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Abstract 739 The combination of a calcineurin inhibitor and methotrexate has been the standard of care in graft-vs.-host disease (GVHD) prophylaxis for over 25 years, with resultant rates of grade II-IV acute GVHD between 30–50%. The mTOR inhibitor, sirolimus, has demonstrated promise in a number of Phase II trials as an immunosuppressant used for GVHD prophylaxis. The BMT CTN, sponsored by the NHLBI and NCI, conducted a multicenter, randomized controlled trial comparing the combination of tacrolimus and sirolimus (Tac/Sir) with tacrolimus and methotrexate (Tac/Mtx) as GVHD prophylaxis after matched, related donor (MRD) hematopoietic stem cell transplantation (HSCT). Methods: Eligible patients were between ages 2 – 60 years, and had acute leukemia in remission, myelodysplasia or chronic myeloid leukemia in chronic or accelerated phase. All had adequate organ function, and a 6/6 HLA-A, B, DRB1 matched sibling donor. 304 patients were randomly assigned to either Tac/Sir (n = 151) or Tac/Mtx (n = 153) as GVHD prophylaxis after TBI-based conditioning and MRD HSCT. An intent-to-treat analysis was performed on the primary endpoint of Grade II-IV GVHD-free survival 114 days from randomization. Ten subjects who received busulfan-based conditioning and were previously reported were excluded from analysis. Three subjects who did not undergo HSCT are included in the primary analysis, but not secondary analyses. Results: Treatment groups were well balanced. The median age of participants was 44 years (range 13 – 59) and 83% had acute leukemia. Neutrophil and platelet engraftment were both faster in the Tac/Sir group (14 vs. 16 days, p < 0.001; 16 vs. 19 days, p = 0.03, respectively), but this did not affect the time to first hospital discharge (20 vs. 21 days, p = 0.37). The incidence of grade II-IV and grade III-IV acute GVHD at 100 days were lower in the Tac/Sir group (26 vs. 34%, p = 0.17; 8 vs. 15%, p = 0.05). Day 100 treatment-related mortality was no different between groups (7 vs. 7%, p = 0.43). The primary endpoint of 114-day acute GVHD-free survival was not statistically different between groups (67 vs. 62%, p = 0.38, Figure). The cumulative incidence of relapse at 2 years from transplantation was not different between groups (27 vs. 30%, p = 0.81). The competing-risk cumulative incidence of chronic GVHD was higher in the Tac/Sir arm (54 vs. 43%, p =0.044). Overall toxicities were not different between groups, with two notable exceptions. The peak and average OMAS oral mucositis scores were lower in the Tac/Sir arm (peak 0.70 vs. 0.96, p < 0.001; average 0.31 vs. 0.47, p < 0.001), however, there was an increased rate of the endothelial injury syndromes, veno-occlusive disease (11 vs. 4%, p = 0.03), and thrombotic microangiopathy (5 vs. 1%, p = 0.05) in the Tac/Sir arm. Causes of death were not different between groups. At 2 years from transplantation, disease-free (DFS) and overall survival (OS) were not different between study arms (DFS 53 vs. 53%, p = 0.76; OS 60 vs. 61%, p = 0.44). Conclusions: No difference in 114-day acute GVHD-free survival was noted between treatment arms. Compared with Tac/Mtx in MRD HSCT, Tac/Sir is associated with more rapid engraftment, less severe acute GVHD and oral mucositis, excess chronic GVHD and endothelial injury syndromes, and similar long-term outcomes. Understanding the trade-offs between regimens, Tac/Sir can be used as an alternative to Tac/Mtx in MRD HSCT. Disclosures: Cutler: Pfizer, inc: Research Funding; Astellas, Inc: Consultancy, Research Funding. Off Label Use: Sirolimus - Prevention of GVHD Tacrolimus - Prevention of GVHD. Waller:Outsuka: Research Funding.

