Mixed Chimerism Research Articles

VLA-4 Agonist Enhances the Capacity of HSC Engraftment and Immune Reconstitution

Poor donor cell implantation and delayed hematopoietic reconstitution are important factors affecting the prognosis of patients after allogeneic hematopoietic stem cell transplantation (Allo-HSCT). However, our understanding of the critical endogenous signals that reconstitute hematopoiesis in transplant patients remains limited，which is essential for achieving targeted interventions and preventing donor-recipient chimeric states after transplantation. Here, we revealed the reconstitution dynamics of human allogeneic hematopoietic stem and progenitor cells (HSPCs) and immune cell subpopulations in the post-transplantation mixed donor chimerism (MDC) to fully donor chimerism (FDC) state at single-cell resolution. The hematopoietic stem cell (HSC) implantation process involves extensive interactions with immune cells mainly through surface Kcnq5, Itga4 (encoding a subunit of VLA-4), Tfrc, CD44 receptor, and ligands for Rps19, B2m, etc. Further identification of VLA-4(integrin α4β1) as a potential regulatory target for HSC implantation by single-cell sequencing of a stable allogeneic bone marrow transplantation (Allo-BMT) MDC-FDC mouse model. Grafts Itgα4 and Itgβ1 deletion impair donor implantation and further diminish the efficacy of donor lymphocyte infusion (DLI) following Allo-BMT, rather than directly affecting DLI. Critically, we identified a VLA-4 agonist that promotes donor implantation in the mixed chimeric mouse model of Allo-BMT in a time-dependent manner, with the most pronounced promotion of early T cell implantation in particular. Moreover, it was found to enhance the self-renewal capacity of HSC and lymphoid reconstitution in competitive transplantation and human umbilical cord blood transplantation xenograft experiments. Fortunately, VLA-4 agonist does not exacerbate the acute graft-versus-host disease(aGVHD) process while maintaining the graft-versus-leukemia (GVL) effect. VLA-4 agonist not only upregulates of both Itgα4 and Itgβ1 expression, but also promotes the ERK/2 phosphorylation pathway, which affects HSC biological functions, and this process can be inhibited by ERK kinase inhibitors. These results provide insights into the regenerative process of transplanted HSPCs in human patients，and demonstrate a previously unknown positive regulation of HSC engraftment and immune reconstitution by VLA-4, and allow for a new and simple translational strategy to enhance HSC transplantation.

Risk-Adapted Donor Lymphocyte Infusion after Hematopoietic Cell Transplant in Pediatric and Young Adult Patients Is Associated with Low Risk of Graft Versus Host Disease

Introduction: Donor lymphocyte infusion (DLI) is a form of adoptive immunotherapy administered to enhance the graft-versus-leukemia (GVL) effect after an allogeneic hematopoietic cell transplantation (HCT). DLI is frequently chosen for its ease of administration and low risk of graft ablation as compared with other chemotherapeutic options in patients experiencing mixed chimerism (MC) or disease relapse. But in patients with acute leukemia experiencing relapse, clinical responses are seen in fewer than 20-30%. Success is also limited by the occurrence of acute and chronic graft-versus-host disease (GVHD). We had previously described our risk-adapted strategy of administering DLIs based on chimerism and donor source (Rujkijyanont P, et al. Blood Cancer J 2013). To determine the efficacy and toxicity of DLIs and to identify potential factors influencing the outcomes, we conducted a retrospective single center study including patients who received DLIs after HCT. Methods: We collected data on all HCTs and conventional DLIs that had been administered to patients from 1993 to 2022 for any indication at our center. Each HCT was considered as an independent event. Numeric variables were represented by median and range and compared using the Wilcoxon-Mann-Whitney test. Categorical variables were compared using the Fisher exact test. Competing-risk regression models using the Fine-Gray method were developed to evaluate risk factors for acute GVHD and chronic GVHD. Death and relapse were considered as competing risk events for these GVHD outcomes. Follow-up data were censored at subsequent HCT, 2 years after the first DLI, 5 years after transplant, or the last follow-up. Logistic regression analysis was performed to assess factors associated with the response to DLI within 180 days after the first DLI. Results: During the study period, 236 patients received 252 HCTs followed by DLIs (median age at HCT 9.2 [range 0.2-25.1] years). An additional 743 patients received 806 HCTs with no subsequent DLI (median age at HCT 10.3 [range 0.1-27.2] years). Dosing most frequently used was 1 × 10 5, 1 × 10 6 and 1 × 10 7 CD3+ cells/kg, for haploidentical donor (HAPLO), matched unrelated donor (MUD) and matched sibling donor (MSD) recipients, respectively. Initial doses were occasionally lower based on risk of GVHD and were escalated as tolerated and based on response. After receiving DLI, 19.4% developed acute GVHD, and 9.2% developed chronic GVHD with no significant differences observed between MSD, MUD and HAPLO recipients. After adjusting for donor source, longer duration between HCT and DLI was associated with a significantly decreased risk of both acute (HR 0.97 P<0.001) and chronic GVHD (HR 0.98 P=0.04). A higher CD3+ cell dose was also significantly associated with an increased risk of acute GVHD (HR 1.03 P<0.001) but not chronic GVHD (HR 0.96 P=0.12). The overall response rate after DLI for treating MC or graft failure was 45.2% for MSD, 32.6% for MUD and 41.4% for HAPLO recipients. Logistic regression analysis revealed leukemia patients had a higher response rate after receiving a DLI for mixed chimerism from MSD (OR=19.5 P=0.002) and HAPLO (OR=9.03 P=0.042), but not from MUD HCT (OR=0.65 P= 0.57), compared to patients with non-malignant disorders. We also evaluated the response to DLI for frank disease relapse in 15 patients. The response rate after DLI for disease relapse was 50% (1/2) for MSD, 44.4% (4/9) for HAPLO, but 0% (0/4) for MUD recipients. Conclusions: In our analysis, the incidence of GVHD was not higher in pediatric and young adult HCT recipients who received DLI as compared to those who did not. Overall, the risk of developing acute and chronic GVHD after DLI was low and comparable between different donor sources. The risk of GVHD decreased further away from HCT and increased with higher DLI dose. The response rate for treating MC and disease relapse remains less than 50%. Thus, DLIs may be safely administered to pediatric and young adult patients after HCT. The therapeutic effect may be modulated by adjusting the dose, timing and frequency of DLI administration.

