ALBERT

Description: The optimal source of donor hematopoietic stem cells (HSC) is controversial. Granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood (G-PB) has replaced bone marrow (BM) as the most common allograft source in adults but is associated with donor morbidity and higher rates of chronic graft versus host disease (GVHD) compared to BM. The CXCR4 antagonist plerixafor (Px) mobilizes HSC into the PB (Px-PB) faster than G-CSF and preliminary data suggest both quantitative and qualitative differences in allograft content that may impact clinical outcomes. We sought to assess the efficacy and safety of transplanted allografts collected following mobilization with Px alone in HLA-identical sibling transplantation. This was a Phase II, two-strata, multi-center prospective trial (NCT01696461) to evaluate Px-PB allografts prior to reduced intensity conditioning (RIC) and myeloablative conditioning (MAC) based hematopoietic cell transplantation (HCT). Patients aged 18-65 years with an HLA-ID sibling donor and a hematological malignancy suitable for HCT were eligible. The primary objective was to determine the proportion of donors whose cells could be successfully mobilized and collected with a sufficient CD34+ cell dose using Px as the sole mobilizing agent. Px mobilization was considered successful if ≥ 2.0x10^6 CD34+ cells/kg recipient weight were collected in no more than two leukapheresis (LP) collections. All donors receiving Px were included in the analysis of the primary objective based on the intention-to-treat principle. Secondary objectives included the incidence of acute and chronic adverse events in donors, rates of hematopoietic engraftment, donor chimerism, rates of acute and chronic GVHD, non-relapse mortality (NRM), progression free survival (PFS) and overall survival (OS) for the recipients. From July 2013 to December 2014, 64 donor/recipient pairs were enrolled at 12 centers. Donors received Px at 240μg/kg subcutaneously 4 hours prior to LP. LP was performed processing at least 4X blood volume for up to two consecutive days (a third day was allowed for low CD34+ cell yields after 2 LP procedures) to achieve a target CD34+ cell dose of ≥ 4.0 x 10^6/kg recipient weight with a minimum goal of ≥ 2.0 x 10^6/kg. All allografts were cryopreserved. GVHD prophylaxis included cyclosporine or tacrolimus in combination with methotrexate, mycophenolate mofetil, or sirolimus. G-CSF was given routinely post HCT only to MAC recipients. Patient demographics are provided in Table 1. The median donor age was 56 years (18-65). 64% of the donors were male. Donors underwent one (23%), two (72%), or three (5%) LP procedures. 63 of 64 (98%) donors achieved the primary objective. The median total CD34+ cell dose/kg recipient weight collected within 2 days was 4.6 (0.9-9.6). Maximal donor toxicity following Px injection and LP was grades 0 (30%), 1 (52%), 2 (17%), and 3 (2%). Bloating, flatulence, abdominal pain, headache, paresthesisas, injection site reaction, and dizziness were the most commonly observed toxicities. Bone pain was not observed. The one grade 3 toxicity was a vasovagal episode felt related to LP and unlikely to Px. Toxicities typically resolved within a week of LP. The median follow up is 6.3 months. Median days to ANC (〉0.5 x10^9/L) and Platelet count (〉20 x 10^9/L) recovery were 13.5 (10-148) and 19 (1-76) after MAC and 14.5 (0-25) and 18 (0-141) after RIC, respectively. The cumulative incidence of acute GVHD grades 2-4 and 3-4 at day 100 were 47% (95% CI: 30-64) and 9% (95% CI: 2-22) after MAC and 19% (95% CI: 6-38) and 5% (95% CI: 0-18) after RIC. Probability of NRM at day 100 was 4% (95% CI: 0-13) and 0% after MAC and RIC, respectively. The probability of OS at day 100 was 97% (95% CI: 88-100) and 90% (95% CI: 78-98) after MAC and RIC, respectively. In conclusion, this is the first multi-center trial to demonstrate that as an alternative to G-CSF, Plerixafor rapidly, safely, and effectively mobilizes sufficient numbers of CD34+ cells from HLA-ID sibling donors for HCT following both RIC and MAC regimens. Engraftment was generally prompt and early results of secondary endpoints in recipients are encouraging. Longer follow-up and more extensive analysis of donor allografts and recipient outcomes will be presented at the time of the meeting. Research support was provided in part by Genzyme, a Sanofi Company. Table 1. Characteristics of recipients Table 1. Characteristics of recipients Disclosures Chen: Bayer: Consultancy, Research Funding. Devine:Genzyme: Research Funding.

