ALBERT

Description: Introduction Disease relapse is the leading cause of death in secondary AML (sAML), which evolves from antecedent hematologic disorders like myelodysplastic syndrome (MDS) or myeloproliferative neoplasms (MPNs) or following exposure to chemotherapy. Persistence of therapy-resistant leukemia stem cells (LSC) harboring enhanced survival and self-renewal capacity has been linked to high relapse rates in sAML. Previously, we showed that missplicing of a stem cell regulatory gene, GSK3 b, and splice isoform switching favoring pro-survival BCL2 family isoform expression promoted generation of therapy-resistant LSC (Abrahamsson et al PNAS 2009; Goff et al Cell Stem Cell 2013). However, whether aberrant pre-mRNA splicing promotes sAML LSC generation, in the absence of mutation, and if pharmacological splicing modulation impairs LSC maintenance, in a mutation-independent manner, has not been elucidated. Methods and Results Comparative RNA-sequencing and gene set enrichment analyses revealed significant alterations in splicing factor gene expression in purified progenitors from untreated sAML compared with normal samples. In addition, using an isoform-specific alignment algorithm, we established a sAML LSC splice isoform expression signature that identified increased expression of select transcripts, e.g. CD82 and PTK2B. Thus, we investigated the LSC inhibitory efficacy of a stable, potent splicing modulatory agent, 17S -FD-895, in humanized AML LSC stromal co-culture and primagraft assays. Notably, there was a dose-dependent reduction in AML LSC (n=4) survival and self-renewal after in vitro 17S -FD-895 treatment, with a favorable therapeutic index compared to normal controls (n=3, p

Description: Introduction Human bone marrow aging is typified by decreased cellularity, stem cell exhaustion and myeloid lineage bias that may set the stage for development of myeloid malignancies. Secondary AML (sAML) arises from prior myelodysplastic syndromes (MDS) or myeloproliferative neoplasms (MPN), and occurs in patients with an average age of 〉65. Because of the typically advanced age of this population, patients currently have few effective treatment options available after leukemic transformation. We and others have recently identified a key role for enzymatic RNA editing activity in cancer progression, and in particular in leukemia stem cell (LSC) generation. In hematopoietic stem and progenitor cells, adenosine deaminase acting on dsRNA-1 (ADAR1) is the most abundantly expressed RNA editing gene. However, the role of abnormal RNA editing activity has not been elucidated in healthy human bone marrow aging and age-related MDS with a high risk of transforming to sAML. Therefore, we established whole transcriptome-based RNA editing signatures of benign versus malignant bone marrow progenitor cell aging, which provides novel RNA-based functionally relevant biomarkers of aging, MDS and progression to sAML. Methods Whole transcriptome sequencing (RNA-Seq) was performed on FACS-purified hematopoietic stem (CD34+CD38-Lin- HSC) and progenitor cells (CD34+CD38+Lin- HPC) from aged (average age = 65.9 y/o) versus young (average age = 25.8 y/o) adult healthy bone marrow samples, and in leukemia stem cells (LSC) from patients with sAML (average age = 71.4 y/o) and MDS (average age = 63.8 y/o). Comparative gene set enrichment analyses (GSEA) and RNA editing profiles were identified for normal and malignant progenitor cell aging. Results Aberrant RNA editing activity has recently been shown to be induced in multiple cancers, and has been implicated as a malignant reprogramming factor. Comparative whole transcriptome RNA sequencing (RNA-seq) and single nucleotide variant analyses revealed widespread increases in RNA editing rates in aged versus young HPC, and in human sAML LSC compared with age-matched normal progenitors. Moreover, RNA editing rates, represented as adenosine (A) to inosine/guanosine (G) changes at known RNA editing loci, were increased in sAML compared with MDS progenitors. The differential expression of certain sites is as high as 70%, which can be readily detected by RESS-qPCR. These data suggest that during aging niche-dependent RNA editing deregulation contributes to MDS progression to sAML. Interestingly, the highly edited loci in sAML LSC were distinct from loci that were differentially edited in aged versus young HPC, suggesting that pro-inflammatory conditions in sAML may trigger RNA editing of a unique set of transcripts, including predominantly RNA processing-related gene products and transcription factors. Notably, several loci in transcripts of APOBEC3C/D that we previously found were associated with blast crisis transformation of chronic myeloid leukemia also displayed enhanced editing in sAML LSC, but not aged versus young HPC. Conclusions Detection of aberrant RNA processing provides novel biomarkers as well as potential therapeutic targets for sAML LSC eradication with implications for treatment of a variety of human malignancies and other age-related disorders. We have identified commonly RNA-edited transcripts in multiple hematologic malignancies, which could be developed clinically and as companion diagnostic targets for LSC-targeted therapeutics. Disclosures Jamieson: CTI Biopharma: Research Funding; Johnson & Johnson: Research Funding; GlaxoSmithKline: Research Funding.

