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Despite the increased usage of post-transplant cyclophosphamide (PTCy) in allogeneic hematopoietic stem cell transplantation (allo-HSCT), our knowledge of immune reconstitution post-allo-HSCT in the setting of PTCy is limited. Adequate immune reconstitution is the key to a successful transplant. In this study, we aim to investigate the effect of PTCy on the reconstitution of each immune component; more focus was placed on the immunophenotype and functions of T cells. Using blood samples from patients who underwent allo-HSCT under regimens containing PTCy (n = 23) versus those who received no PTCy (n = 14), we examined the impact of PTCy on the post-transplant immune response. We demonstrated a distinct T cell immune signature between PTCy versus non-PTCy group. PTCy significantly delayed T cell reconstitution and affected the T cell subsets by increasing regulatory T cells (Treg) while reducing naïve T cells. In addition, we observed remarkable enhancement of multiple inhibitory receptors (TIGIT, PD-1, TIM-3, CD38, CD39) on both CD4+ and CD8+ T cells on day 30 post-transplantation in patients who received PTCy. Importantly, upregulation of PD-1 on CD8 T cells was persistent through day 180 and these T cells were less functional, manifested by reduced cytokine production upon anti-CD3/CD28 stimulation. Furthermore, we found a significant correlation of T cell immune phenotypes to clinical outcome (disease relapse and GVHD) in patients who received PTCy. Our novel findings provide critical information to understand the mechanism of how PTCy impacts immune reconstitution in allo-HSCT and may subsequently lead to optimization of our clinical practice using this treatment.

High-dose cytarabine (HiDAC) consolidation for acute myeloid leukemia (AML) induces transient profound myelosuppression and potential morbidity/mortality. PrE0901 was a phase I multi-center trial evaluating the safety/toxicity of eltrombopag in AML patients receiving HiDAC consolidation. We used a standard 3 + 3 design employing a unique dose-escalation/de-escalation strategy. One hundred four patients were screened, 54 declined participation, 35 were deemed medically ineligible, and 14 were treated on study. Three patients were treated in cohorts 1-4 and two were treated in cohort 5. Eltrombopag + HiDAC was well-tolerated and no dose-limiting toxicities were observed. Median time to platelet recovery of all patients treated was 22.5 (range 16-43) days. Observationally, eltrombopag 150 mg once daily starting on day 3 of consolidation demonstrated the fastest and most consistent platelet recovery (median 19 days). Further investigation is needed to define the optimal role, dose, and schedule of eltrombopag in the treatment of chemotherapy associated myelosuppression.

Summary Decitabine is a DNA‐hypomethylating agent that has been widely applied for the treatment of acute myeloid leukaemia (AML) patients who are elderly or unfit for intensive therapy. Although effective, the complete response rate to decitabine is only around 30% and the overall survival remains poor. Emerging data support that regulation of DNA methylation is critical to control immune cell development, differentiation and activation. We hypothesize that defining how decitabine influences the immune responses in AML will facilitate the development of novel immune‐based leukaemia therapeutics. Here, we performed phenotypic and functional immune analysis on clinical samples from AML patients receiving decitabine treatment and demonstrated a significant impact of decitabine on the immune system. T‐cell expression of inhibitory molecules was upregulated and the ability of CD8 T cells to produce cytokines was decreased upon decitabine treatment. Importantly, in an unbiased comprehensive analysis, we identified a unique immune signature containing a cluster of key immune markers that clearly separate patients who achieved complete remission after decitabine from those who failed to do so. Therefore, this immune signature has a strong predictive value for clinical response. Collectively, our study suggests that immune‐based analyses may predict clinical response to decitabine and provide a therapeutic strategy to improve the treatment of AML.

We analyzed late fatal infections (LFIs) in allogeneic stem cell transplantation (HCT) recipients reported to the Center for International Blood and Marrow Transplant Research. We analyzed the incidence, infection types, and risk factors contributing to LFI in 10,336 adult and 5088 pediatric subjects surviving for ≥2 years after first HCT without relapse. Among 2245 adult and 377 pediatric patients who died, infections were a primary or contributory cause of death in 687 (31%) and 110 (29%), respectively. At 12 years post-HCT, the cumulative incidence of LFIs was 6.4% (95% confidence interval [CI], 5.8% to 7.0%) in adults, compared with 1.8% (95% CI, 1.4% to 2.3%) in pediatric subjects; P < .001). In adults, the 2 most significant risks for developing LFI were increasing age (20 to 39, 40 to 54, and ≥55 years versus 18 to 19 years) with hazard ratios (HRs) of 3.12 (95% CI, 1.33 to 7.32), 3.86 (95% CI, 1.66 to 8.95), and 5.49 (95% CI, 2.32 to 12.99) and a history of chronic graft-versus-host disease GVHD (cGVHD) with ongoing immunosuppression at 2 years post-HCT compared with no history of GVHD with (HR, 3.87; 95% CI, 2.59 to 5.78). In pediatric subjects, the 3 most significant risks for developing LFI were a history of cGVHD with ongoing immunosuppression (HR, 9.49; 95% CI, 4.39 to 20.51) or without ongoing immunosuppression (HR, 2.7; 95% CI, 1.05 to 7.43) at 2 years post-HCT compared with no history of GVHD, diagnosis of inherited abnormalities of erythrocyte function compared with diagnosis of acute myelogenous leukemia (HR, 2.30; 95% CI, 1.19 to 4.42), and age >10 years (HR, 1.92; 95% CI, 1.15 to 3.2). This study emphasizes the importance of continued vigilance for late infections after HCT and institution of support strategies aimed at decreasing the risk of cGVHD. ispartof: BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION vol:25 issue:2 pages:362-368 ispartof: location:United States status: published

Acute myeloid leukemia (AML) relapse after allogeneic stem cell transplant (alloSCT) remains a major therapeutic challenge. While patients with longer remission after initial alloSCT are recommended to receive cell therapy (CT) such as 2ndalloSCT or donor lymphocyte infusion (DLI), survival for patients who relapse within 6 months of alloSCT has been dismal. We evaluated the outcomes of AML relapse after alloSCT to assess the impact of different treatments on long-term survival. One hundred and seventy-two patients with AML underwent alloSCT at the Penn State Cancer Institute from January 2014 to August 2019. Sixty-nine patients relapsed (median age, 60 years; range, 10–75). Of these, 4 patients underwent 2ndalloSCT, and 26 received DLI. One-year overall survival (OS) in all cases was 20.3% (95% CI: 11.8–30.4%). Patients with ECOG performance status (PS) 0–2 at relapse showed a better 1-year OS than those with PS 3–4. Median OS for patients who received chemotherapy only or chemotherapy with CT was 74 or 173.5 days, respectively (p

To improve the outcome of relapsed/refractory acute myeloid leukemia (AML), a randomized phase II trial of three novel regimens was conducted. Ninety patients were enrolled and were in first relapse or were refractory to induction/re-induction chemotherapy. They were randomized to the following regimens: carboplatin-topotecan (CT), each by continuous infusion for five days; alvocidib (formerly flavopiridol), cytarabine, and mitoxantrone (FLAM) in a timed sequential regimen; or sirolimus combined with mitoxantrone, etoposide, and cytarabine (S-MEC). The primary objective was attainment of a complete remission (CR). A Simon two-stage design was used for each of the three arms. The median age of the patients in the FLAM arm was older at 62 years compared to 55 years for the CT arm and the S-MEC arm. The overall response was 14% in the CT arm (5/35, 90% CI 7%−35%), 28% in the FLAM arm (10/36, 90% CI, 16%−43%), and 16% in the S-MEC arm (3/19, 90% CI, 4%−36%). There were nine treatment-related deaths, seven of which occurred in the FLAM arm with four of these in elderly patients. We conclude that the FLAM regimen had an encouraging response rate and should be considered for further clinical development but should be used with caution in elderly patients.

We recently conducted a randomized double-blind study in which we demonstrated that moderate/severe chronic graft-versus-host disease (cGVHD) but not cGVHD-free survival was reduced in patients receiving anti-T lymphocyte globulin (ATLG) versus placebo. In a companion study we performed immunophenotypic analysis to determine the impact of ATLG on immune reconstitution (IR) and to correlate IR with clinical outcomes. The randomized study (n = 254) included patients (aged 18 to 65 years) who underwent myeloablative transplants for acute myeloid leukemia, myelodysplastic syndrome, or acute lymphoblastic leukemia from HLA-matched unrelated donors. Ninety-one patients consented for the companion IR study (ATLG = 44, placebo = 47). Blood samples were collected on days 30, 100, 180, and 360 after hematopoietic cell transplantation (HCT), and multiparameter flow cytometry was performed in a blinded fashion. Reconstitution of CD3+ and CD4+ T cells was delayed up to 6 months post-HCT in the ATLG arm, whereas absolute regulatory T cell (Treg) (CD4+25+127-) numbers were lower only in the first 100 days. Analysis of the CD4+ Treg and conventional T cells (Tconv) (CD4+25–127+) compartments showed a profound absence of naive Tregs and Tconv in the first 100 days post-HCT, with very slow recovery for 1 year. B cell and natural killer cell recovery were similar in each arm. Higher absolute counts of CD3+, CD4+, CD8+ T, Tregs, and Tconv were associated with improved overall survival, progression-free survival, and nonrelapse mortality but not moderate/severe cGVHD. Although ATLG delays CD3+ and CD4+ T cell recovery post-transplant, it has a relative Treg sparing effect after the early post-HCT period, with possible implications for protection from cGVHD. ATLG severely compromises the generation of naive CD4+ cells (Treg and Tconv), potentially affecting the diversity of the TCR repertoire and T cell responses against malignancy and infection.