Abstract 3009FN2 Objective: To determine toxicity and efficacy of adding fludarabine (FLU) to a regimen of total body irradiation (TBI), anti-thymocyte globulin (ATG) and cyclophosphamide (CY), with de-escalation of the CY dose. The goal is to minimize CY-related toxicities while preserving (or improving) engraftment and survival in patients with SAA receiving an unrelated donor marrow transplant. Patients and Methods: Since May 2006, the Blood and Marrow Transplant Clinical Trials Network (BMT CTN), sponsored by the NHLBI and NCI, has conducted a prospective Phase I/II clinical trial of unrelated donor marrow transplantation in SAA (BMT CTN 0301). Patients with SAA are eligible if they are < 65 years, have adequate organ function, and have an available unrelated marrow donor matched 7 of 8 HLA-A, B, C, DRB1 loci. Patients with Fanconi anemia and other marrow failure syndromes are excluded. All patients receive TBI 200 cGy, ATG (either thymoglobulin: 3 mg/kg IV or ATGAM 30 mg/kg IV daily × 3, days –4 to –2), FLU (30 mg/m2 IV daily × 4, days –5 to –2). The Phase I portion of the trial sequentially tested four CY dose levels: 150 mg/kg (administered days –4 to –2); 100 mg/kg (days –3 to –2); 50 mg/kg (day –2); and, 0 mg/kg, and allowed enrollment of up to six patients at each CY dose level unless toxicity or graft failure boundaries were crossed. In the Phase II portion of the trial, patients enroll onto the optimal CY dose level, which is chosen using adaptive Bayesian criteria to rank desirability of the CY doses. All patients are followed for two years after transplantation. Early stopping guidelines are used to monitor for graft failure and early transplant-related mortality through day 100. Results: Twenty-one patients accrued to the Phase I portion of the trial. CY dose level 0 mg/kg was closed after all three enrolled patients developed secondary graft failure. All received a second allograft. One patient remains alive at 23 months after the first transplantation. The other two died from adult respiratory distress syndrome (ARDS) at 114 days after the first transplant and idiopathic pneumonia at 200 days after the first transplant. Review of Phase I results for the other three dose levels suggested CY dose 150 mg/kg as the optimal dose level for Phase II testing. However, after an additional eight patients were treated at this dose, the level was closed to further accrual because of excess toxicity. Seven of the 14 patients receiving 150 mg/kg of CY (and 7 of the last 8 enrolled) died. Causes of death were cardiac/pulmonary/multi-organ failure (n=4), ARDS (n=2), and parainfluenza virus type 3 pneumonia (n=1). Bayesian evaluation of the two remaining dose levels indicates very similar desirability scores and accrual continues at both the CY 100 mg/kg and 50 mg/kg levels. As of July 15, 2011, a total of 61 patients have been enrolled, 17 on the two closed levels, 33 on the 100 mg/kg level and 11 on the 50 mg/kg level. Conclusions: Early analysis of this trial, made necessary by these unexpected severe adverse events, revealed two important findings among patients receiving low-dose TBI, ATG, and FLU at the doses outlined: 1) CY dose 0 mg/kg is associated with higher than expected graft failure; and, 2) CY dose 150 mg/kg is associated with excess transplant-related toxicity. Neither CY dose level should be tested further in the context of the regimen used in this trial. To date, the two intermediate CY dose levels (100 mg/kg and 50 mg/kg) have not crossed the graft failure or fatality stopping boundaries and accrual is in progress. Disclosures: Pulsipher: Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 661 Objective: To compare 131Iodine-Tositumomab/BEAM to Rituximab/BEAM as the conditioning regimen followed by autologous stem cell transplantation for patients with relapsed chemotherapy sensitive DLBCL. Patients and Methods: The Blood and Marrow Transplant Clinical Trials Network (BMT CTN 0401) study sponsored by the National Heart, Lung, and Blood Institute and the National Cancer Institute enrolled 224 patients between 1/06 and 7/09. Eligible patients were age 18–80 years, had a Karnofsky performance score > 70%, persistent or recurrent DLBCL, chemotherapy sensitive disease, and had received 1–3 prior chemotherapy regimens. Patients with transformed DLBCL were excluded. Patients were randomized to receive 131Iodine Tositumomab (dosimetric dose of 5 mCi on day −19 and therapeutic dose of 75 cGy on day −12), carmustine 300 mg/m2 (day −6), etoposide 100 mg/m2 twice daily × 4 (days −5 to −2), cytarabine 100 mg/m2 twice daily × 4 (days −5 to −2), and melphalan 140 mg/m2 (day −1) (Bexxar/BEAM, n=111) vs. rituximab 375 mg/m2 on days −19 and −12 with the BEAM regimen (R/BEAM, n=113). All drugs were given intravenously. The median age at the time of transplant was 56.8 years in the Bexxar/BEAM and 58.8 years in the R/BEAM arm. All 224 patients were included in the intent to treat analysis for the primary endpoint of 2-year PFS. Twelve patients were not transplanted and two patients were ineligible based upon incorrect pathologic subtype and therefore were not included in further analyses Results: The median follow-up of the patients was 25.5 months (mo) (range 13.8– 47.0) in the Bexxar/ BEAM and 24.7 mos (range 4.7 – 58.6) in the R/BEAM arms, respectively. The primary end point of 2-year PFS was 47.9% (95% CI: 38.2%, 57.0%) for Bexxar/BEAM and 48.6% (95% CI: 38.6%, 57.8%) for R/BEAM (p= 0.94). The 2-year OS of all randomized patients was 61.0% (95% CI: 50.9%, 69.6%) for Bexxar/BEAM and 65.6% (95% CI: 55.3%, 74.1%) for R/BEAM (p= 