Clinical Characteristics, Management, and Allogeneic Hematopoietic Cell Transplantation of Patients with Toll-like Receptor 8 Gain-of-Function Mutations

Toll-like receptor 8 gain-of-function (TLR8 GOF) is a recently described inborn error of immunity due to germline or somatic mutations in the TLR8 gene and characterized by severe neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases, bone marrow failure. Treatment of TLR8 GOF has been challenging to date, and guidance for optimal therapy is lacking. We describe the clinical characteristics and management of 10 patients with TLR8 GOF variants, including the first female patient and further clinical outcome on the 6 patients initially reported with this disease with a focus on the role of allogeneic hematopoietic cell transplantation (HCT). All but one patient were male ranging from 0.5 months to 28 years at age of symptom onset. Eight patients had somatic mutations with variant allele frequencies of 7-26%; two patients had germline mutations. The 9 male patients all had severe neutropenia and varying degrees of anemia and/or thrombocytopenia. Oral ulcers, recurrent infections, hepatosplenomegaly, and hypogammaglobulinemia were common. Immune phenotype was variable, ranging from severe lymphopenia to marked T cell lymphoproliferation. Several patients had an inverted CD4:CD8 T cell ratio and skewing of T cell subsets to the terminal effector memory phenotype. Increased double-negative T cells were also observed. All patients had low class switched memory B cells. Bone marrows were hypo- to hypercellular with marked myeloid hypoplasia, and in most cases, with increased T cells, large granular lymphocytes, and/or lymphoid aggregates. Neutropenia was initially responsive to granulocyte colony stimulating factor (GCSF) +/- steroids in several patients but ultimately become refractory to therapy. Immunomodulatory agents and chemotherapy were unsuccessful. Six patients underwent allogeneic HCT; two patients died at 8 and 18 years without definitive therapy; and one patient was lost to follow-up. The female patient had germline disease and presented with pure red blood cell aplasia at 2 weeks of life and is scheduled to undergo HCT. Transplant data were available for 5 of 6 patients who underwent HCT. Age at transplant was 3 to 21 years. One patient who received a graft from a 7/8 HLA-matched unrelated donor developed severe veno-occlusive disease and secondary graft failure. The patient underwent a second transplant and ultimately died. Another patient received a matched sibling donor HCT while critically ill from Candida lusitaniae and mucormycosis infection with associated multisystem organ failure. The patient engrafted with 100% donor chimerism but died on day +15 post-HCT. The remaining 3 patients are alive and well 8 months to 2 years post-HCT. Two patients received grafts from HLA-matched donors (1 sibling and 1 unrelated) with reduced intensity conditioning and cyclophosphamide, mycophenolate mofetil and vorinostat for graft-versus-host disease (GVHD) prophylaxis. The third patient underwent a haploidentical HCT with myeloablative conditioning and post-transplant cyclophosphamide. Post-HCT complications included acute skin and/or gastrointestinal GVHD that was responsive to therapy in all patients, and severe lung GVHD in one patient. There were no major infectious complications. All three patients had full donor chimerism in myeloid and T cells with complete resolution of disease phenotype at last follow-up. The patient for whom detailed transplant data is not available received a haploidentical HCT. He developed mixed myeloid chimerism for which he received multiple donor lymphocyte infusions and was alive at last contact >3 years post-HCT. TLR8 GOF should be considered in male and female patients with severe, unexplained neutropenia refractory to GCSF, and particular care should be taken when investigating for mutations given the disease-causing variant allele frequencies as low as 7%. Neutropenia and immune dysregulation were refractory to all therapies in our cohort, and TLR8 GOF was fatal in two patients who did not receive definitive therapy. Allogeneic HCT is curative and should be considered without delay for medically suitable patients.

Application Gene Variation Detection before and after Hematopoietic Stem Cell Transplantation to Predict the Relapse of Hematopoietic Neoplasm Patients after Transplantation: Retrospective Data from a Single Center in China

Objective: To evaluate the changes of gene variation in patients with hematological neoplasms at the time of diagnosis and after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and the relationship between gene mutation and recurrence after transplantation. Methods: 76 patients with hematological neoplasms were analyzed retrospectively, including 69 acute myeloid leukemia, 21 acute lymphoblastic leukemia, 5 myelodysplastic syndrome, 1 mixed acute leukemia and 1 chronic myelomonocytic leukemia. The median age was 37 (7-63) years, with 52 males and 45 females. Among 97 patients, 21 were matched sibling donor HSCT (MDS-HSCT), 14 were matched unrelated donor HSCT (MUS-HSCT), and 62 were haploidentical HSCT (haplo-HSCT). There were 88 cases of myeloablative conditioning regimen and 9 cases of non-myeloablative conditioning regimen. All patients underwent next generation sequencing (NGS) of hematological neoplasms variation genes at the time of diagnosis and 28 days after transplantation. Results: Primary and Secondary variations were detected in 89 of 97 patients during diagnosis. Common variations include CEBPA (18/97), NRAS (13/97), FLT3 (12/97), DNMT3A (11/97), TP53 (9/97), etc. After transplantation, only 29 patients had primary and Secondary variations, including ABCB1 (30/97), CYP2C19 (13/97); The number of other variations did not exceed 3 cases. The above NGS detection results suggest that the number of primary and Secondary variations after transplantation is significantly reduced (p=0.019), and ABCB1 and CYP2C19 are the main variation forms. The membrane related protein encoded by ABCB1 is a member of the ATP binding cassette (ABC) transporter superfamily, and CYP2C19 is a drug metabolizing enzyme, both of which are involved in drug metabolism. Single Nucleotide Polymorphisms (SNP) variations were detected in 52 of 97 patients at diagnosis, and GATA2 (27/97) and TET2 (25/97) variations were the most common variation forms. SNP variations of GATA2 and TET2 increased significantly after transplantation, 59/97 and 54/97 respectively. The increase of GATA2/TET2 double variation was more significant after transplantation (19/97 vs. 46/97, P<0.001). The increased variation frequency of GATA2 and TET2 after transplantation may be related to the mixed chimerism at the early stage of transplantation, which needs to be observed for a long time. Furthermore, we analyzed the relationship between gene variation and recurrence of 97 patients with hematological neoplasms at the time of diagnosis and after allo-HSCT. Eleven of 97 patients recurred after transplantation. The single factor competitive risk model showed that the presence of primary and Secondary variations after transplantation was an independent risk factor for recurrence (P=0.017) (Figure 1). The primary and secondary variations before transplantation, SNP variations before and after transplantation had no effect on recurrence after transplantation. The schemes of transplantation in 29 patients with primary and secondary variations after transplantation were analyzed, including 9 cases of MSD-HSCT, 14 cases of haplo-HSCT, and 6 cases of MUD-HSCT. It suggests that relative donors are more likely to have primary and secondary variations related to diseases. We used Fisher's Exact Test to analyze the relationship between ASXL1, TP53, GATA2, RUNX1 and other high-risk variations at the time of diagnosis/after transplantation and recurrence, and did not find that the presence of these gene variations would increase the risk of recurrence after transplantation. Conclusion: The NGS gene variation detection before and after transplantation can predict the recurrence after transplantation. Large samples and long-term monitoring are also needed to find a more accurate variation pattern to predict the recurrence of hematological neoplasms after transplantation.

