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Background: In July of 2016, CMMI started a five year bundled payment program called OCM. Beneficiaries are attributed to practices providing their cancer care for a 6 month episode triggered by the administration or distribution of specified cancer drugs. The model provides risk adjustments to the target price based on risk factors such as age, chemotherapy and the receipt of certain treatments (radiation or bone marrow transplant). Target prices are adjusted by geographic region, novel therapy use, and a trend factor. Multiple Myeloma was identified in our practice as a cancer type with high variance on expenditures after the first Performance Period within the model (July 2016-December 2016). Chemotherapy represented 52% of total episode expenditures with oral chemotherapy and hormone therapy representing 24%. The average cost of lenalidomide for one year is $115,000. The model adjusts for novel therapies, including new drugs approved by the FDA after December 31, 2014 with status lasting two years. However, literature demonstrates that this does not fully adjust for the high costs of novel therapies (Muldoon et at., Health Affairs, 2018). Unlike solid tumors, Multiple Myeloma staging may not improve risk adjustment and target price. Methods: We analyzed the total cost of care of beneficiaries who triggered an OCM episode for Performance Period 1 (PP1). Beneficiaries were identified by diagnosis of Multiple Myeloma, and then segregated into cohorts of those who received lenalidomide and/or pomalidomide and those who did not. Observed vs. Expected (O/E) target price for each episode was determined for both cohorts comparing the actual episode expenditures and the target price per episode calculated by the Oncology Care Model. A two sample t-test was conducted followed by a linear regression to determine relation between drug days prescribed and O/E. Results: There were 125 attributed beneficiaries with a Multiple Myeloma diagnosis who triggered an episode during PP1. The average O/E of the cohort which received the chemotherapy, Cohort A, was 1.624 compared to 0.986 for those that did not, Cohort B. The difference in average O/E in the two cohorts was 39% higher in Cohort A, p Discussion: This is the first report on the impact of lenalidomide and pomalidomide on the total cost of care in an OCM practice. The results demonstrate the lack of adequate adjustment to the CMS target price for episodes in which one or both of these drugs were prescribed. Lenalidomide and pomalidomide are first and second line drugs used both for induction and maintenance. Both drugs are frequently used for prolonged periods of time in patients and trigger more than one episode in OCM. Therefore, the use of these agents greatly affects the total cost of care against a target price that is not adequately adjusted. Academic Medical Centers that care for larger populations of multiple myeloma patients may be disproportionately affected and this will impede their success under the OCM methodology. Additional analysis similar to this will inform CMMI as to further refinements to the OCM adjusters. Disclosures No relevant conflicts of interest to declare.

The best conditioning regimen before allogeneic transplantation for high-risk diffuse large B-cell lymphoma (DLBCL) remains to be clarified. We analyzed data from 396 recipients of allotransplants for DLBCL receiving myeloablative (MAC; n = 165), reduced intensity (RIC; n = 143), or nonmyeloablative conditioning (NMAC; n = 88) regimens. Acute and chronic GVHD rates were similar across the groups. Five-year nonrelapse mortality (NRM) was higher in MAC than RIC and NMAC (56% vs 47% vs 36%; P = .007). Five-year relapse/progression was lower in MAC than in RIC/NMAC (26% vs 38% vs 40%; P = .031). Five-year progression-free survival (15%-25%) and overall survival (18%-26%) did not differ significantly between the cohorts. In multivariate analysis, NMAC and more recent transplant year were associated with lower NRM, whereas a lower Karnofsky performance score (< 90), prior relapse resistant to therapy, and use of unrelated donors were associated with higher NRM. NMAC transplants, no prior use of rituximab, and prior relapse resistant to therapy were associated with a greater risk of relapse/progression. In conclusion, allotransplantation with RIC or NMAC induces long-term progression-free survival in selected DLBCL patients with a lower risk of NRM but with higher risk of lymphoma progression or relapse.

Several risk factors are associated with increased mortality in patients with chronic graft-versus-host disease (cGVHD), but there is considerable variability in the reported factors. Therefore, we evaluated patient, transplantation, and cGVHD characteristics to develop a risk score in 5343 patients with cGVHD. Ten variables were identified as being significant in multivariate analysis of overall survival and nonrelapse mortality (NRM): age, prior acute GVHD, time from transplantation to cGVHD, donor type, disease status at transplantation, GVHD prophylaxis, gender mismatch, serum bilirubin, Karnofsky score, and platelet count. These 10 variables were used to build a cGVHD risk score, and 6 risk groups (RGs) were identified. The 5-year NRM was 5% (1%-9%) in RG1, 20% (19%-23%) in RG2, 33% (29%-37%) in RG3, 43% (40%-46%) in RG4, 63% (53%-74%) in RG5, and 72% (59%-85%) in RG6. The 5-year overall survival was highest at 91% (95% confidence interval [CI]:85%-97%) in RG1, followed by 67% (65%-69%) in RG2, 51% (46%-55%) in RG3, 40% (37%-43%) in RG4, 21% (12%-30%) in RG5, and 4% (0%-9%) in RG6 (all P < .01). This analysis demonstrates the usefulness of data from a large registry to develop risk-score categories for major transplantation outcomes. Validation of this cGVHD risk score is needed in a different population to ensure its broad applicability.

We report the retrospective outcomes of unrelated donor (URD) transplants in 169 patients with acute lymphoblastic leukemia (ALL) in first complete remission (CR1) who received transplants between 1995 and 2004. Median age was 33 years (range, 16-59 years). A total of 50% had a white blood cell count (WBC) more than 30 × 109/L, 18% extramedullary disease, 42% achieved CR more than 8 weeks from diagnosis, 25% had adverse cytogenetics, and 19% had T-cell leukemia. A total of 41% were HLA well-matched, 41% partially matched with their donors, and 18% were HLA-mismatched. At 54-month median follow-up, incidences of acute grade 2-IV, III to IV, and chronic graft-versus-host disease were 50%, 25%, and 43%, respectively. Five-year treatment-related mortality (TRM), relapse, and overall survival were 42%, 20%, and 39%, respectively. In multivariate analyses, TRM was significantly higher with HLA-mismatched donors and T-cell depletion. Relapse risk was higher if the diagnostic WBC was more than 100 × 109/L. Factors associated with poorer survival included WBC more than 100 × 109/L, more than 8 weeks to CR1, cytomegalovirus seropositivity, HLA mismatching, and T-cell depletion. Nearly 40% of adults with ALL in CR1 survive 5 years after URD transplantation. Relapse risks were modest; TRM is the major cause of treatment failure. Selecting closely HLA-matched URD and reducing TRM should improve results.

