Nonmyeloablative Hematopoietic Stem Cell Transplantation Research Articles

Cyclosporine A trough concentrations are associated with acute GvHD after non-myeloablative allogeneic hematopoietic cell transplantation.

Low plasma CsA concentrations (<300–350 ng/mL) early following allogeneic hematopoietic stem cell transplantation (HSCT) is associated with an increased risk of developing acute graft-versus-host disease (aGvHD). Nevertheless, the current optimal target trough concentration for CsA following HSCT is considered to be 200–400 ng/mL. Here, we performed a retrospective analysis of a homogeneous group of 129 patients who received HSCT after non-myeloablative conditioning, and we analyzed the impact of CsA trough concentration measured during the first four weeks (CsA W1-4) on the incidence aGvHD, relapse-free survival (RFS), non-relapse mortality (NRM), overall survival (OS), and toxicity. The 180-day incidence of grade II-IV aGvHD was 25% (32/129 patients). In multivariate analysis the incidence of grade II-IV aGvHD was significantly lower among patients with a CsA W1-4 concentration ≥350 ng/mL compared to patients with a concentration <350 ng/mL (18% versus 38%, respectively; P = 0.007), with a hazard ration (HR) of 0.38 (95% CI: 0.19–0.77). In contrast, we found no correlation between CsA trough concentration and RFS, NRM, or OS. Moreover, we found an increased incidence of hypomagnesemia at higher CsA concentrations, but no difference in the incidence of acute renal toxicity, hepatic toxicity, or electrolyte imbalance. Interestingly, 30% of patients experienced hyponatremia with no apparent cause other than the use of CsA, with urinalysis suggesting SIADH as the underlying cause. Our findings suggest that a CsA trough concentration of 350–500 ng/mL might be more appropriate in the first month following non-myeloablative HSCT.

Effect of Nonmyeloablative Hematopoietic Stem Cell Transplantation vs Continued Disease-Modifying Therapy on Disease Progression in Patients With Relapsing-Remitting Multiple Sclerosis

Hematopoietic stem cell transplantation (HSCT) represents a potentially useful approach to slow or prevent progressive disability in relapsing-remitting multiple sclerosis (MS). To compare the effect of nonmyeloablative HSCT vs disease-modifying therapy (DMT) on disease progression. Between September 20, 2005, and July 7, 2016, a total of 110 patients with relapsing-remitting MS, at least 2 relapses while receiving DMT in the prior year, and an Expanded Disability Status Scale (EDSS; score range, 0-10 [10 = worst neurologic disability]) score of 2.0 to 6.0 were randomized at 4 US, European, and South American centers. Final follow-up occurred in January 2018 and database lock in February 2018. Patients were randomized to receive HSCT along with cyclophosphamide (200 mg/kg) and antithymocyte globulin (6 mg/kg) (n = 55) or DMT of higher efficacy or a different class than DMT taken during the previous year (n = 55). The primary end point was disease progression, defined as an EDSS score increase after at least 1 year of 1.0 point or more (minimal clinically important difference, 0.5) on 2 evaluations 6 months apart, with differences in time to progression estimated as hazard ratios. Among 110 randomized patients (73 [66%] women; mean age, 36 [SD, 8.6] years), 103 remained in the trial, with 98 evaluated at 1 year and 23 evaluated yearly for 5 years (median follow-up, 2 years; mean, 2.8 years). Disease progression occurred in 3 patients in the HSCT group and 34 patients in the DMT group. Median time to progression could not be calculated in the HSCT group because of too few events; it was 24 months (interquartile range, 18-48 months) in the DMT group (hazard ratio, 0.07; 95% CI, 0.02-0.24; P < .001). During the first year, mean EDSS scores decreased (improved) from 3.38 to 2.36 in the HSCT group and increased (worsened) from 3.31 to 3.98 in the DMT group (between-group mean difference, -1.7; 95% CI, -2.03 to -1.29; P < .001). There were no deaths and no patients who received HSCT developed nonhematopoietic grade 4 toxicities (such as myocardial infarction, sepsis, or other disabling or potential life-threatening events). In this preliminary study of patients with relapsing-remitting MS, nonmyeloablative HSCT, compared with DMT, resulted in prolonged time to disease progression. Further research is needed to replicate these findings and to assess long-term outcomes and safety. ClinicalTrials.gov Identifier: NCT00273364.

Hematopoietic Stem Cell Transplantation and Disease Progression in Relapsing-Remitting MS

To compare nonmyeloablative hematopoietic stem cell transplantation (HSCT) to standard multiple sclerosis (MS) disease-modifying therapies (DMTs),

Patients´ experiences and perspectives of challenges and needs related to nonmyeloablative stem cell transplantation: Involving patients in developing a targeted rehabilitation programme.

