allo-SCT For Acute Myeloid Leukemia Research Articles

Deciphering Antigen Specificity of Protective Regulatory T Cell Clones in Patients after Allogeneic Stem Cell Transplantation

Introduction: Clonal expansion of donor regulatory T cells (Tregs) in recipients of allogeneic stem cell transplantations (allo-SCT) is associated with the absence of acute graft-versus-host disease (aGVHD). Therefore, we hypothesize that antigen-specific donor Tregs prevent aGVHD. In this project, we aim to identify the cognate antigen of expanded Treg clones in patients after allo-SCT. Methods: We recruited 177 consecutive patients undergoing allo-SCT for acute myeloid leukemia without severe infection and CMV-reactivation for this study. Healthy adults matching patients' sex and age served as the control group. We sorted CD3 +CD4 +CD25 hiCD127 low Tregs from peripheral blood mononuclear cell samples collected early after transplantation (21 to 36 days, median 27 days), and we performed single-cell RNA sequencing (scRNAseq) to profile Treg transcriptome including both TCR alpha and beta chain. To determine the antigen specificity of a given TCR, we adapted a cytokine-capturing antigen-screening system (Lee and Meyerson, Sci Immunol, 2021). We built the system by 1) expressing designated TCRαβ chains in Jurkat cells, and 2) expressing cell-surface interleukin-2 capture mAb, corresponding MHC-II molecules, and CD74-fused epitope libraries in HEK293T cells serving as antigen-presenting cells (APCs). By co-culturing both modified cell lines, we can perform high-throughput screening of pooled candidates. To obtain epitope-encoding minigenes, we either used RNA fragmentation to generate candidates randomly or customized oligonucleotide synthesis to encode the tiled human proteome. To scale down the diversity of the synthetic library, we utilized NetMHCIIpan (DTU Health Tech) for MHC-II binding affinity prediction. By sequencing the CD74-fused minigene after screening out APCs with the highest cytokine intensity on the cell surface, we can decode the peptide sequence recognized by the given TCR. We have validated the cytokine-capturing antigen-screening system using a CMV-specific TCR and its cognate antigen. Results: In total, we analyzed six patients with and six patients without aGVHD. ScRNAseq showed that the FOXP3 + Treg population in patients without aGVHD shared more transcriptional similarities with healthy control as compared to patients with aGVHD. Due to lymphocytopenia, Tregs are extremely low in numbers early after allo-SCT, but clustering analysis revealed highly clonal expanded populations of Tregs with various degrees of activation. Notably, one patient without aGVHD was found to have a distinct Treg-dominant CD4 population, and a markedly abundant Treg clone (>15% of the entire Treg repertoire) was found to be parts of both FOXP3 + and FOXP3 - clusters. We then transduced the most abundant TCR into JM-T, the TCR-deficient Jurkat cell. We used an RNA-fragmented randomized library for the first screening, in which we identified one shared epitope in three independent screenings of high frequency. We then moved on to perform screenings for synthesized candidate pools predicted by NetMHCIIpan, representing human proteome and common post-allo-SCT infections. Currently, we are analyzing sequencing reads from parallel screenings and attempting direct expansion of IL-2 + sort-outs. Conclusion: By expanding our established screening system, we aim to elucidate the antigens of multiple clonal expanded TCR candidates from the same patient. Identification of specific antigens leading to clonal Treg expansion and protection from GVHD holds the potential for the development of targeted cell therapies.

Assessing the Impact of Marginalization on Survival for Patients Undergoing Allogeneic Stem Cell Transplant in Ontario, Canada