0.38). Patients in complete remission after salvage chemotherapy (CR2) had an improved 2-yr OS compared to patients with primary induction failure (PIF) or chemosensitive relapse (p= 0.005). However, there were no differences in any group by treatment arm. 2-yr OS for CR2 patients with Bexxar/BEAM was 76.9% (95% CI: 62.9%, 86.1%) compared to 79.9% (95% CI: 64.7%, 89.1%) with R/BEAM (p= 0.61). The most common cause of failure was progression/relapse of the lymphoma with a cumulative incidence of relapse/progression at 2 yrs post transplant of 45.0% (95% CI: 35.2%, 54.8%) in the Bexxar/BEAM arm and 48.1% (95% CI: 38.1%, 58.1%) in the R/BEAM arm (p= 0.69). The treatment related mortality was 4.9% (95% CI: 0.8%, 9.0%) in the Bexxar/BEAM and 4.1% (95% CI: 0.2%, 8.0%) in the R/BEAM arms at 2 years post transplant (p= 0.97). Engraftment was similar with neutrophils to > 500/ul in 96.1% (95% CI: 92.2%, 100%) of Bexxar/BEAM and 93.5% (95% CI: 88.6%, 98.4%) of R/BEAM patients by day +28 (p= 0.40). Platelet recovery to > 20,000/ul with no transfusion by day +100 was present in 84.5% (95% CI: 77.4%, 91.6%) of the Bexxar/BEAM and 81.3% (95% CI: 73.9%, 88.7%) of the R/BEAM patients (p= 0.58). The median maximum mucositis score (by OMAS scale) was higher in the Bexxar/BEAM patients at 0.72 compared to 0.31 in the R/BEAM patients (p < 0.0001). One case of myelodysplastic syndrome (MDS) was reported in each arm of the trial and one additional case of acute myelogenous leukemia (AML) was reported in the R/BEAM arm. By exploratory analyses, immune reconstitution as measured by levels of quantitative immunoglobulins and B and T-lymphocyte subsets was not different between the two randomized arms at baseline, day +100, day +365, or day +730. Conclusions: The Bexxar/BEAM and the R/BEAM regimens produced similar 2-yr PFS and OS for patients with chemotherapy sensitive relapsed DLBCL. No differences in engraftment or other toxicities were apparent other than an increase in mucositis with the Bexxar/BEAM regimen. No significant difference in the risk of MDS or AML could be detected with the current follow up. Disclosures: Vose: Genentech: Research Funding; Pharmacyclics: Research Funding; SBio: Research Funding; Exelixis: Research Funding; BMS: Research Funding; Celgene: Research Funding; Millenium: Research Funding; GSK: Research Funding. Off Label Use: 131 Iodine Tositumomab combined with BEAM chemotherapy as a transplant preparative regimen for diffuse large B-cell lymphoma is an off label use. Burns:Novartis: Research Funding. Press:Roche/Genentech: Consultancy, Honoraria; Spectrum: Consultancy, Honoraria. Fenske:Seattle Genetics: Consultancy, Honoraria; Spectrum Pharmaceuticals: Consultancy, Honoraria; Millennium (Takeda) Pharmaceuticals: Research Funding.

Abstract 1 Background: Randomized trials demonstrated that filgrastim-mobilized PBSC compared to BM from HLA-identical siblings improved engraftment kinetics, increased risks of acute and chronic GVHD, but also decreased relapse and improved survival in patients with high risk leukemia. Retrospective analyses of unrelated donor transplants did not appreciate the same PBSC protective effect. Patients and Methods: The BMT CTN, sponsored by the NHLBI and NCI, conducted a Phase III, randomized, multicenter, trial of unrelated donor PBSC versus BM. The primary objective was to compare two-year survival probabilities in the two study arms using an intent-to-treat analysis. Both patients and donors provided informed consent. Fifty centers in the U.S. and Canada enrolled patients between January, 2004 and September 2009. Median follow up is 36 months (interquartile range 25 – 37 months). Randomization was performed in a 1:1 ratio to either PBSC or BM and stratified by transplant center and disease risk. Of the 278 subjects randomized to BM, 5% had no transplant, and 4.3% crossed over to PBSC; of the 273 randomized to PBSC, 4% had no transplant, and 0.4% crossed over to BM, so subjects on both arms had greater than 90% compliance with the assigned therapy. Patient primary disease (AML, ALL, CML, MDS, CMML, and MF), disease risk, gender, age, race, ethnicity, CMV serology, performance status, comorbidity, organ function, conditioning regimen, GVHD prophylaxis, use of growth factors, and donor characteristics were all well balanced between the two groups. Overall, 90% were adults over age 20, 47% had AML, 28% had high risk disease, 48% were conditioned with cyclophosphamide plus total body irradiation, and 71% received tacrolimus plus methotrexate for GVHD prophylaxis. Results: There were no observed differences in outcomes between the two groups except for a higher incidence of overall chronic GVHD (see Table) and more common chronic extensive GVHD with PBSC (46% vs. 31%). There were no survival differences according to graft sources in planned subset analyses of low and high risk malignancy or in those received HLA-matched or mismatched grafts. Primary causes of death were relapse in 54% vs. 49%, graft failure in 7% vs. 0%, acute or chronic GVHD in 22% vs. 34%, others in 16% vs. 16% of the BM and PBSC arms, respectively. Conclusion: This large randomized trial shows that PBSC from unrelated donors is associated with higher rates of chronic GVHD compared to BM, although rates of acute GVHD, relapse, non-relapse mortality and overall survival are similar. Disclosures: Off Label Use: Cyclophosphamide, busulfan, melphalan, fludarabine, anti-thymocyte globulin, irradiation were used to eradicate malignancy. Tacrolimus, cyclosporine, methotrexate were used for GVHD prophylaxis. Westervelt:Novartis: Speakers Bureau.