To explore patients' experiences and perspectives of their challenges and needs regarding their return to everyday life after allogeneic nonmyeloablative haematopoietic stem cell transplantation (NMA-HSCT). NMA-HSCT can cure patients with malignant blood diseases, but during the following years, the majority of patients suffer from serious side effects and complications. Hence, it is a major challenge for patients treated with NMA-HSCT to rehabilitate, maintain physical and psychosocial functioning and return to a life in restored balance. The design was qualitative using the interpretive description methodology, and the theoretical framework symbolic interactionism inspired the interview guide and analysis. Between April to May 2017, five focus group interviews were conducted with 15 outpatients in a haematological ward in Denmark. The patients were treated with NMA-HSCT 8-30months prior to the interviews. The study adheres to the Consolidated Criteria for Reporting Qualitative Research guidelines. The impaired functioning was the overarching theme and seemed to be the trigger entailing rehabilitation needs related to the following main themes: realising decline, adapting to changes, the meaning of motivation and reliance on relations. These findings seemed to affect and influence the patients' struggle for a return to an everyday life like before being diagnosed or just before undergoing NMA-HSCT. This study adds to our understanding of important elements to consider when developing a targeted rehabilitation programme. Based on our findings, the rehabilitation programme should encompass: extensive variation regarding how to address the impaired functioning through individualised approaches, multimodal interventions, interventions through several months with varying intensity, an interdisciplinary team approach supporting motivation and visualisation of every progress by tangible goal setting, communication regarding hope and support, extended supportive care for patients living alone and finally increased adherence through social sessions with relatives and fellow patients.

Non-myeloablative hematopoietic stem cell transplantation (HSCT) is superior to disease modifying drug (DMD) treatment in highly active Relapsing Remitting Multiple Sclerosis (RRMS): interim results of the Multiple Sclerosis International Stem cell Transplant (MIST) Randomized Trial (S36.004)

Non-myeloablative hematopoietic stem cell transplantation (HSCT) is superior to disease modifying drug (DMD) treatment in highly active Relapsing Remitting Multiple Sclerosis (RRMS): interim results of the Multiple Sclerosis International Stem cell Transplant (MIST) Randomized Trial (S36.004)

Pain and Opioid Use after Reversal of Sickle Cell Disease Following Hematopoietic Stem Cell Transplant

Introduction: Pain is the most common symptom of sickle cell disease (SCD). Hematopoietic stem cell transplant (HSCT) is currently the only curative therapy for SCD; however, some patients continue to report pain requiring opioid treatment despite hematologic cure following successful HSCT. Knowledge of factors associated with continued pain and opioid use may help reduce post-HSCT pain-related morbidity. We analyzed data from a cohort of SCD patients who have been successfully treated by nonmyeloablative HLA-matched sibling allogeneic HSCT. Our goal was to investigate pre-HSCT factors that are associated with continued pain and opioid use at 12 months after a successful HSCT.Methods: Detailed data on the clinical course, pain, opioid use, and laboratory values were collected from medical records at 3 months pre-HSCT and 12 months post-HSCT (n=35). Patient Reported Outcomes Measurement Information System (PROMIS) measures were available in a subgroup of these patients (n=20), also collected at the same time points. Patients with pre-HSCT pain were classified using Analgesic, Anesthetic, and Addiction Clinical Trial Translations Innovations Opportunities and Networks-American Pain Society Pain Taxonomy (AAPT) guidelines into one of three pain categories: 1. Chronic pain without contributory SCD complication; 2. Chronic pain explained by contributory SCD complication, eg. avascular necrosis; 3. Chronic pain with a mixed pain phenotype (eg. some pain explained by a contributory SCD complication but also pain occurring in unrelated sites). Patients who only experienced acute episodic pain were grouped separately (Episodic pain only).Results: The median age of the cohort was 30.5 years (range: 16-65 years) and 21 (60%) patients were male. Pre-HSCT, all patients experienced intermittent episodes of pain. Forty-six% of the patients experienced acute episodic pain only, 26% also had chronic pain with a contributory SCD complication and 28% also had chronic pain without contributory SCD complication or a mixed pain phenotype. Post-HSCT, median pain-related admissions decreased significantly (from 3 admissions/year (range 0-24) pre-HSCT to 0 (range 0-3) post-HSCT; (p<0.001)). There were significant reductions in the prescription of both short- and long-acting opioids pre- vs post-HSCT (91% to 40% for short acting and 40% to 14% for long acting opioids; p<0.001). While 14 out of 35 (40%) reported pain at 12 months post-HSCT, examination of continued opioid use patterns across the AAPT groups showed that in the group of chronic pain patients without contributory SCD complications, 90% continued to use opioids to manage their pain whereas none of the patients in the episodic pain only group received opioids post-HSCT and for the group of patients with chronic pain related to a SCD complication, 50% stopped using opioids post-transplant.Analysis of PROMIS data, showed significant improvement in the subscales of Pain Intensity (p=0.03), Pain Impact (p=0.007), Satisfaction with Social Role (p= 0.03), and Physical Function (p= 0.004) following HSCT but no significant changes were seen for Fatigue (p=0.09), Anxiety, Depression or Sleep Disturbance (which may be explained by the small sample size not having enough variance). However, when comparing patients with persistent pain and opioid use without related SCD complications (n=6) versus those with resolved pain post-HSCT (n=9), pre-HSCT Anxiety ratings were significantly higher in the group with unexplainable, persistent pain (p=0.05). For those same groups, additional pre-HSCT factors that were associated with unexplainable, persistent pain post-HSCT were higher burden of pain as defined by higher median pain admissions/year (2 (0-10) vs. 5.5 (3-9); p=0.04); higher overall numerical pain rating on a 0-10 scale (2 (0-5) vs. 6 (5-7); p=0.02) and treatment with long acting opioids (0% vs. 86%; p<0.001).Conclusions: SCD patients with higher pre-HSCT pain burden (more pain admissions and higher pain intensity ratings), long-acting opioid use and certain psychological traits (anxious disposition) may be at risk for persistent pain and continued opioid use post-HSCT. Future studies should evaluate outcomes beyond 12 months of follow up, further characterize underlying mechanisms and evaluate potential interventions to reduce pain and opioid use related morbidities in this subset of patients. DisclosuresNo relevant conflicts of interest to declare.