Description: Background: Nonmyeloablative conditioning with 2-Gy TBI alone or in combination with fludarabine (FLU/TBI) and HLA-matched related donor peripheral blood allografts followed by cyclosporine (CSP) and mycophenolate mofetil (MMF) for the prophylaxis of graft-versus-host disease (GVHD) is an effective therapy for many hematologic malignancies with reliable engraftment and moderate toxicity. The major causes of non-relapse mortality (NRM) are the development of acute and chronic GVHD. Several studies have demonstrated that tacrolimus may offer advantages compared with CSP for the prevention of GVHD in patients treated with myeloablative conditioning. The combination of tacrolimus and MMF, which has been used for GVHD prophylaxis after myeloablative hematopoietic cell transplantation (HCT), was well tolerated with low toxicity. Pilot data suggested an improved and perhaps superior GVHD prophylaxis with tacrolimus/MMF compared to our extensive historical experience using CSP/MMF with nonmyeloablative HCT. The purpose of this study is to evaluate the incidence of grade III-IV and II-IV acute GVHD, extensive chronic GVHD, along with the rate of NRM, relapse/progression, and overall survival after nonmyeloablative conditioning and post-grafting immunosuppression with tacrolimus and MMF. Methods: In a phase II multicenter clinical trial we evaluated the effect of post grafting immunosuppression with tacrolimus and MMF for the prophylaxis of GVHD following nonmyeloablative conditioning with 2-Gy TBI alone or in combination with 90mg/m2 FLU (FLU/TBI) for patients with hematologic malignancies. Patients at low risk of graft rejection (preceding autologous HCT within 6 months) received TBI alone (n=50) while the remaining patients received FLU/TBI conditioning (n=100). Tacrolimus was administered orally (0.06 mg/kg, Q12 hr) from days -3 to +56 and in the absence of GVHD tapered off by day +180. Tacrolimus was targeted to 15-20 ng/ml for the first 28 days and 10-20 ng/ml subsequently while on full dose. MMF was given orally (15 mg/kg, Q12 hr) from day 0 until day 27. Results: 150 patients were enrolled from 2004 to 2013 and received peripheral blood stem cells (median doses of 8.1×106 CD34+ cells/kg and 3.5×108 CD3+ cells/kg) from HLA-matched related donors. Diagnosis at transplant included AML (n=42), ALL (n=6), CLL (n=2), MDS/MPD (n=12), NHL (n=25), HL (n=8), and MM (n=55). Median patient age was 56 (range 19-74) years. Sixty-one percent of patients had an HCT comorbidity index (HCT-CI) score of greater than 2. Five percent of patients had failed a prior autologous HCT in FLU/TBI group. Median follow-up was 5.2 years. One graft failure was observed in the FLU/TBI group and no patients rejected their graft. The early NRM at day 100 was 1%. The cumulative incidences of grade II-IV and grade III-IV acute GVHD at 120 days were 26% (FLU/TBI 25%; TBI 28%) and 4% (FLU/TBI 2%; TBI 8%), respectively. Only one patient developed grade IV acute GVHD. Forty-eight percent of patients had chronic GVHD by 5 years (FLU/TBI 44%; TBI 54%). Five-year NRM was low at 12%. The overall cumulative incidence of relapse/progression at 5 years was 52%. Five-year overall and progression-free survivals were 51% and 37%, respectively. Conclusions: Post-grafting immunosuppression and GVHD prophylaxis with tacrolimus/MMF resulted in a low risk of acute and chronic GVHD, which compares favorably with our experience in a concurrent trial using CSP/MMF with FLU/TBI conditioning (46% grades II-IV acute and 72% chronic GVHD with CSP/MMF, respectively; BBMT, 2013, 19: 1340-1347). Furthermore, we recently reported that the active metabolite of MMF (MPA) concentration at steady state (MPA Css) was lower in patients who received concomitant CSP than patients receiving tacrolimus. Low total MPA Css was associated with an increased risk of severe acute GVHD following nonmyeloablative HCT (BBMT 2013, 19: 1159-1166). Together these data warrant consideration of a randomized phase III trial to investigate the role of tacrolimus/MMF versus CSP/MMF in nonmyeloablative HCT. Figure 1. Cumulative incidences of grade II to IV acute GVHD (A) and chronic GVHD (B) Figure 1. Cumulative incidences of grade II to IV acute GVHD (A) and chronic GVHD (B) Disclosures Maloney: Seattle Genetics: Honoraria; Juno Therapeutics: Research Funding; Janssen Scientific Affairs: Honoraria; Roche/Genentech: Honoraria.