Allogeneic Bone Marrow Transplantation from HLA-Identical or Haploidentical Donors for Children and Adolescents with Sickle Cell Disease: A Feasible Curative Option

The only curative option widely available for sickle cell disease (SCD) is bone marrow transplantation (BMT). Since few patients have a healthy matched sibling donor (MSD), haploidentical (haplo) donors have expanded the donor pool to approximately 90% of the patients. The addition of thiotepa to the Johns Hopkins conditioning therapy backbone dor haplo BMT showed durable engraftment and excellent overall survival. However, graft rejection has remained an important obstacle. Our challenge in these patients has been to augment the conditioning therapy to provide adequate engraftment, while maintaining a low transplant-related mortality. Methods: Retrospective data of all 26 patients with SCD followed by our group after MSD (n=7) and haplo (n=19) BMT performed in four different institutions. With a MSD, conditioning was rATG 4.5mg/kg, Busulfan (Bu, AUC 4,500), Fludarabine (Flu, 120mg/m²) or rATG, Bu, Cyclophosphamide (Cy, 200mg/kg) and with haplo donors, rATG 4.5mg/kg, Thiotepa 10mg/kg, Flu 150mg/m², Cy 29 mg/kg and TBI 2Gy. Patients were prepared to BMT with intense hypertransfusion/bleeding or erythrocytapheresis to keep HbS<30 and Hb~10g/dL, reticulocytes <10% for 2-3 months prior to BMT, with intensive chelation to keep ferritin <1,000ng/mL. The presence of donor specific antibodies would exclude the patient from undergoing BMT. Due to two consecutive secondary graft failures (no detectable donor cells despite initial engraftment) and two falling mixed chimerism among the first 13 patients undergoing haplo, the doses of the conditioning therapy were increased to Cy 50 mg/kg and TBI 4Gy in a single fraction, as previously used in our patients with aplastic anemia undergoing BMT. Six consecutive patients received this augmented conditioning therapy. All patients had serial chimerism measured by STR. The graft-versus-host disease (GVHD) prophylaxis included Cyclosporine - Methotrexate in MSD and post-transplant Cy/mesna, mycophenolate mofetil and sirolimus in haplo. Results: Between September 2016 and April 2023, 26 patients underwent BMT, with a median age of 11 years (4-20), half of them females, 7 with a MSD and 19 an haplo (4 siblings, 6 fathers, and 9 mothers); 77% of the donors had sickle cell trait. The graft was bone marrow in all but one patient with Sb thal and Hodgkin's lymphoma. The median follow-up was 15.5 months. All patients engrafted. BMT with MSD had excellent outcomes and stable chimerism. Among the first 13 patients undergoing haplo, secondary graft failure occurred very suddenly in two patients (CD34 cell doses of 2 and 8x10 6 CD34/kg), both male teenagers; two additional patients had a significant drop in chimerism up to 30% donor cells but responded to serial donor leukocyte infusions 1-5 x10 6 CD3/kg (2 and 6) and developed very mild skin GVHD. The other patients maintained > 89-100% donor cells. Thereafter, with the augmented conditioning regimen, all six consecutive patients have stable 100% donor chimerism. Acute II-III GVHD was observed in 34% and chronic GVHD in 15% of the patients. Viral reactivations (CMV, HHV6, BKV, HHV7 and EBV) were observed in all patients. Overall survival is 100% and rejection-free survival is 83% at 1 year (Figures 1, 2). Conclusions: Allogeneic BMT with an HLA-identical or haploidentical family donor was feasible to treat SCD with an excellent overall survival. With a larger cohort and longer follow-up, the long-term effects of this strategy will be thoroughly studied.

Acceptance, Awareness, Attitude, and Practices Toward Heart Transplantation: A Saudi Based Survey.

The awareness of brain death and heart donation (HD) among the Saudi population remains limited, coupled with negative attitudes toward heart donation, resulting in a significant gap between the demand for donor hearts and the available supply. This study aimed to comprehensively understand the current perceptions, attitudes, and practices of the Saudi population regarding HD, as well as identify the obstacles. The ultimate goal was to strengthen the local donor pool. A cross-sectional study was conducted from March to May 2023, employing a self-administered internet survey. The survey collected demographic information, assessed awareness, attitudes, and practices related to HD, and was completed by 1820 participants from various regions in Saudi Arabia. Data was analyzed using SPSS version 25 (SPSS Inc., Chicago, Illinois, USA). Chi-square test, Independent-samples t-test, one way analysis of variance test (ANOVA) and Spearman correlation coefficient was performed with the significance level set at p < 0.05. A significant portion of the population (out of 1820 participants) lacked organ donation cards and were uncertain about the registration process. Participants displayed a moderate level of knowledge about HD, with roughly half holding unfavorable attitudes toward HD. A considerable percentage of participants 62.0% were unwilling to register as heart donors, but a majority (79.9%) were willing to contribute by disseminating information about HD. The study identified significant associations between knowledge scores and several factors, including age (p = 0.002), career (p = 0.000), possession of an organ donation card (p = 0.000), and a history of transplantation or organ donation among relatives (p = 0.000). A significant relationship was observed between attitude scores and several factors, including career (p = 0.001), Saudi region (p = 0.025), possession of an organ donation card (p = 0.000), and a history of transplantation or organ donation among relatives (p = 0.000). The study highlights the urgent need for increased awareness to bolster the number of local heart donors. The involvement of healthcare professionals and social campaigns is essential to enhance public knowledge and potentially boost the willingness of individuals to become donors.

Treosulfan Exposure Predicts Thalassemia-Free Survival in Patients with Beta Thalassemia Major Undergoing Allogeneic Hematopoietic Cell Transplantation.