Immune tolerance induction by nonmyeloablative haploidentical HSCT combining T-cell depletion and posttransplant cyclophosphamide.

The establishment of safe approaches to attain durable donor-type chimerism and immune tolerance toward donor antigens represents a major challenge in transplantation biology. Haploidentical hematopoietic stem cell transplantation (HSCT) is currently used for cancer therapy either as a T-cell-depleted megadose HSCT following myeloablative conditioning or with T-cell-replete HSCT following nonmyeloablative conditioning (NMAC) and high-dose posttransplant cyclophosphamide (PTCY). The latter approach suffers from a significant rate of chronic graft-versus-host disease (GVHD), despite prolonged immunosuppression. The use of T-depleted grafts, although free of GVHD risk, is not effective after NMAC because of graft rejection. We now demonstrate in mice conditioned with NMAC that combining the power of high-dose PTCY with T-cell-depleted megadose HSCT can overcome this barrier. This approach was evaluated in 2 patients with multiple myeloma and 1 patient with Hodgkin lymphoma. The first myeloma patient now followed for 25 months, exhibited full donor-type chimerism in the myeloid and B-cell lineages and mixed chimerism in the T-cell compartment. The second myeloma patient failed to attain chimerism. Notably, the low toxicity of this protocol enabled a subsequent successful fully myeloablative haploidentical HSCT in this patient. The third patients was conditioned with slightly higher total body irradiation and engrafted promptly. All patients remain in remission without GVHD. Both engrafted patients were able to control cytomegalovirus reactivation. Enzyme-linked immunospot analysis revealed immune tolerance toward donor cells. Our results demonstrate a novel and safer nonmyeloablative haplo-HSCT offering a platform for immune tolerance induction as a prelude to cell therapy and organ transplantation.

OR10 Analysis of engraftment kinetics of donor-type chimerism in lineage-specific cell populations after allogeneic hematopoietic stem cell transplantation

Aim The aim was to monitor the engraftment patterns and outcome of the graft by quantitative lineage chimerism testing based on short tandem repeats (STR), focusing on a longitudinal analysis post allogeneic myeloablative (MA) and nonmyeloablative (NMA) hematopoietic stem cell transplantation (HSCT). Methods The study was conducted on a cohort of 45 patients who received an allogeneic HSCT. All grafts were from a HLA ‘full house’ matched related donor. Post-transplant peripheral blood samples were collected from the recipient at D + 21, D + 30, D + 60, D + 90, D + 120, D + 180 and D + 365. Whole blood mononuclear cells was fractionated into CD3+, CD15 + and CD56 + cells using magnetic beads with sequential positive selection before extracting DNA. Sixteen STR (microsatellite) loci (D8S1179, D21S11, D7S820, CSS1PO, D3S1358, THOI, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, Amelogenin, FGA and D5S818) were amplified by multiplex PCR and the amplicon resolved on DNA sequencer. Results Post-transplant at D + 21 the percentage of donor T-cell chimerism was 100% in 17.9% cases whereas, the myeloid and NK cell donor chimerism was found to be 65% and 47.3% respectively. Overall the NK cell chimerism pattern closely approximated myeloid cell chimerism, while T cell recovery was distinctly different. In the MA category the donor chimerism reached >90% earlier than the NMA group. At D + 21 in the MA cohort, the average percentage of donor cells in CD3+, CD56+, CD15 + lineages was 84.12%, 93.92% and 97.61% whereas in the NMA cohort it was 79.4%, 85.7% and 90.99% respectively. The donor chimerism in MA group at D + 90 was >97% in all lineages, whereas in NMA group the donor cells in the three lineages were CD3 90.5%, CD56 92.8% and CD15 98.9%. The percentage of CD15 cells was higher at all-time points as compared to CD56 and CD3. In relapse the T cell chimerism showed maximum variation while myeloid cells demonstrated a more stable graph. No definite chimerism pattern was detected in the three lineages in ac GVHD. Conclusions This study establishes that, longitudinal chimerism testing is an important parameter for surveillance of engraftment and provides important information on disease recurrence or graft loss in HSCT. As a monitoring approach, it exploits the ability of the STR technique to compare numerical results between samples.