Description: Endothelial dysfunction has been shown to be associated with severe complications and increased NRM after alloSCT. Endothelial risk markers, such as angiopoetin-2 (ANG2) serum levels and thrombomodulin single nucleotide polymorphisms can be used for pre-transplant prediction of endothelium-related complications after alloSCT (Blood 2011;118:1685; J Clin Oncol. The aim of the present study was to develop a tool for prediction of endothelial dysfunction prior to alloSCT that can be easily used in clinical practice. For this purpose, we focused on routine parameters which are used to diagnose transplant-associated thrombotic microangiopathy (TMA), which is an endothelial complication associated with high NRM. Based on the fact that TMA is defined by high creatinine, high lactate dehydrogenase (LDH), low thrombocyte counts, schistocytes, and loss of haptoglobin, we hypothesized that the simplified formula termed 'Endothelial Activation and Stress Index (EASIX)' might be valuable for predicting TMA, NRM and overall survival (OS) after alloSCT. Design: The capacity of pre-transplant EASIX ("EASIX-pre") obtained directly prior to conditioning for alloSCT for predicting TMA was tested retrospectively in 771 consecutive adult patients undergoing alloSCT in Heidelberg between 2001 and 2013 (training cohort) using cause-specific Cox regression analysis. The correlation of EASIX-pre with pre-transplant ANG2 and suppressor of tumorigenicity-2 (ST2) serum levels was assessed by Kruskal-Wallis test / Pearson correlation. The prognostic strength of EASIX-pre for NRM, time to relapse (TTR) and OS was calculated in the training cohort and in three independent validation cohorts (Berlin adults n=386, Seattle adults n=450 and Cincinnati children n=247) by calculating the prediction error (integrated Brier score), concordance index, and calibration index. Different intensities of conditioning (MAC+RIC+non-MAC) as well as all donor types and all degrees of HLA-matching were included. Hazard ratios (HR) were estimated to illustrate the effect of a two-fold change in EASIX-pre. Results: In the training cohort, EASIX-pre was a significant risk factor for TMA in univariable (HR=1.24, p=0.03) and in multivariable models including age, disease score, ATG, donor sex, recipient sex, graft source, diagnosis and statin intake as covariates (HR 1.28, p=0.02). EASIX-pre correlated with pre-transplant serum levels of the endothelium-related markers ANG2 and ST2. Increasing EASIX-pre was significantly associated with increasing NRM (uni: HR 1.22, p

Description: INTRODUCTION: Emerging data indicate myeloid-derived suppressor cells (MDSCs) adversely impact outcomes of patients with a variety of malignancies. However, it is less clear how MDSCs affect acute myeloid leukemia (AML) disease biology or the response of AML patients to treatment. Here, we studied a cohort of people with AML undergoing hematopoietic cell transplantation (HCT) in first complete remission (CR1) to examine the relationship between the monocytic subtype of MDSCs (M-MDSCs), pre-transplant measurable residual disease (MRD) status, and outcome after HCT. PATIENTS AND METHODS: Adults ≥18 years of age were included if they underwent first allogeneic HCT with myeloablative or nonmyeloablative conditioning for AML in CR1 from April 2006 until October 2014. Prospective MRD testing in bone marrow aspirates was performed routinely via 10-color multiparameter flow cytometry (MFC) as part of the pre-transplant work-up. Pre-transplant MFC data were used retrospectively to quantify M-MDSCs as the proportion of monocytes (identified by side scatter properties and CD14 positivity) with low or negative expression of HLA-DR. M-MDSC frequency was then expressed as a percentage of the total number of viable cells in the sample. Available combinations of antigens measured by MFC did not allow for the identification of other MDSC subsets (e.g. granulocytic MDSCs). RESULTS: We identified 349 adults undergoing allogeneic HCT for AML in CR1 for whom pre-transplant MRD testing was performed and for whom follow-up data were available. Of these, 8 had to be excluded because of insufficient MFC events available for identification of M-MDSCs. In the remaining 341 patients, M-MDSC frequency ranged from