From 2007 to 2011, 66 patients with primary myelofibrosis or myelofibrosis (MF) preceded by essential thrombocythemia or polycythemia vera were enrolled into a prospective phase 2 clinical trial of reduced-intensity allogeneic hematopoietic stem cell transplantation (AHSCT), Myeloproliferative Disorder Research Consortium 101 trial. The study included patients with sibling donors (n = 32) receiving fludarabine/melphalan (FluMel) as a preparative regimen and patients with unrelated donors (n = 34) receiving conditioning with FluMel plus anti-thymocyte globulin (ATG). Patient characteristics in the 2 cohorts were similar. Engraftment occurred in 97% of siblings and 76% of unrelated transplants, whereas secondary graft failure occurred in 3% and 12%, respectively. With a median follow-up of 25 months for patients alive, the overall survival (OS) was 75% in the sibling group (median not reached) and 32% in the unrelated group (median OS: 6 months, 95% confidence interval [CI]: 3, 25) (hazard ratio 3.9, 95% CI: 1.8,8.9) (P < .001). Nonrelapse mortality was 22% in sibling and 59% in unrelated AHSCT. Survival correlated with type of donor, but not with the degree of histocompatibility match, age, or JAK2V617F status. In patients with MF with sibling donors, AHSCT is an effective therapy, whereas AHSCT from unrelated donors with FluMel/ATG conditioning led to a high rate of graft failure and limited survival. This trial was registered at www.clinicaltrials.gov as #NCT00572897.

The purposes of the research reported here were first to explore a murine model for human placental and umbilical cord blood transplantation and second to evaluate the engraftment ability of ex vivo cultured hematopoietic cells. Murine near-term fetal and neonatal peripheral blood (FNPB) cells, genetically marked with the human multiple drug resistance transgene (MDR1) were used for syngeneic transplants into sublethally irradiated adult mice. Donor cells were transplanted either fresh and untreated, or after ex vivo culture in the presence of the hematopoietic growth factors recombinant murine stem cell factor, recombinant human interleukin-3 (rHu IL-3), and rHu IL-6, in a liquid culture system. To evaluate, count, and characterize FNPB progenitor cell-derived colonies, neonatal mouse mononuclear cells were cultured directly in methylcellulose with growth factors. To assess their ex vivo expansion ability, FNPB mononuclear cells were first cultured in liquid medium for 3 to 8 days and then transferred to semisolid assay plates. Evaluation of the cell counts after liquid culture showed a 1.4- to 11.6-fold increase, and the numbers of colonies observed in methylcellulose were similar to those produced by fresh FNPB cells. Donor-type engraftment was demonstrated by polymerase chain reaction (PCR) amplification of the human MDR1 transgene in the peripheral blood of all surviving animals (5 of 7 recipients of the fresh, and 3 of 8 recipients of the ex vivo–cultured cells) 2 to 4 months after transplantation. The proportion of donor leukocytes in the peripheral blood of the recipients (chimerism) was evaluated using fluorescence in situ hybridization (FISH) analysis 4 to 6 months after transplantation and ranged from 2% to 26%. In addition, bone marrow cultures were obtained from two recipient animals: one had received fresh-untreated cells and was evaluated 8 months after transplant, the other had received ex vivo cultured cells and was tested 14 months after grafting. The derived hematopoietic colonies were tested by PCR and the transgene was detected, conclusively proving long-term engraftment of donor cells. These results indicate that FNPB transplants can be successfully performed in sublethally irradiated mice with and without ex vivo culture. Long-term donor-type engraftment with sustained chimerism has been demonstrated. Thus, murine neonatal blood grafts can be used as an animal model for cord blood transplantation for gene therapy studies where complete myeloablation is not desirable and partial replacement of defective marrow may be sufficient. Furthermore, the possibility of numerically expanding hematopoietic progenitor cells contained in neonatal blood without affecting their engraftment ability could facilitate use of cord blood grafts in adult recipients.

Cell dose is a major limitation for umbilical cord blood (UCB) transplantation because units containing a minimum of 2.5 x 10(7) total nucleated cells (TNC)/kilogram patient body weight are frequently not available. The transplantation of 2 partially HLA-matched UCB units has been adopted as a simple approach for increasing the TNC.We sought to determine whether the relative safety and efficacy of this approach was comparable with a single UCB transplantation. Included are adults with acute leukemia who received transplants with 1 (n =106) or 2 (n =303) UCB units. All UCB units for single UCB transplantations contained TNC ≥ 2.5 x 10(7)/kg. For double UCB transplantations, the total TNC for units 1 and 2 were2.5 x 10(7)/kg but in approximately half of these transplantations, 1 of the 2 units contained2.5 x 10(7) TNC/kg. Adjusting for factors associated with outcomes, risks of neutrophil recovery (odds ratio 0.83, P =.59), transplantation-related mortality (hazard ratio [HR] 0.91, P= .63), relapse (HR 0.90, P= .64), and overall mortality (HR 0.93, P= .62) was similar after double UCB and adequate dose single UCB transplantations. These data support double UCB unit transplantation for acute leukemia when an adequately dosed single UCB unit is not available thereby extending access to nearly all patients.Efficacy of transplanting adequately dosed 1- or 2-cord blood units.

The success of reduced intensity conditioning (RIC) transplantation is largely dependent on alloimmune effects. It is critical to determine whether immune modulation with anti–T-cell antibody infusion abrogates the therapeutic benefits of transplantation. We examined 1676 adults undergoing RIC transplantation for hematologic malignancies. All patients received alkylating agent plus fludarabine; 792 received allografts from a human leukocyte antigen-matched sibling, 884 from a 7 or 8 of 8 HLA-matched unrelated donor. Using Cox regression, outcomes after in vivo T-cell depletion (n = 584 antithymocyte globulin [ATG]; n = 213 alemtuzumab) were compared with T cell– replete (n = 879) transplantation. Grade 2 to 4 acute GVHD was lower with alemtuzumab compared with ATG or T cell– replete regimens (19% vs 38% vs 40%, P < .0001) and chronic GVHD, lower with alemtuzumab, and ATG regimens compared with T-replete approaches (24% vs 40% vs 52%, P < .0001). However, relapse was more frequent with alemtuzumab and ATG compared with T cell–replete regimens (49%, 51%, and 38%, respectively, P < .001). Disease-free survival was lower with alemtuzumab and ATG compared with T cell–replete regimens (30%, 25%, and 39%, respectively, P < .001). Corresponding probabilities of overall survival were 50%, 38%, and 46% (P = .008). These data suggest adopting a cautious approach to routine use of in vivo T-cell depletion with RIC regimens.

