterwards, but achieved normal values only at 18 months (median count, 465/μl). CD4+/CD45RA+ naïve T cells were significantly impaired over the first year, but appeared at increasing counts at 18 months (median count, 157/μl) and beyond. CD19+ B cells were severely suppressed for the entire first semester (median count at 3 months, 5/μl), increased to moderate normal counts by 9 months, and showed a marked elevation exceeding normal range between 12 (median count, 985/μl) and 18 months (median count, 1501/μl). Notably, that was consistent with delayed achievement of normal serum IgG levels at 15 months (median, 1130 mg/dl) and normal IgA levels at 24 months (median, 133.5 mg/dl). Our results differ from previously reported data regarding immune reconstitution post UCBT, mainly due to the absence of in vivo T cell depletion in our series. By elimination of ATG from conditioning, we did not observe rapid recovery of B cells in the early post-transplant period. Moreover, B cells remained severely depressed for 6 months, a finding that may be accounted for by the increased incidence of acute GvHD in our series. On the other hand, T cell reconstitution was relatively faster, and a stable rise of naïve CD4+ T cells suggestive of thymopoietic recovery was noted after the first year from transplant. In conclusion, immune reconstitution patterns following UCBT may be divergent depending on the conditioning regimen. Disclosures:No relevant conflicts of interest to declare.

Reduced Intensity Versus Myeloablative Conditioning in Adults with Acute Myeloid Leukemia Transplanted with Umbilical Cord Blood

AbstractAbstract 3531 Background:Reduced intensity conditioning (RIC) umbilical cord blood (UCB) transplantation is increasingly used in hematopoietic stem cell transplantation (HCT) for older and medically unfit patients. Data on the efficacy of HCT after RIC relative to myeloablative conditioning (MAC) are limited. We compared the outcomes of acute myeloid leukemia (AML) patients > 18 yrs who received UCB grafts after either RIC or MAC. Methods:One hundred nineteen adult patients with AML in complete remission (CR) underwent an UCB transplant after RIC (n=74, 62%) or MAC (n=45, 38%) between January 2001 and December 2009. Conditioning was either reduced intensity and consisted of cyclophosphamide 50 mg/kg, fludarabine 200 mg/m2 and total body irradiation (TBI) 200 cGy or myelablative and consisted for cyclophosphamide 120 mg/kg, fludarabine 75mg/m2 and TBI 1200–1320cGy. All patients received cyclosporine (day -3 to day +180) and mycophenolate mofetil (day -3 to day +45) post-HCT immunosuppression and hematopoietic growth factor. Use of RIC was reserved for patients >45 years (n=37, 82%) or pre-existing severe co-morbidities (n=8, 18%). The two groups were similar except RIC had more preceding myelodysplastic syndrome (RIC=28% vs. MAC=4%, p<0.01) and older age (median, RIC=55yrs vs. MAC=33yrs; p<0.01). Remission status (CR1; RIC=65% vs. MAC=56%, p=0.9), CR1 duration ≥ 1 yr for CR2 HCT (RIC=46% vs. MAC=45% p=0.9) and the HCT-comorbidity index were similar (score >2; RIC=50% vs. MAC=40%, p=0.1). Furthermore, most patients received double UCB graft (RIC=85% vs. MAC=89%, p=0.5) with a maximum HLA disparity of 4/6 and 5/6 in 52% and 34% of RIC recipients vs. 62% and 29% of MAC recipients (p=0.5). Results:Median follow-up for survivors was 4.2 (RIC) and 3.5 years (MAC). The 3-year leukemia-free survival (LFS) was 31% vs. 55% (p=0.02) and 3-year relapse incidence was 43% vs. 9% (p<0.01) in recipients of RIC and MAC, respectively (Figure). One-year transplant related mortality (TRM) was similar (RIC=16% vs. MAC=24%; p=0.55). In multivariate analysis (Table), RIC recipients and those in CR2 with short CR1 duration had higher risk of relapse and poorer LFS. No factors predicted TRM. [Display omitted] Table:Multivariate analysis of outcomesOutcome measureRelative risk95% CIpRelapse incidenceRIC vs. MAC4.72.0–10.4<0.01CR11.00CR2 w/ CR1< 1 yr1.91.0–3.80.06CR2 w/ CR1>=1 yr0.50.2–1.50.2Leukemia free survivalRIC vs. MAC2.31.3–4.0<0.01CR11.00CR2 w/ CR1< 1 yr1.91.1–3.40.03CR2 w/ CR1>=1 yr0.70.3–1.40.33 Conclusion:UCB with RIC extends the use of allogeneic HCT for older and less fit patients with limited TRM. However, the increased relapse risk and poorer LFS in the RIC setting indicates the need for novel strategies to improve leukemia control post-transplantation, especially in CR2 patients with short CR1 duration. Disclosures:Wagner:CORD:USE: Membership on an entity's Board of Directors or advisory committees; VidaCord: Membership on an entity's Board of Directors or advisory committees. Weisdorf:Genzyme: Research Funding; Hospira.