Unlike unrelated donor registries, transplant centers lack uniform approaches to related donor assessment and deferral. To test whether related donors are at increased risk for donation-related toxicities, we conducted a prospective observational trial of 11,942 related and unrelated donors aged 18-60 years. Bone marrow (BM) was collected at 37 transplant and 78 National Marrow Donor Program centers, and peripheral blood stem cells (PBSC) were collected at 42 transplant and 87 unrelated donor centers in North America. Possible presence of medical comorbidities was verified prior to donation, and standardized pain and toxicity measures were assessed pre-donation, peri-donation, and one year following. Multivariate analyses showed similar experiences for BM collection in related and unrelated donors; however, related stem cell donors had increased risk of moderate [odds ratios (ORs) 1.42; P

Purpose Despite the achievement of complete remission with chemotherapy in patients with acute myeloid leukemia (AML), relapse is common and the majority of patients will die of their disease. Patients who achieve a remission after refractory or relapsed disease as well as elderly patients have a very high rate of relapse even if they achieve a complete remission. A phase 3 randomized ECOG-ACRIN-led intergroup study was conducted to determine whether post-remission therapy with the farnesyl transferase inhibitor, tipifarnib (R115777), improved the disease-free survival (DFS) of adult patients with AML in complete remission (CR), at high risk for relapse. Patients and methods Adult patients with AML in remission after salvage therapy and/or over age 60 in first remission were enrolled in this study. They were randomly assigned to treatment with tipifarnib or observation (control). The primary objective was to compare the disease-free survival (DFS) between the two arms based on intention to treat, which includes all randomized patients. Results One hundred and forty-four patients were enrolled on the study. Median DFS was 8.9 vs 5.3 months, for tipifarnib vs observation (one-sided p = 0.026) and did not cross the pre-specified boundary to call the study positive. For the 134 eligible patients, median DFS was 10.8 vs 5.3 months for those randomized to tipifarnib vs observation (one-sided p = 0.008). Moreover in an ad hoc evaluation of all women (n = 71) median DFS was 12.1 vs 3.9 months for tipifarnib vs observation (one-sided p = 0.0004) while median OS was 26.5 vs 8.4 months respectively (one-sided p = 0.001). Conclusion This study was not able to demonstrate a benefit to tipifarnib as maintenance therapy in patients with AML in remission. While subsets of patients may indeed benefit, additional studies would be needed to elucidate that benefit which is unlikely given that other seemingly better options have since become available.

Recipient chimerism increase has been used to predict leukemia relapse in post-hematopoietic cell transplant (HCT) patients with conventional GVHD prophylaxis. However, the value of recipient chimerism increase in patients with post-transplant cyclophosphamide (PTCy) is not clear. We compared PTCy to conventional GVHD prophylaxis (non-PTCy) patients regarding engraftment kinetics and the clinical significance of the 2 chimerism parameters, increasing mixed chimerism (IMC) and degree of recipient chimerism increase at the first event (Δ increase). We studied both total and T-cell-specific chimerism. While leukemia relapse is manifested by an increase in total cell recipient chimerism, an increase in T-cell-specific recipient chimerism may be more impactful in predicting relapse because of the effect of increased T-cell-specific chimerism on the graft-versus-leukemia effect. A total of 220 patients (PTCy: 44, non-PTCy: 176) with AML, MDS, and ALL underwent HCT at our institution from January 2014 to September 2020 and were included in this study (Table). Chimerism was tested at least monthly for the first 3 months, followed by every 3 months up until 1-year post-HCT, and then every 6-12 months thereafter. Short tandem repeat or quantitative PCR were used when percent recipient chimerism was ≥5% and PTCy patients achieved complete donor chimerism (CC) in total cells earlier at a deeper level (>99%) as compared to non-PTCy patients. Deeper total cell CC (>99%) was achieved in 79.5% of PTCy vs. 51% of non-PTCy patients at day 250, while CC (>95%) was achieved in almost 90% of patients in both groups within 100 days (Figure 1A and B). In comparison, the percentage of PTCy patients achieving T-cell-specific CC was significantly higher at day 250 post-HCT: CC (>95%/>99%) was 79.7%/68.4% in PTCy patients vs. 56.1%/37.5% in non-PTCy patients (Figure 1C and D). To evaluate their impact in predicting relapse, IMC was stratified into no IMC, 1 IMC (≥1 nonconsecutive IMC), and 2 IMC (≥2 consecutive IMC), and degree of recipient chimerism increase at the first event (Δ increase) was stratified into Two IMC (total), 1 IMC (T-cell), and 2 IMC (T-cell) groups were associated with shorter DFS in non-PTCy patients but not in PTCy patients (Figure 2). One and 2 IMC groups (both total and T-cell) were associated with relapse risk in non-PTCy patients. Furthermore, 1 IMC (T-cell) in non-PTCy patients showed a strong association in relapse risk (HR 7.0 (95%CI 2.83-17.8) p Δ increase ≥1% (total and T-cell) and Δ increase ≥0.1% (T-cell) were associated with shorter DFS in non-PTCy patients, while only Δ increase ≥1% (T-cell) only showed a trend towards shorter DFS in PTCy patients (Figure 3). The Cox regression model showed Δ increase ≥1% in both total, and T-cell chimerism was associated with relapse risk in non-PTCy patients (HR 6.4 (95%CI 2.9-14.2) p This is one of the most extensive studies investigating engraftment kinetics and the association of total and T-cell recipient chimerism increase to predict leukemia relapse in PTCy and non-PTCy HCT recipients. We found that PTCy HCT recipients achieved deeper engraftment earlier as compared to non-PTCy recipients. In addition, the two chimerism parameters (IMC and Δ increase) are less reliable in predicting relapse in PTCy than non-PTCy recipients. However, other factors, such as disease type, conditioning regimen, and donor HLA disparity, may have affected engraftment kinetics and the significance of chimerism parameters. Further investigations are warranted to elucidate the impact of the engraftment kinetics and recipient chimerism increase to predict relapse, especially in the PTCy setting. Figure 1 Figure 1. Disclosures Rakszawski: SeaGen: Speakers Bureau. Naik: Takeda: Other: Virtual Advisory Board Member ; Sanofi: Other: Virtual Advisory Board Member ; Kite: Other: Virtual Advisory Board Member. Rybka: Spark Therapeutics: Consultancy; Merck: Consultancy. Claxton: Astellas: Other: Clinical Trial; Novartis: Research Funding; Astex: Research Funding; Cyclacel: Research Funding; Daiichi Sankyo: Research Funding; Incyte: Research Funding.