A toxicity-reduced conditioning regimen with treosulfan, fludarabine, and thiotepa in patients with high-risk β-thalassemia major has significantly improved hematopoietic stem cell transplantation (HCT) outcomes. However, complications resulting from regimen-related toxicities (RRTs), mixed chimerism, and graft rejection remain a challenge. We evaluated the dose-exposure-response relationship of treosulfan and its active metabolite S, S-EBDM, in a uniform cohort of patients with β-thalassemia major to identify whether therapeutic drug monitoring (TDM) and dose adjustment of treosulfan is feasible. Plasma treosulfan/S, S-EBDM levels were measured in 77 patients using a validated liquid chromatography with tandem mass spectrometry method, and the pharmacokinetic parameters were estimated using nlmixr2. The influence of treosulfan and S, S-EBDM exposure, and GSTA1/NQO1 polymorphisms on graft rejection, RRTs, chimerism status, and 1-year overall survival (OS), and thalassemia-free survival (TFS) were assessed. We observed that treosulfan exposure was lower in patients with graft rejection than those without (1,655 vs. 2,037 mg•h/L, P = 0.07). Pharmacodynamic modeling analysis to identify therapeutic cutoff revealed that treosulfan exposure ≥1,660 mg•hour/L was significantly associated with better 1-year TFS (97% vs. 81%, P = 0.02) and a trend to better 1-year OS (90% vs. 69%, P = 0.07). Further, multivariate analysis adjusting for known pre-HCT risk factors also revealed treosulfan exposure <1,660 mg•h/L (hazard ratio (HR) = 3.23; 95% confidence interval (CI) = 1.12-9.34; P = 0.03) and GSTA1*B variant genotype (HR = 3.75; 95% CI = 1.04-13.47; P = 0.04) to be independent predictors for inferior 1-year TFS. We conclude that lower treosulfan exposure increases the risk of graft rejection and early transplant-related mortality affecting TFS. As no RRTs were observed with increasing treosulfan exposure, TDM-based dose adjustment could be feasible and beneficial.

A novel flow-cytometric based method to assess post-HSCT donorchimerism exploiting RNA hybridization.

Analysis of donor-recipient chimerism after hematopoietic stem cell transplantation (HSCT) is of pivotal importance for patient's clinical management, especially in the context of mixed chimerism. Patients are routinely monitored for chimerism in sorted subsets of peripheral blood cells. However, measurement of chimerism in sorted immune cell subsets is technically challenging and time consuming. We here propose a novel, flow cytometry-based approach to detect donor cell chimerism in sex-mismatched HSCT. We exploit RNA PrimeFlow™ system, based on RNA hybridization, to detect mRNA from a lysine demethylase encoded by Y chromosome, KDM5D. This approach allows to distinguish male and female derived cells with around 1% sensitivity. The procedure can be coupled with multiparametric immunophenotyping to assess chimerism in specific immune cell subsets without the need for prior FACS-sorting. We apply this method to a cohort of HSCT patients (n = 10) and we show that it is consistent with standard PCR-based method. We also show that different T lymphocyte subsets display variable degrees of donor chimerism, especially in CD8+ T cell compartment where we observe an enrichment for recipient chimerism in central memory T cells. This method can be exploited to advance current knowledge on immune reconstitution focusing on specific subsets avoiding prior FACS-sorting.

Analysis of the Impact of Whole Blood and T-Cell Donor Chimerism after Allogeneic Stem Cell Transplant

Background: Allogeneic stem cell transplant (alloSCT) is one of the few currently available therapies to cure acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Positive outcomes are dependent on engraftment of donor cells to provide hematopoiesis as well as a graft-versus-tumor effect. Measurement of whole blood (WB) and T-cell (TC) chimerism at routine intervals post alloSCT allows for monitoring of engraftment. Development of mixed donor chimerism (MC) may indicate impending graft failure or disease relapse. The ideal frequency for chimerism measurements as well as the role of WB versus TC chimerism remains unclear. In this study, we analyzed the impact of WB versus TC mixed chimerism on survival outcomes in patients with AML and MDS. Furthermore, we studied baseline factors associated with the development of MC. Methods: In this retrospective analysis, adults with AML or MDS who received an alloSCT between January 1, 2016 and February 1, 2022 that had at least 1 donor chimerism measured on Day 30 (D30), Day 60 (D60), Day 90 (D90) at Juravinski Hospital and Cancer Centre (Hamilton, ON, Canada). Baseline demographic and transplant-related characteristics were collected. AML and MDS diagnoses were based on WHO 2016 classification of myeloid neoplasm and acute leukemia. Conditioning intensities were classified according to CIBMTR definitions. A total of 271 transplants were completed during the study period for AML or MDS. 128 were excluded from the analysis (chimerism testing not performed (n=121), second transplants (n=7)). We analyzed a total of 143 patients with median age of 63 years (19 - 76) who were transplanted for AML (n=105) and MDS (n=38). Donors were HLA-matched siblings (MRD, n=26), unrelated donors (MUD (8/8 or 7/8), n=85) or haploidentical related donor (MMRD, n=32). All patients except one received peripheral blood stem cell grafts. For alloSCT, 20 % of patients received myeloablative conditioning (MAC) and 80% received reduced-intensity conditioning (RIC). Complete chimerism (CC) was defined as ≥95%, whereas MC was defined as ≤95%. All chimerism measurements were measured using a PowerPlex® 16 System (Promega) (Hamilton Regional Laboratory Medicine Program, Hamilton, ON). Results: In our cohort, OS at 2 years was 57% and RFS 49%. Of the baseline factors, only disease-risk based on ELN 2022 for RFS with a hazard ratio (HR) of 1.43, 95% CI [0.83, 2.49] for adverse risk and HR 0.53 95% CI [0.23, 1.03] (p=0.03) for favourable risk. WB MC was detected in 22%, 22%, 23% of patients on D30, D60, and D90, respectively. TC MC was detected in 48%, 46%, and 43% of patients on D30, D60, and D90, respectively. WB MC had a strong negative impact on RFS, with a HR of 2.81, 95% CI [1.69, 4.66; p&amp;lt;0.001] on D30, 3.07, 95% CI [1.67, 5.66; p&amp;lt;0.001] on D60 and 4.77, 95% CI [2.47, 9.22; p&amp;lt;0.001] on D90. The influence of TC MC on RFS was also significant at all time points. Of 101 patients who were alive and did not have a relapse prior to D90 and had D30 chimerism and D90 chimerism evaluated, the patients who had a mixed WB and TC chimerism on D90 had a HR of 6.13, 95% CI [2.23, 16.85] and HR 2.73, 95% CI [1.24, 5.98] for RFS, which was statistically significant even after adjusting for known information of D30 WB/TC chimerism status (Table 1). WB MC had a strong negative impact on OS with a HR of 2.67, 95% CI (1.54, 4.64; p=0.001) on D30, 2.81, 95% CI (1.47, 5.38; p=0.002) on D60 and 4.78, 95% CI (2.47, 9.24; p&amp;lt;0.001) on D90. Again, the later time points remained statistically significant, when adjusting for known D30 chimerism status. The influence of TC MC on OS was also significant at all time points, however only a non-significant trend remained at later time points when adjusting for D30 TC chimerism (Table 2). We analyzed baseline factors predictive for D30 chimerism. For WB chimerism, only donor source remained statistically significant (p=0.038) with MMRD having a lower HR for MC 0.39, 95% CI [0.11, 1.41] and MRD having a higher HR for MC 2.33, 95% CI [0.9 - 5.99]. Conclusions: Our data confirm that a mixed chimerism as early as D30 post-alloSCT can have a detrimental impact on outcomes (RFS and OS) in alloSCT. Patients receiving haploidentical alloSCT had a lower risk to develop mixed chimerism in our cohort. These dismal outcomes of patients with mixed chimerism in our cohort provides a strong rationale to study interventions such as novel maintenance strategies or donor-lymphocyte infusions in this patient group.