Umbilical cord blood (UCB) offers many potential advantages as a source of hematopoietic stem cells (HSCs) for allogeneic transplantation, including ease of collection, rapid availability, flexibility of HLA-matching, lower rates of GvHD and potentially lower relapse rates. However, the low HSC content of UCB compared to other graft sources results in a prolonged time to engraftment, and higher rates of graft failure and early mortality. Pulse ex vivo exposure of HSCs to 16,16-dimethyl PGE2 (FT1050) has been demonstrated to enhance HSC engraftment potential, which could benefit clinical UCB transplant. FT1050 modulation promotes multiple mechanisms, including increased proliferation, reduced apoptosis, and improved migration and homing [North 2007&2009; Hoggatt 2009]. Improved HSC homing is mediated by induction of CXCR4 gene expression leading to increased cell surface CXCR4. Further optimization of the UCB modulation process demonstrated that incubation with 10µM FT1050 for 2 hrs at 37C resulted in a maximal biological response of the FT1050-UCB (ProHema®). A Phase 1 trial was performed to evaluate the safety of FT1050-UCB paired with an unmanipulated UCB unit in reduced-intensity double UCBT (dUCBT) [Cutler 2013]. We observed durable, multi-lineage engraftment of FT1050-UCB with acceptable safety. Earlier neutrophil engraftment was observed relative to historical controls (median 17.5 vs. 21 days (historical control), p=0.045), coupled with preferential engraftment of the FT1050-UCB unit in 10 of 12 subjects. A Phase 2 multi-center clinical trial of FT1050-UCB in adult patients undergoing dUCBT for hematologic malignancies was then initiated. Subjects are randomized 2:1 to FT1050-UCB-containing vs. standard dUCBT after high-dose conditioning. The primary endpoint is a categorical analysis of neutrophil engraftment using a pre-specified control median. Data on the initial 11 subjects, of which 8 were randomized to receive FT1050-UCB, continue to demonstrate acceptable safety with adverse events attributed to FT1050-UCB limited primarily to common infusion-related side effects. Of the 8 FT1050-UCB subjects, 1 died prior to neutrophil engraftment, with the remaining 7 subjects engrafting at a median of 28 days vs. 31 days for the 3 control subjects. With median overall follow-up of 16.1 months, 4 of 8 subjects on the FT1050-UCB arm are alive with a median survival not reached (> 11.0 months). 1 of 3 control subjects is alive with median survival of 6.0 months. During the clinical translation process, the media used during FT1050 modulation of UCB was identified as a key variable. Standard UCB washing media, consisting of a nutrient-free saline solution of low molecular weight dextran and human serum albumin (LMD/HSA), is used clinically to stabilize fragile cells post-thaw by reducing lysis. This media was used in the Phase 1 trial and to initiate Phase 2. Early during the Phase 2 trial, we identified a novel cell-stabilizing nutrient-rich formulation (NRM), containing glucose, amino acids and other HSC-supporting nutrients that promoted full FT1050 modulation of UCB and increased cell viability. The expression of key FT1050-pathway genes was significantly higher with NRM compared to intermediate levels observed with LMD/HSA. Modulation of human CD34+ (hCD34+) cells with FT1050 in NRM led to an 8-fold increase over LMD/HSA in induced CXCR4 gene expression (20-fold total), which translated to significantly increased surface CXCR4 protein. In vivo homing models demonstrated that UCB CD34+ cells modulated with FT1050 in NRM resulted in a 2.2-fold homing increase relative to vehicle (p < 0.001) compared to a 1.6-fold increase with LMD/HSA (p = 0.002), with a significant difference between the two media conditions (p = 0.04). A xenotransplantation study in NSG mice with hCD34+ cells modulated with FT1050 in either NRM or LMD/HSA demonstrated a 2-fold increase in circulating hCD45+ cells 12-weeks post-transplant with NRM (p = 0.007; unpaired t-test). These findings supported the incorporation of NRM into the FT1050-UCB manufacturing process in order to further improve its clinical engraftment potential. Enrollment of a 60-patient Phase 2 trial has been initiated that incorporates this manufacturing change. Disclosures Shoemaker: Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics: Employment. Guerrettaz:Fate Therapeutics: Employment. Robbins:Fate Therapeutics: Employment. Medcalf:Fate Therapeutics: Employment. Wolchko:Fate Therapeutics: Employment, Equity Ownership. Ferraro:Fate Therapeutics: Employment. Multani:Fate Therapeutics: Employment.

Background Plerixafor (Mozobil®, Genzyme, Cambridge, MA) is approved for hematopoietic progenitor cell (HPC) mobilization into peripheral blood (PB) in combination with granulocyte colony stimulating factor (G-CSF) at ∼11 hours (hr) prior to apheresis initiation. Since apheresis facilities typically open at 8-9 AM, this 11 hr interval requires plerixafor dosing between 9-10 pm, impractical unless the patient self-administers the drug. No studies have examined mobilization kinetics beyond 15 hr in the target MM and NHL patient population. This is the first study in this target population to examine a total interval time of 17-18 hr post-plerixafor, important because, practically, leukapheresis may not be initiated until 10-11 AM. Even if initiated earlier between 8-9 AM, a standard leukapheresis typically lasts ∼3 hr. Therefore, it is important to rule out a significant decrease in PB [CD34+] extending through this interval. Study Design and Methods A single-center, prospective cohort, IRB-approved study where 11 patients with NHL and MM underwent HPC mobilization from March 2010 to October 2011. Patients met the same entry criteria specified in the initial studies leading to FDA approval. Plerixafor 240 ug/kg was administered at 5pm on day 4 of AM G-CSF 10 ug/kg. PB [CD34+] and [CD34+CD38-] concentrations were enumerated every 2 hours from 5PM to 7AM and immediately pre-apheresis on day 5, for a total interval time of 17-18 hr post-plerixafor. Leukapheresis (3 total blood volumes) was performed if the 7 am peripheral blood CD34+ concentration was ≥10/uL. Data was analyzed used mixed model analysis of repeated measures. Results 9 of 11 subjects , including all 5 patients who had received 3-9 cycles of lenalidomide, achieved a CD34+ product count of >5x106/kg with a single leukapheresis. All 9 patients (in contrast to the other 2) had a pre-plerixafor PB CD34+ concentration > 10/uL. PB [CD34+] did not differ between 10-18 hours post-plerixafor (p≈0.8). In contrast, PB [CD34+CD38-] increased from 10 to 18 hours post-plerixafor (p=0.03). 10 subjects underwent transplant with a median CD34+ dose of 6.0x106/kg (range 3.8-10.8x 106/kg) and engrafted within normal time frames. Using post- to (5PM) pre-plerixafor [CD34+] ratios to compare efficacy of plerixafor kinetics, the median ratio of the 17-18hr post/pre-plerixafor [CD34+] was 4.0 (range 1.8-6.8), not significantly different (p=0.09) from that of the peak post/pre-plerixafor [CD34+] of 4.7 (range 1.9-9). The 3 subjects with lowest mobilization had diabetes, but the peak post/pre [CD34+] ratio was not adversely affected. Two of these three reached their peak [CD34+] mobilization at 8hr post-plerixafor, whereas all other donors reached their peak [CD34+] ≥ 10hr post-plerixafor. The correlation coefficient of 0.54 between the PB [CD34+] and [CD34+CD38-] was weak. Conclusions In MM and NHL patients with adequate pre-plerixafor CD34+ concentration, which includes those with prior lenalidomide, leukapheresis initiated 17-18 hours post-plerixafor may not impair CD34+ product yield and may increase more primitive CD34+CD38- yield. Patient with risk factors for poor mobilization, such as diabetes or salvage chemotherapy, may be an exception. The three donors with post-plerixafor PB [CD34+] < 100/uL all had diabetes, which has been established to impair G-CSF induced stem cell mobilization. Our data supports murine data that plerixafor overcomes sympathetic nervous system-related defects in mobilization, since the peak post/pre plerixafor [CD34+] ratio was > 4 in all 3 patients. Interestingly, the donor with the second highest mobilization also had diabetes but a low peak/pre [CD34+] ratio of 2.9 (i.e. excellent mobilization with G-CSF alone); her mobilization impairment from diabetes may have been overcome by concurrent use (for asthma) of the β2-adrenergic agonist albuterol, previously associated with high mobilization. Consistent with previous studies, the 2 patients with poor [CD34+] mobilization reached their peak PB [CD34+] < 11 hours post-plerixafor. Finally, given the weak correlation between PB [CD34+] and [CD34+CD38-] , the utility of using PB [CD34+CD38-] to help determine the optimal time for collection may be worth exploring, given the correlation between CD34+CD38- graft content and long-term hematopoietic reconstitution in autologous transplantation. Disclosures: Off Label Use: Plerixafor administered up to 18 hours prior to apheresis initiation, rather than the FDA-approved 11 hours.