Double Umbilical Cord Blood Transplant Shown as Viable Alternative for Hematologic Malignancy

Double Umbilical Cord Blood Transplant Shown as Viable Alternative for Hematologic Malignancy

Death by Edible Mushroom: First Report of Volvariella volvacea as an Etiologic Agent of Invasive Disease in a Patient following Double Umbilical Cord Blood Transplantation

We describe a case of invasive fungal infection caused by Volvariella volvacea following double umbilical cord blood transplantation (UCBT). Although infections caused by several mushroom species have been documented, we believe this to be the first published report of invasive infection with Volvariella volvacea, an edible mushroom belonging to Agaricales.

Double umbilical cord blood transplantation with reduced intensity conditioning and sirolimus-based GVHD prophylaxis

The main limitations to umbilical cord blood (UCB) transplantation (UCBT) in adults are delayed engraftment, poor immunological reconstitution and high rates of non-relapse mortality (NRM). Double UCBT (DUCBT) has been used to circumvent the issue of low cell dose, but acute GVHD remains a significant problem. We describe our experience in 32 subjects, who underwent DUCBT after reduced-intensity conditioning with fludarabine/melphalan/antithymocyte globulin and who received sirolimus and tacrolimus to prevent acute GVHD. Engraftment of neutrophils occurred in all patients at a median of 21 days, and platelet engraftment occurred at a median of 42 days. Three subjects had grade II-IV acute GVHD (9.4%) and chronic GVHD occurred in four subjects (cumulative incidence 12.5%). No deaths were caused by GVHD and NRM at 100 days was 12.5%. At 2 years, NRM, PFS and OS were 34.4, 31.2 and 53.1%, respectively. As expected, immunologic reconstitution was slow, but PFS and OS were associated with reconstitution of CD4(+) and CD8(+) lymphocyte subsets, suggesting that recovery of adaptive immunity is required for the prevention of infection and relapse after transplantation. In summary, sirolimus and tacrolimus provide excellent GVHD prophylaxis in DUCBT, and this regimen is associated with low NRM after DUCBT.

The best endpoint for acute GVHD treatment trials

AbstractThe optimal primary endpoint for acute graft-versus-host disease (GVHD) therapeutic trials has not been established. In a retrospective analysis, we examined the response of 864 patients who received prednisone 60 mg/m2/d for 14 days, followed by an 8-week taper, as initial therapy for acute GVHD from 1990-2007 at the University of Minnesota. Patients received grafts of human leukocyte antigen–matched sibling bone marrow (BM) or peripheral blood (PB; n = 315), partially matched sibling BM or PB (n = 24), unrelated donor BM or PB (n = 313), single (n = 89) or double (n = 123) umbilical cord blood. Day 28 responses were similar to day 56 responses and better than day 14 responses in predicting transplantation-related mortality (TRM). In multiple regression analysis, patients with no response at day 28 were 2.78 times (95% CI, 2.17-3.56 times; P < .001) more likely to experience TRM before 2 years than patients with a response. Other factors associated with significantly worse 2-year TRM include older age, high-risk disease, severe GVHD, and partially matched related BM/PB. No other differences in response by donor source were observed. These data suggest that day 28 is the best early endpoint for acute GVHD therapeutic trials in predicting 2-year TRM.

Double umbilical cord blood transplantation for children and adolescents

Umbilical cord blood transplantation (UCBT) with two units has been conducted with promising results in adults to overcome the limitation of low cell numbers. In an attempt to improve the outcomes, double UCBT was performed in children and adolescents. Sixty-one patients, including 44 acute leukemia, and 17 other hematologic diseases, received double UCBT. Donor-type engraftment achieved in 82% of patients. Except one patient with persistent mixed chimerism of two units, other 49 patients showed dominancy of one unit and only the CFU-GM was significant factor influencing dominancy. The event-free survival (EFS) of leukemia and other hematologic disease were 59% and 53%, respectively, and the EFS of acute leukemia patients who received transplant in first or second CR (68.6%) was significantly better than in those with advanced disease (22.2%) (P = 0.007). Among the factors influencing outcomes, low cell dose difference between two units (TNC difference/TNC of large unit <15%) were associated with higher TRM, relapse, and lower EFS. Double UCBT was a promising modality of transplant in children and adolescence. However, engraftment and other results were not so satisfactory yet. To improve the outcomes, development of new selection guideline, probably including cell dose difference between two units and technology to enhance engraftment and reduce transplantation-related mortality are warranted.