Background Allogeneic hematopoietic stem cell transplants (allo-SCT) are potentially life-saving interventions used to treat hematologic disorders. Recent studies have shown that living in rural areas may predict worse outcomes after allo-SCT. Marginalization, a term that encompasses social and health-related factors affecting individuals, is predictive of adverse health outcomes. The Ontario Marginalization Index (ON-Marg), an administrative index using variables obtained from census data, was used to identify marginalization status (quintile 1-least marginalized; quintile 5-most marginalized) encompassing four domains: residential instability (area-level concentrations of people who experience high rates of family or housing instability), material deprivation (area-level concentrations of inability for individuals and communities to access and attain basic material needs), dependency (area-level concentrations of people who do not have income from employment), and ethnic concentration (area-level concentrations of people who are recent immigrants and/or a visible minority). In a publicly-funded healthcare context, it is important to know how marginalization impacts survival for patients undergoing allo-SCT. Methods We performed a retrospective population-based study using administrative healthcare databases from Ontario, Canada. Patients were included if they were 18 years or older at the time of allo-SCT, and had undergone allo-SCT for acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) or acute lymphoblastic leukemia (ALL) between 2010 and 2022. Patients were excluded if they were a non-Ontario resident at the time of allo-SCT and as a result lacked ON-Marg data. Patients who received allo-SCT for a different indication were excluded. The primary outcome of this study was 2-year overall survival (OS) from time of transplant to death or end of the study period, based on ON-Marg quintiles. Multivariable Cox regression analysis was used to identify baseline characteristics associated with OS, with ON-Marg as the main exposure. Results A total of 1961 patients underwent allo-SCT for AML/MDS/ALL. The median age of patients undergoing allo-SCT was 55 years (IQR 43-63), and 55% of patients were male. 40% of patients had high aggregated diagnosis group (ADG) comorbidity burden, and a majority of patients (64%) lived within 50 kilometers (km) from the transplant center, with a mean distance of 33 km in the entire study population. The distribution of patients across each quintile of marginalization was not always uniform. For ethnic concentration, 24% of patients were in quintile 5, with 17% of patients in quintile 1. For dependency, 19% of patients were in quintile 5, and 25% of patients were in quintile 1. During the follow-up period, 43% of patients (n = 842) died. 2-year OS was not significantly different across all quintiles of the ethnic concentration domain of marginalization ( Figure 1A). 2-year OS was worse (HR 1.27, 95% CI 1.02-1.57) for patients in quintile 4 compared to quintile 1 of the dependency domain of marginalization ( Figure 1B). This effect persisted in multivariable logistic regression analyses accounting for other demographic variables ( Table 1). Increased age and a higher comorbidity index were significantly associated with worse survival across all domains. Patients with ALL had worse outcomes compared to patients with AML/MDS across all domains of marginalization. There was also a trend toward patients living >200 km from the transplant center having an increased risk of death compared to patients living less than 50 km from the center (HR 1.24, 95% CI 1.00-1.54). Conclusion This study shows that 2-year OS after allo-SCT for AML/MDS/ALL was not affected by marginalization quintile of ethnic concentration. This is in keeping with other published data suggesting that outcomes after allo-SCT are not impacted by socioeconomic factors such as race. Patients in quintile 4 of dependency had worse outcomes than patients in quintile 1, suggesting that employment and income support are crucial to successful patient outcomes. A majority of patients who underwent allo-SCT lived in close proximity to the transplant center. Transplant outcomes in general do not appear to vary based on marginalization status, and it is therefore crucial that all patients who may benefit receive allo-SCT regardless of demographics or distance to treatment centers.

A national study of allogeneic stem cell transplantation for adults with acute myeloid leukemia: Do gender and age above 70 years matter?

e19020 Background: Allogeneic stem cell transplantation (alloSCT) is increasingly being offered to older patients with acute myeloid leukemia (AML) due to availability of reduced-intensity conditioning regimens and better supportive care. Though studies have shown improving alloSCT survival outcomes in older age groups, it is still not known if older age confers higher mortality and morbidity during transplant hospitalization. We queried a national inpatient database to evaluate in-hospital mortality and resource utilization during alloSCT for AML in patients >70 years compared to a younger age group. Methods: We conducted a retrospective study of alloSCT for AML patients ≥ 18 years using the 2018 National Inpatient Sample database. Hospitalizations were selected using International Classification of Disease, tenth revision (ICD-10) codes. Demographics, comorbidities, transplant, and outcome variables were compared between a reference age group (18-70 years) and a comparator group (> 70 years). Study outcomes were in-hospital mortality, length of stay (LOS) and transplant complications. Regression models were fit to assess the association between predictors and outcomes. Results: During the study period, 2610 alloSCT met the inclusion criteria. This cohort consisted of 50% males and 77% Caucasians. Only 7% of these alloSCT were performed for patients > 70 years. Patients >70 yrs had a lower proportion of females compared to the reference age group (32% versus 52%, p<0.01). There were no significant differences in racial distribution, Charlson comorbidity index or median income between the 2 age groups. In-hospital mortality rates for the reference age group versus >70 years were 3% and 5% respectively (p=0.35). In a multivariable model, we found no association between age >70 years and in-hospital transplant mortality. Acute graft versus host disease was more frequent among the reference age group (14% versus 2%, p=0.04), but there were no differences in LOS or rates of graft failure, respiratory failure, sepsis, and acute renal failure between the 2 groups. Conclusions: Despite similar in-hospital transplant outcomes as their younger counterparts, patients >70 years comprised only a small proportion of alloSCT performed for AML in our study. Females were more disproportionately affected by this age disparity. More studies are necessary to identify barriers to alloSCT for older AML patients, more so for older females.