Abstract 4534 Myelofibrosis is a clonal myeloproliferative disorder, characterized by ineffective hematopoesis and bone marrow fibrosis. The disease can present de novo or secondary to an antecedent chronic myeloproliferative neoplasm such as essential thrombocythemia (ET) or polycythemia vera (PV). Allogeneic hematopoetic stem cell transplantation (AHSCT) is a curative approach for myelofibrosis, and produces durable donor engraftment and regression of myelofibrosis in approximately 50% of eligible patients. We retrospectively evaluated 31 consecutive patients that were treated for MF with AHSCT at our institution between 1998 and 2011. Thirty-one patients, 33 to 70 years of age, with a median age of 47, males 17 and females 14 with primary myelofibrosis or myelofibrosis evolving from antecedent polycythemia vera 4 (12%) or essential thrombocythemia 10 (32%) received AHSCT. Seven (22%) patients had transformed to acute myeloid leukemia prior to transplant. JAK 2 V617F was present in 15 patients, 9 patients were JAK 2 wild type, 5 patients were not tested and 2 were unknown. Patients received AHSCT from related (n= 11) or unrelated (n= 20) donors. Fourteen patients (45%) had a normal karyotype, 16 patients (51%) had an abnormal karyotype and one was unknown. Twenty eight patients received reduced intensity conditioning regimen and three received a fully ablative transplant. The source of stem cells was marrow in 4 patients and peripheral blood in 27 patients. Median time to neutrophil engraftment was 13 days in 28 patients (90%) and three patients had primary graft failure and were re-transplanted. One of the patients receiving a second AHSCT is alive and in complete hematological remission 19 months post-transplant.Twenty-eight patients showed 95–100% donor chimerism at day 30. Two patients had mixed chimerism at day 90. Twenty-six patients (83%) survive more than 100 days, 3 patients died (10%) from relapsed/progressive disease, and 5 (16%) from TRM. However at a median follow up of 31 months post-transplant 15 patients (48%)survived, 11 (45%)with chronic myelofibrosis, and 4 out of 7(57%)who had transformed to AML prior to transplant. Of note all five patients who were greater than 65 years of age at the time of transplant engrafted and enjoyed full hematological and clinical remission, one of these individuals subsequently died of a solid tumor. In addition, one of the patients with AML had an extramedullary relapsed 36 months post-transplant. One of the unanticipated complications observed in these patients who underwent AHSCT was massive peripheral edema in some cases anasarca of unclear etiology that required diuresis and albumin infusions and that occasionally preceded the date of engraftment and persisted for several weeks after engraftment. We conclude that AHSCT is an important therapeutic modality for not only PMF patients 65 years of age and individuals with blastic transformation of MF. Disclosures: No relevant conflicts of interest to declare.

Abstract 211 The adverse impact of chronic GVHD (cGvHD) on health and quality of life is especially critical in children because of their longer life expectancy and problems impacting growth and development. Although risk-factors for developing cGvHD in children are reported, little is known about risk factors for non-relapse mortality (NRM) in children with cGvHD. Identification of predictors for mortality in children with cGvHD could permit risk-adapted therapy, help plan for clinical trials and assist in counseling. We performed a multivariate analysis using data from CIBMTR to identify transplant- and cGvHD-related risk factors for NRM and survival in a cohort of 1117 subjects aged 0–20 years, transplanted from related donors, unrelated donors (URD), or unrelated cord blood (UCB) in 1995–2004 for acute myelogenous leukemia (AML), acute lymphoid leukemia (ALL), chronic myelogenous leukemia (CML) or myelodysplastic syndrome (MDS). Median age was 12 years. Characteristics of cGvHD at diagnosis were: progressive onset (49%), total bilirubin >2 mg/dL (16%), thrombocytopenia (platelets 10 years; (2) transplant from an HLA-partially-matched or -mismatched URD; (3) advanced disease at transplant; (4) KPS/L Disclosures: No relevant conflicts of interest to declare.

