Haploidentical Stem Cell Research Articles

Incorporation of Posttransplant Cyclophosphamide (PCy) As Part of Standard Immunoprophylaxis (IP) for All Allogeneic Transplants: A Retrospective, Single Institution Study

Background: The incorporation of PCy in IP has allowed transplantation of stem cells from haploidentical (HI) family members such that nearly all patients have a potential donor. Thus far, HI stem cell transplantation with PCy appears to yield comparable results to matched unrelated (MUD) and matched sibling donors (MSD) who have been treated with conventional GVHD regimens, but with less chronic GVHD (cGVHD). Particularly in light of the low incidence of cGVHD, which has not been achieved with other IP strategies after T cell-replete products, PCy is being investigated after MUD and MSD transplantation where complications from cGVHD remain the major cause of non-relapse mortality. A recent study from the BMTCTN showed that in patients conditioned with reduced intensity regimens and who received MSD and MUD stem cells, the addition of PCy to standard IP (SIP) was superior to either bortezomib or maravoric in the composite endpoint of graft-versus-host disease-free, relapse-free survival (GRFS). However, this study did not include patients who received ablative conditioning regimens and did not report on the percentage of patients who were disease-free and off immunosuppression (DFOI) at 1 year after transplant. In the present study, we have compared our experience with the addition of PCy for essentially all allogeneic stem cell transplants treated over a 2 year period with the results of patients treated with SIP in the prior two year span. Outcomes of interest included one-year overall survival (OS) and one-year GRFS as well as the percentage of patients DFOI at one year. Methods: With the exception of patients receiving umbilical cord blood transplants, beginning in April 2016, all but two patients who received allogeneic transplants were given mobilized peripheral blood stem cells and then treated with PCy on days +3 and +4 followed by tacrolimus and mycophenolate on day 5. In the absence of GVHD, mycophenolate was stopped at days +35-50 and tacrolimus was tapered beginning after day +100 unless there was low donor chimerism or a suspicion of relapse in which case tacrolimus could be tapered sooner. In order to have at least one-year follow-up, the last patient included in the study was treated before April 2018. During this time period, MSD were prioritized over MUD which in turn were chosen over haploidentical donors. For comparison, we looked at the prior 2 year period (2014-2016) in which patients were treated with SIP (including ATG in patients who received MUD stem cells). Because of a higher percentage of patients with an advanced disease risk index (DRI) in the years 2014-2016, we restricted our analysis in the SIP cohort to those patients with low and intermediate risk disease but included all patients in the more recent period who received PCy. Results: There were 68 patients treated in the PCy group, including 2 patients who received PCy after HI transplants in the years 2014 and 2015. After eliminating patients with high DRI there were 40 patients in the earlier SIP cohort of patients. The resulting patient groups were similar with respect to median age (53) and diagnosis (approximately 80% of patients with AML and ALL). There was a slightly higher percentage of patients in the SIP group with hematopoietic cell transplantation-comorbidity index scores of 3 or more (52.5 vs 48.5). In the PCy group the number of patients with early, intermediate and advanced DRI were 2, 53 and 13, whereas in the (modified) SIP category 2 patients had a low DRI and 38 had intermediate DRI. In the PCy group, HI donors comprised 26.5% of the total compared to 19.1% MSD and 54.4% MUD donors. In the SIP group, MSD and MUD donors accounted for 30% and 70% of the donors. One-year percentages of OS, GRFS and DFOI were 79.4, 47.1 and 44.1 in the PCy group compared to 72, 45 and 35 in the SIP cohort. If the analysis of the PCy group is limited to the 50 patients with MSD and MUD donors (as in the SIP cohort), the one-year OS, GRFS and DFOI are 88, 52 and 52. Conclusions: PCy in combination with SIP resulted in at least comparable results as SIP despite the inclusion of 19% of patients with a high DRI and 26.5% HI donors. The results with the addition of PCy are excellent in patients with MSD and MUD donors with more than half of the patients GRFS and DFOI at one year. Future studies on GVHD prophylaxis should report DFOI as the latter status may be the best platform for posttransplant strategies aimed at eliminating minimal residual disease and for improving QOL. Disclosures No relevant conflicts of interest to declare.

Haploidentical Hematopoietic Stem Cell Transplantation Compared with HLA-Matched Stem Cell Transplantation for Refractory or Relapsed Aggressive Non-Hodgkin Lymphoma