Description: Although HCT offers the best potential for cure for patients with high risk leukemia and MDS, the procedure may not be an option for all patients due to the toxicity of the conditioning regimen. Reduced-intensity conditioning (RIC) allo-HCT regimens are associated with lower non-relapse mortality (NRM), but patients receiving these regimens have a higher risk of relapse. Radioimmunotherapy (RIT) can potentially deliver high doses of targeted radiation while minimizing toxicity to normal tissue. When combined with RIC allo-HCT, RIT may improve responses without increasing toxicity associated with myeloablative conditioning. We evaluated the safety and efficacy of yttrium 90 (90Y)-anti-CD45 antibody (MAb; BC8) followed by a standard RIC regimen with fludarabine (Flu) and 2 Gy total body irradiation (TBI) as a means of developing an improved HCT strategy for high-risk acute leukemia or MDS patients. We used CD45 as a target due to its ubiquitous expression on hematopoietic stem cells including the leukemic blasts. We used the high-energy (2.2 MeVmax) beta-emitter 90Y, which has a relatively short half-life (2.7 days) to eliminate targeted malignant cells. This phase I dose-escalation trial (NCT01300572) was designed to estimate the safety, feasibility and maximum tolerated dose (MTD) of 90Y-BC8-DOTA MAb when combined with Flu and 2 Gy TBI followed by HLA‐matched, related or unrelated allo-HCT for patients with high-risk leukemia or MDS. The MTD was defined as the radiation absorbed dose delivered by 90Y-BC8-DOTA MAb associated with a true dose-limiting toxicity (DLT) rate of 25%, where a DLT is defined as Bearman grade III/IV regimen-related toxicity. Doses of 90Y were escalated in increments of 2 Gy depending on the occurrence of DLT. Inclusion required patients to have advanced leukemia or high-risk MDS (defined as primary refractory or relapsed AML/ALL, secondary AML, MDS expressed as RAEB or CMML). To determine the dose of 90Y-BC8-DOTA, patients first underwent a biodistribution step using a trace-labeled infusion of 111Indium (111In)-BC8-DOTA followed by gamma-camera imaging. On pre-HCT day -12, patients received 90Y-BC8-DOTA at a prescribed radiation dose calculated from the trace-labeled 111In-BC8-DOTA biodistribution, followed by Flu (30 mg/m2/day) on days -4 to -2. TBI (2 Gy) was administered on day 0, prior to G-CSF mobilized donor PBSC infusion. GVHD prophylaxis consisted of mycophenolate mofetil and cyclosporine. Fifteen patients, median age of 62 (range 37-76), were treated (10 with advanced AML, 5 with high-risk MDS). At time of HCT, 9 patients had refractory active disease while 6 were in remission with minimal residual disease (Table 1). The patients received 22.8 to 151.2 mCi of 90Y, delivering an average of 10.5 Gy to marrow, 70 Gy to spleen, and 17 Gy to liver. Although a maximum dose of 28 Gy was delivered to the liver, no DLT was observed. Therefore, the MTD could not be estimated. Treatment led to complete remission in 13 patients (87%), 2 patients had persistent disease after HCT. All patients engrafted with a median donor-derived CD3 and CD33 chimerism both 100% by day 28 after HCT. Ten patients (67%) developed grade II-IV acute GVHD (grade II: n=7; III: n=2; IV: n=1). Five patients (33%) developed chronic GVHD. Six patients relapsed, 5 of whom subsequently died due to progression of disease. The median time to relapse among these 6 patients was 59 days (range, 6- 351 days). One patient died from stage IV steroid-refractory GVHD. One patient died in remission from acute renal failure at 7 months after HCT. Eight patients (53%) are surviving with a median follow-up of 1.8 (range, 0.9-5.9) years. Estimated overall survival at one and two years were 66% and 46%, respectively, with progression-free survival estimated to be 46% at each of these time points. The 1-year estimate of relapse was 41% (Fig. 1). The inclusion of 90Y-BC8-DOTA into a RIC allo-HCT regimen is feasible, tolerable and no DLTs were observed. The efficacy of this approach is promising considering the high-risk leukemia/MDS patients with active disease enrolled. Current studies are evaluating the use of an alpha emitter, astatine-211, which is short-lived (t ½ = 7.2 hours) and provides high-energy radiation, conjugated to anti-CD45 MAb BC8 as part of an HCT conditioning regimen for patients with advanced AML, ALL, or high-risk MDS, in place of 90Y with the goal of continuing to improve outcomes using RIT for allo-HCT (NCT03128034). Disclosures Orozco: Actinium Pharmaceuticals: Research Funding. Green:Juno Therapeutics: Patents & Royalties, Research Funding. Gopal:Incyte: Consultancy; Pfizer: Research Funding; Gilead: Consultancy, Research Funding; Teva: Research Funding; Aptevo: Consultancy; Merck: Research Funding; Janssen: Consultancy, Research Funding; Spectrum: Research Funding; Takeda: Research Funding; BMS: Research Funding; Seattle Genetics: Consultancy, Research Funding; Brim: Consultancy; Asana: Consultancy. Pagel:Pharmacyclics, an AbbVie Company: Consultancy; Gilead: Consultancy.