Significance of Monitoring Minimal Residual Disease by Flow Cytometry in Acute Leukemia Patients Underwent Nonmyeloablative Allo-HSCT

To explore the value of dynamically monitoring minimal residual disease (MRD) by flow cytometry before and after non-myeloablative allo-HSCT (NST) for prediction of acute leukemia(AL) relapse after transplantation. The clinical data of 51 AL patients underwent NST were analyzed retrospectively in Department of Hematology of Affiliated Hospital of Academy of Military Medical Sciences from January 2011 to December 2015. All AL patients achieved the morphologic complete remission of bone marrow before transplantation. The bone marrow samples were collected for monitoring of MRD within 35 days before transplant, every month till 3 months after transplant, every 3 months till 24 months after transplant, and then every 6 months after 2 years of transplant. According to the MRD cutoff value of 0.2%, the AL patients were divided into high level MRD group (18 cases) which was defined as MRD≥0.2% after transplantantion at least for 1 time, and low level MRD group (33 cases) which was defined as MRD<0.2% after transplant all the time. 2 year cumulative relapse rate in 2 groups were compared. Two-year relapse rates were 6.1% and 50% in low-level MRD group and high-level MRD group post NST(P=0.001)respectively. Multivariate analysis indicated that the risk of relapse in high level MRD group was 5.84 times of low level MRD group(P=0.036). MRD≥0.2% post transplant was an independent risk factor for leukemia relapse post NST. The mortality rate was 81.8% and 46.3%(P<0.05) in relapse and non-relapse groups respectively. Dynamically monitoring MRD by FCM is a crucial tool for early relapse estimation of acute leukemia in adult patients after allogeneic nonmyeloablative hematopoietic stem cell transplantation. MRD≥0.2% after transplant can be used as a early valuable evidence for predicting relapse and guiding active medical intervention.

423 - Nonmyeloablative Hematopoietic Stem Cell Transplantation (HSCT) Improves Cardiac Function in Patients with Sickle Cell Disease (SCD)

423 - Nonmyeloablative Hematopoietic Stem Cell Transplantation (HSCT) Improves Cardiac Function in Patients with Sickle Cell Disease (SCD)

Prognostic Factors of CLL Patients Undergoing Reduced Intensity Allogeneic Hematopoietic Stem Cell Transplantation in the Immunochemotherapy Era

Background: Allogeneic hematopoietic stem cell transplantation (HCT) is the only curative therapy for chronic lymphocytic leukemia (CLL). With this analysis we intended to identify prognostic factors in patients undergoing reduced-intensity conditioning (RIC) or non-myeloablative (NMA) HCT at our center in the immunochemotherapy era.Methods: This retrospective chart review included all CLL patients who underwent RIC or NMA conditioned HCT at Memorial Sloan Kettering Cancer Center (MSKCC) in the immunochemotherapy era between 09/2006 and 10/2014. We then analyzed whether pre-HCT factors including age, disease response status at the time of HCT, presence of Richter's transformation, 17p deletion or TP53 mutation, use of anti-thymocyte globulin (ATG), number of prior lines of treatment, and time from diagnosis to HCT, were associated with overall survival (OS), progression-free survival (PFS), non-progression mortality, and grade 2-4 acute graft versus host disease (aGVHD). Univariate factors were evaluated via log-rank or Gray's test as appropriate, while Cox regression models were used to explore the adjusted effect of multiple factors.Results: Thirty-five patients undergoing RIC or NMA HCT with a median age of 53 years (range 36.3-69.0) were analyzed. The patients had a median of 4 prior lines of therapy (range 1-10) and a median time from diagnosis to HCT of 65.8 months (range 7.5-159.0). With a median follow-up for survivors of 58.8 months (95% CI 17.0-NA), the 5-year PFS and OS were 32.4% (95% CI 17.4-48.4) and 49.4% (95% CI 31.2-65.2), respectively (Figure 1). Treatment-related mortality was 20.0% (95% CI 8.7-34.7) and 31.8% (95% CI 17.0-47.6) at 1 and 2 years, respectively. Chemosensitive disease, defined by complete or partial remission per contemporary International Workshop CLL (iwCLL) response criteria at the time of HCT, was associated with improved 3-year OS of 68.0% (95% CI 46.1-82.5) compared to patients with chemorefractory disease to last line of therapy (stable or progressive disease) with a 3-year OS of 15.0% (95% CI: 1.0-45.7, p = 0.002, Figure 2). Additionally, patients with ≤4 lines of therapy prior to HCT experienced superior 5-year OS of 61.2% (95% CI 37.5-78.2) contrasted to 20.0% (95% CI: 3.1-47.5) in patients with >4 prior lines of therapy (p = 0.008, Figure 3). This difference approached statistical significance in multivariate analysis (HR 2.43, 95% CI 0.91-6.50; p = 0.076) adjusted for chemosensitivity. Furthermore, when adjusted for the number of prior lines of therapy, chemorefractory patients remained at greater risk of death in multivariate analysis (HR 3.29, 95% CI: 1.14-9.48, p = 0.027). Other factors including: age, history of transformed disease, the presence of 17p or TP53 deletion/mutation, time from diagnosis to HCT, or use of ATG with NMA/RIC did not impact PFS or OS.Conclusion: This is the first study to report independent prognostic impact of the number of lines of therapy prior to NMA/RIC HCT on OS for CLL patients in the post-immunochemotherapy era. We confer findings from other groups including: the prognostic significance of chemosensitivity at the time of HCT as well as HCT overcoming poor-risk cytogenetics associated with 17p chromosomal aberrations. Our data is limited by lack of patients previously exposed to recently FDA approved novel kinase or BCL2 inhibitors. Nevertheless, given our reported data, HCT should still be considered as the only therapy with curative potential for poor-risk patients earlier in their disease course, while chemosensitivity is maintained and prior to the accumulation of multiple lines of therapies. [Display omitted] [Display omitted] [Display omitted] DisclosuresPerales:Incyte Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Low Serum Galectin-1 Levels Predict Future Lymphoma Development in HIV-Positive Patients