BACKGROUND: Our group recently reported on the results of Blood and Marrow Transplant (BMT) Clinical Trials Network (CTN) 1101 a randomized comparison between double umbilical cord blood (dUCB) and haploidentical bone marrow (haplo) with post-transplant cyclophosphamide (ptCy) in the nonmyeloablative setting that showed similar progression free survival (PFS) between the two treatment groups, but lower non-relapse mortality (NRM) and better of overall survival (OS) in the haplo arm. In this secondary analysis we sought to investigate if transplant center experience with haplo and or cord blood HCT had an impact on outcomes. PATIENTS AND METHODS: All patient randomized in BMT CTN 1101 were included. In order to determine the transplant center experience with either haplo or dUCB we queried the Center for International Blood and Marrow Transplant Research (CIBMTR) for number of transplants with each platform in the year prior to initiation of the study. Centers were then grouped as dUCB center (> 10 dUCB, n=117, 10 centers), Haplo center (>10 haplo and ≤10 dUCB, n=110, 2 centers), and ≤10 haplo and ≤10 dUCB HCTs (other center, n=140, 21 centers). Further analysis considered the alternative cut-off for haplo (> 5 vs ≤ 5) experience, and considered the outcomes based on the donor experience vs. others (e.g. dUCB > 10 vs. ≤ 10; haplo > 5 vs. ≤ 5). RESULTS: The effect of center experience on HCT outcomes shown in Figure, below . After adjusting for age, Karnofsky performance score and, disease risk index we found that there was no difference in outcomes between haplo and dUCB for centers that were experienced with dUCB or had limited to no experience with either dUCB or haplo. In contrast, in centers that were primarily experienced with haplo had better outcomes with this donor type, as compared to dUCB. The higher risk of treatment failure (relapse or death) and overall mortality in dUCB in haplo experienced centers was driven by significantly higher risk of relapse. We then considered the transplant experience with each of the donor types separately. In transplant centers that had performed > 10 dUCB, there were similar outcomes for recipients of both dUCB and haplo. Similarly, centers that had ≤ 5 haplo HCTs had no difference in outcomes between donor types suggesting an overlap with centers that had performed > 10 dUCB HCTs. Overall mortality was higher among dUCB recipients in centers that had performed ≤ 10 dUCB. Notably, the hazard ratio of non-relapse mortality favored haplo in all four donor experience type of transplant center, albeit not statistically significant. CONCLUSION: Except for dUCB recipients in centers with < 10 dUCB/year had worse overall mortality, primarily driven by relapse, the transplant center experience in the year prior to the initiation of BMT CTN 1101 had limited impact on the outcomes of this randomized clinical trial. Figure 1 Figure 1. Disclosures Brunstein: NANT: Research Funding; FATE: Research Funding; GamidaCell: Research Funding; BlueRock: Research Funding; AlloVir: Consultancy. Costa: Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria. Cutler: Deciphera: Consultancy; Cimeio: Consultancy; Editas: Consultancy; Kadmon: Consultancy; Pfizer: Consultancy; Mallinckrodt: Consultancy; CareDx: Consultancy; Incyte: Consultancy; Omeros: Consultancy; Syndax: Consultancy; Mesoblast: Consultancy; Jazz: Consultancy. Horowitz: Sobi: Research Funding; Regeneron: Research Funding; Orca Biosystems: Research Funding; Tscan: Research Funding; Xenikos: Research Funding; Miltenyi Biotech: Research Funding; Stemcyte: Research Funding; Medac: Research Funding; Vor Biopharma: Research Funding; Kite/Gilead: Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Kiadis: Research Funding; Seattle Genetics: Research Funding; Mesoblast: Research Funding; GlaxoSmithKline: Research Funding; Sanofi: Research Funding; Pfizer, Inc: Research Funding; Omeros: Research Funding; Magenta: Consultancy, Research Funding; Jazz Pharmaceuticals: Research Funding; Vertex: Research Funding; Genentech: Research Funding; Takeda: Research Funding; Novartis: Research Funding; Shire: Research Funding; Gamida Cell: Research Funding; Daiicho Sankyo: Research Funding; CSL Behring: Research Funding; Chimerix: Research Funding; Bristol-Myers Squibb: Research Funding; bluebird bio: Research Funding; Astellas: Research Funding; Amgen: Research Funding; Allovir: Consultancy; Actinium: Research Funding. Horwitz: Gamida Cell: Research Funding. McGuirk: Novartis: Research Funding; Magenta Therapeutics: Consultancy, Honoraria, Research Funding; EcoR1 Capital: Consultancy; Pluristem Therapeutics: Research Funding; Novartis: Research Funding; Bellicum Pharmaceuticals: Research Funding; Astelllas Pharma: Research Funding; Gamida Cell: Research Funding; Fresenius Biotech: Research Funding; Allovir: Consultancy, Honoraria, Research Funding; Juno Therapeutics: Consultancy, Honoraria, Research Funding; Kite/ Gilead: Consultancy, Honoraria, Other: travel accommodations, expense, Kite a Gilead company, Research Funding, Speakers Bureau. Rezvani: US Department of Justice: Consultancy; Kaleido: Other: One-time scientific advisory board; Nohla Therapeutics: Other: One-time scientific advisory board; Pharmacyclics-Abbvie: Research Funding. Rybka: Spark Therapeutics: Consultancy; Merck: Consultancy. Vasu: Kiadis, Inc.: Research Funding; Boehringer Ingelheim: Other: Travel support; Seattle Genetics: Other: travel support; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees.

Acute myeloid leukemia (AML) patients often undergo allogeneic hematopoietic cell transplantation (alloHCT) in first complete remission (CR). We examined the effect of depth of clinical response, including incomplete count recovery (CRi) and/or measurable residual disease (MRD), in patients from the Center for International Blood and Marrow Transplantation Research (CIBMTR) registry. We identified 2492 adult patients (1799 CR and 693 CRi) who underwent alloHCT between January 1, 2007 and December 31, 2015. The primary outcome was overall survival (OS). Multivariable analysis was performed to adjust for patient-, disease-, and transplant-related factors. Baseline characteristics were similar. Patients in CRi compared to those in CR had an increased likelihood of death (HR: 1.27; 95% confidence interval: 1.13-1.43). Compared to CR, CRi was significantly associated with increased non-relapse mortality (NRM), shorter disease-free survival (DFS), and a trend toward increased relapse. Detectable MRD was associated with shorter OS, shorter DFS, higher NRM, and increased relapse compared to absence of MRD. The deleterious effects of CRi and MRD were independent. In this large CIBMTR cohort, survival outcomes differ among AML patients based on depth of CR and presence of MRD at the time of alloHCT. Further studies should focus on optimizing post-alloHCT outcomes for patients with responses less than CR.

Background Acute myeloid leukemia (AML) relapse after allogeneic stem cell transplant (allo SCT) remains a major therapeutic challenge. While patients with longer remission after initial allo SCT are recommended to receive either 2nd allo SCT or donor lymphocyte infusion (2nd allo SCT/DLI), survival probability for patients relapsing within 6 months of SCT are dismal. We evaluated the outcomes of AML relapse after allo SCT to assess the impact of 2nd allo SCT/DLI on long term survival. Methods and Results One hundred and seventy-two patients with AML underwent allo SCT at Penn State Cancer Institute from Jan 2014 to Aug 2019. Sixty-nine patients relapsed after allo SCT (median age, 60 years; range, 10-75). Of these, 4 patients underwent 2nd allo SCT and 26 received DLI. Overall survival (OS) was defined as days from the relapse and evaluated with Kaplan-Meier survival curves and log rank tests. One-year OS in all cases was 22.4% (95% CI: 13.3-32.9%). Patients with ECOG performance status (PS) 0-2 at relapse (n=60) showed a better 1-year OS compared to those with ECOG PS 3-4 (n=9) (24 vs 11.1%, p Conclusions Relapsed AML after allo SCT treated with high intensity chemotherapy followed by 2nd allo SCT/DLI showed a better OS. Similar benefit was shown even for patients who relapsed within 6 months of allo SCT.

The development of reduced-intensity approaches for allogeneic hematopoietic cell transplantation has resulted in growing numbers of older related donors (RDs) of peripheral blood stem cells (PBSCs). The effects of age on donation efficacy, toxicity, and long-term recovery in RDs are poorly understood. To address this we analyzed hematologic variables, pain, donation-related symptoms, and recovery in 1211 PBSC RDs aged 18 to 79 enrolled in the Related Donor Safety Study. RDs aged > 60 had a lower median CD34+ level before apheresis compared with younger RDs (age > 60, 59 × 106/L; age 41 to 60, 81 × 106/L; age 18 to 40, 121 × 106/L; P < .001). This resulted in older donors undergoing more apheresis procedures (49% versus 30% ≥ 2 collections, P < .001) and higher collection volumes (52% versus 32% > 24 L, P < .001), leading to high percentages of donors aged > 60 with postcollection thrombocytopenia

Background Clofarabine, a second-generation nucleoside analogue, was studied in combination with etoposide and mitoxantrone in acute leukemia. Patients and Methods In the phase I portion of this study clofarabine was given 20 or 25 mg/m2 daily for 5 days (Days 2-6) with etoposide 100 mg/m2 from day 1 to 5 and mitoxantrone 8 mg/m2 from day 1 to 3. The dose-limiting toxicity was myelosuppression, and dose level 1, with clofarabine 20 mg/m2 daily for 5 days was identified as the phase 2 dose. In total, 22 patients with relapsed or refractory acute myeloid leukemia (n = 18) and acute lymphocytic leukemia (n = 4) were treated. Results Five of 22 patients (23%) achieved complete response (CR), and 3 (13%) achieved CR with incomplete platelet recovery; an overall response rate of 36%. Median overall survival was 167 days (range, 22-1327 days). For 2 patients this regimen represented an effective bridge to allogeneic stem cell transplantation. Conclusion Clofarabine in combination with etoposide and mitoxantrone is tolerable and shows significant activity in relapsed and refractory acute leukemia in adults.