Non-Genotoxic Conditioning for Hematopoietic Stem Cell Transplant through Engineered Stem Cell Antibody Paired Evasion (ESCAPE)

While ex vivo gene editing of CD34 + hematopoietic stem and progenitor cells (HSPCs) offers potentially curative therapies for patients with serious diseases, such as sickle cell anemia, significant autologous hematopoietic stem cell (HSC) transplant-related challenges remain, notably the requirement of myeloablative conditioning using chemotherapeutic agents such as busulfan. To address this challenge, we have developed a non-genotoxic conditioning strategy with our Engineered Stem Cell Antibody Paired Evasion (ESCAPE) approach where we aim to engineer the epitope of the HSC surface protein CD117 (cKIT) using base editors, in such a way that the engineered protein retains the normal CD117 receptor function, but escapes recognition by a cognate monoclonal antibody that recognizes the wildtype CD117 protein. The ESCAPE-CD117 edit can be combined with a therapeutic target edit to generate a multiplex-edited engineered HSC (eHSC). We previously reported that through large parallel antibody and antigen screening efforts, we could identify mAb: edit pairs in which the engineered epitope evades mAb-mediated blocking of the WT CD117 receptor-ligand interaction while maintaining normal CD117 receptor biology and HSPC function and differentiation potential in vitro and in vivo. To further characterize binding properties of our lead CD117 variant to the natural ligand SCF, we determined the 3-dimensional structure of the receptor-ligand complex using cryo-EM. Structural overlay between engineered CD117:SCF and WT CD117: SCF complexes showed high similarity, with root mean square deviation (RMSD) of 0.848-Å, indicating that our epitope engineering did not affect the structure of CD117 nor its binding to its ligand. We have previously demonstrated in vivo proof-of-concept data supporting selection of engrafted multiplex-edited eHSCs after a single dose of mAb in non-competitive and competitive transplant settings in multiple rodent xenotransplantation models. We next sought to examine whether mAb conditioning could achieve long-term, multilineage hematopoietic reconstitution of multiplex-edited eHSCs and potentially therapeutic mixed chimerism levels. To this end, we humanized an immunocompromised mouse model with unedited mobilized peripheral blood (mPB) hCD34 + cells to achieve stable human bone marrow chimerism and then administered a single dose of mAb followed by a second transplant from the same donor of eHSCs harboring both ESCAPE and therapeutic edits targeting the HBG1/2 promoter. Animals receiving CD117 mAb demonstrated significant engraftment by cells harboring our ESCAPE multiplex edits as detected by NGS of bulk BM (~50% multiplex editing) and within the sorted marrow CD34 + cell population (~60% multiplex editing), while the isotype control treated animals only showed 2-5% multiplex editing within the said compartments. We also isolated CD15 + myeloid cells and GlyA + erythroid cells from the bone marrow of the mAb treated animals, which also harbored &amp;gt;50% multiplex editing as detected by NGS. Furthermore, we could detect what is believed to be therapeutically relevant levels of HbF induction (&amp;gt;30%) in human GlyA + sorted bone marrow cells, suggesting that we could achieve therapeutic benefit with a single dose of mAb as the conditioning agent. In conclusion, our structural data showed a high level of structural similarity between our engineered CD117 epitope and the WT protein. Our in vitro assays and in vivo rodent xenotransplantation models showed enrichment of engineered HSCs upon mAb treatment. Altogether, our data suggest the feasibility of achieving therapeutically relevant levels of mixed bone marrow chimerism with our non-genotoxic conditioning ESCAPE platform. This approach may remove the need for patients to undergo systemic genotoxic conditioning prior to transplant and further unlock the promise of base editing therapies for the treatment of patients suffering from serious hematologic diseases.

Haploidentical Aß T Cell Depleted HSCT Represents an Alternative Treatment Option in Pediatric and Adult Patients with Sickle Cell Disease (SCD)

Background: Sickle cell disease (SCD) is the most prevalent inherited hemoglobinopathy worldwide, which is associated with considerable morbidity and early mortality. Although HSCT with a MD offers a curative treatment option with an excellent 2-y OS of 95%, availability of MDs is limited and therefore haploidentical HSCT is increasingly explored. Here we update our in vitro T-depleted haploidentical HSCT approach, which is currently explored in a multicenter international trial (NCT04201210). Methods: 29 pts with SCD or SCD-ß Thal (median age: 18 years; range: 2-32) received either a CD3 +/CD19 + (n=9) or aß/CD19 + (n=20) T-haplo-HSCT and were compared with 20 SCD-pts receiving a BM graft from a MSD (median age: 23 years; range: 9-39) at our center. Indication for T-haplo HSCT pts included severe or moderate SCD related complications such as recurrent pain crisis (&amp;gt;5/year), acute chest syndrome, neurological events, osteonecrosis or nephropathy, transfusion-refractory allo-immunization. After exchange transfusion, almost identical conditioning regimens consisting of treosulfan, thiotepa, fludarabine (FTT) and ATG-Grafalon were applied in almost all pts, with the only difference in the timing of ATG (upfront in T-haplo-HSCT, prior to day 0 in MSD-HSCT). Immunosuppression (IST) was maintained for a minimum of 180 days consisting of calcineurin inhibitors (mainly tacrolimus) and MMF. Results: The OS and DFS for MSD and T-haplo-HSCT was 95% and 83%, respectively (Table 1). The median follow-up was 49 months for MSD (range 5-118) and 67 months for T-haplo-HSCT (range 0.5-135). Neutrophil engraftment was achieved after a median of 29 days for MSD pts and 17 days for T-haplo-HSCT with a median of 2.8 x 10 8 TNC/kg (range: 0.65-4.11) and 12 x 10 6 CD3 +/CD19 + or ab/CD19 + depleted CD34 + cells/kg (range: 4.8-49.2), respectively. One pt received two T-haplo HSCT due to primary graft failure after HHV6 infection with effective engraftment after the second transplantation. One pt required a stem cell boost of the same donor with slow subsequent engraftment and one pt died of infectious complications after the fourth T-haplo HSCT. A mixed chimerism &amp;lt;80% was observed in 1/20 MSD-HSCT (median 94.9%; range 40.5-100%) and in 2/29 T-haplo-HSCT pts (median 97.8%; range: 35.7 -100%). No severe cases of &amp;gt;°III aGVD was observed in both populations. MSD pts reached T cell counts &amp;gt;200/µl on day +62 (median; range: 24-293) and T-haplo-HSCT pts on day +136 (median; range: 25-758). Viral reactivation occurred both in T-haplo HSCT (20/29) and in MSD-BMT (9/20). However, one pt in the T-haplo group developed kidney failure after prolonged BKV infection and four of the T-haplo pts died of complications associated with severe CMV or HHV6 infections that were accompanied by hyperinflammation, macrophage activation and acute respiratory distress despite antiviral and anti-inflammatory treatment. One pt of the MSD group died of late treatment related toxicity in combination with preexisting SCD comorbidity. The last fatality occurred in 2020 with now 16 additional pts being transplanted safely, not least also because of a consistent letermovir prophylaxis. Overall, the treosulfan-based conditioning regimen was well tolerated with only one VOD/SOS and one CNS infarction being observed in this high-risk population. 4/20 pts in the MSD group and none in the T-haplo group developed PRES early after conditioning. Conclusions: The safety and efficacy data of this pilot series of T-haplo-HSCT in SCD reveal that T-haplo-HSCT in advanced stage pediatric and adult SCD patients lacking a MSD is feasible. However, diligent viral monitoring and early treatment of viral reactivation, in particular HHV6 is mandatory as otherwise fatal infections may develop.