Clearance of CMV viremia and survival after double umbilical cord blood transplantation in adults depends on reconstitution of thymopoiesis

Umbilical cord blood grafts are increasingly used as sources of hematopoietic stem cells in adults. Data regarding the outcome of this approach in adults are consistent with delayed and insufficient immune reconstitution resulting in high infection-related morbidity and mortality. Using cytomegalovirus (CMV)-specific immunity as a paradigm, we evaluated the status, mechanism, and clinical implications of immune reconstitution in adults with hematologic malignancies undergoing unrelated double unit cord blood transplantation. Our data indicate that CD8(+) T cells capable of secreting interferon-gamma (IFN-gamma) in a CMV-specific enzyme-linked immunosorbent spot (ELISpot) assay are detectable at 8 weeks after transplantation, before reconstitution of thymopoiesis, but fail to clear CMV viremia. Clearance of CMV viremia occurs later and depends on the recovery of CD4(+)CD45RA(+) T cells, reconstitution of thymopoiesis, and attainment of T-cell receptor rearrangement excision circle (TREC) levels of 2000 or more copies/mug DNA. In addition, overall survival was significantly higher in patients who displayed thymic regeneration and attainment of TREC levels of 2000 or more copies/mug DNA (P = .005). These results indicate that reconstitution of thymopoiesis is critical for long-term clinical outcome in adult recipients of umbilical cord blood transplant. The trial was prospectively registered at http://www.clinicaltrials.gov (NCT00133367).

Observation on therapeutic effect of double unrelated umbilical cord blood transplantation for treatment of leukemia due to chronic benzene poisoning in adults

目的 探讨双份非血缘关系脐血移植(UCBT)治疗成人慢性苯中毒性白血病的有效性及可行性.方法 对4例成人慢性苯中毒性白血病患者[急性非淋巴细胞白血病(ANLL-M2)2例、慢性粒细胞白血病(CML)-慢性期1例、CML-急变期1例]进行双份UCBT,其中人类组织相容性抗原(HLA)配型全相合1例,516位点相合2例,4/6位点相合1例.输入的单个核细胞数(MNC)为5.5×107～6.8×107/kg,CD34+细胞数为2.6×105～4.8×105/kg.预处理方案:在经典的白消胺(BU)/环磷酰胺(CY)预处理方案基础上加用抗胸腺细胞球蛋白(ATG);应用环孢素A(CsA)、赛尼哌、霉酚酸酯(MMF)+短程氨甲蝶呤(MTX)预防移植物抗宿主病(GVHD);采用肝素联合丹参、低分子右旋糖酐预防肝静脉闭塞病(HVOD).结果 3例植入成功,患者中性粒细胞绝对数恢复至≥0.5×109/L的时间分别为移植后15、30、18 d,血小板数≥20×109/L的时间分别为移植后31、65、40 d;DNA短串联重复序列多态性(DNA-STR)分析显示,仅有1份脐血植入,另1份被排斥,未发生GVHD.至今3例患者分别长期无病存活达69、70和39个月,其中1例HLA配型4/6位点相合、CML-急变期患者于移植后42 d恢复自身造血.结论 双份UCBT治疗成人慢性苯中毒性白血病是可行有效的。

Disease-specific hematopoietic cell transplantation: nonmyeloablative conditioning regimen for dyskeratosis congenita

Dyskeratosis congenita (DC) is characterized by reticular skin pigmentation, oral leukoplakia and abnormal nails. Patients with DC have very short telomeres and approximately one-half have mutations in telomere biology genes. A majority of patients with DC develop BM failure (BMF). Hematopoietic cell transplantation (HCT) represents the only known cure for BMF in DC, but poses significant toxicities. We report six patients who underwent allogeneic HCT with a novel nonmyeloablative conditioning regimen specifically designed for DC patients. Graft sources included related PBSCs (1), unrelated BM (2) and unrelated double umbilical cord blood (3). Complete donor engraftment was achieved in five of six patients. One patient had initial autologous hematopoietic recovery, which was followed by a second transplant that resulted in 88% donor chimerism. With a median follow-up of 26.5 months, four patients are alive, three of whom were recipients of unrelated grafts. We conclude with this small study that encouraging short-term survival can be achieved with HCT in patients with DC using a preparative regimen designed to promote donor engraftment and minimize life-threatening disease-specific complications such as pulmonary fibrosis. Long-term follow-up will be crucial with respect to individualized patient care with each of the transplanted individuals.

Donor-Derived Second Hematologic Malignancies after Cord Blood Transplantation

Double umbilical cord blood transplantation (UCBT) with a reduced-intensity conditioning regimen is an effective strategy for adult patients without a matched donor. The risk of second malignancies in these patients has not yet been established, however. In the present study, 98 adults with a hematologic malignancy underwent double UCBT. Seventy patients received a reduced-intensity conditioning regimen of fludarabine 30 mg/m(2)/day for 6 days, melphalan 100 mg/m(2)/day for 1 day, and rabbit antithymocyte globulin 1.5 mg/kg/day for 4 days, and 28 patients received a myeloablative total body radiation-containing conditioning regimen. Sixty-three patients received sirolimus-based graft-versus-host disease (GVHD) prophylaxis, and 35 patients received non-sirolimus-based GVHD prophylaxis. The median patient age was 48 years (range, 19-67 years). Eighteen patients developed a second malignancy at a median of 134 days after transplantation. Sixteen patients had lymphoma, and 2 patients had myelodysplasic syndrome/myeloproliferative disorder (MDS/MPD). Sixteen of these second malignancies (both cases of MDS/MPD and 14 of the lymphomas) were donor-derived; the origins of the others were not determined. GVHD prophylaxis, HLA matching, primary disease, age, total nucleated cell dose, and CD34(+) cell dose were not associated with a higher rate of second malignancy. Second myelogenous malignancies of donor origin occur after double UCBT, suggesting that a search for donor origin should be performed in all patients with suspected relapse.