Background T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) and programmed cell death protein 1 (PD-1) are important inhibitory receptors that associate with T cell exhaustion in acute myeloid leukemia (AML). In this study, we aimed to determine the underlying transcriptional mechanisms regulating these inhibitory pathways. Specifically, we investigated the role of transcription factor B lymphocyte-induced maturation protein 1 (Blimp-1) in T cell response and transcriptional regulation of TIGIT and PD-1 in AML. Methods Peripheral blood samples collected from patients with AML were used in this study. Blimp-1 expression was examined by flow cytometry. The correlation of Blimp-1 expression to clinical characteristics of AML patients was analyzed. Phenotypic and functional studies of Blimp-1-expressing T cells were performed using flow cytometry-based assays. Luciferase reporter assays and ChIP assays were applied to assess direct binding and transcription activity of Blimp-1. Using siRNA to silence Blimp-1, we further elucidated the regulatory role of Blimp-1 in the TIGIT and PD-1 expression and T cell immune response. Results Blimp-1 expression is elevated in T cells from AML patients. Consistent with exhaustion, Blimp-1+ T cells upregulate multiple inhibitory receptors including PD-1 and TIGIT. In addition, they are functionally impaired manifested by low cytokine production and decreased cytotoxicity capacity. Importantly, the functional defect is reversed by inhibition of Blimp-1 via siRNA knockdown. Furthermore, Blimp-1 binds to the promoters of PD-1 and TIGIT and positively regulates their expression. Conclusions Our study demonstrates an important inhibitory effect of Blimp-1 on T cell response in AML; thus, targeting Blimp-1 and its regulated molecules to improve the immune response may provide effective leukemia therapeutics. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0486-z) contains supplementary material, which is available to authorized users.

Introduction: Non-myeloablative conditioning regimens enable elderly and younger patients with significant comorbidities to undergo allogenic stem cell transplant (allo SCT). These regimens allow for the benefit of graft-versus-leukemia (GVL) effect with reduced toxicity. A retrospective analysis of 94 patients with acute myeloid leukemia (AML) who received a non-myeloablative transplant with fludarabine (25 mg/m2 days -7 through -3)/cyclophosphamide (1000 mg/m2 days -7 and -6) (Flu/Cy) conditioning was conducted to evaluate patient outcomes. Methods: Ninety-four patients with AML underwent non-myeloablative allo SCT with Flu/Cy conditioning at Penn State Cancer Institute between 2007 and 2015 were retrospectively analyzed. The patients were deemed ineligible for myeloablative or reduced intensity conditioning transplants due to advanced age or significant comorbidities. Most unrelated donor patients (n=69) received tacrolimus/sirolimus/methotrexate as graft-versus-host disease (GVHD) prophylaxis. Overall survival (OS) and disease-free survival (DFS) were assessed with Kaplan-Meier survival curves and log rank tests, and non-relapse mortality (NRM) and relapse rates (RR) were assessed with cumulative incidence of competing events and Gray test. Cox proportional hazard regression with stepwise selection was used for multivariate analysis. Results: Patients with AML underwent Flu/Cy conditioning for allo SCT from either a matched related donor (MRD) (n =16), matched unrelated donor (MUD) (n=54), or mismatched unrelated donor (MMUD) (n=24). Evaluation of AML status immediately prior to allo SCT identified 79 patients in complete remission, 10 patients with non-remission, and 5 patients with primary induction failure. There were 11 patients with hematopoietic cell transplantation specific comorbidity index (HCT-CI): 0, 14 patients with HCT-CI: 1-2, and 69 patients with HCT-CI: ≥3. Post-transplant analysis identified: 48 patients with grade 0, 31 patients with grade I, and 15 patients with grade II-IV acute graft-versus-host disease (aGVHD). Furthermore, 52 patients were without chronic GVHD (cGVHD), 15 experienced limited cGVHD, and 13 experienced extensive cGVHD. One-year OS and DFS were 41.3% (95% CI: 31.3-51%) and 27.7% (95% CI: 18.7-37.4%), respectively. One-year NRM and RR were 12.9% (95% CI: 0.68-21%) and 59.5% (95% CI: 48.2-69.1%). There were no statistically significant differences in NRM between patients with varying comorbidity indices. One-year OS for patients in complete remission versus non-remission prior to transplant was 45.3% versus 20.0% (p Conclusions: The primary endpoint for this retrospective analysis was OS following non-myeloablative allo SCT for AML utilizing Flu/Cy conditioning. Although RR was high (59.5%), this conditioning regimen resulted in rapid stem cell recovery and low NRM. There were no significant differences in NRM between patients with lower (0, or 1-2) and higher (≥3) HCT-CI and patients receiving MRD, MUD or MMUD. This confirms that Flu/Cy conditioning is safe, especially for patients with multiple comorbidities and receiving MMUD. This analysis also identified a significant improvement in OS for patients with grade I as compared to grade II-IV aGVHD. Also, patients with cGVHD showed a significantly reduced RR. Collectively, the improved survival for patients with grade I aGVHD and cGVHD may be associated with a more robust GVL effect. Additionally, patients who achieve a higher chimerism showed a reduced RR. This study highlights the tolerability of Flu/Cy and the importance of balancing GVHD and GVL effects; future studies are needed to evaluate the addition of maintenance therapies to overcome higher RR occurring with this regimen. Disclosures Naik: Celgene: Other: Advisory board. Zheng:Pfizer: Research Funding. Claxton:Daiichi Sankyo Co. and Ambit Biosciences Corp, Astellas Pharma, Novartis Pharmaceuticals, Incyte Corporation, Cyclacel Pharmaceuticals, Inc, Celegene Corporation, Medimmune, Inc, Merck Sharp & Dohme Corp., Gilead Sciences, Inc.: Research Funding.

// Emmanuel K. Teye 1, 2 , Abigail Sido 1, 2 , Ping Xin 1, 2 , Niklas K. Finnberg 3 , Prashanth Gokare 3 , Yuka I. Kawasawa 4, 5, 6 , Anna C. Salzberg 4, 5, 6 , Sara Shimko 1, 2 , Michael Bayerl 7 , W. Christopher Ehmann 1, 2 , David F. Claxton 1, 2 , Witold B. Rybka 1, 2, 7 , Joseph J. Drabick 1, 2 , Hong-Gang Wang 8 , Thomas Abraham 9, 10 , Wafik S. El-Deiry 3 , Robert A. Brodsky 11 , Raymond J.Hohl 1, 2 , Jeffrey J. Pu 1, 2, 7 1 Penn State Hershey Cancer Institute, Penn State University College of Medicine, Hershey, Pennsylvania, USA 2 Department of Medicine, Penn State University College of Medicine, Hershey, Pennsylvania, USA 3 Department of Hematology/Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA 4 Institute for Personalized Medicine, Penn State University College of Medicine, Hershey, Pennsylvania, USA 5 Department of Pharmacology, Penn State University College of Medicine, Hershey, Pennsylvania, USA 6 Department of Biochemistry and Molecular Biology, Penn State University College of Medicine, Hershey, Pennsylvania, USA 7 Department of Pathology, Penn State University College of Medicine, Hershey, Pennsylvania, USA 8 Department of Pediatrics, Penn State University College of Medicine, Hershey, Pennsylvania, USA 9 Department of Neural and Behavioral Science, Pennsylvania State University, Hershey, Pennsylvania, USA 10 Microscopy Imaging Facility, Pennsylvania State University, Hershey, Pennsylvania, USA 11 Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Correspondence to: Jeffrey J. Pu, email: jeffreypu@gmail.com Keywords: PIGN gene expression aberration, MDS, AML with myelodysplasia-related changes (AML-MRC), genomic instability, leukemogenesis Received: November 25, 2016 Accepted: January 11, 2017 Published: February 07, 2017 ABSTRACT Previous studies have linked increased frequency of glycosylphosphatidylinositol-anchor protein (GPI-AP) deficiency with genomic instability and the risk of carcinogenesis. However, the underlying mechanism is still not clear. A randomForest analysis of the gene expression array data from 55 MDS patients (GSE4619) demonstrated a significant (p = 0.0007) correlation (Pearson r =-0.4068) between GPI-anchor biosynthesis gene expression and genomic instability, in which PIGN, a gene participating in GPI-AP biosynthesis, was ranked as the third most important in predicting risk of MDS progression. Furthermore, we observed that PIGN gene expression aberrations (increased transcriptional activity but diminished to no protein production) were associated with increased frequency of GPI-AP deficiency in leukemic cells during leukemic transformation/progression. PIGN gene expression aberrations were attributed to partial intron retentions between exons 14 and 15 resulting in frameshifts and premature termination which were confirmed by examining the RNA-seq data from a group of AML patients (phs001027.v1.p1). PIGN gene expression aberration correlated with the elevation of genomic instability marker expression that was independent of the TP53 regulatory pathway. Suppression/elimination of PIGN protein expression caused a similar pattern of genomic instability that was rescued by PIGN restoration. Finally, we found that PIGN bound to the spindle assembly checkpoint protein, MAD1, and regulated its expression during the cell cycle. In conclusion, PIGN gene is crucial in regulating mitotic integrity to maintain chromosomal stability and prevents leukemic transformation/progression.