Pre-Emptive Donor Lymphocyte Infusion As Optimal Treatment for Relapsed Acute Myeloid Leukemias and Myelodysplastic Syndromes Following Allogeneic Stem Cell Transplantation: Insights from a French-Italian Study on 134 Patients

Disease relapse after allogeneic stem cell transplantation (allo-SCT) is the main challenge in achieving a cure for acute myeloid leukemias (AMLs) and myelodysplastic syndromes (MDSs). The treatment of disease relapse can be offered in case of full hematological recurrence or in the presence of minimal residual disease (MRD) positivity and/or mixed chimerism. The latter approach is defined as pre-emptive therapy. Overall, treatment strategies may include chemotherapy, molecular-target drugs, hypomethylating agents (HMAs), donor lymphocyte infusions (DLIs) or even a second allo-SCT. We retrospectively analyzed a cohort of 553 AML and MDS adult patients consecutively allotransplanted between January 1, 2015, and December 31, 2021, at Saint-Antoine University Hospital, Paris (France) (n=420) and Spedali Civili di Brescia, Brescia (Italy) (n=133). 134/553 (24%) patients relapsed and were included in this study. 103/134 (77%) were subsequently treated. Among patients who received treatment, 40/103 (39%) were treated with a DLI-based regimen. In this group, 9 received DLIs alone, while the others also received HMAs (4 cases), HMAs+ venetoclax (12 cases), FLT3-inhibitors (3 cases), intensive chemotherapy (5 cases), a second allo-SCT (5 cases), or other therapies (2 cases). The remaining 63/103 (61%) patients were treated with regimens that did not include DLIs. Of these, 10 received HMAs, 27 HMAs+ venetoclax, 4 FLT3-inhibitors, 6 intensive chemotherapy, 9 a second allo-SCT, and 7 other therapies. With a median follow up of 1.6 years, the estimated 1-, 2- and 5-year OS rate of the 134 patients who relapsed after allo-SCT was 32%, 18 %, and 11 %, respectively. The OS of patients treated after allo-SCT relapse was 40 %, 20%, and 15% at 1, 2 and 5 years compared with 6 %, 3%, and 0 % for patients who did not receive therapy, respectively ( p&amp;lt;0.01).Moreover, the OS was significantly better in patients treated in a pre-emptive setting than in those treated for a morphological relapse (OS at 1, 2 and 5 years was 60%, 36% and 30%, respectively, for patients treated in a pre-emptive setting versus 26%, 12%, and 6%, respectively, for patients treated in hematological relapse ( p&amp;lt;0.01) (Figure 1A). Focusing on post-relapse treatment, patients who received DLI-based regimens had a 1-, 2- and 5-year OS of 55%, 32% and 32%, respectively, compared to 27%, 16% and 7% for patients treated with other therapies ( p&amp;lt;0.01). Among patients who received DLIs, 50% of patients were treated in the pre-emptive setting. Conversely, in the group of patients treated without DLI, only 22% were treated in the pre-emptive setting, reflecting the more advanced phase of relapse in this group. Finally, when patients were analyzed by the treatment strategy (pre-emptive therapy vs therapy in hematological relapse) and DLI administration (yes vs no), the best outcome was achieved when the relapse was promptly detected and pre-emptive therapy was started, particularly if a DLI was administered. Indeed, the 1-year OS of patients treated in the pre-emptive setting was 67% for those receiving a DLI and 54% for those treated without DLI, while for patients with hematologic relapse, the 1-year OS was 43% for those treated with the DLI-based regimen and 17% for those treated without DLI ( p&amp;lt;0.01) (Figure 1B). On multivariate analysis, treatment with DLI after relapse, and pre-emptive setting were independent factors associated with better OS ( p=0.03 and p&amp;lt;0.01), respectively. Our data showed that relapse treatment with pre-emptive therapies is associated with improved outcomes and this effect is more evident when combined with a DLI . These results confirm the primary role of the graft- versus leukemia effect in the treatment of relapse and underscore the importance of its early identification through MRD monitoring.

Initial Results of a Phase 1 Trial of TSC-100 and TSC-101, Engineered T Cell Therapies That Target Minor Histocompatibility Antigens to Prevent Relapse after Allogeneic Hematopoietic Cell Transplantation