Description: A randomized 3-arm Phase II trial involving 208 older or medically infirm patients (pts) with hematological malignancies given unrelated HCT after minimal intensity conditioning demonstrated that adding sirolimus to tacrolimus and MMF resulted in less grades II-IV GVHD, less steroid use, and less CMV reactivation (Hematologica 2014; 99(10); 1624). Based on this trial we designed a Phase III multi-site trial comparing triple therapy with sirolimus/MMF/CSP (Arm2) to the standard immunosuppressive regimen of MMF/CSP (Arm1). The primary objective of the trial was to compare the respective incidences of grades II-IV acute GVHD. Secondary objectives included comparing non-relapse mortality, survival, and progression-free survival. Pts in both Arms received CSP 5 mg/kg bid starting on day -3 through day 96 with a taper through day 150. In the first 28 days after HCT CSP was targeted to 400 and 350ng/ml in Arm1 and Arm2, respectively and 120-360ng/ml after day 28 in both arms. Arm 1: MMF was given daily at 15 mg/kg Q8 hours until day +30, reduced to 15 mg/kg Q12 hours until day 150, then tapered through day 180. Arm 2: MMF was given daily at 15 mg/kg Q8 hours until day +30, reduced to 15 mg/kg Q12 hours until day 40, then discontinued (no taper). In addition to the MMF/CSP, sirolimus was administered starting on day -3 at 2.0 mg/day through day 150 with a target level of 3-12ng/ml, with tapering off by day 180. The target enrollment was 300 pts with a built-in interim analysis for futility. At the time of the interim analysis, 158 pts ineligible for high-dose conditioning had been enrolled (Nov. 2010 to Jan. 2016: Arm1 n=74, Arm2 n=84) Their median age was 62 (range 18-79) yrs. The median HCT comorbidity index (HCT-CI) was 3 (range 0-10). Five pts had 6 previous allogeneic HCT and 32 pts (20%) had 36 previous autologous HCT. All pts were matched for HLA-DRB1 and -DQB1 at the allele level: 8 had single allele mismatches at HLA-A, -B or -C and the remainder (n=150) were fully HLA-matched. Diagnoses included AML (n=64), MDS/MPD (n=30), NHL (n=23), MM (n=13), CLL (n=13), ALL (n=11), HL (n=2), and CML (n=2). Randomization was stratified by transplant center. Unmodified PBSC grafts contained a median of 8.0 ×106 CD34 and 2.9 × 108 CD3 cells/kg. Conditioning consisted of fludarabine 90mg/m2 and 2-3 Gy TBI. Sustained donor engraftment occurred in 99% of pts. The median follow-up of surviving pts was 24 (range, 1-65) months. Table 1 and Figures 1 and 2 summarize results. The day-100 cumulative incidences of grades II-IV acute GVHD were in Arm1: 53%, and Arm2: 25% (p=0.0001) and the grades III-IV acute GVHD were in Arm1: 8%, Arm2: 2% (p=0.04). The 1-year cumulative incidence of chronic extensive GVHD was similar between Arm1: 49%, and Arm2: 48% (p=0.94). The 1-year cumulative incidence of non-relapse mortality was lower in Arm2 (Arm1: 15% and Arm2: 5%; p=0.007), while relapse/progression was similar at 1 year for Arm1: 21%, and Arm2: 19% (p=0.86). The 1-year overall and progression-free survivals for Arm1 vs. Arm2 were: 72% vs. 85% (p=0.03), and 65% vs. 77% (p=0.08); respectively. T-cell (CD3) donor chimerism was lower in Arm 2 on day 28 (median, Arm1 85%; Arm2 80%; p=0.05) with no differences seen in granulocyte (CD33: Arm1 98%, Arm2 95%) or NK cell (CD56: Arm1 96%, Arm2 97%) donor chimerisms. In summary, the interim analysis showed that adding sirolimus to MMF and CSP not only reduced the risks of grades II-IV but also of grades III-IV acute GVHD and of non-relapse mortality without increasing the risk of relapse or progressive malignancy. Based on these findings and the significantly improved survival, the DSMB recommended the trial be closed. This triple immunosuppressive regimen should, therefore, be considered in the future as the standard of care in pts given unrelated donor grafts after minimal intensity conditioning. Disclosures Pulsipher: Novartis: Consultancy, Other: Study Steering Committee; Chimerix: Consultancy; Jazz Pharmaceutical: Consultancy; Medac: Other: Housing support for conference.