Abstract 37 The primary objective of CALGB 100104 was to determine if maintenance lenalidomide would prolong time to progression (TTP) after single AHSCT for multiple myeloma. Eligibility included: Stage I-III multiple myeloma, ≤ 1 year from diagnosis, ≥ 2 months of induction with stable disease or better and age < 70 years. AHSCT regimen was melphalan 200 mg/m2. Patients (pts) with stable disease or better were randomized double-blinded at day 100–110 post-AHSCT to lenalidomide or placebo, after stratification by diagnostic β2-microglobulin (β2M) level and prior thalidomide or lenalidomide therapy. Starting dose was 10 mg/day, escalated to 15 mg/day after 3 months and continued until disease progression. Drug was stopped and dose reduced according to the development of toxicity. Drug was held for ≥ Gr 3 toxicity, restarted at resolution to ≤ Gr 2 and de-escalated by 5 mg or maintained as tolerated at 15, 10, 5 mg daily or 5 mg daily for 21 of 28 days per month. All pts required some form of anticoagulation including aspirin, warfarin or heparin compounds. There was no consolidation therapy. Results: 568 pts were enrolled before AHSCT (04/15/05-07/03/09) from 47 centers. Of 108 pts (19%) not randomized, reasons were: progressive disease/no response 16%, adverse events (AEs) 5%, died during therapy 2%, refusal 26 %, other disease 1%, other therapy 4 %, other reasons 33%, unknown 14%. Pt characteristics in the lenalidomide arm and placebo arm respectively were: median age (range) 58 (29-70) and 57 (39-70); male gender 48% and 52%; β2M >2.5 mg/L, 28% and 27%. For 554 pts with complete data, induction regimens were thalidomide based (27%), lenalidomide based (22%), bortezomib based (20%), bortezomib and thalidomide based (12%), bortezomib and lenalidomide based (9%), dexamethasone based (4%), lenalidomide and thalidomide (3%), lenalidomide, thalidomide and bortezomib (1%), other (1%) and missing (1%); hence 74% of pts received either lenalidomide or thalidomide prior to enrollment. The primary endpoint of the study, TTP was met in a planned protocol interim analysis in the 3rd quarter of 2009 and the study results were released on 12/17/09. This updated 3rd interim analysis for TTP includes further events up until 12/17/09 after which study pts were un-blinded. This interim analysis is based on 460 randomized pts with approximately 33% of the required number of events (progression or death before progression) observed. The median follow-up is 17.5 months from ASHCT. The number of events among 231 pts randomized to lenalidomide was 44 compared to 91 among 229 pts randomized to placebo. The one-sided unadjusted P-value was Conclusions: Long term administration of lenalidomide is feasible. When compared to placebo controls, lenalidomide initiated at day 100–110 post-AHSCT in multiple myeloma patients significantly delays TTP. Disclosures: McCarthy: Celgene: Honoraria, Research Funding. Off Label Use: Lenalidomide maintenance therapy for myeloma following autologous hematopoietic cell transplant. Anderson:Millenium: Consultancy, Honoraria; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy; Acetylon: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Hurd:Celgene: Research Funding. Giralt:Celgene: Honoraria, Speakers Bureau; Millenium: Honoraria, Speakers Bureau. Stadtmauer:Celgene: Speakers Bureau. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Vij:Celgene: Honoraria, Speakers Bureau. Callander:Millenium: Research Funding. Maziarz:Millenium: Speakers Bureau; Genzyme: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Landau:Millenium: Membership on an entity's Board of Directors or advisory committees. Martin:Celgene: Speakers Bureau; Millenium: Speakers Bureau; Novartis: Speakers Bureau. Qazilbash:Celgene: Speakers Bureau. Shea:Millenium: Consultancy, Research Funding.

Abstract 680 Cytogenetics play an essential role in determining the prognosis of patients with AML. However, there is still no validated cytogenetics grouping scheme that specifically applies to patients undergoing allogeneic stem cell transplantation, which hampers accurate prognostication and risk stratification. We studied 821 adult patients (median age 41, range 18–74) who underwent SCT between 1999 and 2004 for AML (excluding APL) in CR1 or CR2 and who were reported to the CIBMTR from centers with >20 patients meeting study criteria. 75% of patients received a myeloablative conditioning. 496 patients had a normal karyotype. The primary cytogenetics reports were manually reviewed for 92% of the patients with an abnormal karyotype. We compared the ability of the 6 existing grouping schemes (MRC, CALGB, EORTC/GIMEMA, SWOG/ECOG, DFCI, and Monosomal Karyotype (MK) classifications) to stratify patients, using both the Akaike Information Criterion in multivariable models and the C-statistic. Among all existing schemes, the DFCI system provided a marginally superior stratification for overall and leukemia-free survival. We also built a new classification using individual cytogenetic abnormalities in a Cox model that included other significant covariates (performance status, therapy-related disease, conditioning intensity, graft source, donor match, duration of CR1, and gender match). This CIBMTR scheme (see Table), which is similar to the DFCI scheme except for the inclusion of patients with t(8;21) in the intermediate group, could stratify patients into 3 groups with similar treatment-related mortality but significantly different overall survival, leukemia-free survival, and incidence of relapse. This scheme appeared to apply to both patients in CR1 and in CR2 (see FiguresF2). This transplant-specific scheme could be adopted for prognostication purposes and to stratify patients with karyotypic abnormalities entering transplantation clinical trials. Future studies may clarify the relative outcome of patients with t(8;21) and refine this scheme with the inclusion of molecular abnormalities. Disclosures: No relevant conflicts of interest to declare.

Few data are available on the outcomes of patients who fail to engraft following URD transplantation. We describe the results of 122 patients reported to the National Marrow Donor Program (NMDP) between 1990 and 2005, who received a second bone marrow (BM) or peripheral blood (PB) URD transplant after failing to achieve an absolute neutrophil count of ≥ 500/mm3 without evidence of recurrent disease. Patients were transplanted for leukemia (n=83), myelodysplastic disorders (n=16), severe aplastic anemia (n=20) and other disease (n=3). The median age at first transplant was 29 years. Approximately one third were in each age range, < 20 (33%), 20–40 (37%) and >40 (29%). The initial transplant was BM in 84% of patients while 51% received BM for the subsequent transplant. One hundred ten of these patients received two transplants, 11 received three and one received 4 transplants. Twenty four used a different donor whereas 98 used the original donor for the second transplant; 28 of these used cells that had been cryopreserved at the initial transplant date. Nearly two thirds of the patients received reduced intensity conditioning or no conditioning for the second transplant. Sixty one percent had ATG as a component of their second transplant. The median interval from first transplant to second transplant was 48 (range, 18–126) days. The median nucleated cell dose was 2 × 108/kg (range, Outcome N eval Probability (95% CI) *cumulative incidence Neutrophil engraftment* 122 @ 28 days 43 (34–52) % @100 days 48 (39–57) % 30 day mortality 122 39 (31–48) % 100 day mortality 122 75 (67–82) % Overall survival (OS) @ 1 year 122 11 (6–17) % Treatment related mortality (TRM) @ 1 year* 99 86 (79–92) % Disease free survival (DFS) @ 1 year 99 7 (2–12) % Of the 122 patients analyzed, only 10 remain alive. The most common causes of death were graft failure or rejection (33%), organ failure/interstitial pnuemoniatis (22%), infection (15%), GVHD (14%) and recurrent disease (6%). Of the 37 patients who had persistent graft failure 21 (57%) died from infectious causes. We observed no differences between patients who received a graft from the same donor vs. different donor, fresh vs. cryopreserved cells, or small initial graft size vs. large initial graft size. Patients who received a T-cell depleted graft at the time of their first transplant had significantly worse survival at 1 year after their second transplant, 4% (95% CI, 0–11) compared to those patients who had a non T-cell depleted initial graft, 16% (95 % CI, 8–25)(P = 0.02). It appears that a second salvage transplant strategy may be effective in a small group of patients, although the overall DFS at 1 year of only 7% is disappointing. To improve these results, efforts might focus on early identification and treatment of those at highest risk for non engraftment to determine whether such a strategy can alter the natural history we have described.