Impact of Human T-Cell Lymphotropic Virus Infection on Survival Following Stem Cell Transplantation

Human T-Cell Lymphotropic Virus (HTLV) Types 1 or 2 are delta retroviruses that may lead to complications including adult T-cell leukemia-lymphoma (ATLL) and HTLV-1-associated myelopathy (HAM). There is concern that immunosuppression may result in progression from an asymptomatic carrier state to ATLL or HAM. Data on the safety of stem cell transplantation (SCT) in patients with HTLV-1/2 infection are lacking. The Center for International Blood and Marrow Transplant Research (CIBMTR) was queried for all patients who tested positive for HTLV infection in the pre-transplantation workup and underwent either autologous SCT (AutoSCT) or allogeneic SCT (AlloSCT) between 2008-2017. The primary outcome was overall survival up to four years post-SCT, assessed using the Kaplan-Meier statistic. All analyses were univariate and did not adjust for differences in disease, disease stage or patients characteristics. In those who underwent AutoSCT, 54 (0.5%) patients were HTLV-positive and 9,837 were HTLV-negative. In those who underwent AlloSCT, 126 (0.7%) patients were HTLV-positive and 18,084 were HTLV-negative. In the HTLV-negative group, a larger proportion underwent AlloSCT for acute myeloid leukemia in the HTLV-negative group, while Non-Hodgkin lymphoma/other leukemia was more common in the HTLV-positive group (P<0.001). Additional patient demographics are listed in Table 1. No difference in overall survival (p=0.661) or non-relapse mortality (p=0.522) was noted following AutoSCT based on HTLV status. Following AlloSCT, univariate analysis showed the HTLV-positive patients had a statistically significant lower overall survival (Figure 1; p=0.013) and higher non-relapse mortality at year four (Figure 2; p=0.023). In conclusion, survival after AutoSCT seems unaffected by HTLV status. While the underlying indications for AlloSCT differed in HTLV-infected patients, decreased overall survival and increased non-relapse mortality were seen in HTLV-positive patients. Reasons for increased non-relapse mortality may include increased infectious complications or graft-versus-host disease. Further studies are needed to understand the relative contributions of these factors. Disclosures Janakiram: Takeda, Fate, Nektar: Research Funding. Sloan:Stemline: Consultancy; Abbvie: Consultancy.

Thoracic Air Leak Syndrome: A Rare and Fatal Complication of Allogeneic Stem Cell Transplant

Thoracic air leak syndrome (TALS) is a rare complication of allogeneic stem cell transplant (Allo-SCT) and is associated with a poor prognosis. We report a case of a 67-year-old male, after 20 months of Allo-SCT for acute myeloid leukemia, who presented with fever and acute worsening dyspnea on a background of a 4-month history of slowly progressive dyspnea. He was initially diagnosed with parainfluenza pneumonia and later developed TALS as a result of bronchiolitis obliterans (BO) caused by chronic graft-versus-host disease (cGVHD). Herein, we highlight the importance of early recognition and treatment of non-infectious pulmonary complications of Allo-SCT.

Combination Lenalidomide and Azacitidine: A Novel Salvage Therapy in Patients Who Relapse After Allogeneic Stem-Cell Transplantation for Acute Myeloid Leukemia

PURPOSESalvage options for patients who relapse after allogeneic stem-cell transplantation (allo-SCT) for acute myeloid leukemia (AML) and myelodysplasia (MDS) remain limited, and novel treatment strategies are required. Both lenalidomide (LEN) and azacitidine (AZA) possess significant antitumor activity effect in AML. Administration of LEN post-transplantation is associated with excessive rates of graft-versus-host disease (GVHD), but AZA has been shown to ameliorate GVHD in murine transplantation models. We therefore examined the tolerability and activity of combined LEN/AZA administration in post-transplantation relapse.PATIENTS AND METHODSTwenty-nine patients who had relapsed after allo-SCT for AML (n = 24) or MDS (n = 5) were treated with sequential AZA (75 mg/m2 for 7 days) followed by escalating doses of LEN on days 10 to 30. Dose allocation and maximum tolerated dose (MTD) estimation were guided by a modified Bayesian continuous reassessment method (CRM).RESULTSSequential AZA and LEN therapy was well tolerated. The MTD of post-transplantation LEN, in combination with AZA, was determined as 25 mg daily. Three patients developed grade 2 to 4 GVHD. There was no GVHD-related mortality. Seven of 15 (47%) patients achieved a major clinical response after LEN/AZA therapy. CD8+ T cells demonstrated impaired interferon-γ/tumor necrosis factor–α production at relapse, which was not reversed during LEN/AZA administration.CONCLUSIONWe conclude LEN can be administered safely post-allograft in conjunction with AZA, and this combination demonstrates clinical activity in relapsed AML/MDS without reversing biologic features of T-cell exhaustion. The use of a CRM model delivered improved efficiency in MTD assessment and provided additional flexibility. Combined LEN/AZA therapy represents a novel and active salvage therapy in patients who had relapsed post-allograft.