Background: Non-Hodgkin's lymphoma (NHL) is a malignant hematological tumor that accounts for approximately 90% of lymphoma in China. About 2/3 of NHL patients can be treated or even cure, but still about one-third will die from refractory or relapsed disease, especially high-risk patients. In these cases, continued conventional chemotherapy will not benefit, and may even lead to significant chemo-related toxicity and reduce the quality of life. Allogeneic hematopoietic stem cell transplantation (allo-SCT) is an effective treatment for whom with chemoresistent, recurrence after autologous transplantation or without autologous transplantation indication. However, less than 30% of patients can find HLA-matched relative donors. hematopoietic stem cell transplantation (haplo-HSCT) enables more than 90% of patients to find suitable donors in the shortest time, bringing good news to patients without HLA-matched donors.Therefore, we compare the efficacy of haploidentical stem-cell transplantation (haplo-SCT) for patients with refractory relapsed(R/R) aggressive non-Hodgkin lymphoma (NHL) with those who contemporaneously receiving HLA-matched SCT in myeloablative conditioning settings. Methods: One hundred and fifty-one patients who had undergone haplo-SCT (n=81) or HLA-matched SCT (n=70, sibling or unrelated) in our center between January 2006 and December2018 were enrolled. A median age at alloSCT was 30(5-59) years old. All patients received a myeloablative conditioning (MAC) consisting of total body irradiation (12 Gy) combined cyclophosphamide or busulphan plus cyclophosphamide,and followed by infusion of granulocyte-colony stimulating factor-primed bone marrow (G-BM) and/or peripheral blood stem cells without in vitro T cell depletion. In the case of haplo-SCT and HLA-matched unrelated donor for SCT, the GVHD prophylaxis consisted of antithymocyte globulin, cyclosporine A, mycophenolate mofetil and a short course of methotrexate. The clinical effect, hematopoietic reconstitution, and transplant-related complications were retrospective analyzed. Results: One hundred and forty-five (96%) patients engrafted with a median time to neutrophil and platelet recovery of 12 and 15 days, respectively. At a median follow-up of 20 months, 66 of 151 patients were alive (43.7%) and 67 (44.4%) were dead (39 disease recurrence, 27 transplantation-related mortality (TRM)). Between the haplo-SCT and HLA-matched SCT group, the corresponding progress free survival (PFS) rate was 60.5% and 54.3% (P = .938), respectively; and overall survival (OS) rate were 67.9% and 55.7% (P = .460), respectively. The cumulative incidences of relapse (RI) were 36.6% and 37.7% (P = .836), and those of NRM were 20.0 % and 24.7% (P = .530), respectively. And cumulative incidences of chronic GVHD were 33.3% and 30.0%(P=.407), respectively. These data showed no difference in every major HSCT endpoint between two groups. Multivariate analysis suggested that occurrence of grade Ⅲ-Ⅳ aGVHD had a significant worse outcome, primary chemorefractory was the strongest factors for relapse. Conclusion: The haplo-SCT group obtained the OS and PFS similar to HLA-matched SCT. And there is no higher incidence of GVHD observed in haplo group. Our results indicate that the outcomes of haplo-SCT are equivalent to HLA-matched SCT, and that MAC followed by haplo-SCT can be an acceptable and feasible alternative for patients with R/R NHL who having no access to a HLA-matched donor. Figure Disclosures No relevant conflicts of interest to declare.

Risk Factors and Different Therapeutic Patterns of Late-Onset Hemorrhagic Cystitis after Haploidentical Allogeneic Stem Cell Transplantation

Introduction Late-onset hemorrhagic cystitis (LOHC) is a common complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with an incidence ranging from 6.5% to 70% and leads to prolonged hospitalization and even death (Silva Lde P et al, Haematologica, 2010; LAM et al, Transpl Infect Dis, 2017). The pathogenesis of LOHC remains obscure, but in our previous study, viral infections and acute graft-versus-host disease (aGVHD) have frequently been shown to be associated with its development (Han et al., Am. J. Hematol, 2014). Therapeutic strategies for LOHC are still not standardized due to the complicated clinical background and given the paradoxical phenomenon that immunosuppressive agents, such as steroids, are necessary for aGVHD but cause an immunosuppressive state. We aimed to investigate the incidence, risk factors and outcomes of LOHC after allogeneic stem cell transplantation. Additionally, for the first time, we propose four therapeutic patterns and their impact on prognosis of LOHC to aid in clinical decision making. Methods This retrospective, nested, case-control study reviewed data from 2158 patients who received allo-HSCT from January 2014 to December 2018. In total, 364 (16.87%) patients were diagnosed with LOHC. Individual matching was performed by randomly selecting 3 controls from the same cohort for each identified LOHC patient according to the time of allo-HSCT. Standard basic measures included hyperhydration, forced diuresis, insertion of a vesical catheter for intermittent or continuous bladder irrigation and evacuation of clots. Patients with grade III-IV LOHC were divided into four groups according to immunosuppressant use (steroid, MMF and Basiliximab) 1 week before and after the onset of LOHC: an intensified-intensified (I-I) group, intensified-not intensified (I-N) group, not intensified-intensified (N-I) group and not intensified-not intensified (N-N) group. "Intensified" was defined as an increase in any immunosuppressant, and "not intensified" was defined as maintenance or tapering of all immunosuppressants. Data concerning the baseline characteristics, incidence, treatment patterns and outcomes were recorded. Results In total, 364 patients developed LOHC at a median of 29 days (range, 23-37.75 days), with a cumulative incidence of 16.87%. AML (HR =0.493 95% CI, 0.244-0.997; p=0.049), AA (HR =0.444, 95% CI,0.18-1.096; p=0.078), HLA match(HR =0.149, 95% CI, 0.036-0.616; p=0.009), days to platelet engraftment(HR =1.023, 95% CI, 0.998-1.049;p=0.069)and CMV viremia (HR =2.365, 95% CI, 1.179,4.733;p=0.015)were associated with LOHC. Only HLA match (HR =0.111, 95% CI, 0.014-0.878; p=0.037)remained significant in the multivariate analysis. 6.5%, 12.9%, 48.39% and 32.26% patients with III-IV LOHC were divided into I-I, I-N, N-I and N-N groups, respectively. There were no significant differences in overall survival (p=0.05) and the incidence of CMV viremia (p=0.187) among the four groups. The incidence of relapse (p=0.007) and the incidence of aGVHD (p=0.01) was highest in the I-I group, followed by the N-I, I-N and N-N groups. 50% of the patients in N-I group showed the improvement of LOHC, which is significantly highest among the four groups(p=0.000). However, no statistical significance was found regarding the CMV viremia turning shade or the improvement of aGVHD. Non-relapse mortality (NRM) was observed in 25% of patients without resolution of LOHC. NRM was 0% among patients without intensified immunosuppression but was 25%, 0% and 50% among those with intensified immunosuppression before LOHC, after LOHC and before and after LOHC, respectively. Conclusion The independent risk factors for LOHC after allo-HSCT were HLA mismatch. Avoiding intensified immunosuppression that damages endothelium or reactivates the related virus may improve transplant outcome. The N-N pattern could reduce the incidence of CMV viremia or aGVHD in the LOHC patients, and the N-I pattern might be more promising as a therapeutic strategy for LOHC. Disclosures No relevant conflicts of interest to declare. Disclosures No relevant conflicts of interest to declare.