Abstract 2877 FLC assay measures circulating unbound k and l light chains and is of diagnostic and prognostic value in plasma cell disorders. Normalization of FLC ratio is considered a higher level of complete response (CR) in MM but this has not been well validated prospectively. HLC is a novel antibody based assay that targets the unique junctional epitopes between the heavy chain and light chain constant regions of intact immunoglobulin (Ig) molecules. It separately measures in pairs the light chain types of each immunoglobulin class generating ratios of monoclonal Ig/background polyclonal Ig concentrations (i.e IgGκ/IgGλ, IgAκ/IgAλ and IgMκ/IgMλ); potentially simplifying assessment of monoclonal protein response. To assess the prognostic impact of these assays and to correlate them with electrophoretic/immunofixation (SPE/IFE) assessment, we analyzed 497 stored serum samples from patients enrolled in the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) 0102 clinical trial, sponsored by the NHLBI and NCI, of tandem autologous (autoHCT) vs. tandem autoHCT- allogeneic (alloHCT). Samples were collected prior to the first autoHCT and analyzed for FLC (Freelite™) and HLC (Hevylite™) at The Binding Site, UK. Corresponding disease status determination was centrally performed by an expert data review committee using uniform response criteria and correlated with FLC and HLC results. HLC remission was defined as normalization of composite heavy and light chain ratios across all 3 measured heavy/light chain pairs or the normalization of clonal isotype with normal ratios of uninvolved pairs. The demographics of 497 patients with HLC/FLC samples at baseline were concordant with the main study and showed similar patient proportions from the 2 study arms. Of the 211 pts with baseline SPE response better than or equal to a very good partial response (>VGPR), 188 also had an HLC remission (Sensitivity=89%). Comparison of the HLC remission with the >VGPR disease state also identified a specificity of 52%, positive predictive value (PPV) of 58% and a negative predictive value (NPV) of 87%. Similarly, all 56 patients in complete remission (CR) by SPE/IFE were in HLC remission. Compared with conventional CR assessment, sensitivity of HLC remission was 100%, specificity 39%, PPV and NPV values of 17% and 100% respectively. FLC remission correlated with >VGPR disease state had a sensitivity of 47%, specificity of 81% and a PPV and NPV of 64% and 67% respectively. Multivariate models for post transplant progression free survival (PFS) indicated that baseline disease response (>VGPR vs. partial remission (PR) orVGPR disease state did not impact PFS (HR 0.96, p=0.83). Abnormal HLC after induction therapy has a high negative predictive value (100%) for identifying patients not achieving CR by uniform response criteria and is also associated with shorter PFS after transplant. FLC ratio normalization had no impact suggesting that stringent CR criteria may need further validation. Disclosures: Krishnan: Celgene: Speakers Bureau; Millenium: Speakers Bureau. Alvi:the binding site: Employment. Harding:The Binding Site Group Ltd: Employment. Maloney:Cellgene: Consultancy.

Abstract 655 Allogeneic hematopoietic cell transplantation (HCT) is the most effective means to prevent relapse in patients (pts) with AML in complete remission (CR). However, quality of life and overall survival (OS) are often affected by both acute and chronic graft versus host disease (GVHD). GVHD is most effectively prevented by ex vivo T cell depletion (TCD) of the allograft, but has been limited in its use by logistical difficulties, lack of an FDA-approved method, and concerns regarding potential risk of graft rejection, post transplant infections, and leukemic relapse. Most reported TCD studies represent single centers, multiple disease types and processing methods with varying degrees of TCD, all of which affect outcome. Therefore we designed a trial using a single processing method providing extensive TCD that did not require post transplant GVHD prophylaxis involving adult pts with AML in first or second CR. We hypothesized that the undesired side effects of TCD HCT would be reduced if combined with a conditioning regimen that was highly immunosuppressive and anti-leukemic. The primary objective was to achieve a disease-free survival (DFS) rate at 6 months (mos) post transplant that exceeded 75%. Secondary objectives included assessments of engraftment, transplant related mortality (TRM), GVHD, relapse, and performance of a single TCD method (CD34+ cell selection using the Miltenyi CliniMACS device) at participating centers. From 10/2005 to 12/2008, 47 pts were enrolled and 44 transplanted at 8 different centers. Median age was 48.5 years (range 21-59) with 28 female and 16 male pts. Of 37 AML CR1 pts, 49% had an unfavorable cytogenetic or molecular risk profile. The conditioning regimen consisted of hyperfractionated total body irradiation (1375cGy in 11 fractions) with partial lung shielding, thiotepa (10mg/kg), cyclophosphamide (120mg/kg), and rabbit antithymocyte globulin (2.5mg/kg). The donors, all HLA-identical siblings, were given G-CSF for mobilization and scheduled to undergo at least 2 leukapheresis procedures to ensure a graft with a high CD34+ cell content. All allografts were CD34-enriched and were targeted to contain ≥ 5×10e6 CD34+ cells/kg and < 1.0×10e5 CD3+ cells/kg. The median CD34+ and CD3+ doses achieved were 8.1 × 10e6/kg (range 2.4-46.2) and 0.07 × 10e5/kg (range 0.01-0.85), respectively. The majority (81%) of pts received the targeted CD34+ cell dose and no pt received > 1.0×10e5 CD3+ cells/kg. No pharmacological GVHD prophylaxis was given post transplant. There were no significant toxicities related to infusion of the CD34 enriched allografts. The most common grade 3-5 regimen-related toxicities included grades 3 or 4 mucositis (39%) and grades 3-5 pulmonary abnormalities (11%). Only 1 pt experienced hepatic veno-occlusive disease. All pts engrafted rapidly with a median time to neutrophil recovery (ANC > 500/ul) of 11 days (range 9-19). There was 1 secondary graft failure. The assessed outcomes are shown below.Estimate (95% Confidence Interval)Outcome100 Days6 Months12 MonthsAcute GVHD II-IV20.5% (8.7 – 23.3%)Acute GVHD III-IV4.5% (0 – 10.6%)Chronic GVHD17.7% (5.8-29.6%)Extensive Chronic GVHD7.6% (0-15.7%)TRM17.8% (5.8-29.8%)Overall Relapse18.2% (5.9-30.5%)Relapse 1st CR9.6% (0- 19.8%%)Relapse 2nd CR64.3% (27.5-100%)DFS81.3% (66.1-90.2%)64.0% (46.5-77.1%)DFS 1st CR89.2% (73.7-95.8%)72.1% (53.0-84.6%)OS74.3% (57.3-85.4%) The absolute peripheral CD4+ cell count remained on average below 200/ul until day +365. Donor cell chimerism increased in the CD3+ cell compartment through day +365. There were 14 deaths. The most common causes of death were relapse (N=5) and pulmonary toxicity (N=4). The median follow-up of survivors is 489 days (range 96-776). There was no difference in OS or DFS for pts above or below the median age of 48.5 years. We conclude that TCD HCT following myeloablative chemoradiotherapy can be performed in a multi-center setting using a single TCD method without additional post transplant prophylaxis with excellent DFS and OS, consistent engraftment, low TRM, and low incidence of relapse even in pts with unfavorable risk AML in CR1. The low incidences of acute and chronic GVHD in the absence of post transplant prophylaxis were particularly encouraging. A follow-up study of TCD HCT in AML recipients of unrelated donor allografts is being planned by the BMT CTN Disclosures: No relevant conflicts of interest to declare.