Monitoring TIGIT/DNAM-1 and PVR/PVRL2 Immune Checkpoint Expression Levels in Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia.

After allogeneic stem cell transplantation (alloSCT), several immune checkpoints play an important role in the antileukemic immune response in the bone marrow (BM) microenvironment. However, immune checkpoint expression levels in the BM have not been reported after alloSCT in patients with acute myeloid leukemia (AML). We investigated the clinical impact of immune checkpoint expression in BM samples after alloSCT for AML. Higher expression of T cell immunoreceptor with Ig and ITIM domains (TIGIT) was associated with a decreased incidence of acute graft-versus-host disease (P = .048) and poor overall (P = .046) and progression-free survival (P = 0.024). In addition, higher expression of TIGIT at engraftment after alloSCT was correlated with a decreased number of natural killer cells in BM (P = .019). Monitoring TIGIT expression in the BM could be useful for predicting outcome after alloSCT for AML. Our findings raise the possibility that blockade of TIGIT would improve survival.

Pre-Conditioning Serum Leptin Levels Predict Early Relapse Rates of High Risk AML after Allogeneic Stem Cell Transplantation

Background: Allogeneic stem cell transplantation (alloSCT) is an effective treatment for many hematologic diseases including intermediate- and high-risk acute myeloid leukemia (AML). Relapse of the original disease continues to be the most common cause of treatment failure following alloSCT for AML. The factors governing relapse and non-relapse mortality (NRM) following alloSCT are poorly understood. We have previously reported that weight loss prior to conditioning therapy is a risk factor for relapse of myeloid diseases following alloSCT (1). The peptide hormone Leptin is a key regulator of food intake and body weight (2). Moreover, it has known roles in regulation of metabolism and immune homeostasis (3). This retrospective study evaluates Leptin as a predictor of survival, relapse and NRM following alloSCT for hematologic malignancies.Methods: Clinical data and Leptin serum levels measured prior to start of conditioning were collected retrospectively in a cohort of patients transplanted for acute leukemias (AML and ALL, n=206). Findings from this cohort were validated in an independent validation cohort of AML patients transplanted in another institution (n=367). Distribution of serum Leptin levels was highly skewed and thus log2-transformation was applied.Results: We identified low serum Leptin as an independent predictor for early relapse following alloSCT for intermediate and advanced stage acute leukemia (AML and ALL) as defined by the EBMT score (4). Multivariate analysis of Leptin and known covariables (age at transplantation, HLA mismatch, conditioning intensity, in-vivo T cell depletion with ATG, recipient and donor sex) revealed a hazard ratio (HR) for relapse of 0.75 per log2 increase (0.59-0.96, p=0.020) for Leptin. We observed an increased risk of NRM with higher leptin serum levels with an HR of 1.63 (1.15-2.31, p=0.006) while no statistically significant effect on OS was found. In order to visualize the effect of the continuous variable on the distribution of survival times, the quartiles of serum Leptin levels with regards to relapse, NRM and OS are shown in Figure 1. This effect on relapse was similar in intermediate and advanced stage AML patients from an independent cohort with an HR of 0.84 (0.72-0.97, p=0.020) while no statistically significant effects on NRM and OS were found. Pre-conditioning serum Leptin was confirmed as a predictor for early relapse by fitting the Cox model to the validation data.Conclusion: Low pre-conditioning serum Leptin levels appear to be an independent risk factor for early relapse following alloSCT for intermediate and advanced stage AML. Pre-conditioning serum Leptin should be explored to guide nutritional intervention in AML patients undergoing alloSCT in order to improve transplant success.ReferencesDietrich S, Radujkovic A, Stolzel F, Falk CS, Benner A, Schaich M, et al. Pretransplant metabolic distress predicts relapse of acute myeloid leukemia after allogeneic stem cell transplantation. Transplantation. 2015;99(5):1065-71.Elmquist JK, Elias CF, Saper CB. From lesions to leptin: hypothalamic control of food intake and body weight. Neuron. 1999;22(2):221-32.Procaccini C, La Rocca C, Carbone F, De Rosa V, Galgani M, Matarese G. Leptin as immune mediator: Interaction between neuroendocrine and immune system. Developmental and comparative immunology. 2017;66:120-9.Gratwohl A, Stern M, Brand R, Apperley J, Baldomero H, de Witte T, et al. Risk score for outcome after allogeneic hematopoietic stem cell transplantation: a retrospective analysis. Cancer. 2009;115(20):4715-26. [Display omitted] DisclosuresBeelen:Medac: Consultancy, Other: Travel Support.