Description: Background: Regimens using post-transplant cyclophosphamide (CY) have been developed to provide potent in vivo T cell depletion for patients undergoing human leukocyte antigen (HLA)-haploidentical hematopoietic cell transplantation (HCT). Luznik, O'Donnell and colleagues (BBMT 2008) reported that when this immune suppression strategy is coupled with non-myeloablative conditioning (fludarabine 150 mg/m2, CY 29 mg/kg, 2 Gy total body irradiation) followed by marrow transplantation, it was well-tolerated with low rates of non-relapse mortality (NRM; 15% at 1 year). However, the 2 year overall survival (OS) and event-free survival were low at 36% and 26%, respectively, due to high relapse rates (51% at 1 year). One explanation could be that while post-HCT CY promoted low rates of acute graft-versus-host disease (GVHD), it also eliminated early T and natural killer (NK) cell clones important for disease surveillance. Based on this hypothesis, we developed a next-generation Phase I/II clinical trial incorporating a boost of donor NK cells on day +7 as an attempt to prevent relapse after transplant. In this study, CY 50 mg/kg as a single dose on day +3 was used for T cell depletion. Methods: Forty patients (pediatric, n=14; adult, n=26) with median age of 45 (range 8-75) years having ALL (n=11), AML (n=9), MDS (n=6), HL (n=6), MM (n=4), NHL (n=3), and CLL (n=1) underwent non-myeloablative transplantation using related, HLA-haploidentical marrow donors on this prospective clinical trial. Patients were high risk due to underlying disease, potential for relapse, and/or risk for transplant-related mortality (TRM). Most patients were heavily pre-treated, with median time from cancer diagnosis to transplant being 2 (0.3 - 12.1) years, including 18 patients having 26 prior HCTs (auto, n=14; allo, n=12). Twenty-five patients (63%) had HCT-CI scores ≥ 3 indicating high risk for TRM. In order to obtain NK cells, non-mobilized peripheral blood mononuclear cells were collected from donors on day +6 using apheresis and stored overnight. NK cells were isolated on day +7 using the Miltenyi CliniMACS system (CD3 depletion followed by CD56 selection) and were administered as a single, fresh infusion that same day without prior culturing or expansion. The Phase I dose-finding study (n=11) enrolled at 2 NK doses [2.5 or 5 x 106/kg +/- 20%, respectively], with extended enrollment at the 2nd dose level for Phase II (n=29) with 83% of patients meeting NK dose parameters. NK cell products had a median log T cell depletion of 5.4 (4.1-7.1), median NK recovery of 54% (33-68%), and median NK purity of 92% (74-99%). Excellent viability (〉95%) was seen in all NK products. Results: One patient developed chest pain associated with NK cell infusion; otherwise all other patients tolerated their NK cell infusions well without fevers or other adverse reactions. Full donor chimerism (〉95% CD3) was seen in 83% of patients at last follow-up, while 18% and 10% experienced graft rejection or graft failure, respectively. Cumulative incidence of grades 2-3 and grade 3 (no grade 4 seen) acute GVHD occurred in 36% and 8% of patients, respectively, at day +100. Of the 39 evaluable patients, 16% developed chronic extensive GVHD at 1 year. Relapse or progression occurred in 31% of patients by 1 year after HCT. With a median follow-up of 1.5 years (range, 0.1 - 4.9 years), 14 patients have died from relapse/progression (n=11) or infection/VOD (n=3), giving a probability of OS, relapse/progression-free survival (PFS), and NRM at 1 year of 73%, 62%, and 8%, respectively, and 2 year OS and relapse/PFS of 63% and 46%, respectively (Fig 1). Summary: We have demonstrated the safety of infusing donor NK cells early after HCT in a group of heavily-treated patients with high-risk hematological malignancies. In many patients, disease-free survival was possible with the aid of this prophylactic infusion of donor NK cells in combination with allogeneic HCT. These results provide a promising platform to further augment NK cell alloreactivity in the post-HCT setting to prevent relapse and disease progression. Figure 1 Incidence of Overall Survival and Relapse/Progression-Free Survival Figure 1. Incidence of Overall Survival and Relapse/Progression-Free Survival Disclosures Hari: Merck: Research Funding; BMS: Honoraria.