Randomization in clinical trials involving comparison of patients receiving an allogeneic HSCT versus other treatments is challenged by limitated donor availability. Biologic assignment relies on the availability of suitable donors to determine assignment to allogeneic HSCT. BMT CTN 0102 is a phase III multicenter clinical trial that compares tandem autologous HSCT (auto-auto) to an autologous HSCT followed by a nonmyeloablative allogeneic HSCT (auto-allo) for the treatment of multiple myeloma after initial systemic therapy. Transplant arm assignment was determined by the availability of an HLA-matched sibling donor. Patients were stratified into high and standard risk groups. Absence of chromosome 13 deletion by standard karyotyping and beta-2 microglobulin (B2M) ≤ 4mg/L were defined as standard-risk disease (SRD). The trial was powered to detect differences in 3-year progression-free survival in patients with SRD. A total of 710 patients from 43 centers in the U.S. were enrolled from December 2001 to March 2007. Data from 596 patients with SRD were available for this analysis (sibling donor availability for other 28 SRD patients is not yet determined); 30% (n=178) were assigned to the auto-allo arm. The median ages were 55 and 52 years for the auto-auto and auto-allo arms, respectively (p=0.01). The proportions of African-American patients were 17% and 9% in the auto-auto and auto-allo arms, respectively (p=0.006). Most patients were Durie Salmon Stage III (66% and 70%, p=0.6), in partial remission (86% and 83%, p=0.6) and had Karnofsky Performance Scores ≥ 90 (76% and 77%, p=0.5) at time of study entry. Treatment arms were balanced for B2M, baseline cardiac and hepatic functions and CMV serological status. Median times from diagnosis, initiation of systemic therapy and stem cell mobilization to first transplant were 213, 191 and 27 days, respectively and the same in both arms. The interval from the first to second transplant was 98 days for the auto-auto arm and 105 days for the auto-allo arm (p=0.02). We conclude that biologic assignment resulted in two reasonably comparable treatment groups, although statistically significant differences in age, race and interval between first and second transplant were identified. The differences in age between the groups is related to donor availability, since patients younger than 55 years had on average significantly more living siblings than older patients. Importantly, full-sibling donor availability did not account for differences in race distribution. Because the clinical significance of all detectable differences is unknown, the planned statistical analysis will adjust for these discrepancies. These results confirm that biologic assignment is feasible and might be considered when standard randomization is prohibitive, as in allogeneic HSCT clinical trials.

A multi-center, randomized, double blind trial was performed to determine the impact of FLU (400 mg daily in adults) vs. VORI (200 mg twice daily in adults) on the prevention of IFIs in standard risk allogeneic BMT patients receiving full-intensity conditioning regimens. 600 patients with AML (n=230), ALL (n=122), CML (n=103), MDS (n=98), lymphoma (n=43) and other diseases (n=4) were randomized; 295 to FLU, 305 to VORI between 2003 and 2006 (1 not transplanted). Study drugs were given for 100 days; in those receiving prednisone at a dose of ≥1 mg/kg/day at day 100 or for recipients of T cell depleted grafts with CD4+ counts 18 years; 55% male; 95% with HLA A, B, and DRB1 matched donor; stem cell graft was related donor marrow or PBSC in 56%. There were no significant differences between the two arms in patient, disease type or risk, or transplant characteristics. Rates of engraftment, acute or chronic GVHD, non-fungal infections, expected or unexpected severe adverse events, and rates of premature study drug withdrawal were similar in both arms (p=NS). At a median follow-up of 12 months, overall survival was 80% at 6 months and 67% at 12 months. A blinded data review committee reviewed source documents of all reported fungal infections, deaths, patients with a positive GM and those given empirical antifungal therapy. There were 25 proven, 30 probable, 15 presumptive, and 74 possible IFIs. The cumulative rates of proven, probable and presumptive IFI were similar in the two arms: 10.6% for FLU and 6.6% for VORI at 6 months (p=0.11) and 13.1% and 11.6% at 12 months (p=0.50), respectively. Microbiologically documented IFIs at 6 months in each arm (FLU and VORI) were caused by Aspergillus (16 and 7, p=0.05), Candida (3 and 3), Zygomycetes (3 and 2), and other (1 and 1). Fungal-free survival rates were similar: 76% for FLU and 78% for VORI at 6 months (p=NS) and 65% and 63% at 12 months (p=NS), respectively. Event-free and overall survival rates also were similar in both arms at 6 and 12 months (p=NS). There were no differences in fungal-free survival rates in patients who received prophylactic fluconazole or voriconazole when intensive monitoring and early empirical therapy were employed in standard risk allogeneic BMT recipients.