The impact of HLA-matching on reduced intensity conditioning regimen unrelated donor allogeneic stem cell transplantation for acute myeloid leukemia in patients above 50years-a report from the EBMT acute leukemia working party.

BackgroundData comparing fully matched and mismatched-unrelated-donor (M- and mM-URD) allogeneic hematopoietic stem cell transplant (allo-SCT) following reduced intensity conditioning regimens for acute myeloid leukemia are limited.MethodsWe retrospectively compared the outcome of 3398 patients above the age of 50 years who underwent 10/10 M-URD (n = 2567), 9/10 (n = 723), or 8/10 (n = 108) mM-URD allo-SCT for acute myeloid leukemia after reduced intensity conditioning regimen between 2000 and 2013. The Kaplan-Meier estimator, the cumulative incidence function, and Cox proportional hazards regression models were used where appropriate.ResultsHLA matching had no impact on engraftment (p = 0.31). In univariate analysis, in comparison to 10/10 M-URD, mM-URD was associated with higher incidence of grade II–IV acute graft-versus-host disease (GVHD) (p = 0.0002), similar rates of chronic GVHD (p = 0.138) but increased incidence of its extensive form (p = 0.047). Compared to 10/10 M-URD, patients transplanted in the first complete remission (CR1) with a 9 or an 8/10 mM-URD had decreased 2-year leukemia free (LFS) (p = 0.005) and overall survivals (OS) (56.7, 46.1, and 50.2 %, respectively, p = 0.005), while outcomes were comparable between all groups for patients transplanted beyond CR1. In multivariate analysis, 9/10 versus 10/10 URD was associated with higher non-relapse mortality (HR 1.34, p = 0.001), similar risk of relapse and chronic GVHD and inferior LFS (HR 1.25, p = 0.0001), and OS (HR 1.27, p = 0.0001). There was no difference in adjusted transplant outcomes between 9/10 and 8/10 mM-URD.ConclusionsReduced intensity conditioned allo-SCT with a 10/10 M-URD remains the preferable option for AML patients above the age of 50 years. The use of a 9/10 or an 8/10 mM-URD in patients not having a fully matched donor represents an alternative therapeutic option that should be compared to other alternative donor transplant strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0295-9) contains supplementary material, which is available to authorized users.

Extramedullary Leukemia Relapse In Patients With Acute Myeloid Leukemia Allogeneic Stem Cell Transplantation: Risk Factors and Prognosis

BackgroundThe management of extramedullary relapse (EM) of acute myeloid leukemia (AML) after allogeneic stem cell transplant (allo-SCT) is not standardized. We present a single institution experience, describing the risk factors, outcomes and management of EM leukemia relapse in AML patients who underwent allo-SCT. MethodsAfter due IRB approval, 351 patients with AML who underwent allo-SCT at Mayo Clinic, Rochester, from 1985 through 2012, were evaluated for EM relapse. All patients had BM biopsies and cytogenetic studies at diagnosis and at relapse. Extramedullary relapse was identified by clinical exams and radiological methods and confirmed where possible by biopsies and cerebrospinal analysis (CSF). Clinical, pathological and transplant related data was retrospectively abstracted. ResultsOf 351 patients, 26 (7%) had EM involvement at initial diagnosis (17 CNS, 9 non-CNS, 3 both). Forty-four (13%) patients had CNS disease detected prior to allo-SCT. Of these, 17 (39%) were detected at AML diagnosis due to clinical signs/symptoms, 11 (25%) were detected to have CNS involvement at the time of AML relapse and 16 (36%) were asymptomatic; diagnosed on routine pre transplant CSF studies. All patients with pretransplant EM disease received appropriate therapy prior to undergoing transplantation.After allo-SCT, 21 (6%; 12 male) of 351 patients sustained an EM relapse (11 CNS alone, 9 non-CNS, 1 both). The non-CNS EM relapse sites were cutaneous, skeletal, renal, breast, testicular, gastrointestinal tract and soft tissue. In 12 patients with CNS relapse, 8 (68%) presented with localizing signs and symptoms and 4 were asymptomatic. The median age at EM relapse was 42 years (range; 2 - 61 years) and the median time to EM relapse was 443 days (range; 90- 6069 days). Salvage therapy offered in 18 patients included, intrathecal chemotherapy (IT) alone in 2, IT + systemic chemotherapy (CTX) in 2, allo-SCT in 1, CTX alone in 6, IT + radiation therapy (XRT) in 1, CTX, IT and XRT in 1, and CTX + XRT in 5 patients, respectively. Three patients were ineligible for further therapy. Median survival after EM relapse was 136 days (18 – 819, days), with 4 patients alive at last followup.At the time of AML diagnosis; 6 (29%) of 21 patients had EM involvement (2 breast, 1 lymph node, 1 CNS and lymph node, 1 cutaneous), 8 (38%) patients had a monocytic differentiation (AML-M4/M5) and 6 of 9 tested, had CD56 expression. Three (14%), 8 (38%) and 10 (48%) patients were risk stratified based on cytogenetic and molecular status (FLT3/NPM1) into favorable, intermediate and high risk, respectively. Subsequently, all 21 patients underwent allo-SCT with 14 patients undergoing matched related donor (MRD) transplants. The transplant conditioning was myeloablative in 16 (76%) patients, of which 11 (69%) received total body irradiation (TBI). Grade II-IV acute graft versus host disease (GVHD) was noted in 11 (52%) patients, while chronic GVHD was noted in 8 (38). On univariate analysis, factors associated with shortened survival after EM relapse included; high risk karyotype at diagnosis (p=0.009), concurrent bone marrow involvement with EM relapse (p=0.01) and reduced intensity conditioning (RIC) (p=0.04). Importantly, CD56 expression (p=0.27), CNS involvement at diagnosis (p=0.76), and chronic GVHD (p=0.383) were not prognostic. On a multivariate analysis, high risk karyotype (p=0.04) and concurrent bone marrow involvement (p=0.04) retained their negative prognostic value. ConclusionsAlthough uncommon, EM relapse after allo-SCT for AML is associated with poor outcomes. Pretransplant CSF screening identifies a significant number of asymptomatic patients with CNS disease. High risk karyotype at diagnosis and concurrent BM involvement with EM relapse are poor prognosticators. Disclosures:No relevant conflicts of interest to declare.