Safety Study and Preliminary Efficacy of Infusion Haploidentical Mesenchymal Stem Cells Derived From Bone Marrow for Treating Recessive Dystrophic Epidermolysis Bullosa

Safety Study and Preliminary Efficacy of Infusion Haploidentical Mesenchymal Stem Cells Derived From Bone Marrow for Treating Recessive Dystrophic Epidermolysis Bullosa

Haploidentical Stem Cell Transplantation With Posttransplant Cyclophosphamide Therapy vs Other Donor Transplantations in Adults With Hematologic Cancers

Use of haploidentical (HAPLO) stem cell transplantation with posttransplant cyclophosphamide is rapidly increasing in adults with hematologic cancers. However, its specific role compared with other transplant strategies has yet to be identified. To synthesize the existing evidence regarding outcomes of stem cell transplantations comparing HAPLO stem cell transplantation and posttransplant cyclophosphamide therapy with transplantations from matched related donors (MRDs), matched unrelated donors (MUDs), or mismatched unrelated donors (MMUDs). PubMed, Cochrane Library, ClinicalTrials.gov, and meeting abstracts were searched for the key words haploidentical and cyclophosphamide from inception through March 1, 2019. Studies comparing HAPLO stem cell transplantation and posttransplant cyclophosphamide therapy with transplantations from other donors in adults with hematologic cancers were eligible for meta-analysis. Pooled odds ratios (ORs) and 95% CIs were calculated using a random-effects model. Main outcomes were all-cause mortality, nonrelapse mortality, and relapse. A total of 30 studies including 22 974 participants were analyzed. HAPLO stem cell transplantation with posttransplant cyclophosphamide therapy was associated with increased all-cause mortality compared with MRDs (OR, 1.17; 95% CI, 1.05-1.30), similar all-cause mortality compared with MUDs (OR, 1.06; 95% CI, 0.96-1.18), and reduced all-cause mortality compared with MMUDs (OR, 0.75; 95% CI, 0.61-0.92). Regarding nonrelapse mortality, HAPLO stem cell transplantation with posttransplant cyclophosphamide was associated with worse outcomes compared with MRDs (OR, 1.20; 95% CI, 1.04-1.40) but better outcomes compared with MUDs (OR, 0.75; 95% CI, 0.61-0.92) and MMUDs (OR, 0.51; 95% CI, 0.25-1.02). In terms of relapse, HAPLO stem cell transplantation with posttransplant cyclophosphamide was associated with similar outcome compared with MRDs (OR, 1.01; 95% CI, 0.86-1.17) and MMUDs (OR, 1.06; 95% CI, 0.77-1.47) but showed increased relapse compared with MUDs (OR, 1.20; 95% CI, 1.03-1.40). Results of this meta-analysis suggest that MRDs, if available, remain the optimal donors regarding mortality and HAPLO stem cell transplantation with posttransplant cyclophosphamide may be preferred over MMUDs. Prospective comparisons with MUDs are needed.

Unmanipulated haploidentical peripheral blood stem cell transplantation for patients with Philadelphia-negative acute lymphoblastic leukaemia in first complete remission

Haploidentical peripheral blood stem cell transplantation (Haplo-PBSCT) is a promising treatment option for patients with Ph-negative acute lymphoblastic leukemia (ALL). In this study, we retrospectively analyzed data from Ph-negative ALL patients who underwent haplo-PBSCT during their first complete remission (CR1), and compared the long-term outcomes between the standard-risk and high-risk patients. The 3-year probability of relapse was 7.6% and 16.7% for the standard- and high-risk group (p = .274). The 3-year probability of disease-free survival (DFS) and overall survival (OS) for the standard-risk versus high-risk groups were 84.6% versus 50% (p = .0063) and 92.3% versus 61.1% (p = .046), respectively. Univariate analysis showed that a diagnosis of high risk with fusion/mutation genes were associated with worse outcomes, which was confirmed by multivariate analysis (p = .016). In summary, haplo-PBSCT may be a promising alternative for patients with Ph-negative ALL in CR1, although the fusion/mutation genes in high-risk patients may relatively impair the long-term efficacy compared with standard-risk patients.