Background: The hypomethylating agent azacitidine (azaC) which can reverse epigenetic silencing, is the first agent demonstrated to alter the natural history of MDS and improve survival in higher-risk patients (Silverman JCO 2002, Fenaux Blood 2007). AzaC also produces comparable rates of response in patients with non-proliferative AML and appears to affect survival (Silverman JCO 2006). Time to response is slow with single agent azaC, requiring a median of 3 to 4 cycles to initial response and the CR + PR rates ranges from 7 to 27%. Vorinostat, a histone deacetylase inhibitor (HDACI) which inhibits class I and II HDAC, has demonstrated single agent activity in patients with MDS and AML with responses of 25% (Garcia-Manero Blood 2006). In vitro the 2 agents are synergistic in reactivating epigenetically silenced genes. The effect is sequence dependent requiring exposure to the hypomethylating agent first followed by the HDACI. This study was designed to test the safety of the combination and to determine the response rate and effects on the MDS/AML clone. Methods: In the phase I component eligible patients were entered into one of eight cohorts with the combination of vorinostat and azaC in a 3+3 dose escalating de-escalating design (see table). Results: As of the data cut for this submission 21 patients are evaluable for toxicity and response. Accrual to all 8 cohorts has been completed, full data will be available for the meeting. Among the 28 patients entered: 20 have MDS and 8 AML with median age 68. Responses among evaluable patients have occurred in 18 of 21 (86%); 9 CR, 2 CRi, (CR+CRi=53%) 7 HI, 2 SD. Median time to response is 2 cycles. Among patients with high risk MDS and AML 10/12 (83%) responded (5CR, 1CRi, 4 HI). Responses including CR occurred, but in 57% of patients the abnormal MDS/AML clone persisted, suggesting a modulating effect of the combination on the clone. A total of 171 cycles have been administered, range 1 to 17 with a mean 5 cycles. Eight patients have come off study for progression (1); relapse (2); co-morbidities (2); or consent withdrawal (3). No grade 3 or 4 non-hematologic toxicities in cycle 1 were observed. Grade 2 fatigue occurred during cycle 1 in 89% of patients in cohorts 2, 3 and 4. Grade 2 anorexia and fatigue occurred in cycle 1 in 31.6% and 57.9% of patients, respectively. The fatigue correlates with the scheduled duration of vorinostat administration with 14 days vorinostat (cohorts 1–4) associated with grade 2 and 7 days (cohorts 5–7) producing grade 1. Some suggestion of cumlative fatigue (grade 3) occurred in cycles 2 and beyond in cohorts 1, 3 and 4 with 14 days of vorinostat. However it did not result in discontinuation of therapy. Correlative biologic studies are underway. Conclusion: The combination of azaC and vorinostat can be safely combined, is well tolerated in repetitive cycles and is active in both lower and higher risk/AML patients with an OR and CR rate superior to azaC alone. AzaC at a dose of 55 mg/m2 appears to be optimal for combination. The phase II study will utilize an optimization design to further identify the optimal biologic/epigenetic effects among the three vorinostat schedules of 3, 7 or 14 days when combined. Cohort No of Pts AzaC dose mg/m2/d 1–7SQ Vorinostat dose Mg/d Total dose AzaC/Vorinostat Adverse Events anorexia/fatigue Response 2 patients withdrew consent before treatmen and were replaced in cohorts. 1 3 55 200 bid × 14d 385/5600 1;0;0/2;1;2 CR;CRi;CR 2 3 55 200 tid × 14d 385/8400 2;2;2/2;2;2 CR;HI;CR 3 3 75 200 tid × 14d 525/8400 1;0;1/2;0;2 NR;CR;CRi 4 3 75 200 bid × 14d 525/5600 2;1;2/2;2;2 IE;CR;HI 5 4 75 300 bid × 7d 525/4200 1;2;1/2;1;1 IE;SD;HI;SD 6 3 55 300 bid × 7d 385/4200 0;0;0/0;0;0 CR;HI;CR 7 5 55 200 bid ×7d 385/2800 0;2;2/0;1;1 IE;IE;CR;HI;HI 8 4 55 300 bid × 3d 385/1800 NA IE;HI;TE;TE

Impact of donor characteristics is well described for standard intensity unrelated donor and matched sibling donor transplants but may differ in recipients of unrelated donor RIC transplants. Less immunosuppressive regimens at transplantation may lead to higher graft failure rates. We examined risk factors affecting graft failure, acute and chronic graft-versus-host disease (GVHD) and survival after RIC unrelated donor transplants in 715 patients with acute (n=394) and chronic leukemia (n=74), myelodysplastic syndrome (n=70) and non-Hodgkin lymphoma (n=177). Graft failure was defined as 95% donor chimerism. 63 patients had

Reduced intensity allogeneic stem cell transplantation has been used increasingly to treat patients with both aggressive and indolent diseases such as low grade NHL and CLL. Most reports have consisted of single institution or registry data. We report here a prospective Cooperative Group study by CALGB in which 45 patients were treated at 11 CALGB institutions. Methods: Patients < 70 years of age with a matched sibling donor and recurrent or refractory low grade NHL or CLL, high-risk CLL in first remission, mantle cell lymphoma or prolymphocytic leukemia were eligible. Patients were required to have CrCl > 40, DLCO > 40%, RNEF > 30% and LFTs < 3 × ULN. Disease must have been stable or responsive to the most recent previous therapy. The preparative regimen included 30 mg/m2 fludarabine × 5, and 1 gm/m2 cyclophosphamide × 3. The graft was G-CSF mobilized PBSC, and tacrolimus and low-dose MTX (5 mg/m2) on days 1,3,and 6 was used for GVHD prevention. Tacrolimus was tapered from day +90 to +150 in the absence of GVHD. Standard infectious disease prophylaxis, supportive care, and G-CSF from day +5 to engraftment were employed. DLI was given for stable or progressive disease, not loss of chimerism alone. Results: 47 pts were enrolled with 44 eligible and evaluable; median age 53; 23 NHL, 14 CLL, 2 PLL, 4 mantle cell and one unknown. 27% developed ≥ grade 2 acute GVHD and 47% (18% extensive) chronic GVHD. TRM at 6 months was 2% and was 7% overall. Mortality at 2.6 year f/u is 25% due to GVH, sepsis, or 2nd malignancy (9%) and progressive disease (7 pts,16%). Estimated 2 yr DFS is 71% (CI 54–82) and OS is 76% (CI 60–87). Grade 4 AEs included 4 pneumonia/ pneumonitis, 1 PE, 1 VOD and 1 second malignancy (pancreatic CA). Documented infectious events included 32% viral, 41% bacterial, and 20% fugal. For T cell (CD3), the median chimerism at day 180 was 98% and 99% in peripheral blood and bone marrow; > 90% chimerism was present in 64% and 66% respectively. For unfractionated, the median chimerism at day 180 was 99% and 98% in peripheral blood and bone marrow; > 90% chimerism was present in 65% and 70% respectively. Conclusion: These encouraging results indicate that a high CR and DFS rate can be obtained with low TRM without use of anti-CD20 therapy in a Cooperative Group trial of matched related allogeneic transplant in patients with low grade NHL or CLL. Appropriate patients should be considered for such therapy when standard treatments are no longer capable of achieving prolonged remissions. Overall and Disease-Free Survival Overall and Disease-Free Survival