High-dose melphalan (HD-Mel) is considered the current standard of care among the preparative regimens used in autologous peripheral blood stem cell transplantation (SCT) for multiple myeloma (MM), but optimal time and schedule of administration is not defined. We retrospectively analyzed outcomes and toxicities of HD-Mel administered on day -2 vs. day -1 before autologous stem cells infusion. A total of 138 consecutive MM patients treated at Penn State Hershey Cancer Institute between 2007 and 2010 were included in this study. No difference in time to hematopoietic recovery, common SCT-related toxicities, and clinical outcomes was seen between patients who received HD-Mel on day -2 (group A, n = 47), and those who received it on day -1 (group B, n = 91). Prompt and full hematopoietic recovery occurred even when stem cells were infused between 8 and 24 h after completion of chemotherapy. In the absence of prospective and randomized data, we conclude that a single I.V. infusion of HD-Mel on day -1 is a safe and effective practice, and the so-called 'day of rest' before the transplant appears not to be necessary.

Treatment for elderly AML patients who are unfit for intensive chemotherapy remains challenging. Induction with hypomethylating agents (HMA) are the most commonly used approach, however the rate of complete remission (CR) is only around 20% and median survival of 7-12 months. Clearly how to improve the clinical outcome in this patient population is an unmet need. Immunotherapy is promising in cancer treatment. A recent study demonstrated that HMA enhanced PD-1 pathway in MDS and AML patients (Yang et al., Leukemia 2014), suggesting a role of combining HMA and immunotherapy in leukemia therapeutics. To optimize this strategy, it is crucial to understand the interaction between HMA and immune response in AML. Here we performed both phenotypic and functional analysis on clinical samples collected from AML patients undergoing treatment of decitabine (DAC), a broadly used HMA. We aim to determine 1) how DAC influences the immune system; and 2) whether the immune status in AML patients predict the response to DAC treatment. Total of 22 peripheral blood samples from 11 AML patients receiving DAC were examined. Samples were collected at the time of prior to and 1-month post DAC treatment. We conducted a comprehensive phenotypic analysis of immune markers characterizing each immune component (T cells, B cells, NK, NKT, monocytes, myeloid-derived suppressor cells, and dendritic cells) and expression patterns of co-stimulatory or inhibitory molecules. In addition, perforin, granzyme B and cytokine release in response to anti-CD3/CD28 stimulation were examined to determine the functional status of T cells. A total of 45 markers separated in 8 overlapped staining panels were tested by flow cytometry. We first assessed the impact of DAC on the immune response in AML. Comparing the paired samples prior to vs. post treatment from the same patients, we observed an increase of the percentage of NK cells, as well as their expression of granzyme B and perforin upon DAC treatment. In contrast, DAC appears to cause CD8 T cell suppression as CD8 T cells from post-DAC samples showed higher PD-1 expression whereas lower IFN-γ production. We next investigated the predictive value of immune status for the response to DAC treatment. Among the 18 evaluable samples, we defined 10 responders (CR/Cri/PR) vs. 8 non-responders (treatment failure) based on the ELN 2017 recommendations. The correlation of immune characteristics to the clinical response was carefully examined. We started with an unsupervised principal component analysis (PCA), which revealed a distinguished pattern between the responders and non-responders (Fig. 1A). This encouraging observation suggests an association of immune signature to clinical outcome. Subsequent analysis demonstrated a major contribution of CD8 T cells to the association. We then focused on the phenotypic and functional analysis of CD8 T cells in responders vs. non-responders. We found a significantly higher percentage of terminal differentiated cells in non-responders compared with that of responders. Importantly, in the perspective of phenotypes, hierarchical clustering on markers of CD8 T cells divided samples into two major clades, 7 of 8 samples (87.5%) were responders on one branch and 7 of 10 samples (70%) were non-responders on the other. Most responders expressed lower frequency of inhibitory receptors and higher frequency of stimulatory receptors, whereas non-responders showed the opposite trend (Fig. 1B). The significant difference of these receptors was validated in paired t test analysis. Functional studies also showed more IFN-γ production in responders. Interestingly the intracellular staining of perforin and granzyme B was higher in non-responders, likely due to exhausted T cells that are unable to release these particles extracellularly. Collectively, our study discovered the vital role of the immune signature in predicting clinical outcome in AML. High functional CD8 T cell status, manifested by more capability of IFN-γ production, enhanced stimulatory molecule and low inhibitory receptor expression, associated with effective clinical response to DAC treatment. In addition, CD8 T cell function was down-regulated upon DAC treatment. Our results provide a strong rationale for integrating immunotherapy into HMA treatment in AML, also highlight the importance of immune signature analysis in the future trial design for this clinical setting. Disclosures No relevant conflicts of interest to declare.

High-dose melphalan is considered the current standard of care among the preparative regimens used in peripheral blood autologous SCT (ASCT) for multiple myeloma (MM). We report the results of a single ASCT in 79 MM patients using the BU/CY conditioning regimen, with BU 1 mg/kg p.o. or 0.8 mg/kg i.v. every 6 h x 16 doses, and CY 60 mg/kg per day i.v. for 2 days. ASCT was carried out in first (62%) or subsequent remission/refractory disease (38%). For an overall RR of 86%, 48 and 20 patients achieved PR and CR, respectively. At a median follow-up of 41 months (range 2-132 months), the estimated median OS and PFS were 45 months (95% confidence interval (CI)=38-92) and 20 months (95% CI=15-25), respectively. The BU/CY regimen was well tolerated, and transplant-related mortality was 4%. Clinical outcomes of the BU/CY regimen are not superior to those obtained in historical controls with high-dose melphalan followed by a single ASCT. Therefore, considering even the greater complexity of administration of the BU/CY regimen compared with that of single-agent melphalan, we believe the latter should remain the conditioning regimen of choice for ASCT in MM.

e18001 Background: Umbilical cord blood (UCB) is rich in hematopoietic stem cells. Because of its loose HLA-matching requirement and abundant resource available, UCB transplantation (UCBT) is emerg...

Background Few therapeutic options are available for severe, life-threatening, refractory autoimmune hemolytic anemia. Case report A 53-year-old 110-kg man was seen with acute onset of symptomatic severe anemia with syncope, unstable angina, and jaundice. His nadir Hct was 8.3 percent with a peak total bilirubin of 44 mg per dL. The DAT was positive but the IAT was negative. Elution studies demonstrated an IgG pan-agglutinin antibody reactive at 37 degrees C. Treatment with high-dose corticosteroids and IVIG was instituted. An accessory spleen measuring 2 cm was identified and surgically removed, but the patient continued to have intense hemolysis. Cyclophosphamide at 200 mg per day was started. Apheresis with a staphylococcal protein A immunoadsorption column (Prosorba, Cypress Bioscience, Inc.) was initiated on Day 18 and was performed twice weekly for a total of six treatments. Cyclophosphamide was continued for a total of 14 days. His transfusion requirement ceased by the third immunoadsorption treatment. Forty units of RBCs were required over 23 days in an attempt to maintain a Hct greater than or equal to 15 percent. Conclusion Refractory autoimmune hemolysis can be a life-threatening event. The patient did not achieve a response until after several different therapeutic modalities were instituted, including plasmapheresis with a staphylococcal protein A column (Prosorba). A complete response continues to be durable for more than 1 year after therapy.

A multistep HDC regimen was designed as first-line chemotherapy for MBC. Twenty-four patients with MBC and no previous chemotherapy for metastatic disease were treated with high-dose cyclophosphamide (5000 mg/m 2 ), and etoposide (1000 mg/m 2 ) (CyVP16), followed by granulocyte colony-stimulating factor (G-CSF). Peripheral blood stem cells (PBSCs) were collected. Subsequently patients received cyclophosphamide (6000 mg/m 2 ), thiotepa (500 mg/m 2 ) and carboplatin (800 mg/m 2 ) (CTCb) with hematopoietic rescue. Upon recovery from hematopoietic and gastrointestinal toxicity three cycles of doxorubicin (50 mg/m 2 ) and taxol (150 mg/m 2 ) were delivered. After CyVP16 42% of patients developed neutropenic fevers. There was one documented episode of bacteremia. Patients received CTCb 32 days after starting CyVP16. After CTCb the median number of days to ANC >5 × 10 9 /1 was 10 and to a platelet count >20 × 10 9 /1 was 14. Neutropenic fevers developed in 16 patients. There were no hemorrhagic episodes. A total of 69 cycles of doxorubicin and taxol were delivered (87% of planned). The median time from PBSC infusion to the first cycle was 38 days. The median time to the second cycle was 27 days and to the last cycle was 24 days. One patient developed congestive heart failure. Two episodes of neutropenic fevers were observed. No toxicity-related deaths were observed. Grafts are stable at 6 months post transplantation. This multistep regimen is feasible with acceptable toxicity.