Leukemia Cell Mobilization with Plerixafor Plus G-CSF with Busulfan/Fludarabine and Allogeneic Hematopoietic Cell Transplantation in Patients with AML/MDS Is Associated with Decreased Complete Chimerism and GvHD, and Increased Risk of Relapse

AbstractAbstract ▪360▪This icon denotes a clinically relevant abstract Background:The goal of preparative regimen in allogeneic stem cell transplant (allo-SCT) is to eradicate leukemia cells. Relapse remains the major cause of treatment failure in patients receiving allo-SCT for acute myeloid leukemia (AML) and myelodysplasia (MDS). There is accumulating evidence that specific niche interactions between the bone marrow stroma and leukemic cells provide protection from cytotoxic chemotherapy. We hypothesized that disruption of those stromal-leukemia interactions using a CXCR4 inhibitor, plerixafor, in combination with granulocyte-colony stimulating factor (G-CSF), may promote release of leukemic cells from the niche and improve progression-free survival post transplant. Patients and Methods:In a phase 1/2 investigation, we treated 41 AML/MDS patients (34 AML and 7 MDS) with a median age of 55 years (range, 31–65 years) with plerixafor (doses of 0, 80, 160 or 240 mcg/kg daily for 4 days starting on day -7) plus G-CSF (10 mcg/kg daily for 6 days starting on day -9) along with the busulfan/fludarabine (Bu/Flu) conditioning regimen (fixed-dose Bu 130mg/m2 IV daily on days -6 to -3 plus Flu 40mg/m2 IV daily on days -6 to -3). Graft-versus-host disease (GvHD) prophylaxis consisted of tacrolimus plus mini-methotrexate. Results:In the phase 1 portion, we determined that pre-treatment with plerixafor plus fixed dose G-CSF is safe when used with the Bu/Flu conditioning regimen. There were no dose limiting toxicities and the highest dose of plerixafor used in the phase 1 study (240 mcg/kg) was selected for the phase 2 extension. The treatment was successful at preferentially mobilizing leukemic cells, as measured by fluorescent in situ hybridization (FISH), with a median fold increase of 22.5 (range, 2.3–73.8) for FISH-positive cells and 6.7 (range, 1.5–19.1) for FISH-negative cells (difference p-value=0.005). This mobilization effect has not been observed when plerixafor was used alone, without G-CSF, in the treatment of AML.1 The plerixafor plus G-CSF combination increased circulating WBCs and CD34+ cells (for 240mcg/kg dose, median fold increase of 6.3 and 36.8 respectively at day -6), as well as CXCR4+ cells. We found that the increase in circulating blasts and CD34+ cells positively correlated with baseline percentage of bone marrow blasts and peripheral blood WBC/blast count. The clinical effects were examined in patients receiving first transplants for AML/MDS (n=39), and outcomes were compared to historical data from 483 patients receiving Bu/Flu alone prior to allo-SCT. There was no difference in time to engraftment between the two groups. Significantly fewer patients in this study group had complete chimerism at day 30 (28%) when compared to historical data (55%, p-value=0.002). The largest difference was seen in lymphoid chimerism, with <80% donor-derived lymphoid cells at day 30 in 56% in the study group vs. 23% in historical data (p-value=0.001). Median follow-up among surviving patients was 16 months (range 6–28) in the study group and 54 months (range 5–126) in the historical dataset. There was a lower frequency of grade 3–4 acute GvHD in the study group vs. historical data (0% vs. 7%, p-value=0.06), and the rate of extensive chronic GvHD was also lower in the study group (HR at 1 year=0.3, p-value=0.06). Despite the intended biological effect of mobilizing and then eliminating leukemia cells, the cumulative incidence of disease progression for patients transplanted in first remission (CR1) was 47% in the study group vs. 25% in the historical comparison (p-value=0.03) and 62% vs. 40% for those not in CR1 at the time of transplant (p-value=0.07). Overall survival at 2 years was 48% vs. 67% for study and historical patients in CR1 (p-value=0.2), and 34% vs. 47% for patients not in CR1 (p-value=0.5). Conclusions:In summary, our findings demonstrate that the combination of plerixafor plus G-CSF can be safely added to the Bu/Flu conditioning regimen, and preferentially mobilizes leukemic cells. However, the rate of leukemia relapse was higher in these patients compared to historical data. The etiology of this finding is not yet clear, and may be the result of reduced graft-versus-leukemia effect as evidenced by the reduced lymphoid chimerism and rate of GvHD. Disclosures:Off Label Use: Plerixafor plus G-CSF as a part of conditioning in allogenic transplant for AML/MDS. Champlin:Sanofi/Genzyme: Research Funding; Otsuka: Research Funding.