Efficacy analysis of haploidentical allogeneic hematopoietic stem cell transplantation combined with third-party umbilical cord blood infusion in treatment of high-risk lymphoblastic malignancies

Objective To evaluate the efficacy of haploidentical allogeneic hematopoietic stem cell transplantation (Haplo-HSCT) combined with third-party umbilical cord blood (UCB) infusion in treatment of high-risk lymphoblastic malignancies. Methods The clinical data of 20 patients with high-risk lymphoblastic malignancies who received Haplo-HSCT from April 2012 to April 2015 in Shanghai General Hospital were retrospectively analyzed, which were compared with the data from 15 patients who underwent matched unrelated donor HSCT (MUD-HSCT) or 14 matched sibling donor HSCT (MSD-HSCT) during the same period. The efficacy of Haplo-HSCT combined with UCB infusion in treatment of high-risk lymphoblastic malignancies was evaluated. The preparative regimen mainly consisted of teniposide, cyclophosphamide and total body irradiation (TBI). Graft versus host disease (GVHD) preparative regimen included cyclosporine and a short term of methotrexate. The patients who received Haplo-HSCT combined with UCB infusion and MUD-HSCT were treated with antithymocyte globulin (ATG). Results After the transplantation, one patient in MUD-HSCT group and one in MSD-HSCT group died within 21 days, and other patients were engrafted successfully. The median time of neutrophil engraftment was 13 days (10-18 d), 12 days (9-16 d) and 12 days (9-14 d) in Haplo-HSCT + UCB group, MUD-HSCT group and MSD-HSCT group, respectively; the median time of platelets engraftment was 11 days (9-18 d), 12 days (10-23 d) and 12 days (9-14 d), respectively. There were 10, 3, 3 cases of grade Ⅱ-Ⅳ acute GVHD at day 100 in the three groups, respectively, and there were 6, 4, 3 cases of chronic GVHD in the three groups, respectively. The 2-year cumulative incidence of relapse was 40.6%, 66.2% and 26.7%, respectively. The predicted 2-year overall survival rate was 37.9%, 42.9% and 55.4%, respectively. All these data had no significant difference (all P > 0.05). Conclusion The efficacy of Haplo-HSCT combined with UCB infusion is similar to that of MUD-HSCT or MSD-HSCT in treatment of high-risk lymphoblastic malignancies, which should be recommended to the patients with high-risk lymphoblastic malignancies and without matched donors. Key words: Lymphoblastic malignancies; Allogeneic hematopoietic stem cell transplantation; Fetal blood

Reduced-intensity conditioning haploidentical peripheral blood hematopoietic stem cell transplantation without T-cell depletion in vitro for treatment of relapsed Hodgkin lymphoma after autologous hematopoietic stem cell transplantation: report of one case

Reduced-intensity conditioning haploidentical peripheral blood hematopoietic stem cell transplantation without T-cell depletion in vitro for treatment of relapsed Hodgkin lymphoma after autologous hematopoietic stem cell transplantation: report of one case

Relationship of Cell Compositions in Allografts with Outcomes after Haploidentical Transplantation for Acquired Severe Aplastic Anemia: Effects of CD34+ and CD14+ Cell Doses.

Background:The dose of certain cell types in allografts affects engraftment kinetics and clinical outcomes after allogeneic stem cell transplantation (SCT). Hence, the present study investigated the association of cell compositions in allografts with outcomes after unmanipulated haploidentical SCT (haplo-SCT) for patients with acquired severe aplastic anemia (SAA).Methods:A total of 131 patients with SAA who underwent haplo-SCT were retrospectively enrolled. Cell subsets in allografts were determined using flow cytometry. To analyze the association of cellular compositions and outcomes, Mann–Whitney U nonparametric tests were conducted for patient age, sex, weight, human leukocyte antigen mismatched loci, ABO-matched status, patient ABO blood type, donor-recipient sex match, donor-recipient relationship, and each graft component. Multivariate analysis was performed using logistic regression to determine independent influence factors involving dichotomous variables selected from the univariate analysis.Results:A total of 126 patients (97.7%) achieved neutrophil engraftment, and 121 patients (95.7%) achieved platelet engraftment. At 100 days after transplantation, the cumulative incidence of II–IV acute graft-versus-host disease (GVHD) was 32.6%. After a median follow-up of 842 (range: 124–4110) days for surviving patients, the cumulative incidence of total chronic GVHD at 3 years after transplantation was 33.7%. The probability of overall survival at 3 years was 83.0%. Multivariate analysis showed that higher total doses of CD14+ (P = 0.018) and CD34+ cells (P < 0.001) were associated with a successful platelet engraftment. A successful platelet was associated with superior survival (P < 0.001). No correlation of other cell components with outcomes was observed.Conclusions:These results provide evidence and explain that higher doses of CD34+ and CD14+ cells in haploidentical allografts positively affect platelet engraftment, contributing to superior survival for patients with SAA.