Transplantation of marrow from unrelated donors is associated with an increased incidence and severity of graft-versus-host disease (GVHD). In an attempt to minimize GVHD without compromising engraftment, unrelated donor marrow was depleted of lymphocytes by counterflow centrifugal elutriation (CCE), and a fixed dose of 0.5 x 10(6) CD3+ T cells/kg, as measured in real time by flow cytometry, was added back to the graft. Patients received cyclosporine (CYA) and corticosteroids for GVHD prophylaxis and to facilitate engraftment. In the first cohort (study I), 7 patients received busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) (CY) and one patient received CY (200 mg/kg) + 1260 cGy fractionated TBI. Of 6 who were evaluable for both engraftment and rejection, 4 rejected their graft. The study was terminated, and the protocol was modified (study II) by the addition of antithymocyte globulin (ATG) to the pre-BMT and post-BMT therapy. Twelve patients received CY + TBI as above plus ATG given pre-BMT and post-BMT. Ten of twelve who received ATG engrafted. Twelve patients from studies I and II were evaluable for acute GVHD. Two developed grade I acute GVHD. Two patients developed grade II acute GVHD, 2 patients developed grade III GVHD, and 1 patient developed grade IV acute GVHD. Two of three cases of acute GVHD (grade II) occurred later than day 100 after BMT concomitant with reduction of immunosuppressive therapy. The rate of engraftment was significantly higher in study II (p = .054). In numbers of CD34+ cells infused, numbers of CFU-GM infused, and numbers of nucleated cells did not correlate with engraftment. We conclude that (a) in contrast to the results seen in recipients of marrow from HLA-matched sibling donors, the depletion of unrelated donor marrow of all but 0.5 x 10(6) CD34+ cells/kg together with CYA + corticosteroids was not sufficient to facilitate engraftment. The use of a more immunosuppressive regimen containing TBI and ATG appeared to improve engraftment. (b) The reduction of the graft T cell dose to 0.5 x 10(6) CD34+ cells/kg resulted in a higher incidence of acute GVHD than that seen in recipients of marrow from genotypically identical donors whose marrow was similarly processed.

The combination of busulfan and cyclophosphamide has seldom been employed as a conditioning regimen for patients with lymphoma. Twenty patients with relapsed or refractory lymphoma were treated with busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) (BU/CY) followed by peripheral blood stem cell rescue in 19 patients or autologous bone marrow in one patient. There were 12 females and eight males, with a median age of 48 years (range 30-65). Four patients had Hodgkin's disease, and 16 patients had non-Hodgkin's lymphoma. Disease status at the time of BU/CY was: first relapse in 10 patients (four patients with chemosensitive disease and six patients with chemoresistant disease), primary refractory disease in six patients, and more advanced disease in four patients. Excessive treatment-related toxicity was not noted. There were no cases of interstitial pneumonitis, but three cases of veno-occlusive disease occurred. At 2 years, the estimated overall survival and event-free survival are 50% and 33%. We concluded that BU/CY seems to have sufficient antilymphoma activity, is devoid of excessive toxicity and warrants further investigation in this patient population.

We evaluated early and late hematopoietic reconstitution in 27 patients with advanced lymphoma, Hodgkin's disease, and breast or ovarian cancer after treatment using high-dose/myeloablative conditioning regimens and autologous peripheral blood stem cell PBSC) transplantation. Eighteen patients (67%) received G-CSF 5 micrograms/kg/day following chemotherapy and nine (33%) were mobilized using G-CSF alone. Each patient had 7 x 10(8) mononuclear cells (MNC) per kg collected. G-CSF was administered post-PBSC infusion. While all patients showed prompt granulocyte recovery by day 14, platelet recovery failed to occur in our (15%) heavily pretreated patients with non-Hodgkin's lymphoma. Retrospective analysis in 17 patients revealed that the infused number of CD34 surface antigen-positive cells correlated with time to granulocyte (r = 0.59, P = 0.012) and platelet (r = 0.58, P = 0.021) recovery. Patients receiving the higher numbers of CD34+ cells had consistently better hematologic parameters at 11 times examined. At 180 days post-transplant, the median Hb level was 124 g/l vs 88 g/l (P = 0.004); platelet count was 202 x 10(9)/l vs 25 x 10(9)/l (P = 0.004); and neutrophil count was 3100 x 10(6)/l vs 1400 x 10(6)/l (P = 0.15). Hemoglobin strongly correlated with the CD34+ cell dose at 360 days (r = 0.90, P = 0.01). We conclude that graft CD34+ cell content appears to be an indicator of the quality of late as well as early hematopoietic function.

Human herpesvirus 6 activity (HHV-6) was studied in 15 allogeneic and 11 autologous marrow transplantation patients. After transplantation, HHV-6 was isolated from the peripheral blood mononuclear cells of 12 of 26 patients (6 allogeneic and 6 autologous). All isolates were variant B. Eleven of 26 and 12 of 19 patients showed salivary shedding of HHV-6 DNA before and after transplantation, respectively. The antibody titer increased in 7 of 26 patients. Thus, 23 of 26 patients showed evidence of active HHV-6 infection either by virus isolation, salivary shedding, or increases in antibody titers. The fraction of saliva specimens positive in 19 patients was negatively associated with their antibody titers (P= .005). The proportion of cultures positive increased after transplantation (P = .007). Sinusitis was associated with HHV-6 isolation in autologous recipients (P= .002). In allogeneic patients, active human cytomegalovirus infection was associated with HHV-6 isolation (P = .04). No association was observed between HHV-6 infection and GVHD, pneumonia, delay in engraftment, or marrow suppression. Of the 120 clinical events analyzed in 26 patients, HHV-6 was defined as a probable cause of 16 events in 9 patients based on the propinquity of HHV-6 activity and the clinical event plus the absence of other identified causes of the event.

Introduction: High-dose cytarabine consolidation chemotherapy for acute myeloid leukemia (AML) induces profound transient myelosuppression. Thrombocytopenia is a limiting factor in chemotherapy administration for maintaining dose intensity and schedule as thrombocytopenic-related hemorrhage may contribute to significant morbidity and mortality. Therapeutic and prophylactic platelet (plt) transfusions remain the treatment of choice but the benefits typically last only 3-7 days, are expensive, time consuming, and may induce alloimmunization and transmit infection. PrE0901 was a phase I multi-center study to evaluate the safety and toxicity of eltrombopag, an orally bioavailable thrombopoietin (TPO) receptor agonist, for plt recovery in AML patients (pts) receiving consolidation therapy. Methods: The primary objectives were to determine the safety, tolerability, and optimal dosing of eltrombopag while describing the kinetics of plt recovery in AML pts receiving intensive consolidation. Plt recovery was defined as a sustained plt count of ≥ 20 x 109/L following the nadir with no plt transfusions in prior 7 days or a continued upward trend in plt count for at least 2 successive measurements despite no plt transfusions in 48 hours. We used a standard 3+3 design utilizing a novel dose escalation/de-escalation strategy for pts in either first or second complete remission, ≥18 and ≤70 years of age, who had an ECOG performance status of 0-2. Cytarabine was administered at 3 g/m2 (1.5 g/m2 for pts >60 years) IV over 3 hours twice daily on days 1,3,5. Eltrombopag at assigned dose was administered with a first cycle of cytarabine only. Planned eltrombopag dose levels -2, -1, 1, 2, 3, 4, and 5 were 50, 100, 150, 150, 150, 200, and 300 mg, respectively (Table 1). Eltrombopag was to start on days X, X, 3, -1, -5, -5, and -5 relative to day of cytarabine start and levels -2, -1, 1, 2, 3, 4, and 5, respectively, with day X being variable depending on timing of observed dose limiting toxicities (DLTs). The eltrombopag was continued until plt recovery or for up to 35 consecutive days, whichever occurred first. An exploratory objective was to determine if eltrombopag had an effect on TPO and EPO blood concentrations in this setting. Results: Accrual began on July 31, 2012 and the trial closed on January 8, 2016 upon recommendation after an independent Data and Monitoring Committee reviewed a separate trial of eltrombopag therapy in MDS which demonstrated futility. 104 pts were screened. 54 declined participation and 35 were deemed medically ineligible. A total of 15 eligible pts were enrolled; 14 were treated on study with one pt being withdrawn prior to starting treatment due to infection. Three pts were treated at each of dose levels 1-4 and two were treated on dose level 5. No DLTs were observed. Median time to plt recovery of all pts treated on study was 22.5 (range 16-43) days. Pts in cohort 1 dosed with eltrombopag 150 mg daily starting on day 3 of consolidation demonstrated the fastest plt recovery (median =19 days; range 19, 19, 19) compared to cohort 2 who received 150 mg daily starting on day -1 of consolidation and recovered the slowest (median 23 days; range 16, 23, 43). Dose escalation in terms of starting eltrombopag further in advance of chemotherapy exposure as within cohort 3 (150 mg starting day -5) was associated with a median plt recovery of 27 (range 22, 27, 27) days. Eltrombopag dose intensification with cohort 4 at 200 mg daily and cohort 5 at 300 mg daily was associated with median plt recoveries of 24 (range 18, 24, 26) and 23 (range 19, 27) days, respectively. Kinetics of plt recovery for all cohorts are represented in Figure 1 and duration of eltrombopag exposure for each individual patient is represented in Table 2. Conclusions: Endogenous cytokine (TPO and EPO) levels varied inversely with plt and hematocrit values and did not appear to be affected by eltrombopag dose / schedule (details to be provided at ASH Annual Mtg). Unusually high screen fail numbers warrant examination to guide future trial design in the post-remission setting. The addition of eltrombopag to high-dose cytarabine consolidation therapy was well-tolerated and no DLTs were observed. The results for cohort 1 [eltrombopag 150 mg daily starting on day 3 of consolidation] demonstrated the fastest plt recovery. Further investigation is needed to define the optimal role, dose, and schedule of eltrombopag in the treatment of chemotherapy associated myelosuppression. Disclosures Strickland: Alexion Pharmaceuticals: Consultancy; Ambit: Consultancy; Baxalta: Consultancy; Boehringer Ingelheim: Consultancy, Research Funding; CTI Biopharma: Consultancy; Daiichi Sankyo: Consultancy; Sunesis Pharmaceuticals: Consultancy, Research Funding; Abbvie: Research Funding; Astellas Pharma: Research Funding; Celator: Research Funding; Cyclacel: Research Funding; GlaxoSmithKline: Research Funding; Karyopharm Therapeutica: Research Funding; Sanofi: Research Funding.