T cell depletion (TCD) of donor allogeneic blood or marrow grafts can reduce the incidence and severity of acute graft-versus-host disease (GvHD) but may place the patient at risk for disease relapse. This multicenter randomized trial examined the impact of selective depletion of αβ T cells (αβTCD) + cyclosporine immunosuppression in recipients of unrelated marrow grafts vs. T-replete marrow grafts + methotrexate and cyclosporine immunosuppression. Previously reported data from this trial have shown that 3-year disease-free survival (DFS) did not differ between these groups, although the αβTCD group showed improved neutrophil recovery, decreased incidence of acute GvHD, and reduced grade III-IV toxicities. In order to further clarify these findings, we examined the impact of each graft manipulation and immunosuppression strategy on lymphocyte subset recovery and associated outcomes. Lymphocyte recovery was assessed at 1, 3, 6, 12, 18, and 24 months post-SCT. Of all patients accrued, only patients that had survived for at least one year and who had at least four separate immunophenotyping assessments were included in this analysis (unmanipulated grafts = 66/206 32.0%; αβTCD grafts 57/203 28.1%). Non-parametric pair wise tests were used to compare recovery of absolute cell counts at each measurement period. We also fit a mixed model to each individual’s data to estimate the rate of change in cell counts in the two groups. The slope estimate for each subject obtained from the mixed model was then used as a variable in models to assess DFS. The Cox proportional hazards test was used to examine associations between recovery of individual cell subsets and DFS. Recovery of total absolute CD3, αβ, γδ, CD3+CD4+, CD3+CD8+ T cell count as well as the absolute B cell count did not significantly differ between groups for at each measurement period. CD3+CD4+CD45RA naive T cell recovery remained below the expected adult normal range for all time points in both groups but was improved in patients who received unmanipulated grafts. Interestingly, NK cell absolute count recovery was significantly improved in patients that received TCD grafts over those who received unmanipulated grafts (p = 0.001) through the first year post-SCT. Analysis of the impact of individual cell subset recovery on DFS revealed that an absolute γδ T cell count greater than 1.75 x 105/ml at any time during the first year predicted for improved DFS regardless of treatment group (p = 0.05) consistent with previous single-institution reports. Although the αβTCD regimen did not result in improved DFS in this trial, these findings show that αβTCD is a reasonable graft engineering strategy that can reduce the incidence of acute GvHD without producing a significant negative impact on lymphocyte subset recovery. Indeed, patients in the αβTCD group showed markedly improved NK recovery. Approaches that are currently in early-phase trials such as post-SCT targeted T and/or NK cell therapies could be used to supplement the αβTCD regimen, potentially improving relapse-free survival.

Background: Allogeneic bone marrow transplantation from related and unrelated donors leads to improvement in longevity and quality of life due to replacement of missing enzyme and engraftment of donor-derived glial cells in patients with LSD. Recently, unrelated umbilical cord blood transplant (UCBT) has yielded encouraging results but due to the rarity of LSDs, there has never been a large series of patients transplanted at a single center. Methods: Between 1995 and 2007, 159 consecutive LSD patients received unrelated UCBT at Duke after busulfan, cyclophosphamide, and antithymocyte globulin myeloablation. Cyclosporine+methylprednisolone (n=125) or cyclosporine+cellcept (n=34) was given for graft-versus-host disease (GvHD) prophylaxis. Cord blood units from 8 US public banks were screened and the unit with high-normal enzyme was selected. Engraftment, enzyme, organ function, neurodevelopmental, neuroimaging, and neurophysiologic studies were performed pre- and post-UCBT. The probabilities of engraftment, overall survival (OS) and GvHD were estimated. Multivariate models for graft and patient factors were analyzed. Results: Median patient age was 1.5 yrs (range 0.05–26.3); 19.5% were CMV seropositive; and 41.5% had a performance status2.1x105/kg, and infused CFU >5.7x104/kg. The probability of day+100 Grade III/IV acute GvHD was 10.3% (95%CI 5%–15%). Chronic GvHD developed in 25 (18%), 11 of whom were extensive. OS at 0.5, 1, 3, and 5 years was 79.0% (95%CI 73%–85%), 71.8% (95%CI 65%–79%), 62.7% (95%CI 55%–71%) and 58.2% (95%CI 50%–67%), respectively. In multivariate analysis, performance status 80–100 (p5.7x104/kg (p=0.02) and matched ethnicity (p=0.05) were independently associated with higher OS. In median follow-up of 4.2 years (range 0.2–11.5), all but 3 engrafted patients maintained donor chimerism >90% and all but 4 normalized enzyme level. In patients with high performance status (80–100), the OS at 1, 3 and 5 years was 88.4% (95%CI 79.6%–97.1%), 83.5% (95%CI 73.0%–94.1%) and 79.5% (95%CI 66.9%–92.1%), respectively. Conclusions: Unrelated Cord blood is an excellent graft source for treatment of patients with these fatal disorders, particularly when transplantation is performed in early stages. Pre-transplant performance status and post-thaw CFU dosing were highly predictive of overall survival.