Introduction. HIV infected individuals have an increased risk of developing lymphoma compared to sex- and age matched non-immunocompromised control population and approximately 2% of HIV infected individuals developed lymphoma (Gopal et al, J Natl cancer Inst 2013). Our group has been among the first who identified novel serum protein markers present at time of HIV diagnosis, which were predictive of subsequent lymphoma development (Vase et al, AIDS 2016). Galectins are important regulators of cell adhesion, apoptosis, cell cycle, and mRNA processing. Galectin-1 (Gal-1) is a known lectin-binding protein able to mediate Th2 skewed microenvironment in lymphomas (Juszczynski et al, Proc Natl Acad Sci U S A 2007; Cedeno-Laurent et al, Blood 2012), and facilitates HIV-infection (Sato et al, Ann N Y Acad Sci 2012). Increased serum Gal-1 levels were correlated to increased tumor burden and adverse clinical features in Hodgkin lymphoma (HL) (Kamper et al, Blood 2011; Ouyang et al, Blood 2013) and low Gal-1 levels were associated with an increased risk of chronic graft-versus-host disease in patients with hematologic malignancies treated with non-myeloablative hematopoietic stem cell transplantation (Petruskevicius et al, BMT 2016). In this study, we investigated whether the serum level of Gal-1 at the time of HIV diagnosis was predictive for subsequent lymphoma development.Methods. We determined the serum levels of Gal-1 in serum samples from19 HIV infected patients collected at the time of HIV diagnosis. Measurements were performed using a time-resolved immunofluorometric assay, as previously described (Petruskevicius et al, BMT 2016). Patients were grouped based on clinical outcomes in (i) future HIV-associated lymphoma (HIV/lymphoma), (ii) future HIV-associated benign lymphadenopathy (HIV/adenopathy), and (iii) no future neoplasia (HIV/no neoplasia), Table 1. Furthermore, serum Gal-1 levels were compared to those of a healthy control group (n=30), as previously reported (Petruskevicius et al, BMT 2016). Gal-1 sample concentrations were calculated by regression anaysis on basis of a standard curve of recombinant Gal-1 at concentrations of 100 to 0.78 ng/mL with 1:4 sample dilutions. Gal-1 levels > 400ng/mL was included in the analyses with a value of 400ng/mL. Estimates of differences between groups were evaluated using Student’s t-test or ANOVA on log transformed data. A ROC analysis was computed to establish cut-off values for serum galectin-1, with respect to development of lymphoma.Results. Overall, the serum Gal-1 level in the HIV cohort was lower than in the healthy control group (p<0.001), Figure 1A. At HIV diagnosis, those HIV patients who would subsequently develop lymphoma had significantly lower levels of serum Gal-1 compared to the remaining cohort, Figure 1B (p=0.017). There was no gender-related difference (p=0.436) and Gal-1 serum levels did not correlate with either CD4 count (p=0.553) or viral load (p=0.600) at time of HIV diagnosis. In this size-limited study population, it was not possible to show any significant difference between HIV/lymphoma, HIV/adenopathy, and HIV/no neoplasia. ROC calculated cut-off of 2.6 ng/mL was able to separate HIV patients with future lymphoma from the remaining HIV patients and controls with a specificity of 78% and sensitivity of 100%. At this cut-off 13 (31%) patients were allocated to the low Gal-1 group, including all future lymphoma patients.Conclusion. HIV infected patients had significant lower serum Gal-1 levels than compared to a healthy control cohort. All HIV infected patients that later developed lymphoma belonged to the subset with lowest serum Gal-1 levels. If confirmed in independent cohorts of HIV patients from the cART era, this observation will support the use of low serum levels of Gal-1 as an early predictive biomarker for subsequent lymphoma development in HIV infected individuals. This may in turn have potential implications on the clinical monitoring strategy of these patients. [Display omitted] [Display omitted] Disclosuresd'Amore:Servier: Honoraria, Other: Advisory Boards; CTI LIfe Sciences: Honoraria, Other: Advisory Boards.