Introduction: To date, engineered T cell therapies have not proven effective for non-B cell hematologic malignancies due to a lack of antigens that spare healthy myeloid cells. Allogeneic hematopoietic cell transplantation (HCT) remains the best curative option for these malignancies, yet ~40% of patients relapse post-HCT with high mortality after relapse. A potential solution to prevent relapse is to target hematopoietic lineage-specific minor histocompatibility antigens (MiHAs) that are genetically mismatched between HCT patients and donors. These mismatches enable engineered T cells to selectively eliminate residual patient hematopoietic cells, normal or malignant, and spare donor cells. TSC-100 and TSC-101 are allogeneic, donor derived T-cell receptor-engineered T (TCR-T) cells that target MiHAs HA-1 and HA-2 respectively, both presented on HLA-A*02:01. By choosing HCT patients who are HLA-A*02:01 positive and donors who are either HLA-A*02:01 or MiHA negative, TSC-100 or TSC-101 are designed to eliminate residual patient hematopoietic cells post-HCT and thus prevent relapse (figure). Methods: Study TSCAN-001 (NCT05473910) is a multi-center, multi-arm non-randomized, controlled Phase 1 study evaluating the feasibility, safety and preliminary efficacy of TSC-100 and TSC-101. Adults with AML, MDS or ALL eligible for reduced intensity conditioning (RIC)-based haploidentical donor transplantation from HLA or MiHA mismatched donors are enrolled. HLA-A*02:01 and HA-1 or HA-2-positive patients receive either TSC-100 or TSC-101 after HCT. HLA-A*02:01-negative control arm patients receive HCT alone. Upon count recovery after HCT, patients in treatment arms receive a single dose of TSC-100 or TSC-101 at Dose Level 1 or repeat doses at Dose Levels 2 and 3. Dose escalation follows interval 3+3 rules. Primary endpoints include adverse event profiles and dose-limiting toxicities (DLTs). Secondary endpoints are relapse rates, disease-free and overall survival. Exploratory endpoints include biomarkers of efficacy such as minimal residual disease (MRD) by deep next-generation sequencing (NGS), and donor chimerism using novel high-sensitivity and standard assays. Results: At submission time, 8 patients were enrolled, 5 in treatment arms (3 TSC-101, 2 TSC-100) and 3 in the control arm, with a median of 111 days post-HCT follow-up (range 33-266 days). No DLTs occurred, and patients were enrolled at Dose Level 2 in both TSC-100 and TSC-101 arms. Safety analysis found expected post-HCT adverse events similar in treatment and control arms. Incidence of graft-versus-host disease (GvHD) was similar in control (3 events) and treatment arms (4 events). Serious adverse events of grade 3 GvHD and infections were observed in TSC-101 and control arms (1 event each). No cytokine release syndrome or neurotoxicity occurred after TSC-100/101 and minimal changes in CRP/ ferritin occurred, consistent with general safety of TCR-Ts with low target cell burden. No clinical relapses occurred to date. Translational analysis found peak TSC-100/101 expansion and activation 7-14 days post dosing, with ongoing persistence at longest follow-up of 138 days. In repeat dose cohorts, substantial increases of early expansion were noted after dose 2 of TSC-101/100, supporting the benefit of repeat dosing. Chimerism analysis (table) using a novel high-sensitivity NGS-based AlloHeme assay (limit of detection (LOD) 0.13%) in whole blood, CD33+ or CD3+ subsets found mixed chimerism in 2 out of 2 control patients but complete donor chimerism in 4 out of 4 TSC-100/101 patients after Day 42. Both control patients had declining donor chimerism after Day 100 by AlloHeme and standard STR-based chimerism assays (LOD 1-2%) prompting early withdrawal of immunosuppression in one patient. Marrow MRD analysis by NGS (LOD &amp;lt;0.05%) found 1 control patient was MRD positive pre-HCT and remained MRD positive post-HCT. In contrast, 2 TSC-100/101 patients who were MRD positive pre-HCT turned MRD negative after HCT and TSC-100/101 therapy including a TP53 mutated MDS patient confirmed MRD negative by droplet digital PCR (LOD 0.01%). Conclusions: Targeting MiHAs HA-1 or HA-2 with TSC-100/ 101 following HCT shows early safety and biomarker evidence of efficacy by completing elimination of all detectable patient hematopoietic cells, normal or malignant, thereby reducing relapse risk. Updated results will be presented at the meeting.

Knowledge, Attitudes, and Barriers of Organ Donation in Jeddah City, Saudi Arabia: A Cross-sectional Study.

Organ donation is considered a life-saving treatment for end-stage organ failure worldwide. However, the severe organ shortage in Saudi Arabia creates a gap between patients in need of transplantation and the availability of organs. In Saudi Arabia, a limited number of studies have assessed knowledge and attitudes toward organ donation, especially in Jeddah City. Thus, this study aimed to assess the knowledge, attitudes, and barriers to organ donation and to determine the factors that may hinder or facilitate organ donation. Our findings indicated that almost half of the participants (49.2%) obtained their information from online resources. Fear of the unknown (31.2%) and a lack of knowledge (28.5%) were the main barriers to organ donation. A high percentage of the participants (73%) were unaware of any local or international legislation regarding organ donation. Half of the study participants were willing to donate their organs, with insignificant differences in age, gender, occupation, and education level. Only 5.4% of the respondents had an organ donor card. The study indicated that half of the participants were willing to donate organs, but they were unaware of local and international legislation. Therefore, an intensive education program and campaigns are recommended to increase public awareness about organ donation.

Validation of next-generation sequencing-based chimerism testing for accurate detection and monitoring of engraftment in hematopoietic stem cell transplantation

Allogenic hematopoietic stem cell transplantation (allo-HSCT) is a life-saving treatment for various hematological disorders. The success of allo-HSCT depends on the engraftment of donor cells and the elimination of recipient cells monitored through chimerism testing. We aimed to validate a next-generation sequencing (NGS)-based chimerism assay for engraftment monitoring and to emphasize the importance of including the most prevalent cell subsets in proficiency testing (PT) programs. We evaluated the analytical performance of NGS-based chimerism testing (AlloSeq-HCT and CareDx) with a panel of targeted 202 informative single-nucleotide polymorphisms (SNPs) (i.e., linearity and precision, analytical sensitivity and specificity, system accuracy, and reproducibility). We further compared the performance of our NGS panel with conventional short tandem repeat (STR) analysis in unfractionated whole blood and cell-subset-enriched CD3 and CD66. Our NGS-based chimerism monitoring assay has an impressive detection limit (0.3% host DNA) for minor alleles and analytical specificity (99.9%). Pearson’s correlation between NGS- and STR-based chimerism monitoring showed a linear relationship with a slope of 0.8 and r = 0.973. The concordance of allo-HSCT patients using unfractionated whole blood, CD3, and CD66 was 0.95, 0.96, and 0.54, respectively. Utilization of CD3+ cell subsets for mixed chimerism detection yielded an average of 7.3 ± 7-fold higher donor percentage detection compared to their corresponding unfractionated whole blood samples. The accuracy of the NGS assay achieved a concordance of 98.6% on blinded external quality control STR samples. The reproducibility series showed near 100% concordance with respect to inter-assay, inter-tech, inter-instrument, cell flow kits, and AlloSeq-HCT software versions. Our study provided robust validation of NGS-based chimerism testing for accurate detection and monitoring of engraftment in allo-HSCT patients. By incorporating the cell subsets (CD3 and CD66), the sensitivity and accuracy of engraftment monitoring are significantly improved, making them an essential component of any PT program. Furthermore, the implementation of NGS-based chimerism testing shows potential to streamline high-volume transplant services and improve clinical outcomes by enabling early relapse detection and guiding timely interventions.