Inherited deficiency of purine nucleoside phosphorylase (PNP) is associated with a profound loss of T-cells. This rare clinical condition provides a model for developing specific inhibitors of PNP as therapy for T-cell malignancies. Forodesine is a specific PNP inhibitor that raises plasma 2′-deoxyguanosine (dGuo) and intracellular dGTP levels, leading to T-cell apoptosis. The objective of this study was to determine the efficacy and safety of forodesine in patients with relapsed or refractory T-cell leukemia. Patients received IV forodesine 40 mg/m2 5 days each week (1 cycle) for a total of 6 cycles (8 cycles if dose escalation needed after cycle 2). To date, 21 patients have been treated, 19 of which are T-ALL. Results are based on available data from 13 T-ALL patients. Median number of prior regimens was 4 (range: 1–6). Overall response rate was 38%, including 3 patients with complete response (CR) (23%) and 2 patients with partial response (PR) (15%). Three patients had stable disease and 5 patients had disease progression. Time to progression (TTP) for the 3 CR patients was 80, 119, and 160+ days. One CR patient continues to receive treatment. TTP for the 2 PR patients was 89 and 40 days. Plasma dGuo was elevated in all patients (range: 3.4 to 88.5 μM; predose levels ≤0.004 μM). Treatment was well tolerated. One serious adverse event possibly drug related was CMV pneumonitis in 1 patient. The most common adverse events possibly drug related were nausea (33%), headache (26%), anemia (20%), thrombocytopenia (20%), and weakness (20%). In 1 patient, after 2 doses of forodesine, WBC count dropped dramatically, requiring interruption of treatment for 2 weeks because of tumor lysis syndrome (Figure). After 2 cycles of treatment, the patient achieved a CR. By week 3, platelets and neutrophils recovered, indicating a specificity of forodesine for leukemic cell populations. These study results show that forodesine is active, with minimal toxicity, as a single agent in relapsed or refractory T-cell leukemia. Additional clinical efficacy and safety data will be presented. Effect of Forodesine on Blood Components in One T-All Patient Effect of Forodesine on Blood Components in One T-All Patient

Valproic acid (VPA), acetylates histones, induces apoptosis and synergizes with All Trans Retinoic Acid (ATRA), in promoting G1 arrest and leukemic cell differentiation in vitro. Based on these observations we designed a Phase I/II pilot study of VPA alone and in combination with ATRA in patients with relapsed, refractory or poor risk acute myeloid leukemia (AML). VPA was initiated at 15mg/kg/day (day 1 IV followed by daily po) and escalated weekly by 10mg/kg (max 60 mg/kg/day) targeted to reach a serum concentration of 80–120mg/ml. For the combination, ATRA was to be added initially at 25mg/M2/day (first 3 patients) and titrated, in the absence of toxicity, to 45mg/M2/day, in two divided doses, following a minimum of 7 days of VPA therapy alone. Treatment was planned to continue for 45 days following achievement of a VPA level of >80mg/ml on two separate, consecutive measurements. To date we have treated 8 patients (7 tAML/MDS, 1 de novo AML) with VPA alone. Two of 8 additional patients to be treated with VPA + ATRA (1 tAML/ET; 1 tAML/MDS), are too early in their treatment upon which to report. Of the VPA alone treated patients, Median age was 63 (range 52–78). No Prior therapy: 3/8 pts; 5F:3M; ECOG PS 0 (1), 1 (5), 2 (2);Cytogenetic abnormalities: normal (1); t(3;18) (1); complex karyotype (6), including trisomy 8 (3), abnormalities of 11 (2) or monosomy 7 (2). Baseline bone marrow blast percentage > 50%; (8); median bone marrow cellularity 84% (range 35–90). Treatment: Median duration of treatment 45 days (range 19–99). Therapy discontinued before 45 days; withdrawl (2); death at home on study (day 33 of treatment), (1); reversible grade 3 toxicity(2). VPA levels: Serum concentrations of VPA of > 80mg/ml was achieved in all patients; Hematologic effects: Changes in CBC: increase in WBC, ANC and blast count (BLC)(3), followed by decrease in WBC and BLC (2); increase in WBC/ANC &decrease in BLC (1); decrease in WBC, ANC and BLC (3); &initial decrease in BLC &increase in ANC (1). Marrow: decrease in marrow cellularity (2)( 90% to 30%); loss of detectable trisomy 8 population (1); &decrease in bone marrow blasts (1) (64% to 5%) was observed. One patient reverted back to MDS, lasting 5 months. Side Effects: somnolence (grade 2–4) was observed in 4/8 patients, and was the major cause of withholding treatment. We also observed a constellation of symptoms (weight gain, fever, elevated WBC, shortness of breath, elevated LDH, bone pain) and chest x-ray findings consistent with &reminiscent of APL syndrome, within seven days of achieving the targeted serum VPA levels (> 80mg/ml), in 2 pts. Decadron was started two days (1) and immediately (1) following the onset of symptoms: signs and symptoms abated quickly &resolved subsequently within three days. In retrospect, one additional patient had comparable symptoms, for which VPA was stopped, following admission to an outside hospital for fever, pulmonary infiltrates and altered mental status. Correlative studies: serial assessment of bulk histone acetylation (H3, H4) was performed in all 8 patients. 7/8 patients showed evidence of histone hyperacetylation in mononuclear cells from peripheral blood, during treatment with VPA. Additional patients are being treated with VPA + ATRA. Analysis of these studies will be forthcoming.

The addition of rituximab to mobilization regimens does not adversely affect collection of adequate numbers of CD34+ PBSC and may decrease tumor cell contamination. Prior administration of rituximab may sensitize lymphoma cells to chemotherapy, but its use with high dose chemotherapy has not been studied. In this study, eligible patients had intermediate grade NHL either primary refractory, relapsed or in CR1 with an intermediate/high risk IPI score. Eight patients with a median age of 49 years (28–61) were enrolled in this study. Four patients had relapsed disease, chemosensitive to salvage in 3 patients, and untested in 1 patient. Two patients had primary refractory disease, chemosensitive to salvage. Two patients had intermediate-high risk IPI scores and were in CR-1. One patient in CR-1 declined to undergo transplant following stem cell collection. All patients had received rituximab previously. Stem cell mobilization was rituximab 375mg/M2 followed by CY 2gm/M2/day for 2 doses and filgrastim 10ug/kg/d. High dose chemotherapy consisted of rituximab 375mg/M2 on day -8, carmustine 300mg/M2 on day -7, etoposide 200mg/M2/d on days -6 to -3, cytosine arabinoside 200mg/M2 on days -6 to -3 (8 doses) and melphalan 140mg/M2 on day -2. Patients received filgrastim post transplant. RESULTS: The median CD34+ cell dose collected was 12.5 x 106/kg (1.1 to 33.2). There was one inadequate CD34+ cell collection requiring bone marrow harvesting which yielded 0.72 x 106/kg CD34+ cells. Engraftment of neutrophils and platelets occurred at a median of 8 and 11 days post transplant respectively. Infectious complications included febrile neutropenia in one patient following mobilization and in 6 patients following R-BEAM. Transient grade I–II (common toxicity criteria) hepatic enzyme elevation occurred in 2 patients following R-CY. Following R-BEAM, 3 patients developed grade II-IV LFT elevation without evidence of VOD, which resolved. One patient developed moderate hemorrhagic cystitis which resolved. One patient developed bilateral pulmonary infiltrates of unkown etiology (BAL negative for organisms) which resolved. Immunoglobulin levels remained normal in two patients, but decreased in all other patients for up to a year post transplant. Peripheral blood flow cytometry and T cell subset analysis showed absent B cell populations, decreased CD4 cells and increased CD 8 cells in all patients up to a year post transplant. Five patients are alive with no evidence of disease 381 to 676 days post transplant. Three of these patients developed PET positive lymph nodes that showed florid follicular hyperplasia on biopsy, with no evidence of lymphoma. Two patients had persistent disease following transplant. One patient underwent a non-myeloablative allogeneic transplant and died of progressive disease. CONCLUSIONS: R-CY was well tolerated as a mobilization regimen and led to adequate stem cell collection. The addition of rituximab to the high dose chemotherapy regimen did not appear to increase the incidence of infections or peritransplant complications in this Phase I study. The effect, on disease free survival, of the addition of rituximab to the collection and preparative regimens requires further investigation.