Double Umbilical Cord Blood Transplantation Achieves Universal Engraftment But Is Associated With Considerable Transplant-Related Mortality

The applicability of umbilical cord blood transplantation (UCBT) is limited by inadequate cell dose especially for adult patients. Infusion of 2 partially HLA-matched units has been shown to facilitate engraftment, and allows for transplantation of patients for whom a single unit of sufficient cell dose is not available. However, double UCBT is associated with an increased risk of acute graft-versus-host disease (aGVHD), which may impair the outcomes. From 8/2006 to 7/2009, 21 patients (female:male=10:11) with high-risk acute myeloid (AML, n=16) or lymphoblastic (ALL, n=5) leukemia received double UCBT in our center.

Extending Cord Blood Transplant to Adults: Dealing With Problems and Results Overall

The development of newer strategies to overcome, in particular, the cell dose limitation, has increased the availability of umbilical cord blood (UCB) as a source of hematopoietic stem cells (HSC) for transplantation of adults. Among these strategies is the development of the double UCB, ex vivo, and reduced-intensity transplantation platforms. Several ongoing registry-based and single-institution and multicenter clinical trials are investigating ways to make UCB transplantation safer and to improve the outcomes of adults after UCB transplantation. We review the background data and promising newer strategies that will further expand the utilization of UCB for the treatment of adults.

Engraftment Kinetics After Transplantation of Double Units of Umbilical Cord Blood From Unrelated Donors in Adolescents and Adults with Hematologic Malignancies.

Abstract 1183Poster Board I-205 Objective:To evaluate the clinical therapeutic effects and the early engraftment kinetics of the transplantation of double partially-human leukocyte antigen (HLA)-matched umbilical cord blood (UCB) units in patients with hematologic malignancies from November 2005 to March 2009. Methods:Twenty-one adults and adolescents (median age 21 years [range 10-40 years]; median weight 57 kg [range 31-76 kg]; 16 males and 5 females) with hematologic malignancies were given transplants of double UCB units. Diagnoses included 7 ALL patients, 7 AML, 5 CML, 1 Mix-AL, and 1MDS. 17 (81%) of the patients were refractory to chemotherapy and considered at high risk. All patients received myeloablative conditioning, which included Flu/Cy/TBI for 16 patients, and Bu/CY2±ATG±Ara-C for 5 patients. Graft versus host disease (GVHD) prophylaxis was CSA+MMF. The analytic method used was based on the quantitative amplification of informative polymorphic short tandem repeat (STR) regions in the recipient and donor using polymerase chain reaction (PCR), which detect engraftment and chimerism dynamically. Results:The median nucleated cell dose was 4.93×107 nucleated cell [NC]/kg range:3.26-7.70×107 NC/kg. Eighteen patients (86%) achieved hematopoietic recovery after the double UCB transplantation. The median number of days required to reach an ANC > 0.5×109/L was 20 days (14–35 days), and platelet> 20×109/L was 34.5 days (25 - 49 days). One patient's engraftment was derived from both donors, which were 6/6 HLA matched the recipient's, for six months until her death. The other 17 patients achieved sustained hematopoietic engraftment that was derived from a single dominant donor based on STR-PCR results. The median infused cell dose of the engrafted units was 2.34×107 NC /Kg (ranging from 1.87 to 4.45 ×107NC/kg), and 3.225×107CD3+/Kg (ranging from 0.51 to 13.92×107 CD3+/kg). This compared with 2.17×107 NC/Kg (range: 0.96 - 3.98×107 NC/kg) and 1.71×107CD3+/Kg (range: 0.40 - 10.65×107 CD3+/kg) in the nonsustained unit. The difference in cell doses was not significant(P=0.718 AP=0.073. By STR-PCR, the donor DNA can be detected as early as post-transplantational day 7. Seventeen patients who achieved dominant engraftment had full donor chimera (FDC) at post-transplantational day 14, and this was highly consistency with chimerisms at post-transplantational days 21 and 30. If no unit reached FDC at post-transplantational day 14, the graft would be rejected. Therefore, the result at post-transplantational day 14 could indicate the last chimerism (Kappa =1). Complications: 1) Three patients had UCB graft rejections at early period; two were adolescent ALL, one was MDS-RCMD. Depending on long-term transfusion, they all achieved long-term hematopoiesis recovery after a secondary haploidentical stem cell transplantation at post-DUCBT 33-38 day. 2) Eight/eighteen patients(44.4%)had grade±-IIacute GVHD, 2 in accessible 13 patients developed local (non-extensive) cGVHD. 3) Three patients relapsed (1 was CNSL, and 1 relapsed on cellular level and re-achieved FDC after treatment with imatinib and benzene). 4) All patients had a median follow-up of 12 months (ranging 5 months to 43 months). The one-year disease-free survival rate was 66.64%, with 6 patients died. The one hundred-day transplantation-related mortality was 14.3%; 4 died of invasive fungal infection, 1 died of body exhaustion, and 1 died of serious hepatitis. Conclusion:1) Double-unit UCB can overcome the shortage of cell dose in one-unit UCB, and was proved to be a safe, effective, and promising alternative treatment option with good engraftment potential. 2) The total nucleated cells and CD3 cell dose were not associated with the UCB unit that would predominate. 3) The STR -PCR and capillary electrophoresis techniques can accurately evaluate grafting at an early time after UCBT. Detecting the chimerism at 14 days after UCBT can provide the information on graft implantation. 4) The relapse rate was 14.3% in 18 CB-engrafted patients with high-risk and refractory disease conditions, which suggests that UCBT has a fairly good graft versus leukemia effect. Disclosures:No relevant conflicts of interest to declare.