Prognostic Significance of Detection of Minimal Residual Disease by Multiparametric Flow Cytometry Before Allogeneic Stem Cell Transplantation in Patients with Acute Myeloid Leukemia,

Abstract 4116The main prognostic determinant of the outcome of allogeneic stem cell transplantation (allo-SCT) for acute myeloid leukemia (AML) is disease phase at transplant while the relevance of the assessment of minimal residual disease (MRD) at transplant has not been elucidated. Although molecular markers are highly sensitive for the detection of MRD, they are available in only half of AML cases to date. On the other hand, multiparametric flow cytometry (MFC) offers the feasibility of MRD assessment in virtually all patients. We retrospectively studied the influence of pre-transplant MRD status by MFC on the outcomes of patients with AML in relation to other known prognostic factors.From 01/2003 to 12/2010, 102 AML patients (male/female; 58/44), aged 15–64 (median; 42) years, received allo-SCT in our center at first (CR1, N=69) or subsequent (CR≥2, N=33) morphologic complete remission. Donors were HLA-compatible siblings (N=45), matched unrelated (N=34) or alternative (haploidentical relatives or unrelated cord blood; N=6 and 17, respectively). The conditioning regimen was myeloablative in 85 (83.33%), and reduced-intensity in 17 (16.67%) cases. Data on MRD assessment by four- or five-color MFC before transplant were available in 92 patients. MRD was defined as any percentage of cells with an aberrant antigen expression pattern compared to normal or regenerating marrow. Transplant outcomes associated with MRD in univariate and multivariate analysis were overall (OS) and disease-free (DFS) survival, relapse incidence, and non-relapse-related mortality (NRM). Prognostic parameters included in the statistical model were the following: age and gender of recipient or donor, interval from diagnosis to transplant, de novo versus secondary AML, CR1 versus CR≥2 at transplant, cytogenetic risk group (according to Intergroup ECOG/SWOG definition), recipient and donor cytomegalovirus (CMV) serostatus, donor type, conditioning regimen (myeloablative versus reduced-intensity), degree of HLA match, method of graft-versus-host disease (GVHD) prophylaxis, and the modified European Blood and Marrow Transplantation Group (EBMT) risk score (Hemmati et al, 2011).With a median follow-up of 47 (range, 6–98) months, OS and DFS were 54.2% and 51.72%, respectively. The cumulative incidence of relapse was 24.45%, and of NRM 23.82%. Eighteen out of 92 (19.57%) patients had MRD by MFC before allo-SCT. According to univariate analysis, the presence of MRD showed a negative association with OS (29.63% versus 63.17% at 4 years in patients with or without MRD, respectively, P=0.053) and DFS (28.52% in MRD-positive versus 59.74% in MRD-negative cases at 4 years, P=0.025, Figure 1). Presence of MRD correlated with higher incidence of relapse compared to MRD negativity (52.7% versus 15.8%, respectively, P=0.001) (Figure 2), but did not influence NRM. In multivariate analysis (Cox proportional hazards, backward stepwise selection), the parameters that remained independent adverse risk factors in terms of specific outcomes were: a) For OS: age and male gender of patient, alternative donor, unfavorable karyotype, CR≥2, and secondary AML, b) For DFS: presence of MRD (HR=3.2, P=0.009), CR≥2 (ÇR=3.1, P=0.005), reduced-intensity conditioning (HR=2.6, P=0.034), and unfavorable karyotype (HR=2.06, P=0.074), c) For relapse: presence of MRD (ÇR=3.83, P=0.003), and unfavorable karyotype (HR=3.55, P=0.007), and d) For NRM: age of recipient, CR≥2, and alternative donor. [Display omitted] [Display omitted] In conclusion, the presence of MRD by MFC before allo-SCT for AML is associated with a significantly higher risk of relapse and an inferior DFS, despite morphologic complete remission at transplant. Moreover, pre-transplantation MRD status has independent prognostic significance in addition to the disease phase, cytogenetic risk group, and intensity of the conditioning regimen. Prospective studies are warranted to examine if the adverse impact of MRD on the outcomes of allo-SCT can be reversed by immunologic manipulations aiming at augmenting graft-versus-leukemia (GVL) effect. Disclosures:No relevant conflicts of interest to declare.