Age Impacts Risk of Mixed Chimerism Following Reduced-Intensity Conditioning Hematopoietic Cell Transplantation for Non-Severe Combined Immune Deficiency Inborn Errors of Immunity

Alemtuzumab, fludarabine, and melphalan containing-reduced intensity conditioning (RIC) is commonly used in patients undergoing allogeneic hematopoietic cell transplantation (HCT) for definitive treatment of high-risk inborn errors of immunity (IEI). Although survival is favorable, there is an increased risk of mixed chimerism leading to secondary graft failure. This study evaluated factors associated with the risk of developing mixed chimerism, particularly the influence of age in patients undergoing allogeneic HCT for non-severe combined immune deficiency (SCID) IEI who received a uniform RIC regimen that included intermediate schedule alemtuzumab, fludarabine, and melphalan. We hypothesized that age would impact the incidence of mixed chimerism. We retrospectively reviewed records of patients who underwent HCT for non-SCID IEI with a uniform RIC regimen that included intermediate schedule alemtuzumab (1 mg/kg divided over days -14 to -10), fludarabine (150 mg/m2 or 5 mg/kg if weight <10 kg divided over days -9 to -4), and melphalan (140 mg/m2 or 4.7 mg/kg if weight <10 kg on day -3) between 2010 and 2020 at our institution. Mixed chimerism was defined as <95% donor chimerism on 2 or more consecutive occasions in whole blood. Ninety-three patients who underwent RIC-HCT for non-SCID IEI using intermediate schedule alemtuzumab, fludarabine, and melphalan were categorized into 3 groups: age <1 year, age 1 to 5 years, and age >5 years. Forty-nine patients (52.7%) developed mixed chimerism, at a median of 34 days post-HCT (range, 10 to 1396 days). Mixed chimerism developed in 88.9% (n=16/18) of the age <1 year group, in 57.1% (n=20/35) of the age 1 to 5 years group, and in 35% (n =14/40) of the age >5 years group. Patients age <5 years were significantly more likely to develop mixed chimerism (χ2(3,N=93)=14.8; P=.001). We observed a significantly increased cumulative incidence of developing mixed chimerism associated with age <1 year (P=.0002). Competing risk regression analysis showed a 3-fold higher risk of developing mixed chimerism for age <1 year (subdistribution hazard ratio (HR), 3.05; 95% confidence interval [CI], 1.11 to 8.38; P=.031,) compared to age >5 years and a significantly decreased risk of mixed chimerism in patients who developed acute GVHD prior to any intervention (OR, .24; 95% CI, .09 to .65; P=.005) There were no significant associations between mixed chimerism and graft source, graft type, CD34+ or CD3+ cell dose, HLA match, or underlying disease (hemophagocytic lymphohistiocytosis [HLH] versus non-HLH). Additionally, the need for secondary intervention was evaluated; 27 patients (29.0%) required 1 or more secondary interventions (donor lymphocyte infusion, CD34 boost, or second HCT). Patients age <1 year with mixed chimerism were significantly more likely than patients age >5 years to require secondary intervention for mixed chimerism (P=.004). Our study demonstrates that age <5 years, especially age <1 year, is associated with an increased risk of developing mixed chimerism in patients undergoing RIC-HCT for non-SCID IEI using intermediate-schedule alemtuzumab, fludarabine, and melphalan. Our data suggest tailoring regimen intensity based on age to reduce the incidence of mixed chimerism. Children age <5 years, particularly those age <1 year, require a higher-intensity regimen. Possible strategies include adding thiotepa or using a busulfan-based reduced toxicity regimen.

Can novel methods replace the gold standard chimerism method after allogeneic hematopoietic stem cell transplantation?

After hematopoietic stem cell transplantation, chimerism assay is a useful approach to monitor the success of the transplant and to select the appropriate treatment strategy, such as donor leukocyte infusion or immunosuppressive drug dosage. Short tandem repeat PCR is the method that has been accepted as the gold standard for chimerism. However, it has not yet been sufficient to detect mixed chimerism in patients with minimal residual disease. Simultaneously, recent years have been marked by developing sensitive, high-throughput, and accurate molecular genetic assays. These novel methods have subsequently been adapted for the analysis of post-transplant chimerism. In this review, we discuss the technical features of both novel and conventional gold standard chimerism assays. We also discuss their advantages and disadvantages.

312.2: Combined double thymus transplantation and mixed xenogeneic chimerism for the induction of optimal tolerance and enhancement of immune function

312.2: Combined double thymus transplantation and mixed xenogeneic chimerism for the induction of optimal tolerance and enhancement of immune function

Turkish Validity and Reliability Study of "Intensive Care Nurses" Attitude Scale Toward Brain Death and Organ Transplantation.

This study was carried out to analyze the validity and reliability of the Turkish adaptation of the "Intensive Care Nurses" Attitude Scale towards Brain Death and Organ Transplantation". The research was carried out as a methodological study and 256 nurses were included in the study. Language, content validity, explanatory and confirmatory factor analyzes were used to analyze data. Twenty-one point 5% of nurses stated that they had an organ donation card. It was determined that the Cronbach alpha coefficient of scale was .85, and item-total score correlations were between .143 and .700. It was established that the scale showed three-factor structure and the first factor was named as "Approving Organ Donation", the second factor as "Discomfort" and the third factor as "Improving The Quality of Life". The Turkish version of the scale was found to be an valid and reliable measurement tool.

Genotype, oxidase status, and preceding infection or autoinflammation do not affect allogeneic HCT outcomes for CGD

AbstractChronic granulomatous disease (CGD) is a primary immunodeficiency characterized by life-threatening infections and inflammatory conditions. Hematopoietic cell transplantation (HCT) is the definitive treatment for CGD, but questions remain regarding patient selection and impact of active disease on transplant outcomes. We performed a multi-institutional retrospective and prospective study of 391 patients with CGD treated either conventionally (non-HCT) enrolled from 2004 to 2018 or with HCT from 1996 to 2018. Median follow-up after HCT was 3.7 years with a 3-year overall survival of 82% and event-free survival of 69%. In a multivariate analysis, a Lansky/Karnofsky score <90 and use of HLA-mismatched donors negatively affected survival. Age, genotype, and oxidase status did not affect outcomes. Before HCT, patients had higher infection density, higher frequency of noninfectious lung and liver diseases, and more steroid use than conventionally treated patients; however, these issues did not adversely affect HCT survival. Presence of pre-HCT inflammatory conditions was associated with chronic graft-versus-host disease. Graft failure or receipt of a second HCT occurred in 17.6% of the patients and was associated with melphalan-based conditioning and/or early mixed chimerism. At 3 to 5 years after HCT, patients had improved growth and nutrition, resolved infections and inflammatory disease, and lower rates of antimicrobial prophylaxis or corticosteroid use compared with both their baseline and those of conventionally treated patients. HCT leads to durable resolution of CGD symptoms and lowers the burden of the disease. Patients with active infection or inflammation are candidates for transplants; HCT should be considered before the development of comorbidities that could affect performance status. This trial was registered at www.clinicaltrials.gov as #NCT02082353.