Response of 864 Patients to Steroids as Primary Therapy for Acute Gvhd at a Single Institution: Day 28 Response Is the Best Endpoint to Predict Transplant-Related Mortality.

Abstract 1134Poster Board I-156The optimal primary endpoint for acute GVHD treatment trials has not been established. In a retrospective analysis, we examined the response of 864 patients who received prednisone 60 mg/m2 for 14 days, followed by an 8 week taper, as initial therapy for acute GVHD from 1990-2007 at a single institution. Complete response (CR), partial response (PR), very good partial response (VGPR), and no response (NR) were scored at day 14, day 28 and day 56 after initiation of steroids. To determine the best endpoint, an index of concordance, the c-statistic (C), was performed to estimate the probability that patients with the better GVHD response will have lower transplant related mortality (TRM) than patients with a worse response. Median patient age was 32 (range, 0.2-69) years; 35% were <18 years old. Patients received grafts of HLA-matched sibling bone marrow (BM) or peripheral blood (PB, n=315), partially matched sibling BM or PB (n=24), unrelated donor (URD) BM or PB (n=313), single umbilical cord blood (sUCB, n=89) or double UCB (dUCB, n=123). Prior to initiation of steroid therapy, initial GVHD grades were grade I in 230 (27%), grade II in 504 (58%), grade III in 119 (14%), and grade IV in 11 (1%). Initial GVHD organ involvement was skin only (57%), gut only (17%), liver only (1%) or multiorgan (25%). Day 28 responses were similar to day 56 responses (p=0.14) and better than day 14 responses (p=0.03) in predicting TRM. In multiple regression analysis, patients with NR at day 28 were 2.77 times more likely to have TRM than patients with CR, VGPR or PR while any response favored lower TRM (table). Factors associated with significantly worse 2 year TRM in patients with acute GVHD include: NR to steroids, partially matched BM/PB, high risk disease, older age and skin only GVHD. Factors not associated with TRM include CMV serostatus, conditioning therapy, GVHD prophylaxis, days to steroid treatment, and initial grade of GVHD.Table:Factors associated with 2 year TRM: multiple regression analysisFactorRelative RiskP valueOverall P ValueDay 28 Response (% patients)..<0.001CR (53%)1.0.VGPR (7%)0.630.13PR (4%)1.220.45NR (31%)2.77<0.001Donor Type..<0.01MSD BM/PBSC1.0.URD well matched BM/PBSC1.350.17URD partial matched BM/PBSC1.540.03URD or sibling MM BM/PBSC1.77<0.001Single UCB1.060.83Double UCB0.790.36Disease Risk..0.02Standard1.0.High1.350.02Age1.00.020.02<18 years1.40318 yearsAffected Organs (skin only)..0.05Yes1.0.No1.360.05Grade at Start of Steroid Therapy..0.11I1.0.II1.080.60III-IV1.510.10These data suggest that responses at day 28 or 56 are equally effective endpoints for acute GVHD trials. Day 14 responses cannot as accurately predict TRM. As patients with NR require further therapy in a timely manner, early progression or response by day 28 is the best endpoint to assess efficacy of initial therapy for acute GVHD. Prospective trials are still required to determine the best therapy for different subgroups of patients with acute GVHD, especially those identified to have predictably poor responses and high TRM. DisclosuresNo relevant conflicts of interest to declare.

Outcome of Umbilical Cord Blood Transplantation in 51 Patients with Hematologic Malignancies.