Lymphnode tuberculosis in a 4-year-old boy with relapsed ganglioneuroblastoma: a case report

BackgroundMycobacterium tuberculosis (M. tuberculosis) disease is a generally well-known problem among immunocompromised adults and children. In pediatric oncology, only few cases of M. tuberculosis disease are reported so far.Case presentationWe report a case of concomitant lymphnode tuberculosis in a 4-year-old German boy with relapsed ganglioneuroblastoma. 18 months after the initial diagnosis, relapse with new paravertebral lesions and new lesions in the left lower lobe of the lung and in the perihilar lymphnodes suspicious of metastases of the ganglioneuroblastoma were detected. While relapse in the tumor was confirmed, unexpectedly, pathologic examination revealed morphological diagnosis of lymphnode tuberculosis. The boy was of German background without previous history of tuberculosis exposure. Both, antituberculostatic and relapse treatment were immediately initiated. Three months on, MRI revealed regressive findings in the lung and lymphnodes and partial response in the tumor. The patient underwent second MiBG therapy and haploidentical stem cell transplantation.ConclusionThe diagnosis of lymphnode tuberculosis in a 4-year-old German boy with relapsed ganglioneuroblastoma was only made by chance, but most likely saved his life. Pediatric oncologist should be aware of tuberculosis as the incidence might increase over time and the timely diagnosis of a potentially preventable M. tuberculosis disease is irreplaceable. Further studies are needed to explore the incidence of M. tuberculosis infections and the value of IGRA, testing for latent tuberculosis infection prior to chemotherapy in children with underlying malignancies.

Managing Drug Interactions in Cancer Therapy: A Guide for the Advanced Practitioner

Mrs. P is a 30-year-old woman who presented to our bone marrow transplant program with myelodysplastic syndrome (MDS). She received a haploidentical allogeneic stem cell transplant with a conditioning regimen consisting of busulfan and cyclophosphamide. This treatment was followed by post-transplant immunosuppression for graft-versus-host disease (GVHD) with cyclophosphamide, mycophenolate mofetil (MMF), and tacrolimus (see Table 1 for medication list). Tacrolimus levels were monitored twice a week with adjustment to a goal range of between 5 and 10 ng/mL. We initiated tacrolimus at a dose of 0.03 mg/kg by mouth twice daily (rounded to 2 mg by mouth twice daily). Drug interactions were assessed by the clinical pharmacist prior to admission, routinely with medication changes, and then upon discharge.

Related Haploidentical Peripheral Blood Stem Cell Transplantation as a Rescue after Double Cord Blood Unit Graft Rejection in an Anti-HLA-Immunized Patient

Related Haploidentical Peripheral Blood Stem Cell Transplantation as a Rescue after Double Cord Blood Unit Graft Rejection in an Anti-HLA-Immunized Patient

Complementary Transplantation with Haploidentical Stem Cells and Unrelated Cord Blood in Thalassemia Major

▪BackgroundEffects of haploidentical stem cells (Hap-SC) and unrelated cord blood (UCB) transplant are unsatisfactory in thalassemia major (TM) so far. High rejection and low TM-free survive (TFS) were key clinical issues. Increased rejection often offset the effort of lowered GVHD. The effect of alloreactive clone destruction in post-transplant cyclophosphamide (Cy, PTCy) transplant resulted in low GVHD and high relapse but nice immuno-recovery and immuno-tolerance by keeping antivirus and regulatory T cells. These may just complement the congenital insufficiency, delay engraftment and slow immuno-recovery, of UCB transplantation. Conversely, graft vs. leukemia effect and controllable GVHD from naive T cells of UCB offset the lack above of hap-SC transplant with PTCy. Therefore, we developed a novel complementary transplant with Hap-SC and UCB (CT-hap-CB) for leukemia and TM patients.Patients and methodThirty-six TM patients received CT-hap-CB between December 2013 and June 2016. Median age was 8 (range, 3-24) years old. Median follow-up time was 19 (2-25) months. Conditioning (Regimen, CT-13) included Cy on day-8 to -7, Busulfan on day-6 to -4, Fludarabine on day-6 to -2 and Thiotepa on day-3. GVHD prophylaxis consisted of Cy on day+3 to +4, Mycophenolate mofetil and Tacrolimus from day+6. Noteworthily, UCB was infused on day+6. The latter 26 TM patients received additionally Thymoglobuline on day -11 to -9 (Regimen, CT-14).ResultsPreponderance engraftment of hap-SC, UCB, mixed stem cells (MSC) and rejection in16, 14, 3 and 3 patients, respectively, at the last follow-up. Initial (day+28) full hap-SC, UCB and MSC chimerism occurred in 15, 7 and 12 patients. Cells derived from UCB overtime were dominant instead of initial majority from hap-SC (Fig. 1) in MSC group. 7/10 UBS with centromeric B motif engrafted.Median times to neutrophil ≥ 0.5x109/L, platelet ≥ 20 x109/L and hemoglobin ≥ 80 g/L were day+20.0 (range; 14-47), 11.0 (9-162) and 11 (1-39); 47.0 (34-113), 66.0 (12-227) and 46.0 (13-63); and 24 (1-46), 14 (11-116) and 8 (4-91), respectively, in initial hap-SC (n, 15), UCB (n, 7) and MSC (n, 12) groups. Overall survive, thalassemia free survive (TFS), rejection and mortality related transplant were 91.2%, 85.7%, 5.6% and 8.8%, respectively. Impressively, all of the 26 patients who received CT-14 protocol were alive without TM (Fig. 2). Grade II, III and IV acute GVHD occurred in 3 patients, respectively. One patient died of grade IV GVHD and another died of infection when the parents abandoned therapy after rejecting graft. Chronic GVHD was mild and rare.SummaryCT-hap-CB resulted in high OS and TFS, especially CT-14 leaded to 100% TFS with low GVHD rate. MSC engraftment improved hematopoietic recovery of UCB. KIR and HLA typing impacted what stem cells engraftment. The multicenter study should be developed in the future. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Generation of genetically modified mice using CRISPR/Cas9 and haploid embryonic stem cell systems.