Description: Background A critical barrier to progress in allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been a lack in understanding regarding why some transplant recipients of HLA-matched transplant grafts develop severe graft-versus-host disease (GvHD) while other recipients have relapse of their cancer without GvHD. Patients who develop a modest degree of acute and/or chronic GvHD have less relapse and optimal survival after allogeneic BMT. Thus, a mechanistic understanding of regulation of donor T-cell activation after allo-HSCT is needed. Using mouse models, Desmarets et al. have shown that pre-transplant leukoreduced RBC transfusions can cause recipient immunization against minor histocompatibility antigens (miHA) and activation and expansion of recipient T-cells that recognize donor miHA, contributing to rejection of subsequent allo-HSCT (Blood. 2009; 114:2315). Preliminary data from our lab suggest that leukoreduced RBC transfusions given concurrently with allo-HSCT can also increase post-transplant activation and expansion of donor T-cells, an effect which may lead to increased GvHD after transplant. Here, we have conducted a retrospective study of post-transplant RBC transfusions and acute GvHD (aGvHD) in allo-HSCT patients. We hypothesized that increased numbers of transfusions during the 30-day post-transplant period would be correlated with increased severity of aGvHD in these patients. Methods We conducted a retrospective analysis of RBC transfusion records and aGvHD data collected for 181 adult allo-HSCT patients who received their transplants at Emory University Hospital (EUH) between 2004 and 2009. Nine patients were excluded who died 〈 50 days post-transplant without developing aGvHD, since this was too early to determine aGvHD occurrence. Of the remaining 172 patients studied (median age 48 yrs at time of transplant, range 18-72), 88 (49%) were male and 84 (51%) were female. Patients had received either matched related HSCT (n=69, 40%) or matched unrelated HSCT (n=103, 60%) for treatment of SAA (n=7), BAL (n=2), ALL (n=18), AML (n=69), hemolytic anemia (n=2), CLL (n=6), CML (n=8), HD (n=5), MDS (n=23), myelofibrosis (n=6), MM (n=7) or NHL (n=19). For pts who developed aGvHD, the onset time ranged from 1 to 139 days post-transplant, with a median of 30 days. No aGvHD (grade 0) was diagnosed in 58 pts (34%), while 37 pts (21%) developed grade 1 aGvHD and 77 pts (45%) developed grade 2-4 aGvHD. The number of ABO matched, irradiated RBC units transfused 0 - 30 days post-transplant was tallied for each patient, ranging from 0 (no transfusions, n=13, 7.6% of pts) to 26 units, with an average of 5.6 and median of 4 units. All transfusions during this timeframe were administered at EUH. The median follow up time was 22 months post-transplant (range, 1.1 – 96.1 months). Results Pts were assigned to two groups, those who developed grade 0-1 aGvHD (n=95, 55%) or grade 2-4 aGvHD (n=77, 45%) within 140 days post-transplant. This study did not include analysis of late-onset aGvHD or chronic GvHD past this time point. Patients with grade 2-4 aGvHD had a higher average number of transfusions 0 - 30 days post-transplant compared with patients having grade 0-1 aGvHD (6.5 vs. 4.9 units, p = 0.02). Receiver-operator characteristics (ROC) analysis showed that a cutoff value of 〉 4 transfusions 0 - 30 days post-transplant had 56% sensitivity and 65% specificity to predict development of grade 2-4 aGvHD. When tested by logistic regression in a multivariate model, this cutoff value had a highly significant correlation with grade 2-4 aGvHD, with an odds ratio of 2.83 and p value = 0.0024. Other covariates including patient age, gender, and type of transplant (related vs. unrelated) were not significantly associated with aGvHD outcome. Conclusion Our retrospective analysis identified a significant positive correlation between the number of post-transplant RBC transfusions and severity of aGvHD after allo-HSCT. Additional studies are planned to determine whether RBC transfusions 0 - 30 days post-transplant stimulate allo-reactive T-cells via allo-antigen presentation or by otherwise promoting inflammation, and if one or both of these mechanisms contribute to increased GvHD. If so, it may be possible to develop strategies for optimization of RBC transfusion practices to reduce the risk of severe aGvHD after allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