Abstract 4342 Background and ObjectivesUmbilical cord blood transplantation (UCBT) has recently emerged as a curative approach for patients with hematologic malignancies who lack a suitable HLA-matched donor. We report the outcome of UCBT for 51 hematologic malignancies from a single hospital in Southeast China. Design and MethodsFifty-one cases of UCBT for hematologic malignancies at Anhui Provincial Hospital from March 2000 to May 2009 were retrospectively characterized, including 20 cases of acute lymphoblastic leukemia (ALL), 12 acute myeloid leukemia (AML), 12 chronic myeloid leukemia (CML) (accelerated phase 5, blast crisis 4), 2 mixed acute leukemia(MIX-AL),4 myelodysplastic syndrome (MDS), and one case of non-Hodgkin lymphoma (NHL). Double UCB grafts were used for 27 patients, while single UCB graft was used for 24 patients. All but one UCB grafts were from unrelated donors. The median cell doses infused were 4.37×107TNC/kg (range, 2.41?16.24), 2.96×105 CD34/kg (range, 0.67?29.28) in single UCBT or 4.44×107TNC/kg (range, 2.76?7.70), 2.67×105 CD34/kg (range, 0.8?9.35) in double UCBT. Myeloablative conditioning was given to 46 patients and nonmyeloablative regimen was given to 5 patients according to the standard protocols. Myeloablative regimen consisted of fractionated total body irradiation (TBI) 1,200 cGy, cyclophosphamide 120 mg/kg, cytarabine 8g/m2 (n=26) or of modified BUCY2 consisting of busulfan 16mg/kg orally or 12.8 mg/kg intravenously, cyclophosphamide 120 mg/kg, and rabbit antithymocyte globulin (ATG, Fresenius, Germany) 7.5mg/kg (n=20). Reduced intensity conditioning consisted of fludarabine 160 mg/m2, BU6.4m/kg, ATG 7.5mg/kg and TBI 3Gy. All patients were given a combination of cyclosporin A (CsA) and mycophenolate mofetil (MMF) for graft-versus-host disease (GVHD) prophylaxis. ResultsOf the 51 patients, 34 had advanced stage of disease. The patients' age ranged from 3 to 48 years (median 19 yr) and the body weight ranged from 15 to 84 kg (median 53 kg), respectively. Although one patient with advance-staged chronic myeloid leukemia (CML) died 16 days after UCBT and 6 patients showed primary graft failure, 44 of the 50 patients (88%) achieved engraftment, with neutrophil recovery (≥0.5×109/L) occurring at a median of 19 days (range, 12-39) and platelet recovery (≥20×109/L) occurring at a median of 34 days (range, 21-73), respectively. All of the 44 patients gained complete donor chimerism by PCR analysis of short tandem repeat (STR) sequences on whole blood or bone marrow from day 7 to day 100 after transplantation. Half of the patients (22/44, 50%) developed acute GVHD with 19 cases of grade I/II disease (43.2%) and 3 cases of grade III disease (6.8%). Only five patients (16.1%) developed chronic GVHD, with 2 in skin, 1 in intestinal, 1 in liver, and 1 patient with refractory chronic GVHD with thrombocytopenia. Three patients had relapse (2 year relapse rate of 5.9%) with one in central nervous system, who achieved remission after intrathecal injection of chemotherapy regimen.,one with CML blast crisis achieved remission after oral imatinib,and the third died of relapsed disease. The median follow-up duration of surviving patients was 19 months (range, 3-113) with a 2 year overall survival rate of 54.5%. A total of 20 patients died during the study, with a 2 year transplant related mortality rate of 37.3%. The most common cause of death was severe infection (12) due to bacteria, fungi and/or viruses, and other causes included acute GVHD (1), disease progression (1), veno-occlusive disease of the liver (1), sudden cardiac death (1), cerebral hemorrhage (1), fulminant viral hepatitis (1), early rejection (1),and systemic failure (1). Interpretation and ConclusionsUCBT could be safely and effectively used for patients with hematologic malignancies. The use of double UCB as a strategy extends the availability of transplantation. Prevention and control of infection are essential for long-term survival after UCBT. Disclosures:No relevant conflicts of interest to declare.

Reduced Relapse and Similar Progression-Free Survival After Double Umbilical Cord Blood Transplantation (DUCBT): Comparison of Outcomes Between Sibling, Unrelated Adult and Unrelated DUCB Hematopoietic Stem Cell (HSC) Donors.