A single umbilical cord blood unit (CBU) often doses patients at the threshhold for successful hematopoietic cell transplantation. While both cryopreserved and infused total nucleated cell (TNC) content is somewhat predictive of outcomes, there has been no reliable "potency" assay developed for UCB. Frequently, pre-cryopreserved and post-thaw recoveries are inconsistent and unpredictable. The lack of standardization from cord blood bank to bank and transplant center laboratory to laboratory further complicates interpretation of the data that is currently available. Our program is in the unique position to operate both a public cord blood bank, "The Carolinas Cord Blood Bank" (CCBB) and adult and pediatric blood and marrow transplant programs performing >100 unrelated donor cord blood transplants (UCBT) per year, placing us in the unique position of evaluating cord blood pre-cryopreservation and post-thaw TNC, viability, CD34 and colony forming assays (CFU-GM, CFU-GEMM, BFU-E) in the same laboratory, eliminating the potential for differences in laboratory practices. We have evaluated the results of 218 transplants performed from CBUs obtained from the CCBB and transplanted at our center. These units did not require transfer from the bank to the transplant center (TC) eliminating the possibility that warming of CBUs during shipping could compromise cell potency. In this dataset, the cummulative incidence (CINC) of neutrophil engraftment (ANC 500/uL by day +42) was 79.4% (95% CI 74–84.8) and platelet engraftment (PLT 50K by day 180) was 61.3% (95% CI 54.8–67.8). The CINC of event-free survival (EFS) at day 180 and 1 year was 68.7% (95% CI 62.5–75) and 62.5% (95% CI 56–69.1), respectively. The CI of acute grades II–IV graft-versus-host disease at days 100 and 150 was 10.6% (95% CI 6.5–14.7%). The CI of relapse at 1 year was 8.1% (95% CI 4.4–11.8%). Thus, most deaths occurred from graft failure, infection or regimen related toxicity all of which could be influenced by graft potency. We also expanded our dataset to include an additional 405 transplants where the CBU was shipped from other cord blood banks and transplanted at Duke. In this group, the CINC of neutrophil engraftment was 78.2% (95% CI 75–81.2) and the CI of EFS at day 180 and 1 year was 60–2 (95% CI 56.3–64) and 51% (95% CI 47–55), respectively. We hypothesized that post-thaw CFU and or CD34 would be better predictors of engraftment and survival than TNC. We examined the impact of post-thaw total CFU and CD34 recoveries and dosing on neutrophil engraftment and overall survival in these patients. Dosing of post-thaw CFU/kg infused (median 3.5x10e4/kg) correlated with neutrophil engraftment (p=

Children with AML unable to achieve remission after initial diagnosis or at relapse will not be cured by conventional therapy but may be salvaged by allogeneic hematopoietic stem cell transplantation. Those lacking a related donor may not survive the time that is required to find an unrelated adult donor. Banked, unrelated donor umbilical cord blood (UCB) increases access to transplant by providing a rapidly available source of allogeneic hematopoietic stem cells. Fifty-one pediatric patients with Relapse or Refractory AML were transplanted with Unrelated Cord Blood (UCB) between 1995 and 2005. By low resolution HLA-A and -B plus high-resolution DRB1 typing, UCB units were 3/6 (18%), 4/6 (45%), 5/6 (31%) or 6/6 (6%) matched. The median age was 8.4 years. Ethnic minorities comprised 35.3% of patients. Thirteen patients had received prior auto- or allo-transplants. The median total nucleated cell (TNC) dose infused was 4.4x107/kg. All patients received myeloablative regimens. Patients receiving Melphalan containing regimens (Bu/Mel/ATG or Flu/Mel/ATG or TBI/Mel/ATG) were grouped as Melphalan-Pos (n=33) and the rest as Melphalan-Neg (n=18). GVHD prophylaxis was Cyclosporine plus methylprednisone in 94.8%. Two groups were statistically similar for sex (p=0.69), age (p=0.1), ethnic minority (p=1), HLA matching (p=0.1), CMV status (p=0.68), and cell dose infused (p=0.35). The probabilities of overall survival (OS) were estimated by Kaplan-Meier analysis and compared using log-rank test. Three year probability of OS in Melphalan-Pos group was higher (36%) than the Melphalan-Neg (11%) group although the difference did not reach statistical significance. We restricted subsequent analyses to a subgroup of patients (n=28) who were transplanted between year 2000 and 2005 because the supportive care practices in our program regarding the use of voriconazole, granulocyte transfusions, early detection and treatment of CMV, and VOD prophylaxis and therapy had been standardized since 2000. The 3 year probability of overall survival for Melphalan-Pos patients (n=13) was significantly higher (54%) than the Melphalan-Neg (n=15) patients (7%) (p=0.023). AGVHD (Grade 2–4) was seen in 18% and CGVHD in 6% of patients. The causes of death in Melphalan-Pos vs. Melphalan-Neg patients were relapse (30% vs 46%), infection (8% vs 40%) and organ failure (8% vs 7%). Two Melphalan-Pos and one Melphalan-Neg patient developed graft failure. The median time to ANC > 500 was 21 and 28 days and to Platelet > 50K was 70 and 104 days in Melphalan-Pos and Melphalan-Neg groups, respectively. We conclude that for high risk pediatric patients with relapsed or refractory AML lacking a family donor, UCB transplantation following Melphalan containing cytoreduction results in cure in a significant number of patients.