With the development of high-throughput sequencing technology in the post-genomic era, researchers have concentrated their efforts on elucidating the relationships between genes and their corresponding functions. Recently, important progress has been achieved in the generation of genetically modified mice based on CRISPR/Cas9 and haploid embryonic stem cell (haESC) approaches, which provide new platforms for gene function analysis, human disease modeling, and gene therapy. Here, we review the CRISPR/Cas9 and haESC technology for the generation of genetically modified mice and discuss the key challenges in the application of these approaches.

Optimization of individualized graft composition: CD3/CD19 depletion combined with CD34 selection for haploidentical transplantation.

Excessive T-cell depletion (TCD) is a prerequisite for graft manufacturing in haploidentical stem cell (SC) transplantation by using either CD34 selection or direct TCD such as CD3/CD19 depletion. To optimize graft composition we compared 1) direct or indirect TCD only, 2) a combination of CD3/CD19-depleted with CD34-selected grafts, or 3) TCD twice for depletion improvement based on our 10-year experience with 320 separations in graft manufacturing and quality control. SC recovery was significantly higher (85%, n = 187 vs. 73%, n = 115; p < 0.0001), but TCD was inferior (median log depletion, -3.6 vs. -5.2) for CD3/CD19 depletion compared to CD34 selection, respectively. For end products with less than -2.5 log TCD, a second depletion step led to a successful improvement in TCD. Thawing of grafts showed a high viability and recovery of SCs, but low NK-cell yield. To optimize individualized graft engineering, a calculator was developed to estimate the results of the final graft based on the content of CD34+ and CD3+ cells in the leukapheresis product. Finally, calculated splitting of the starting product followed by CD3/19 depletion together with CD34+ graft manipulation may enable the composition of optimized grafts with high CD34+-cell and minimal T-cell content.

Phase I Trial of IL-21 Ex Vivo Expanded NK Cells Administration to Prevent Disease Relapse after Haploidentical Stem-Cell Transplantation for Myeloid Leukemias