Langerhans cells (LCs) are bone marrow (BM) derived epidermal dendritic cells that represent a critical immunological barrier to the external environment, but little is known about their life cycle. We have recently discovered that in mice, LCs are maintained by local radio-resistant LC precursors that self-renew in quiescent skin throughout life but are replaced by circulating precursors upon inflammation (Merad et al. 2002). We have also shown that host radio-resistant LCs that persist after allogeneic HSCT play a major role in initiating skin GVHD (Merad et al. 2004). To explore the turnover of human LCs, we first asked whether human LCs are maintained by local LC precursors in quiescent skin of patients that have undergone HSCT. We assessed LC turnover from skin biopsies of recipients of sex-mismatched grafts and used X and Y chromosomes as a marker of host or donor origin. Patients skin samples were subjected to fluorescence in situ hybridization using X and Y specific DNA probes followed by immunofluorescence staining of Langerin (a LC specific marker) and nuclei staining. X and Y chromosomes were simultaneously visualized within the nuclei of Langerin-positive cells with high-resolution four-color microscopy. In a preliminary study, we found that in 7 patients with histological signs of skin GVHD, 98+3.4% of LCs were of donor origin. In contrast, in 2 patients that had no histological signs of GVHD, 100% LCs remained of host origin at 9 months and 4 years after myeloablative conditioning and HSCT despite 100% BM chimerism. These data suggest that in the absence of skin inflammation host LCs can persist in the skin for a long time despite complete donor hematopoietic engraftment. Next, we hypothesized that host LCs can persist in the skin because they are radio-resistant and able to proliferate in situ. Indeed, cell cycle analysis of purified LCs and co-staining of skin sections with Ki-67 and Langerin antibodies revealed that 2% of epidermal LCs present in quiescent human skin proliferate in situ, a percentage similar to that found in mice. To follow the behavior of human LCs experimentally, we reconstituted γ-irradiated Rag2 −/−γc−/− newborns mice with human CD34+ cord blood cells to engraft human hemato-lymphopoiesis. Engrafted animals developed functional human B cells, T cells and dendritic cells in the blood and spleen together with structured primary and secondary lymphoid organs. Consistent with our findings in patients, no human LCs were detected in the skin in steady state conditions, despite high levels of human hematopoietic cell engraftment. These results suggest that human LCs are maintained by a local pool of radio-resistant LC precursors that self-renew in situ. Since local LC precursors are eliminated during severe skin injuries, this xeno-transplantation model will be useful to dissect the homing molecules that govern the recruitment of LCs from the blood to the skin and may help develop new therapeutic strategies for the prevention and treatment of clinical GVHD.

Acute GvHD is a frequent complication of non-myeloablative transplantation although it is seen at a lower rate and severity than after myeloablative transplantation. The use of ATG in transplant conditioning produces substantial host immunosuppression. Furthermore, the persistence of ATG in the circulation after transplantation is thought to accomplish an in-vivo T-cell depletion of the stem cell inoculum. Non-myeloablative stem cell transplantation using low dose TBI (200cGy) and fludarabine results in a high rate of full donor chimerism. We previously showed that the addition of ATG to TBI 200 cGy and fludarabine can improve donor engraftment. Whether it also impacts on acute and chronic GvHD and on graft versus tumor effect is less known. Forty-seven patients, not eligible for conventional fully ablative allogeneic SCT by virtue of age or co-morbidities, underwent non-myeloablative allogeneic transplantation using ATG 15 mg/kg/day (equine) or 1.5mg/kg/day (rabbit) days −4 to −1, TBI 200 cGy on a single fraction on day −5, fludarabine 30 mg/kg/day on days −4 to −2. Oral immunosuppression with MMF and CSA were started on day −5. Allografts were unmanipulated peripheral blood progenitor cells collected on day 5 of mobilization with filgrastim 10 mcg/kg/day. Thirty-two patients (68%) had match related donors, 9 (19%) match unrelated (10/10), 1(2%) mismatch related (9/10) and 5 (10.6%) mismatch unrelated donors (9/10). Twenty-five patients had sex matched donors (53.2%). Full donor chimerism was documented in 85% and partial (>50%) in 11% of patients. At a median follow up of 309 days, graft versus host disease grade II–IV developed in 13 patients (27.6%). Fatal GvHD of liver and/or gut occurred in 8 patients (17%). The mean age of patients who developed GvHD was 54 years and 57 for those who did not (p=ns). The corresponding mean ages for their donors were 47 vs. 50 years (p=ns). Seven out of fourteen (50%) unrelated transplants recipients and 6/33 (18%) with related donors develop GVHD (p=0.023). Infectious complications were seen in 8 patients (17%) and were the cause of death in 6 (12%). Thirteen of the 47 patients (27.6%) underwent transplant for AML. Six patients had relapsed/refractory disease at the time of transplant, 3 were in CR2 and 4 in CR1. Eleven patients (84.6%) relapsed with AML after transplant. Previously reported data on non-myeloablative stem cell transplant using 200 cGy of TBI and fludarabine with no ATG showed an incidence of acute GvHD grade II–IV of 64% and 24% GvHD related mortality. Similar rates have been reported with fludarabine and melphalan reduced-intensity conditioning. In our cohort of 47 patients the addition of ATG resulted in decreased incidence of acute GvHD grade II–IV and GvHD related mortality without a high rate of infectious complications. A high incidence of disease relapse in patients with AML may reflect the high proportion of patients with advanced leukemia enrolled in the study. However, it also warrants further investigation to rule out that ATG may hinder graft versus leukemia effect.