Total nucleated cell count (TNC), CD34 and CFU content of cryopreserved cord blood units (CBUs) are measurements of hematopoietic potential previously demonstrated to be predictive of engraftment and/or survival post-transplant. Pre-cryopreservation measurements do not capture downstream events in cord blood banking that could impact CBT outcome: freezing, long-term storage, shipment to the transplant center and thawing. We hypothesized post-thaw enumeration of these measures would be more predictive of outcome. These tests were routinely performed at two of the COBLT transplant centers, at the time of CBU thaw. We examined the correlation of these laboratory measures pre-cryopreservation and post-thaw with engraftment and survival of 153 patients with leukemia and metabolic disorders undergoing myeloablative transplantation. The median age of recipients was 4.4 years (range 0.1 – 17.9) and weight 19.1 kg (range 3.9 – 118.4). Pre-cryopreservation Thaw Yield Median (range) Median (range) Median (range) TNC/kg x 107/kg 6.9 (1.5 – 50.4) 5.7 (1.1 – 31.6) 80.9% (43.0 – 98.9) CD34/kg x 105/kg 2.3 (0.1 – 20.1) 2.0 (0.2 – 15.5) 72.6% (16.1 – 99.6) CFU/kg x 104/kg 20.2 (1.7 – 169.7) 3.0 (0.0 – 31.1) 17.7% (0.0 – 82.7) Trypan blue viability 99 (91 – 100) 98 (83 – 100) — Overall 114/153 engrafted by day +42 with a median day to engraftment of 29 days. In multivariate analysis, post-thaw CFU count and yield were the highest correlates with engraftment (p5% (38% vs. 65%, p =0.05). Importantly, post-thaw yield of TNC and trypan blue viability did not correlate with engraftment. Total hematopoietic content of units (TNC, CD34, and CFU) was strongly predictive of engraftment. However, independently, low CFU counts and poor yield of CFU at time of CBU thaw is a harbinger of poor/nonengraftment and is evidence of deterioration of the product between time of processing and infusion. Low CFU count or poor yield of CFU post-thaw may be used clinically to predict engraftment in patients undergoing CBT. These findings illustrate the need for the development of product release testing criteria for CBUs and should be a priority for the cord blood community.

We and others have previously demonstrated that unrelated cyropreserved banked CB is an effective alternative source of allogeneic stem cells but is limited by delays in myeloid and platelet engraftment and immune reconstitution (Kurtzberg et al, NEJM 1996; Cairo et al, Blood 1997). CB nucleated (nuc) cell count, nucleated RBC and total CFU have been previously demonstrated to be associated with time to hematopoietic reconstitution (Rubinstein et al, NEJM 1998; Stevens et al, Blood 2002). More recently CB CD34+ content has been demonstrated to be significantly correlated to speed of engraftment and survival (Wagner et al, Blood 2002; Styczynski et al, BMT 2004). CB CD34+ subsets (CD38−, CD61+, CD90+) may also be critically important in predicting time to CB hematopoietic reconstitution, ex-vivo CB expansion and/or CB gene therapy. We investigated the relationships of CB CD34+ and CD34+ subsets with CB total CFU, CFU subsets, nuc cell, nuc RBC, ethnicity, gender, birth weight (BWT), gestational age (GA) and type of delivery in over 8,000 CB units. Briefly, CB was collected and processed (Fraser et al, J Hematother 1998) and aliquots were obtained for flow cytometry and in-vitro colony assays. Statistical analysis was based on multiple regression analysis and analysis or variance of logged counts. Approximately 35,000 maternal donors were screened, 20,700 consented, 16,700 CB collected and 8,730 CB units entered long-term cryopreserved storage. Ethnic distribution was 42% Caucasian, 21% Hispanic, 14% African-American, 11% Asian and 12% mixed. CB was collected by vaginal delivery (74%), at 39–40 weeks GA (61%) and 3–4000 Gm BWT (73%). Mean ± SD CD34+ and CD34+/38−, 61+, 90+ were 34±42, 3.4±4.8, 7.3±9.5 and 0.3±1.9 x 105, respectively. Mean SD of total CFU, BFU-E, CFU-GM and CFU-GEMM was 23.2±19.8, 13.3±3.1, 9.3±8.6 and 0.5±1.1, respectively. There was a significant correlation of log CD34+ with log nuc cell count and log nuc RBC count (R= 0.64, p

It has not been possible to determine the singular contribution of naive T lymphocytes to antigen-specific immunity after hematopoietic stem cell transplantation (HSCT), because of the confounding effects of donor-derived antigen-specific T lymphocytes present in most hematopoietic stem cell (HSC) products. Because umbilical cord blood contains only naive T lymphocytes, we longitudinally evaluated the recipients of unrelated cord blood transplantation (UCBT) for the presence of T lymphocytes with specificity for herpesviruses, to determine the contribution of the naive T lymphocytes to antigen-specific immune reconstitution after HSCT. Antigen-specific T lymphocytes were detected early after UCBT (herpes simplex virus on day 29; cytomegalovirus on day 44; varicella zoster virus on day 94). Overall, 66 of 153 UCBT recipients developed antigen-specific T lymphocytes to 1 or more herpesviruses during the evaluation period. The likelihood of developing antigen-specific T lymphocyte function was not associated with immunophenotypic T lymphocyte reconstitution, transplant cell dose, primary disease, or acute and chronic graft-versus-host disease. These results indicate that naive T lymphocytes present in the HSC inoculum can contribute to the generation of antigen-specific T-lymphocyte immunity early after transplantation.