Abstract 662For patients with hematologic malignancies who lack a matched or mismatched unrelated donor, UCBT is an alternative. However, only a minority of larger adolescents and adults are able to find a single UCB with an adequate cell dose. Double UCB unit grafts (DUCB) have previously been shown to extend this HSC option. In order to understand the relative merits of each HSC source, we combined the datasets at the University of Minnesota and Fred Hutchinson Cancer Research Center. Between 2001- and 2008, 536 patients with leukemia (AML, n=211; ALL, n=236; CML, n=70 and MDS, n=19) were transplanted with HSC from a matched sibling (SIB, n=204), 8/8- (MUD, n=152) or 7/8-HLA (MMUD, n=52) matched unrelated donor or DUCB (n=128). All patients received myeloablative conditioning with cyclophosphamide 120 mg/kg and total body irradiation 1200-1320cGy with fludarabine 75mg/m2administered prior to DUCB. All patients received GVHD immunosuppression with a calcineurin inhibitor and either methotrexate (SIB, MUD and MMUD) or mycophenolate mofetil (DUCB). While patient weight and sex distribution and proportion with standard risk disease (STD, AML and ALL in CR1-2, and CML in CP1) were similar, DUCB pts were younger (median age 25 yrs, SIB 40 yrs, MUD 31 yrs, MMUD 31 yrs; p<.01). The proportion of AML and ALL was similar among groups, although more CML patients received a MUD or MMUD. Recipient CMV seropositivity was lower among MUD (44%; SIB 58%; MMUD 60; DUCB 62%, p<.01). The median follow-up of survivors was 3.1 yrs (0.3-8.1). The univariate point estimates at the stated timepoints and multivariate outcomes are summarized in the Table.Outcomes after SIB vs. MUD vs. MMUD vs. DUCBSIB* Ref.MUD* RR (95%CI)MVA p-value†MMUD* RR (95%CI)MVA p-value†DUCB* RR (95%CI)MVA p-value†Neutrophil Recovery at day 4598% 1.097% 0.62 (0.50-0.78). . <0.0198% 0.63 (0.46-0.86). . <0.0187%; 0.29 (0.23-0.37). . <0.01Platelet Recovery at day 10086% 1.081% 0.83 (0.64-1.09). . 0.1875% 0.71 (0.48-1.04). . 0.0845% 0.22 (0.17-0.29). . <0.01Grade II-IV aGVHD at day 10065% 1.080% 1.68 (0.32-2.14). . <0.0185% 2.17 (1.46-3.23). . <0.0160% 1.01 (0.76-1.35). . 0.94cGVHD at 2 year47% 1.043% 1.04 (0.75-1.46). . 0.8048% 1.24 (0.77-2.0). . 0.3726% 0.73 (0.47-1.13). . 0.16TRM at 3 years24% 1.014% 0.92 (0.53-1.60). . 0.7627% 1.83 (0.97-3.42). .0.0634% 2.86 (1.81-4.51). . <0.01Relapse at 5 years43% 1.037% 0.83 (0.58-1.19). . 0.3235% 0.68 (0.39-1.20). . 0.1915% 0.27 (0.16-0.47). . <0.01PFS at 5 years33% 1.048% 0.83 (0.62-1.11). . 0.2038% 1.04 (0.70-1.53). . 0.8551% 1.00 (0.73-1.37). . 0.99*Results are shown as pointwise estimates, relative risk (RR) and the 95% confidence interval. SIB group was the reference (Ref) group for the multivariate analysis (MVA).†P-value shown relates to multivariate analysis.In summary, after a Cy/TBI based myeloablation, DUCB is associated with slower hematopoietic recovery compared to other HSC sources. While incidence of acute GVHD was similar to that observed after SIB, DUCB was associated with less acute and trend toward less chronic GVHD relative to unrelated donors. Despite reduced risk of GVHD after DUCBT, risk of relapse was remarkably low while TRM was elevated, resulting in similar PFS. In the absence of a SIB donor, using either MUD or DUCB yield encouraging PFS and are promising donor options. These results support a) the front line use of HLA 0-2 antigen mismatched DUCB in patients with hematological malignancy, b) development of new strategies to enhance hematopoietic recovery after DUCBT, potentially reducing the risk of TRM, c) studies to understand why relapse rates are reduced with DUCBT and d) quality of life assessments long term between HSC groups, as there appears to be less risk of acute and chronic GVHD after DUCBT. Disclosures:No relevant conflicts of interest to declare.

Early Engraftment Kinetics of Leukocyte Subsets by Means of HLA-Specific Mabs After Double Umbilical Cord Blood Transplantation Show a Very Rapid Induction of Single Complete Donor Chimerism.

Abstract 3338Poster Board III-226Double umbilical cord blood transplantation (UCBT) results in higher engraftment rates as compared to single UCBT in adult patients. Sustained hematopoiesis is usually derived from a single cord blood unit (CBU). So far, the mechanism of predominance of a particular CBU is unresolved. Immunological rejection of one CBU has been proposed, as well as a selective growth advantage of one particular unit. Frequent serial chimerism studies in leukocyte subsets by use of HLA-specific monoclonal antibodies (mAbs) during the first month post transplant might contribute to unravel the mechanism of graft predominance. Methods.Seventeen consecutive patients (pts) with high risk hematological diseases received a double UCBT preceded by a non-myeloablative conditioning regimen (Cy 60 mg/kg/ Flu 160 mg/kg/ TBI 2×2 Gy). CBUs were selected by low resolution typing for HLA-A and –B loci and by allele typing for HLA-DRB1. The minimal required HLA-match grade was 4/6. If discriminating HLA mismatches between the 3 different parties were present, early analysis in leukocyte subpopulations was performed at day 11, 18, 25 and 32 post transplant by flowcytometry using lineage-specific (CD3, CD4, CD8, CD19, CD16/56, CD14, CD33) mAbs in combination with fluorochrome labeled HLA-antigen specific human mAbs. Results of day 32 were compared with routine chimerism analysis (VNTR) of peripheral blood TNC and T-cells. Results.Two pts were non-evaluable for engraftment due to early death and insufficient follow up, respectively. Donor engraftment in 15 pts occurred after a median of 30 days (range: 11-45). At 1 month post transplant, 9 pts showed complete single and 3 showed mixed chimerism by VNTR-analysis. In 3 patients, cells originating from both CBUs were present. Ultimately, complete single donor chimerism was established in 14 pts. Early chimerism studies with HLA-specific mAbs were performed in 8 pts. In all pts results at day 32 corresponded with chimerism analysis performed by VNTR. For 7 other pts no discriminating HLA-mAbs were available. Simultaneous 3-donor-origin detection of T-cells, monocytes and granulocytes based on HLA-disparities was possible in 4 pts. In 3 of them, all subsets showed a similar pattern: a clear predominance of a single CBU in all lineages as from day 11-18 onwards. Furthermore, the disappearing CBU was transiently detectable in these pts at day 11. In the fourth patient, T cells were of recipient origin at day 32 with predominance of a single CBU in all other subsets. The disappearing unit could be followed in another 4 pts, which revealed again a transient appearance of that unit in the various lineages at day 11. Although detectable in a total of 8 pts at day 11, all disappearing CBUs were completely lost within 18 days. The prefreeze TNC did not predict for the prevailing UCB, nor did the number of HLA-mismatches. Conclusions.These results show that double UCBT following a non-myeloablative regimen without ATG is associated with a rapid induction of complete single donor chimerism. The disappearing unit, although early detectable, was completely lost within 18 days in all patients, in whom it could be monitored by HLA-specific mAbs. We observed no disproportional increase of any leukocyte subset of the prevailing CBU nor of host leukocyte subset. This characteristic, uniform, early engraftment pattern in the presence of different HLA-disparities may rather argue for a difference in growth potential between both CBUs than an immunological rejection of the disappearing unit. Disclosures.No relevant conflicts of interest to declare.