Next Generation Veto Cells for Non-Myeloablative Haploidentical HSCT: Combining Anti-Viral and Graft Facilitating Activity

Transplantation of T cell-depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders or can be used to induce immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. We previously demonstrated that this barrier can be overcome using donor-derived naïve CD8 T cells if they are expanded against third-party alloantigens under culture conditions inducing a central memory phenotype. Thus, such anti-3rd party CD8 T cells (Tcm) exhibit marked veto activity and reduced risk for GVHD due to the lower frequency of anti-host clones achieved during the culture period. In the present study, we tested the feasibility of generating veto Tcm by stimulation against well-defined peptides including viral antigens. For proof of concept, we used OT1 mice that express a transgenic (Tg) TCR designed to recognize ovalbumin (OVA) residues 257-264 in the context of H2Kb MHC-I. Prior to harvest of OT1 CD8+ T cells, mice were immunized twice with OVA-peptide. Mice were sacrificed 7 to 14 days after immunization, their spleens and lymph nodes removed and crushed, and magnetic bead sorting utilized to isolate the memory cells (CD8+CD44+). The resulting population was subjected to third-party stimulation by co-culture with irradiated splenocytes generated from spleens of OVA-expressing mice, under cytokine deprivation. hIL-15 (10ng/ml) was added to the culture 60 hrs after culture initiation to induce the cells to express a Tcm like phenotype, as previously described for anti-3rd party Tcm generated from WT naïve CD8 T cells (WT Tcm).When tested in vivo, the OT-1 Tcm (H2Kb) were able to enhance engraftment of allogeneic T cell depleted BMT (H2Kb) in sub-lethally irradiated (5.5 Gy TBI) Balb/c recipients (H2Kd), in analogy to the chimerism induced by WT Tcm. This initial successful experiment was then followed by an experiment, which more closely resembled the human setting, in which the CD8+CD44+ cells were isolated from WT C57BL/6 OVA-immunized mice and subsequently introduced to co-culture with irradiated splenocytes generated from spleens of OVA-expressing mice. Results showed that Tcm grown from such a starting population of CD8+CD44+ cells were also able to achieve marked donor chimerism when administered with megadose of T cell depleted (TCD) alloSCT in sublethally irradiated Balb/c recipients (Fig.1A). Notably, infusion of 5x106 Tcm into sublethally irradiated mice (5.5. GY TBI) without BMT, did not cause any GVHD as measured by weight loss, in contrast to CD8+CD44+ T cells used to generate the Tcm (Fig.1B). These results demonstrate that CD8+CD44+ derived from memory CD8 T cells by expansion against cognate peptides exhibit markedly reduced risk for GVHD compared to freshly isolated memory cells, while retaining their veto activity and inducing tolerance.Finally, based on these proof of concept studies, we were able to translate this approach and generate human anti-viral CD8 veto cells with central memory phenotype. Thus, CD8+CD45RO+ memory T cells were selected by depletion of CD4+CD56+CD45RA+ cells from PBMC of normal donors and then co-cultured with donor DCs pulsed with a viral peptide mixture of three prominent viruses (EBV, CMV and Adenovirus). In three large scale experiments using leukapheresis preparations of normal CMV and EBV positive donors, with GMP grade reagents, more than 1x109 Tcm could be attained by the end of 9 days of culture (average expansion of Tcm=13.5±4 fold) with greater than 90% purity of CD45RO+CD62L+CD8+ T cells (Fig 2).The harvested anti-viral Tcm exhibited more than 3 log depletion of alloreactivity, compared to fresh CD8 T cells, as measured by limit dilution analysis of cytotoxic T cell precursors against host type target cells (Fig. 3).In conclusion, our results suggest that potent veto CD8 Tcm can be generated from the memory pool of donors positive for viral reactivity by stimulation against viral antigens. Such veto Tcm could be most attractive for haploidenitcal transplantation of TCDBM, as they can enable engraftment following non-myeloablative conditioning and at the same time provide anti-viral protection.*E.B.L and N.O.G contributed equally [Display omitted] [Display omitted] [Display omitted] DisclosuresBachar Lustig:Yeda LTD: Patents & Royalties. Or Geva:Yeda LTD: Patents & Royalties. Lask:Yeda LTD: Patents & Royalties. Gidron:Yeda LTD: Patents & Royalties. Kagan:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