Background: As outcomes of haploidentical stem cell transplantation (HaploSCT) have improved, disease relapse represents the most common cause of treatment failure.Methods: We initiated a phase I clinical trial (clinicaltrials.gov NCT01904136) using peripheral blood-derived NK cells expanded ex vivo for 14 days with K562 antigen presenting cells expressing membrane-bound (mb) IL21 to prevent disease relapse after HaploSCT for patients with myeloid malignancies (AML,CML,MDS). We hypothesized that infusion of expanded and activated NK cells would compensate for the lower NK cell function in the early post-transplant period, and higher NK cell numbers would enhance anti-tumor effects of the graft. The primary endpoints were safety and determining the maximum tolerated dose (MTD). Patients were treated with a melphalan-based conditioning regimen (Figure). All patients had a primary bone marrow graft. NK cells were generated from peripheral blood mononuclear cells of the same donor obtained prior to marrow harvest and infused on days -2, +7 and on/after +28. The first infusion was with fresh and the other two were with cryopreserved NK cells. The dose escalation was planned in cohorts of 2 patients starting at 1x105/kg up to 1x109/kg or more if MTD will not be reached. Predictive NK alloreactivity and/or donor KIR B genotyping was preferred but not required to participate on study, however, was evaluated in all patients (Table).Results: Ten patients have been enrolled and treated to date. Of these, 8 patients were beyond Day+30 and were evaluated, 5 with AML (3 in CR1 with intermediate and 1 with high-risk cytogenetics, and 1 in CR2 FLT3+ with persistent MRD by flow cytometry) and 3 with CML (2 in second chronic phase, one with clonal evolution who failed multiple TKIs). The median age was 39 years (range 18-59). Four patients were males and 4 females. The NK cells dose escalation was as follows: 1x105/kg (N=2), 1x106/kg (N=3) and 1x107/kg (N=2). One patient was treated with 1x104/kg before full evaluation of 1x105/kg was completed. All patients achieved primary engraftment (100%). All patients except the one who received the lowest dose (1x104/kg) had sustained engraftment and 100% donor chimerism on Day 30 post-transplant. The median time to neutrophil engraftment was 18 days and to platelet engraftment was 26 days. The first patient had a mixed chimerism, developed secondary graft failure and concurrent parainfluenza pneumonia. He was re-transplanted with a different donor but died of treatment-related mortality (TRM). Of 7 patients evaluable for aGVHD, the maximum aGVHD grade was gr II in 4 patients. No gr III-IV aGVHD or cGVHD was observed. Only 3/7 patients had CMV reactivation (43% compared with 71% in retrospective data with the same treatment without NK cells), 2 requiring a brief period of treatment of approximately 1 month. None developed BK virus hemorrhagic cystitis. All patients achieved CR after transplant. One patient (#2) treated at 1x105/kg NK cell dose relapsed, received salvage treatment and is alive at last follow-up. All other patients are alive and in remission (N=6) after a median follow-up of 6 months (range 1-12.5). NK cell phenotype and function early post-transplant will be presented at the meeting.Conclusions: Doses up to 1x107/kg of ex vivo expanded NK cells using the mbIL-21 method can be safely administered after HaploSCT. Administration of these cells in this setting in not associated with a higher incidence of aGVHD. There was a low rate of viral reactivation, suggesting that the infused NK cells may provide antiviral activity. MTD has not been reached, the study is ongoing.TablePT NrInitialsNK cell dose (/kg)Pt KIR LigandDonorDonor KIR LigandNK Allo-reactivityDonor KIR Haplotype# Cen-B/BKIR ScoreKIR CentromericKIR2DS1Outcome1RB1x104C2/C2, Bw4SonC2, Bw4NoA/A0NeutralCen-A/A-Died2FM1x105C1/C2, Bw4SonC1, Bw4NoA/B2BetterCen-A/B-Relapsed +1203RG1x105C1/C2, Bw4DaughterC1, C2, Bw4NoA/A0NeutralCen-A/A-CR +3744GM1x106C1/C1, Bw4SisterC1, C2, Bw4YesA/B2BetterCen-A/B-CR +1685DS1x106C1/C1BrotherC1, C2, Bw4YesA/A0NeutralCen-A/A-CR +1666MH1x107C1/C2, Bw4SisterC1, Bw4NoA/B2BestCen-B/B+CR +917JG1x106C1/C2, Bw4SisterC1, C2, Bw4NoA/A0NeutralCen-A/A-CR +358DD1x107C1/C1, Bw4FatherC1, Bw4NoA/A0NeutralCen-A/A-CR +879RR3x107C1/C1BrotherC1, C2YesA/B2BetterCen-A, Cen/Tel-B-NE10JC3x107C2/C2, Bw4SonC1/C2, Bw4YesA/B2BetterCen-A/B+NE [Display omitted] DisclosuresLee:Intrexon: Equity Ownership; Ziopharm: Equity Ownership; Cyto-sen: Equity Ownership. Rezvani:Pharmacyclics: Research Funding.

Hemophagocytic syndrome following haploidentical peripheral blood stem cell transplantation with post-transplant cyclophosphamide.

Hemophagocytic syndrome (HPS) is a rare but serious complication after allogeneic transplantation which has been reported to be particularly high after unrelated cord blood transplantation. We report on the incidence, risk factors and outcome of HPS in 51 patients (age 2-64 years) after haploidentical peripheral blood stem cell (PBSC) transplantation with post-transplantation cyclophosphamide (PTCY). The incidence of HPS was 12.2 %, occurring at a median of 18 days. The non-relapse mortality in patients with HPS was 83.3 % compared to 11.6 % in patients without HPS. Complete donor chimerism was documented in all patients with HPS. Definite infective etiology was identified in two patients only. The others were refractory to multiple lines of treatment and 3 patients underwent a second transplant. Even though the symptoms and biochemical markers of HPS showed prompt response in 2/3 patients undergoing a second allograft, they succumbed to infections before haematological recovery. The others succumbed to multi-organ failure or infections. Age < 10 years, transplantation for non-malignant disease and high CD34 content of the graft were identified as risk factors for HPS. Considering the fact that post-transplant HPS is usually a refractory and fatal condition, we discuss further attempts at deciphering the pathogenesis, developing modalities to prevent this complication and improve the outcome.

Establishment and Use of Mouse Haploid ES Cells.

Haploid genetics has facilitated new insights into mammalian pathways and disease mechanisms. Most animal cells are diploid, and mammalian haploid cell cultures have remained elusive for a long time. Recent methodological progress has enabled the routine derivation of haploid stem cell lines from mammalian haploid embryos. Here we provide detailed protocols for the establishment, culture, and manipulation of parthenogenetic and androgenetic haploid embryonic stem cells from mouse embryos.