Background: Chronic GVHD contributes to morbidity and mortality after allogeneic transplantation. Several phase 2 and 3 open label studies suggested that ATLG (formerly referred to as ATG-F) reduces cGVHD without impacting relapse or survival. This report describes the first prospective randomized double blind phase 3 trial to assess the effect of ATLG (Neovii) when added to tacrolimus / methotrexate prophylaxis on cGVHD free survival. Patients and Methods: This study was conducted at 27 sites in the United States and Australia. 260 patients were randomized (132 placebo, 128 ATLG). 6 patients who were randomized never received ATLG/placebo (4 placebo and 2 ATLG). Data presented include the 128 placebo and 126 ATLG patients who underwent treatment and transplant. Patients were ages 18-65 years with a diagnosis of acute myeloid (AML, 64%) or lymphoblastic (ALL, 21%) leukemia in first or subsequent complete remission or myelodysplastic syndrome (MDS, 15%) with less than 10% marrow blasts. Median age was 48 years (18-65) with 55% males. All patients received 8/8 HLA (A, B, C, DR) allele matched unrelated products (80% mobilized peripheral blood, 20% bone marrow). Myeloablative conditioning was with cyclophosphamide and total body irradiation (Cy/TBI, 27%), busulfan and cyclophosphamide (Bu/Cy, 33%), or busulfan and fludarabine (Bu/Flu, 40%). All patients received tacrolimus starting on Day -1 and standard mtx on days 1, 3, 6, and 11 for GVHD prophylaxis. Patients received ATLG or placebo on Days -3, -2, -1 at an ATLG dose of 20 mg/kg per day (60 mg/kg total). The primary endpoint of the study was moderate/severe chronic GVHD free survival. cGVHD was scored by the investigator and then confirmed or overturned by an independent adjudication committee. Secondary endpoints included engraftment, acute GVHD , moderate/severe cGVHD, non-relapse mortality, GVHD and relapse free survival (GRFS), progression free (PFS) and overall survival (OS). The impact of ATLG on immune reconstitution was assessed in 91 patients. Results: Treatment arms were balanced with respect to age, diagnosis, remission status, cytogenetics, graft source (PB or BM), CMV serostatus and conditioning regimen. Median follow up of survivors was 745 days (61, 1425). Regarding the primary endpoint, there was no difference in 2 year moderate/severe cGVHD free survival between ATLG and placebo (48 vs 44%, p = 0.57), nor was there a difference in GRFS (Table 1). Neutrophil engraftment at Day 30 was less in the ATLG arm (85% vs 95%, p=0.00001). Grade 2 or higher infusion reactions were higher in the ATLG arm (16% vs 2.3%, p=0.0001). Reactivation of CMV was similar (27% vs 31%, p=0.58). The Day 180 cumulative incidence of grades 2-4 acute GVHD was lower in the ATLG arm (23 vs 40%, p = 0.003) as was the 2-year cumulative incidence of moderate/severe cGVHD (12 vs 33%, p = 0.000006). However, both 2-year OS and PFS were lower in ATLG treated patients (58 v 76% and 47 vs 67%, respectively) due in part to a higher incidence of relapse (32 vs 19%, p=0.068). Multivariable models confirmed that ATLG was associated with inferior OS, HR 1.85 (1.1-2.9, p=.0075) and PFS, HR 1.63 (1.1-2.41, p=0.015). We then conducted an unplanned post-hoc analysis and discovered a striking influence of conditioning regimen on outcomes. There were no differences in OS or PFS between ATLG and placebo arms in patients receiving Bu/Cy or Bu/Flu but a dramatic difference in patients receiving Cy/TBI conditioning (OS, 88% vs 48% p =0.006 and PFS, 75 vs 29%, p=0.007 in placebo and ATLG arms, respectively). 91 patients (44 ATLG, 47 placebo) participated in the immune reconstitution study. ATLG treatment was associated with lower CD3, CD4, and CD8 counts at Days 30, 100, and 360, respectively. In turn lower CD3, CD4, and CD8 counts assessed as time dependent variables were all associated with inferior OS and PFS as well as higher NRM (data not shown). Conclusion: In this first ever prospective, randomized, double blind trial of ATLG added to tacrolimus /methotrexate in recipients of matched unrelated donor grafts after myeloablative conditioning, there was no significant difference in 2 year moderate/severe cGVHD free survival or GRFS. ATLG did significantly reduce grades 2-4 acute and moderate/severe chronic GVHD. However, overall survival was higher in the placebo arm, driven mainly by patients who received Cy/TBI conditioning. Further analyses are needed to understand the proper role for ATLG in HCT. Disclosures Soiffer: Juno: Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Porter:Genentech: Employment; Novartis: Patents & Royalties, Research Funding. Jagasia:Therakos: Consultancy. Szer:Alnylam: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ritz:Kiadis: Membership on an entity's Board of Directors or advisory committees. Glavin:Neovii Biotech: Employment. Chen:Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding.

There is little data comparing the activity and toxicity of donor lymphocyte therapy with granulocyte (G)-CSF-mobilized cells (G-donor lymphocyte infusion (DLI)) with the conventionally collected DLI (C-DLI) after allogeneic blood or marrow transplantation. We retrospectively evaluated 67 patients to compare the efficacy and toxicity of GCSF-mobilized DLI with C-DLI in the treatment of relapse of malignant disease or poor donor engraftment post transplant. We assessed clinical outcomes that may represent the immunological outcome of DLI. The median OS was 210 days (range 3-2436 days), 291 days (range 17-1491 days) in the G-DLI group (15 patients) and 207.5 days (range 3-2436 days) in the C-DLI group (52 patients). The median PFS time was 72 days (range 8-1491 days) in the G-DLI group vs 82 days (range 1-2436 days) in the C-DLI group. Rates of post DLI GVHD and improvement in donor engraftment were similar in the G-DLI and C-DLI groups. We conclude that G-DLI appears to have similar therapeutic activity to that seen with C-DLI, and where such cells are available they may be substituted for conventional donor lymphocytes.

The CD34+ cell fraction of bone marrow and blood contains the hematopoietic stem cells required for marrow reconstitution following myeloablative therapy. Because they are present in small numbers, accurate quantification is often difficult. We have developed a reproducible and sensitive flow cytometric method for CD34+ enumeration of both bone marrow harvests and peripheral blood stem cell collections. The total numbers of harvested cells are enumerated by particle counting. A measured aliquot is stained with two FITC-labeled anti-CD34 antibodies, one directed against 8G12 and the other against QBend epitope. To eliminate cells committed to mature lineages (lin+), the suspension is counterstained with a cocktail of PE-labeled antibodies including CD3 (T cells), CD19 (B cells), CD11b (neutrophils), and CD14 (monocytes). Particles < μm in diameter are excluded by use of a standard bead gate. Regions are established using unstained U937 cells to set the vertical axis and PE stained U937 cells for the horizontal axis. Because of the low numbers of CD34+ cells, 20,000 events/sample are analyzed. Dilutions of KG-1A tumor cells (CD34+) in U937 cells showed a threshold of detection of 0.1% CD34+ lin- cells. Duplicate samples varied by

Allogeneic bone marrow transplants with major ABO incompatibility may be associated with delayed erythroid engraftment. A case of a male patient with erythroleukemia (blood group O) who received a bone marrow transplant from an HLA-identical sibling (blood group AB) is reported. The bone marrow transplantation was followed by normal myeloid and megakaryocytic engraftment, but pure red cell aplasia was present for more than 230 days after bone marrow transplant. Despite documentation of an elevated endogenous erythropoietin level (360 mU/mL; normal value, < 19 mU/mL) during the period of absent erythropoiesis, erythroid engraftment was observed soon after the initiation of human recombinant erythropoietin at a dose of 50 U per kg daily. This experience suggests that high-dose erythropoietin may stimulate sufficient production of erythroid precursors to overcome circulating inhibitors resulting in the correction of pure red cell aplasia.