Monocytes Induced to Differentiate Into Dendritic Cells After Overnight Transduction with Lentiviral Vectors Co-Expressing GM-CSF/ IL-4 and a Truncated Form of WT1: Preclinical Validation for Immunotherapy of High Risk Leukemia Patients After Allo-SCT

Abstract 2981Wilms tumor 1 (WT1) is a transcription factor reported to be overexpressed in acute leukemia blasts and is a prognostic marker for relapse. WT1 is currently being tested in leukemia immunotherapy trials as a putative leukemia-associated antigen, but repeated WT1 peptide/adjuvant vaccination trials have failed to induce sustained high-avidity CD8+ T cell responses. Immunization against WT1 in the allogeneic stem cell transplantation (allo-SCT) setting will warrant new approaches to counteract possible tolerogenic effects of minimal residual disease and the immunesuppressed milleau of the host.Thus, we are developing dendritic cells that co-express stimulatory cytokines and antigens to be used as vaccines for high risk acute myeloid leukemia (AML) after allo-SCT. We have previously demonstrated that DCs programmed ex vivo with lentiviral vectors (LV) expressing GM-CSF and IL-4 lead to autonomous in vivo differentiation of long-lived “SmartDCs”, which were potent vaccines with long-lasting effects in melanoma models.Here, a tricistronic LV (LV-G242W) co-expressing GM-CSF, IL-4 and truncated form of WT1 (lacking the zinc finger domain) was produced and tested in functional assays. RESULTS: CD14+ monocytes obtained from three different healthy donors were transduced with LV-G242W at a multiplicity of infection of 20 and kept in culture without exogenous addition of cytokines. The number of integrated LV copies in differentiated SmartDCs/tWT1 was in the range of 3–9 per cell. Cytokine production persisted for 3 weeks (GM-CSF: 3–8 ng/mL and IL-4: 0.05–3.0 ng/mL). WT1 overexpression in SmartDCs/tWT1 was confirmed by RT-Q-PCR and cytospin/IHC. Typical DC morphology and expression of the DC markers CD86, HLA-DR and CD80 were stable throughout the culture. Gene expression profiling revealed that SmartDCs/tWT1 expressed higher levels of several members of the interferon stimulated genes (ISG) than conventional DCs, indicative of an activated state. Luminex analyses of cytokines secreted by SMART-DC/tWT1 revealed endogenous expression of TNF-alpha (185–500 pg/mL) and IL-6 (60–123 pg/mL), which promote DC maturation.SmartDCs/ tWT1 and conventional DCs pulsed with WT1 class I peptides were used to stimulate autologous CD3+ T cells. SmartDCs/tWT1 promoted T cell expansion and up-regulation of the activation marker CD137. Evaluation of WT1-specific responses were performed by ELISPOT and streptamer analysis using cell samples obtained from a healthy donor and from an acute myeloid leukemia patient. For both models, SmartDCs/ tWT1 were more effective at promoting immune reactivity than SmartDCs pulsed with peptides, indicating the relevance of the intracellular antigen processing for optimal T cell activation.SmartDCs/tWT1 genetically marked with firefly Luciferase were injected s.c. into NOD/Rag −/−/ IL2gcR−/− mice for in vivo viability and biosafety analyses. SmartDCs/tWT1 engrafted efficiently, maintained viability at the injection site for at least 3 weeks and no signs of malignancies were observed so far.Our results support the feasibility of generation and high immunogenicity of SmartDCs/tWT1 and highlight the potential for those cellular vaccines to boost graft-versus-leukemia after allo-SCT for AML. Disclosures:Ganser:Novartis: Honoraria, Research Funding.