At Least 20% Donor Myeloid Chimerism Is Necessary to Reverse the Sickle Phenotype after Allogeneic Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell transplantation (HSCT) is the only curative option for patients with sickle cell disease (SCD). HLA-matched sibling HSCT has become increasingly successful, and haploidentical approaches are being explored to increase the donor pool. Advances have also been made in gene therapy such that clinical trials are now underway for the first time in patients with SCD. The question of what percentage of donor chimerism is necessary to cure SCD is critical in identifying the target percentage of the transferred globin gene needed to reverse SCD. One study showed that a whole blood donor chimerism level as low as 11% was sufficient to render patients free of SCD1; however, lineage specific chimerism was not performed. We have previously demonstrated that peripheral blood myeloid chimerism reflects the HSC compartment. We sought to determine the percentage of donor myeloid chimerism (DMC) necessary to reverse the sickle phenotype in our patients who underwent HSCT.In protocols testing both HLA-matched and haploidentical nonmyeloablative HSCT in over 70 patients with SCD, we identified 3 who despite robust initial engraftment experienced declining DMC levels over prolonged follow up eventually associated with return of SCD. All 3 patients’ donors had sickle cell trait with HbS ranging from 36-40%. Percentages of DMC and HbS were monitored at day 100 and at least every 6-12 months post-transplant thereafter. The 3 patients initially achieved nearly 100% DMC; however, their chimerism levels slowly decreased over time (see Figure 1). As long as mean DMC was >20%, mean %HbS remained less than 50% (similar to their sickle cell trait donors) and the patients stayed free of SCD-related symptoms. However, mean DMC at 2, 3, 4, and 5 years post-transplant fell below 20%, were 18, 12, 11, and 10%, and were associated with mean %HbS levels of 52.5, 51.6, 57.8, and 64.2%. Further, 2 of the patients had return of their recurrent painful crises, and the third patient with moyamoya syndrome developed severe anemia requiring re-initiation of chronic transfusion therapy.We subsequently developed a mathematical model to test the hypothesis that the percentage of necessary DMC is determined solely by the differences between donor and recipient red blood cell (RBC) half-lives. The model incorporates donor and recipient stem cell kinetics (self renewal and differentiation), which are assumed to be identical, as well as donor and recipient RBC survival times, which are known to be different. The model assumes first order kinetics for erythropoiesis and RBC senescence/destruction. It predicts the fraction of donor RBC as a function of DMC and donor/recipient RBC lifetimes. Although we did not directly measure RBC lifetimes, we identified 4 patients with SCD in the literature where reticulocyte percentages ranged from 9.0 to 21.0% and corresponding RBC half-lives determined by radioactive sodium chromate were 1 to 11 days2. We assumed that in erythropoietic homeostasis, RBC lifetime must be proportional to the inverse of the reticulocyte fraction; and we confirmed this relationship using data from these 4 patients with a good coefficient of determination (R2= 0.91). Therefore we reasoned that (at stable hemoglobin concentration) the reticulocyte fraction is a good proxy for RBC lifetime. Figure 1 depicts a comparison between the measured and our model predicted HbS fractions in our 3 patients transplanted with sickle cell trait donors using a mean sickle RBC lifetime of 7 days. As observed in our 3 patients, our model predicts that DMC of at least 20% is necessary to reverse the sickle phenotype.Therefore, our model predicts that genetic strategies aimed at SCD must achieve correction levels of at least 20% to reverse the sickle phenotype. Further, the mechanism explaining why a minority of donor myeloid cells is adequate is due soley to the vast differences in RBC survival between donor and recipient cells. Lastly, recipient RBC survival is lowest in patients with the highest reticulocyte counts suggesting that gene therapy may be most efficacious in subjects with high reticulocyte percentages as our model predicts that lower levels of correction would be sufficient in this setting. Further studies are indicated to validate our findings.1. Walters MC et al. Biol Blood Marrow Transplant. 2001;7(12):665-673.2. Weinstein IM et al. Blood. 1954;9(12):1155-1164. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Engraftment Efficiency after Intra–Bone Marrow versus Intravenous Transplantation of Bone Marrow Cells in a Canine Nonmyeloablative Dog Leukocyte Antigen-Identical Transplantation Model

An intra–bone marrow (IBM) hematopoietic stem cell transplantation (HSCT) is assumed to optimize the homing process and therefore to improve engraftment as well as hematopoietic recovery compared with conventional i.v. HSCT. This study investigated the feasibility and efficacy of IBM HSCT after nonmyeloablative conditioning in an allogeneic canine HSCT model. Two study cohorts received IBM HSCT of either density gradient (IBM-I, n = 7) or buffy coat (IBM-II, n = 6) enriched bone marrow cells. An historical i.v. HSCT cohort served as control. Before allogeneic HSCT experiments were performed, we investigated the feasibility of IBM HSCT by using technetium-99m marked autologous grafts. Scintigraphic analyses confirmed that most IBM-injected autologous cells remained at the injection sites, independent of the applied volume. In addition, cell migration to other bones occurred. The enrichment process led to different allogeneic graft volumes (IBM-I, 2 × 5 mL; IBM-II, 2 × 25 mL) and significantly lower counts of total nucleated cells in IBM-I grafts compared with IBM-II grafts (1.6 × 108/kg versus 3.8 × 108/kg). After allogeneic HSCT, dogs of the IBM-I group showed a delayed engraftment with lower levels of donor chimerism when compared with IBM-II or to i.v. HSCT. Dogs of the IBM-II group tended to reveal slightly faster early leukocyte engraftment kinetics than intravenously transplanted animals. However, thrombocytopenia was significantly prolonged in both IBM groups when compared with i.v. HSCT. In conclusion, IBM HSCT is feasible in a nonmyeloablative HSCT setting but failed to significantly improve engraftment kinetics and hematopoietic recovery in comparison with conventional i.v. HSCT.