The First Case of Ganciclovir-Resistant Cytomegalovirus Colitis with a 597-600 Deletion inUL97Gene after Stem Cell Transplantation in Korea

Human cytomegalovirus (CMV) infection has been a major concern in hematopoietic stem cell transplant recipients. Ganciclovir (GCV) resistance results mostly from mutations within the protein kinase UL97 gene. The three hot spots for GCV resistance (codons 460, 520, and 590-607) were well known. We describe a case of GCV-resistant CMV colitis caused by a 597-600 deletion in UL97 after haplo-identical peripheral blood stem cell transplantation (h-PBSCT) in a 46 year-old man with myelodysplastic syndrome. On post-PBSCT day 28, CMV antigenemia turned positive. Treatment of GCV was started and continued for 12 weeks but CMV antigenemia did not respond to the treatment and CMV colitis was worsened. The UL97 showed the in-frame deletion between codons 597 and 600 by direct sequencing. The treatment was switched to foscarnet and the antigenemia test was consecutively negative twice, and clinical symptoms improved. Despite the recovery of the patient from CMV colitis, the patient expired post-PBSCT day 146 from acute liver failure, hepatorenal syndrome and septic shock. This case is a first report of a deletion 597-600 in CMV UL97 in Korea. A 597-600 deletion in UL97 was responsible for the GCV resistance while preserving susceptibility to foscarnet. (Ann Clin Microbiol 2015;18:64-67)

Early Multilineage Chimerism Predicts The ‘Winning' Unit In Double Cord Blood Transplantation

BackgroundUmbilical cord blood (UCB) is an established alternative haemopoietic cell source for use in clinical transplantation for life-threatening malignant and non-malignant disorders. One challenge in using UCB in adolescents and adults has been delayed engraftment due to the finite and limited cell dose of a single unit, which has been shown to be a key determinant of engraftment, transplant related mortality and survival. The use of double unit transplantation, pioneered at the University of Minnesota, has become one of the most successful approaches to overcome this limitation to date. This group have also developed a reduced intensity conditioning (RIC) regimen, successfully broadening access to UCB transplant to older patients and those with co-morbidities. The kinetics of granulocyte, T cell and B cell chimerism in this setting require detailed study. MethodsSince 2009, the British Society of Blood and Marrow Transplantation have conducted a prospective, phase II study of UCB transplantation using the Minnesota RIC conditioning regimen (Fludarabine 200mg/m2, Cyclophosphamide 50mg/kg and TBI 2Gy), with Ciclosporin and Mycophenolate Mofetil graft versus host disease prophylaxis. Lineage specific chimerism was performed at days 7, 14, 21, 28, 35, 60, 100, 180, 360 and 720 post transplant and analysed at laboratories associated with participating centres. Results28 consecutive adult trial patients who have received a double unit transplant, have engrafted and have chimerism data up to at least day 35 are included in this analysis. The ‘winning' unit had a median unit:recipient match of 4/6 (range 4-6/6), with a median pre freeze total nucleated cell (TNC) count of 189x107 (range 83-250) and CD34 of 84x105 (range 23-169). The ‘losing' unit had a median unit:recipient match of 5/6 (range 4-6/6), with a median pre freeze total nucleated cell (TNC) count of 183x107 (range 127-303) and CD34 of 55x105 (range 42-95). Despite the low white count early post transplant, peripheral blood (PB) lineage specific chimerism for mononuclear cells (PBMC), T cells and granulocytes was feasible in nearly all patients. B cell chimerism was unsuccessful or not available in 55% of time points. The pattern of early T cell and granulocyte chimerism is summarised in the table.From day 60 onwards, the median granulocyte and T cell chimerism remained 100% winning unit. However, T cell chimerism at day 14 identified the winning unit in all patients with a result at this time point (n=25). Contribution to the B cell compartment was 100% winning unit by day 35, with 83% recipient at day 7 and 24% at day 14 and 4% losing unit at day 7 and 14. ConclusionsLineage specific chimerism is technically feasible in the immediate post transplant period and gives important insights into the kinetics of double cord blood unit engraftment. Although the ‘losing' unit may contribute to the B and T cell compartments in the first 2 weeks, it contributes little after day 21. The winning unit is clearly identifiable by day 14 in all lineages. Early granulocyte recovery (driven by G-CSF) in the RIC setting is seen to be primarily due to autologous recovery until around day 21 after which the winning unit predominates. These data provide an insight into the biology of engraftment that may also inform additional novel strategies such as ex vivo CD34 expansion and adding in haploidentical stem cells. Disclosures:No relevant conflicts of interest to declare.Table% Median contribution (range)Day post transplantRecipientWinning unitLosing unitT CELLS770 (22-94)23 (0-73)5 (0-41)144 (0-30)87 (29-100)0 (0-38)210 (0-35)100 (52-100)0 (0-34)280 (0-24)100 (32-100)0 (0-44)350 (0-3)100 (58-100)0 (0-42)600 (0-4)100 (91-100)0 (0-8)GRANULOCYTES7100 (84-100)0 (0-16)0 (0-10)1455 (1-100)28 (0-99)0 (0-20)210 (0-99)80 (0-100)0 (0-25)280 (0-54)99 (15-100)0 (0-6)350 (0-27)99 (21-100)0 (0-16)600 (0-8)100 (40-100)0 (0-52)