Description: Objectives: Low donor chimerism after hematopoietic cell transplantation (HCT) is associated with increased risk of post transplant relapse. Our group has shown that donor lymphocyte infusion (DLI) was ineffective for converting 90% elicited GVT effects (Blood 2004;103:790). To facilitate effectiveness of DLI for patients (pts) with low or declining donor CD3chimerism, a prospective multicenter trial assessing the safety and efficacy of addingpentostatin was developed. Patients and Methods: Pts at risk for rejection after HCTs, defined as low (5%) donor CD3 chimerism and with stable/in remission disease, were included. Pts were excluded if they had evidence of relapse/progression, ongoing grades II-IV acute (a)GVHD, or extensive chronic (ec)GVHD. Between 2003 and 2014, 36 pts were treated on the study; 35 pts received a total of 41 DLIs following a dose of pentostatin, and 1 pt received pentostatin only. Diagnoses included AML (n=13), NHL (n=4), CLL (n=7), CML (n=2), MDS (n=6), MPD (n=2), MM (n=1), NHL and MDS (n=1). Pts received nonmyeloablative (n= 35) or ablative (n=1) conditioning, followed by PBSCs from HLA-matched related (n=17) or unrelated (n=17) donors, or 1-allele mismatched unrelated donor (n=2). Median age at HCT was 58 (34-72) years. DLI was given at a median of 96 (54-339) days after HCT. A dose of pentostatin was given 2 days before infusing 1x107 CD3 cells/kg (n=26), 2x107 CD3 cells/kg (n=1), or 3x107 CD3 cells/kg (n=14). Per protocol, prophylactic immunosuppression (IS) with cyclosporine and MMF was given after DLI to the last 6 pts on study. Results: Median donor CD3chimerism beforepentostatin/DLI was 28 (5-47)%. Efficacy, defined by increases in donor CD3chimerism〉10% maintained to day 56 post-DLI, was seen in 16 pts (44.4%) with a median CD3 donorchimerism of 64 (48-100)%. There was a trend for better efficacy among the 21 pts who received first treatment within 100 days after transplant (57%) compared to the 15 pts who received first treatment more than 100 days after HCT (27%) (p=0.07). Six pts received 2nd treatment ofpentostatin/DLI; among them 1 responded. Fifteen pts (12 responders) developedaGVHD after DLI [grade I (n=2), II (n=9), III (n=3), IV (n=1)] at a median of 10 (0-83) days after DLI. One pt developed skinGVHD, grade II, afterpentostatin administration and DLI was aborted. Of the 16 pts who developedaGVHD afterpentostatin/DLI, 3 had a prior history of GVHD after transplant. Thirteen pts developedcGVHD, ofwhom 10 developedecGVHD at a median of 112 (13-347) days after DLI. One pt developedecGVHD with no prioraGVHD. Among the 6 pts who received prophylaxis IS after DLI 1 pt developedaGVHD grade II, and none developedcGVHD. However, only 2 of those 6 pts responded. Seventeen pts developed grade 4cytopenia after DLI; 9 pts developed both neutropenia and thrombocytopenia, 4 pts developed neutropenia, and 4 pts developed thrombocytopenia. Among the 13 patients who developed grade 4 neutropenia, 9 (69%) were non-responders. Median time between DLI and lowest ANC count was 22 (2-100) days, and neutropenia lasted for median of 7 (1-52) days. Twelve pts received platelet transfusions started at median of 33 (1-98) days after DLI. Nineteen pts relapsed at a median of 91 (26-777) days after DLI. Among the 20 non-responders 12 relapsed (60%), compared to 7 of 16 responders (44%) (HR 0.64 (0.3-1.6), p=0.35). Ten of the 17 pts (59%) who developedcytopenia relapsed, 6 ofwhom (60%) were non-responders. Median number of significant infections was 2 (0-10) per pt. Twenty-eight pts died at a median of 522 (67-2028) days after DLI. Causes of death were relapse (n=21), respiratory failure/infections (n=4), grade IV GVHD (n=2), or other causes (n=1). Five of the 16 responders (31%) are alive, all in CR. Six of the 20 non-responders (30%) are alive, only 3 in CR (Figure 1). Summary:Pentostatin/DLI increased donor CD3chimerism in pts after HCT if was given within 100 days after transplant, and it was well tolerated. Response was associated with GVHD. Delayed treatment after HCT and prophylactic IS after DLI were associated with decreased efficacy. There was a trend for higher relapse rate among the non-responders and pts who developed cytopenia, but the differences were not statistically significant. Figure Progression Free Survival after pentostatin/DLI Figure. Progression Free Survival after pentostatin/DLI Disclosures Pulsipher: Novartis: Consultancy, Other: Advisory Board, Steering Committee for Phase II Study; Jazz Pharmaceutical: Consultancy, Other: Advisory Board; Chimerix: Consultancy, Other: Advisory Board ; Medac: Other: Travel support for a study group.