Kinetics of Langerhans cell chimerism in the skin of dogs following 2Gy TBI allogeneic hematopoietic stem cell transplantation.

BackgroundLangerhans cells (LC) are bone marrow-derived cells in the skin. The LC donor/recipient chimerism is assumed to influence the incidence and severity of graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT). In nonmyeloablative (NM) HSCT the appearance of acute GVHD is delayed when compared with myeloablative conditioning. Therefore, we examined the development of LC chimerism in a NM canine HSCT model.Methods2 Gy conditioned dogs received bone marrow from dog leukocyte antigen identical littermates. Skin biopsies were obtained pre- and post-transplant. LC isolation was performed by immunomagnetic separation and chimerism analysis by PCR analyzing variable-number-of-tandem-repeat markers with subsequent capillary electrophoresis.ResultsAll dogs engrafted. Compared to peripheral blood chimerism the development of LC chimerism was delayed (earliest at day +56). None of the dogs achieved complete donor LC chimerism, although two dogs manifested a 100 % donor chimerism in peripheral blood at days +91 and +77. Of interest, one dog remained LC chimeric despite loss of donor chimerism in the peripheral blood cells.ConclusionOur study indicates that LC donor chimerism correlates with chimerism development in the peripheral blood but occurs delayed following NM-HSCT.

Dietary created niche for non-myeloablative hematopoietic stem cell transplantation

Dietary created niche for non-myeloablative hematopoietic stem cell transplantation

Impact of outpatient non-myeloablative haematopoietic stem cell transplantation in quality of life vs. conventional therapy

The role of outpatient hematopoietic stem cell transplantation (HSCT) as a therapeutic tool has been strengthened significantly because of the increasing number of patients undergoing this treatment. Due the very nature of this procedure, one of the aspects that should not be overlooked is the quality of life (QOL) of patients undergoing HSCT. Thus, one must consider not only health status after treatment, but also, the psychosocial implications for the patient. This is an observational, longitudinal, and prospective study to assess QOL in patients undergoing outpatient HSCT vs. similar patients receiving medical treatment (MxTx). By applying the COOP/WONKA charts on five occasions (pre-HSCT/initial, post-HSCT/first month, and at 3, 6, and 9 months), thirty-eight patients were analysed, 19 with HSCT and 19 with MxTx with no differences in age, gender or diagnosis. The initial survey found significant differences only in pain perception, which was higher in the HSCT group (p = .08); at the first month, there was a greater tendency for feelings of depression or anxiety in the HSCT group (p = .016), with more limitations in social (p = .003) and daily (p = .044) activities. From 3 months post-HSCT, the results were very similar. The differences persisted only in the area of social activities. Four patients developed graft-versus-host disease with no significant difference in the scores obtained compared to other transplant patients at 3, 6, and 9 months (p = .26) of follow-up.

The role of donor-derived veto cells in nonmyeloablative haploidentical HSCT.

Allogeneic HSCT offers a route for achieving immune tolerance via mixed chimerism and remains the sole curative option for many nonmalignant, autoimmune and metabolic diseases. Present-day improvements of nonmyeloablative protocols are increasing the safety of HSCT, thereby widening the target population and resurrecting the interest of HSCT application as a platform for tolerance induction in organ transplantation. Using high cell doses of T-cell-depleted (TCD) grafts has been shown to circumvent graft-vs-host disease, leaving graft rejection as the main hindrance due to the robust host immunity that remains after mild conditioning. In this review we highlight the advantages of utilizing unique non-alloreactive 'veto' cells, such as anti-third party central memory CD8 T cells (Tcm), to enable induction of mixed chimerism after megadose HSCT under nonmyeloablative conditioning. Co-administration of HSCT with veto Tcm allows for induction of mixed chimerism that was successfully translated into immune tolerance, as demonstrated by engraftment of donor-type skin grafts. These veto Tcm cells have been shown to specifically eradicate anti-donor host T cells, through lymph-node sequestration and deletion, thus sparing host immunity and circumventing the need for life-long immunosuppression. Hence, data indicate that this treatment modality of combined TCD HSCT and adoptive transfer of Tcm veto cells under nonmyeloablative conditions may serve as a valuable tool for treatment of patients with a wide array of disorders such as hemoglobinopathies, autoimmune diseases and as a prelude for organ tolerance.

Nonmyeloablative Hematopoietic Stem Cell Transplantation in Multiple Sclerosis

Nonmyeloablative hematopoietic stem cell transplantation (HSCT) has been hypothesized to reset the immune system and provide long-term benefit in some