Adoptive Transfer of Umbilical Cord Blood (UCB)-Derived Regulatory T Cells (Tregs) to Recipients of Nonmyeloablative Unrelated Double UCB Transplantation.

Abstract 513UCB transplantation (UCBT) is associated with greater risk of graft rejection and, in particular in double UCBT, with graft vs. host disease (GVHD). Our group has shown in mouse models that ex vivo activated and expanded Tregs improve engraftment and reduce and treat GVHD. We here report on the first clinical trial to study the safety of the infusion of UCB Tregs in patients receiving a nonmyeloablative double UCB transplant. We enrolled 19 patients (pts) (age 25-65 yrs; weight 52-133; male 9; CMV seropositive 6) with high risk or advanced leukemia (n=12) and lymphoma/CLL (n=7). All patients received the same nonmyeloablative conditioning consisting of cyclophosphamide 50mg/kg/×1day, fludarabine 40 mg/m2/×5d, and total body irradiation 200 cGy/×1d; immunosuppression was cyclosporine A/mycophenolate mofetil (MMF) in the first 17 and sirolimus (Siro)/MMF in the last 2 pts. Tregs were obtained from a 3rd UCB unit and after CD25+ selection (Miltenyi CliniMACS) were activated and expanded in the presence of anti-CD3/CD28 mAb coated beads and IL-2 for 18±1 days. Treg dose escalation levels were 1, 3, 10, 30 × 105/kg on day +1, and 30 × 105/kg on days +1 and +15 after UCBT. After CD25+ selection the median % CD4+/CD25+ was 62% (range , 11-87%) and following culture was 86% (r, 62-97%). Median fold expansion was 198 (r, 13-1169); median mixed lymphocyte reaction suppression at a 1:4 ratio post-culture was 86% (39-95%). All products achieved lot release. Pts were monitored for 48h post-infusion and no dose limiting or serious adverse event was observed. After infusion an increase in the proportion of peripheral blood comprised of CD4+FoxP3+CD127- cells was observed. Donor Tregs were clearly detected in all pts receiving Tregs that were HLA disparate with the pt and graft donor units (Figure 1). Mixed day 21 donor graft UCB chimerism was observed in 59% (10/19) of pts vs. 35% (38/107) in historical controls. The cumulative incidence of grades II-IV acute GVHD was 47% (95%CI, 24-71) and III-IV 16% (95%CI, 0-33). In pts receiving ≥ 30 × 105/kg (n=14), grades II-IV acute GVHD was 43% (95%CI, 16-70) and III-IV 7% (95%CI, 0-21). Compared to historical controls receiving the same conditioning regimen, the median time to the development of acute GVHD was longer (Figures 2), although not statistically significant. Neutrophil recovery was 95% (95%CI, 79-100) at median of 8.5 days (r, 3-36), platelet recovery >50,000/mL was achieved in 74% (95%CI, 53-95) at a median 43 days (r, 27-57), cumulative incidence of opportunistic infections (fungal + viral) at 100 days was 32% (95%CI, 11-53), treatment related mortality at 100 days was 11% (95%CI, 0-25), and disease free-survival was 40% (95%CI, 15-65%). None differed from historical controls. Based on our results, the co-infusion of ex vivo expanded and activated UCB-derived Treg to recipients of nonmyeloablative UCBT 1) is safe at the tested dose levels, 2) leads to detectable increased in Treg-donor unit derived circulating CD4+FoxP3+CD127- cells, and 3) results in an increased the proportion of mixed chimerism at day +21. Identification of the Treg MTD in the context of Siro/MMF immunosuppression will set the stage for the planned definitive studies that will establish the true impact of Tregs on engraftment and GVHD and for future studies in the treatment of autoimmune disease. [Display omitted] Disclosures:No relevant conflicts of interest to declare.