Description: Background: Graft rejection is a significant complication following allogeneic hematopoietic cell transplantation (HCT). Heavily transfused patients with nonmalignant blood disorders, such as aplastic anemia, thalassemia major, and sickle cell anemia, had rejection probabilities in the range of 5~60% in earlier transplant series. Since rejections were seen despite the use of HLA-identical donors, rejections were attributed to sensitization to minor non-HLA antigens via transfusions. Systemic intensification of regimens with higher doses of chemotherapy or total body irradiation (TBI) lead to worse outcomes due to regimen-related mortality. In our well-established canine model, following 9.2Gy TBI conditioning, only 1.6% (1/62) of untransfused dogs rejected the marrow grafts from their dog leukocyte antigen (DLA)-identical littermate donors. However, 100% (27/27) of the dogs pretreated with three unirradiated transfusions from their respective DLA-identical marrow donors rejected their grafts. We seek to overcome graft rejection by adding monoclonal antibody (MAb)-based targeted radioimmunotherapy (RIT) with astatine-211 (211At) to conditioning with 9.2 Gy TBI. 211At is an alpha-particle-emitting isotope, which has a short path length (0.06 mm), very high energy, and high cytotoxicity of alpha emissions. These properties and the short half-life (7.2 hours) may reduce early toxicities as well as late effects, such as secondary malignancies. The purpose of this study is to determine the quantity of 211At required to provide durable donor engraftment in DLA-identical HCT when 211At-labeled anti-CD45 MAb is used in combination with TBI to condition transfusion-sensitized dogs. Methods: Five recipients were given three preceding transfusions of 50 mL unirradiated whole blood from their respective DLA-identical marrow donors on days -24, -17, and -10, followed by 211At-labeled anti-CD45 MAb on day -3 and 9.2 Gy TBI and a marrow transplant on day 0. The injected 211At activity ranged between 0.188 and 0.304 mCi/kg body weight labeled on 0.5 mg/kg anti-CD45 MAb. Ten percent of the total dose of anti-CD45 MAb was administered as unlabeled MAb prior to infusion of the radiolabeled MAb in order to prevent nonspecific tissue binding of the radiolabeled MAb. Dogs received marrow containing a mean of 5.1 x 108 (range, 1.3-8.3) total nucleated cells/kg and 2.8 x 106 (range, 0.4-5.5) CD34+cells/kg. Dogs did not receive post-grafting immunosuppression. Results: Results are shown in Figure 1. Granulocyte nadirs (median, 20/µL) and platelet nadirs (median, 4500/µL) occurred at a median time of 5 days and 11 days, respectively. Neutrophil engraftment (〉=500/µL, 3 consecutive days) and platelet engraftment (〉=20,000/µL, 7 consecutive days, without transfusion) were achieved at a median of 8 (7-9) days and 20 (18-21) days, respectively. Four dogs achieved 100% donor chimerism in mononuclear cells, granulocytes, and CD3+ T-cells. Only one dog rejected the graft on day 14 after transplantation. That dog received the lowest numbers of CD34+ and marrow cell dose (0.35 x 106 CD34+ and 1.3 x 108 total nucleated cells per kg, respectively), which could be one of the reasons for the graft rejection. After a median follow-up time of 60 (21-182) days, no renal toxicity had been observed. One dog was euthanized on day 60 due to liver toxicity (CTCAE grade 4), but there was no evidence of GVHD on histopathological evaluation. Conclusions: The preliminary results are encouraging and show that 211At-labeled anti-CD45 MAb in combination with TBI as conditioning is successful in establishing sustained DLA-identical grafts in 4/5 dogs, especially when compared to previous studies showing that 100% of the dogs (27/27) rejected their grafts when pretreated with three unirradiated transfusions from their marrow donors. We will increase the sample size and then proceed to study canine models of human blood disorders to further determine the optimal dosing of 211At in disease settings. In view of the potential of RIT, 211At-anti-CD45 RIT in conjunction with TBI may serve as a novel promising strategy to overcome graft rejection. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.