Description: Background Graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplant (allo-HSCT) is caused by CD4+ and CD8+ donor T cells directed against mismatched recipient antigens, presented in the context of donor MHC-II (indirect pathway) and recipient MHC-I (direct pathway). Recently, the presence of 'cross-dressed' CD11c+ antigen presenting cells (APCs) expressing both donor and recipient type MHC-I molecules has been demonstrated in animal organ and HSCT transplant models supporting 'semi-direct' pathway of allo-activation (Wang et al, Blood. 2011).These APCs can efficiently present allo-antigens to both CD4+ and CD8+ T cells and activate immune responses that could lead to allograft rejection or GVHD. Exchange of membrane fragments and associated proteins between cells, termed trogocytosis, generates cross-dressed APCs.We sought to test whether cross-dressed APCs facilitate antigen presentation to donor T cells and initiate GVHD following allo-HSCT. Further, we tested an array of drugs as inhibitors of trogocytosis, to interrupt the semi-direct pathway of allo-antigen presentation. Methods In vivo experiments used a B6(H2Kb) ˆ B10.BR(H2Kk) murine transplant model. Spleens of transplanted mice were analyzed on days 10, 15, 20 post-transplant for presence of cross dressed CD11c+cells, and their expression of CD80, CD86 and MHC-II by flow cytometry. Cross dressed donor CD11c+ FACS sorted cells from recipient spleens were co-cultured with CFSE labeled donor type T-cells for 6 days, and T-cell proliferation was measured as dilution of CFSE by flow cytometry. In vitro experiments used primary MLR consisting of CFSE labeled B6 bone marrow cells co-cultured with PKH26 (membrane dye) labeled B10.BR splenocytes. B6 antigen presenting cells were analyzed by flow cytometry for the presence of CFSE+PKH26+ double positive cells generated by trogocytosis. Pharmacological inhibitors of cytoskeleton function were added to the primary MLR and their effect on trogocytosis as well as T cell proliferation was assessed. Results Cross-dressed donor CD11c+ APCs were generated in vivo following allo-HSCT (Figure 1). Recipient spleens showed that 50%, 28.6% (p=0.01) and 12% (p=0.02) of donor type CD11c+ cells were cross dressed on days 10, 15 and 20 respectively post transplant (n=5). These cross dressed APCs expressed higher levels of co-stimulatory molecules CD80 (p

Description: Abstract 144 In MHC-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT), host antigen specific donor T cells mediate acute and chronic graft-versus-host disease (GvHD). Based upon the radio-protective effects of flagellin, a TLR5 agonist protein (∼50 kDa) extracted from bacterial flagella, we reasoned that flagellin might modulate donor T cells immune responses toward host antigens, reduce GvHD, and improve immune responses to CMV infection in experimental models of allogeneic HSCT. Two 50mg/mouse i.p doses of highly purified flagellin were administered 3 hrs before irradiation and 24 hrs after allo-HSCT in H-2b ^ CB6F1 and H-2k ^ B6 models. GvHD scores were obtained with weekly clinical examination and with histological scoring of intestine, colon, liver and skin at necropsy. Flagellin treatment successfully protected allo-HSCT recipients from acute and chronic GvHDs after transplantation of 5×106 splenocytes and 5×106 T cell depleted (TCD) BM, and significantly increased survival compared to PBS-treated control recipients. Reduced acute GvHD was associated with significant reduction of a) early post-transplant proliferation of donor CD4+ and CD8+ T cells measured by Ki67 and CFSE staining, b) fewer CD62L+, CD69+, CD25+, ICOS-1+ and PD-1+ donor CD4+ and CD8+ T cells compared with the PBS-treated control recipients. Decreased numbers of activated and proliferating donor T cells were associated with significantly reduced pro-inflammatory serum IFN-g, TNF-a, and IL-6 on days 4–10 post transplant in flagellin-treated recipients compared with the PBS-treated recipients. Interestingly, both flagellin-treated recipients and PBS-treated recipients had over 99% donor T cell chimerism at 2 months post transplant. Moreover, MCMV infection on 100+ days post-transplant flagellin-treated mice significantly enhanced anti-viral immunity, including more donor MCMV-peptide-tetramer+ CD8+ T cells in the blood (p