The majority of adult patients with acute leukemia eventually die of their disease, owing to either resistance to or complications of therapy. It is hoped that this situation will improve as chemotherapy is intensified during the initial phases of therapy and as supportive care continues to improve. Maximal dose intensification can be achieved using marrow ablative chemotherapy or radiotherapy followed by hematopoietic stem cell transplantation. Recent reports from centers around the world have shown that this form of therapy is truly effective; increasing proportions of patients with acute leukemia are cured of their disease.

The ECOG-ACRIN Cancer Research Group Led Trial, E2902, was a randomized phase III North American Leukemia Intergroup trial of the Farnesyl Transferase Inhibitor , R115777(Tipifarnib), as maintenance therapy for acute myeloid leukemia (AML). Patients (pts) with AML in remission after requiring salvage therapy (primary induction failure or second or subsequent remission) or over the age of 60 in first remission were eligible. Pts were eligible if they were in a complete remission (CR) or complete remission without platelet recovery (CRp) as confirmed by bone marrow examination within 2 weeks prior to randomization. Consolidation or post remission therapy prior to enrollment was allowed but not required. Pts who had received an allogeneic transplant in this remission were ineligible. Adequate blood counts (absolute neutrophil count >= 1000/mm3 and platelet count >=50,000/mm3), and normal renal, and hepatic function were required within 2 weeks of randomization. Pts were randomized to either receive tipifarnib twice daily (BID) or to observation. Pts were stratified by remission ( CR1 vs > CR1) and whether or not they received consolidation therapy in the current remission. The main objective of this study was to compare disease free survival(DFS) in the two groups. Between August 2004 and December 2009, 144 pts were accrued to the study. The median age of the pts was 69 (range 28-86). Seventy-three pts (51%) were male and 135 pts (94%) were white. The majority of pts enrolled on the study (70%) were in first CR, and had post remission chemotherapy (56%) prior to randomization. Seventy-three pts were enrolled onto the treatment arm (Arm A) and 71 pts were enrolled onto the observation arm (Arm B). When the study was initially opened, pts on Arm A were treated with tipifarnib at a dose of 400 mg BID with dose reductions and interruptions defined per protocol for toxicity. After the first planned interim analysis, based on toxicity data, the ECOG Data Monitoring Committee recommended that the starting dose be decreased to 300 mg BID. While non-hematologic toxicity was acceptable, significant hematologic toxicity was seen in pts treated at an initial dose of either 400 mg or 300 mg BID. Of note, hematologic toxicity was also seen on the observation arm although less frequently than with both treatment doses. The primary endpoint of the study was analysis of DFS on an intent to treat basis. The median DFS for arms A and B are 8.87 months and 5.26 months respectively ( p=0.058, HR 0.760). When restricted to eligible patients only(n=134 ), the DFS for arms A and B are 10.81 vs 5.26 months, respectively ( p=0.019, HR 0.690). An unplanned subgroup analysis was performed by gender. The median DFS for male pts (n=73) is 5.36 vs 5.91 months for arms A and B respectively (p=0.868, HR 1.320) and 12.09 vs 3.91 months for female pts (n=71)(p=0.0002, HR=0.408) In a multivariate Cox proportional hazards model to adjust for baseline clinical variables, there is a significant interaction between treatment and gender. (Table 1) Similar results were noted for OS (Figure 1). ECOG ACRIN Trial E2902 evaluated the potential role of Tipifarnib ( R115777) in patients with AML in remission with a high risk of relapse. While the primary endpoint for improved DFS was not reached when evaluating all randomized pts, when restricted to eligible patients only, there is a statistically significant improvement in DFS seen. Moreover, an unplanned subgroup analysis by gender demonstrates an improvement in DFS and OS for females who were enrolled on the study which persists on multivariate analysis. Possible explanations include the use of flat dosing rather than BSA based dosing on this study. However futher evaluation of patient and disease characteristics, dosing and toxicity profiles of responders and nonresponders is needed. Given what appears to be a potentially clinically relevant benefit, further evaluation of this agent is warranted. Table 1. Multivariate Cox Proportional Hazards Model for DFS: All Randomized Patients Hazard Ratio (95% CI) P value Arm B vs A 2.559(1.547,4.236) 0.00025 Male vs Female 2.005(1.228,3.274) 0.00544 CR1 vs >CR1 0.447 (0.305,0.656) 0.00004 Figure 1. (A) DFS (B) OS in Female Patients Figure 1. (A) DFS (B) OS in Female Patients Disclosures Rowe: BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; BioLineRx Ltd.: Consultancy. Larson:Astellas: Consultancy, Research Funding.

Prognosis of leukemia relapse post allogeneic stem cell transplantation (alloSCT) is poor and effective new treatments are urgently needed. T cells are pivotal in eradicating leukemia through a graft versus leukemia (GVL) effect and leukemia relapse is considered a failure of GVL. T-cell exhaustion is a state of T-cell dysfunction mediated by inhibitory molecules including programmed cell death protein 1 (PD-1) and T-cell immunoglobulin domain and mucin domain 3 (TIM-3). To evaluate whether T-cell exhaustion and inhibitory pathways are involved in leukemia relapse post alloSCT, we performed phenotypic and functional studies on T cells from peripheral blood of acute myeloid leukemia patients receiving alloSCT. Here we report that PD-1hiTIM-3+ cells are strongly associated with leukemia relapse post transplantation. Consistent with exhaustion, PD-1hiTIM-3+ T cells are functionally deficient manifested by reduced production of interleukin 2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). In addition, these cells demonstrate a phenotype consistent with exhausted antigen-experienced T cells by losing TN and TEMRA subsets. Importantly, increase of PD-1hiTIM-3+ cells occurs before clinical diagnosis of leukemia relapse, suggesting their predictive value. Results of our study provide an early diagnostic approach and a therapeutic target for leukemia relapse post transplantation.

Non-myeloablative conditioning has extended the use of allogeneic haematopoietic transplant to many previously ineligible patients. We added the immunosuppressive and antitumour agent sirolimus (rapamycin) to an established transplant regimen of fludarabine 25 mg/m(2) days -7 through -3 and cyclophosphamide 1000 mg/m(2) days -7 and -6, with tacrolimus and methotrexate immunoprophyllaxis. A total of 23 patients with acute myelogenous leukaemia (AML) were treated, with a median age of 59 years (range: 28-72) at transplant. Only seven patients in total were in complete remission prior to transplantation. Nine patients were in chemotherapy-refractory progression and seven were primarily refractory to induction therapy. Six patients received matched sibling, 11 unrelated donor, 1-5/6 matched and five haploidentical (haplo - three of six or four of six matched) transplants. The haplo-recipients also received antithymocyte globulin, all patients engrafted. Only two, both recipients of haploidentical cells, have died from transplant-related causes. Twelve of 23 patients survived at 198-1162-d post-transplant (median 578). Four of 12 survivors relapsed at 83, 88, 243 and 508 d and three were in remission after chemotherapy and donor lymphocyte infusion. Although follow up is short, this data suggests that non-myeloablative haematopoietic cell transplantation with sirolimus (rapamycin)-based immunosuppression may provide disease control over several years in some patients with advanced and poor prognosis AML.

Immunotherapy consisting of withdrawal of immunosuppression and/or donor lymphocyte infusions was initiated in 14 children (10 acute lymphoblastic leukemia, three acute myeloblastic leukemia and one myelodysplastic syndrome) with an increasing amount of autologous DNA (increasing mixed chimerism, inMC) detected after allogeneic hematopoietic cell transplantation (HCT). Two children were in relapse when inMC was detected, 12 remained in CR. Children with overt relapse at the time of cessation of cyclosporine A (CsA) received 'debulking' chemotherapy. One of them developed acute grade III graft-versus-host disease, converted to complete donor chimerism (CC) and achieved remission. Another patient did not respond and died due to disease progression. Among 12 children treated in remission, 11 responded with conversion to CC, seven after CsA withdrawal and four after DLI. One patient did not respond, rejected the graft and died due to pulmonary aspergillosis. In one patient, the response was transient, inMC reappeared and frank relapse occurred. One patient developed isolated CNS relapse despite conversion to CC, but achieved CR after conventional treatment. One child died in CC due to infection. No graft-versus-host disease (GvHD)-related death occurred. A total of 10 patients are alive in remission with median follow-up of 338 days. Our results support the hypothesis that chimerism-guided immunotherapy after alloHCT may prevent progression to hematological relapse.