Description: Targeted therapy is increasingly incorporated into the chemotherapy regimens of adult ALL. While the prognosis of BCR/ABL + (Philadelphia) ALL is well recognized; the significance of CD 20 postivity is unknown. We report the characteristics of 76 patients treated at Emory University between January 1999 and March 2005, divided into 2 groups based on CD 20 expression, as determined by flow cytometry. The CD 20+ patients presented with a higher WBC count and LDH, and lower platelet counts (table). In the CD 20+ group, 65% had pre B cell ALL and 8% had high grade B cell leukemia/lymphoma, whereas the CD 20- group consisted of 30% pre B cell ALL, 24% T cell ALL, and 10% had high grade leukemia/lymphoma. Of the 35 Ph+ ALL patients, 27 were CD20+ (20%) and 18 CD20- (51%). Patients were treated on clinical trials L20, ALL2, and more recently with the Hyper-CVAD regimen (n=32). Only 5 of the CD 20+ patients received rituximab in addition to chemotherapy. With a median follow-up of 320 days, 46% of patients in each group are alive, 35% and 38% relapsed in the CD 20+ and CD 20- group, respectively. In summary, we found that CD 20+ ALL patients present with higher WBC count and LDH, and a lower platelet count; although, due to our small sample size, these differences were not significant. Further follow up is needed to provide mature results on survival and remission. CD 20+ CD 20- p= Total numbers 26 50 Median Age (yrs) 45.2 (18–74) 44 (19–76) NS Median WBC (103/μl) 29.1 10 0.09 Median Platelet (103/μl) 35 65 0.11 Median LDH (IU) 894 500 0.05 Normal Cytogenetics 13 (50%) 21 (42%) NS Ph+ ALL 7 (26%) 18 (35%) NS CR within 60 days 20 (77%) 27 (54%) NS Primary Refractory 2 (8%) 6 (12%) 0.7 Relapse 9 (35%) 19 (38%) NS Alive at last follow up 12(46%) 23 (46%) NS Expired by d270 8 (36%) 11 (25%) NS

Description: Key Points Mismatches in alleles C*03:03/C*03:04 were most frequent (68.7%) among the transplants with a single allele level mismatch in HLA-C. The 7/8 C*03:03/C*03:04 mismatch group was not significantly different from the 8/8 HLA matched transplants in any transplant outcome.

Description: The intestinal microbiota in allogeneic bone marrow transplant (allo-BMT) recipients modulates graft-versus-host disease (GVHD), a systemic inflammatory state initiated by donor T cells that leads to colitis, a key determinant of GVHD severity. Indole or indole derivatives produced by tryptophan metabolism in the intestinal microbiota limit intestinal inflammation caused by diverse stressors, so we tested their capacity to protect against GVHD in murine major histocompatibility complex–mismatched models of allo-BMT. Indole effects were assessed by colonization of allo-BMT recipient mice with tryptophanase positive or negative strains of Escherichia coli, or, alternatively, by exogenous administration of indole-3-carboxaldehyde (ICA), an indole derivative. Treatment with ICA limited gut epithelial damage, reduced transepithelial bacterial translocation, and decreased inflammatory cytokine production, reducing GVHD pathology and GVHD mortality, but did not compromise donor T-cell-mediated graft-versus-leukemia responses. ICA treatment also led to recipient-strain-specific tolerance of engrafted T cells. Transcriptional profiling and gene ontology analysis indicated that ICA administration upregulated genes associated with the type I interferon (IFN1) response, which has been shown to protect against radiation-induced intestinal damage and reduce subsequent GVHD pathology. Accordingly, protective effects of ICA following radiation exposure were abrogated in mice lacking IFN1 signaling. Taken together, these data indicate that indole metabolites produced by the intestinal microbiota act via type I IFNs to limit intestinal inflammation and damage associated with myeloablative chemotherapy or radiation exposure and acute GVHD, but preserve antitumor responses, and may provide a therapeutic option for BMT patients at risk for GVHD.