Antithymocyte globulin (ATG) prevents graft-versus-host disease (GVHD) in allogeneic hematopoietic cell transplantation (HCT). However, variable ATG exposure impacts transplant outcomes. We aimed to develop a population pharmacokinetic (popPK) model for ATG in myeloablative HCT and evaluate the relationship between area under the time-concentration curve (AUC) and mortality to identify the optimal ATG AUC. We studied 200 adult HCT recipients who received myeloablative conditioning (MAC) and a peripheral blood stem cell graft from 7/8 or 8/8 HLA-matched related or unrelated donors. ATG was given on days -2, -1, and 0. All patients received additional GVHD prophylaxis with methotrexate and cyclosporine. Serum concentration of lymphocyte-binding ATG was determined by flow cytometry in 2,140 samples. For the popPK modeling, the cohort was split into a model development cohort (n = 134) and a validation cohort (n = 66). The modeling was performed using Monolix Suite 2024R1. The relationship between model-estimated AUCs and mortality was evaluated in all 200 patients (combined development and validation subcohorts) using a Cox proportional hazards model. The relationship between model-estimated AUCs and cause-specific outcomes (eg, relapse or acute GVHD [aGVHD]) was evaluated using a competing risk analysis. A two-compartment model with parallel linear and target-mediated elimination best described ATG disposition. Population means, and residual standard errors (RSE%) were 11.78 L (2.05%) for the central volume of distribution (V1), 0.20 L/h (4.36%) for clearance (CL), and 2.20 U/L (7.08%) for the initial ATG-binding capacity of lymphocytes in the central compartment. Lean body weight (LBW) positively correlated with V1, CL, and intercompartmental clearance, while pre-ATG absolute lymphocyte count (ALC) positively correlated with R0_initial. Internal and external validation (using the development and validation subcohorts, respectively) confirmed model stability and robustness. The optimal ATG AUC range was 30 to 45 U·day/L. In multivariate analysis, patients whose AUC was within this range had lower mortality than those whose AUC was outside this range (hazard ratio = 0.46, P = .03). The low mortality of patients with AUC within the range of 30 to 45 U·day/L appeared to be due to both low incidence of grade III to IV aGVHD, which was high in patients with AUC <30 U·day/L, and low incidence of relapse, which was high in patients with AUC >45 U·day/L. This novel model described the pharmacokinetics of ATG in adult HCT recipients following MAC. The model identified LBW and ALC as significant covariates for ATG disposition. Furthermore, we identified the optimal ATG AUC (associated with the lowest mortality). These findings provide the foundation for developing an individualized dosing strategy aimed at improving post-HCT survival.

Objective Autologous hematopoietic cell transplantation (HCT) is an effective treatment for a subset of patients with systemic sclerosis (SSc). Unfortunately, relapse is a significant problem, with no available tests to predict relapse. We studied whether relapse is associated with pre- or post-HCT serum levels of SSc-related autoantibodies. Methods The cohort comprised 38 consecutive evaluable patients with SSc who underwent HCT at a single center and were followed for a median of 33 months. Sixteen patients (42%) relapsed at a median of 14 months post-HCT. Autoantibody levels were determined by immunoassays. Results Regarding pre-HCT autoantibodies, in univariate analyses, the cumulative incidence of relapse (CIR) was lower in anti-RNA polymerase III (ARA)-positive than ARA-negative patients (hazard ratio [HR] 0.21, P = 0.04). Conversely, the CIR was higher among patients with positive anti-Ro52, although this difference was not statistically significant (HR 2.90, P = 0.053). The CIR was similar in patients positive and negative for antitopoisomerase antibody (ATA; ie, Scl-70) or antinuclear antibody (ANA). In bivariate analyses that included older age as a risk factor for relapse, pre-HCT ARA was still associated with relapse (HR 0.21, P = 0.04). This was not the case for Ro52 (HR 2.21, P = 0.16). Regarding post-HCT autoantibody level trajectory, there was no significant difference between patients with vs without relapse. Conclusion Positive ARA pre-HCT is associated with reduced relapse risk, and post-HCT autoantibodies do not appear to be associated with relapse risk.

Background: Cytomegalovirus (CMV) reactivation is a major complication after hematopoietic stem cell transplantation (HSCT). Natural killer (NK) cells help control CMV through killer-cell immunoglobulin-like receptors (KIRs) and their HLA ligands, but donor-derived CMV-specific T-cells may confound the interpretation of NK-mediated effects. Methods: We analyzed 276 HLA-matched (10/10) adults receiving ATG-based T-cell-depleted myeloablative HSCT with a known donor and recipient CMV serostatus. The donor and recipient KIR genotypes were scored by the Cooley B-content score (0-4; ≥2 = high). Clinically significant CMV reactivation (plasma viral load > 25,000 IU/mL, the institutional threshold for pre-emptive therapy) was analyzed with Fine-Gray competing-risks regression, stratified by the donor-recipient serostatus. Results: In seronegative-donor/seropositive-recipient (D-R+) pairs (n = 68), a high donor KIR B-content score was associated with a significantly lower reactivation risk (sub-hazard ratio, 0.46; 95% CI, 0.24-0.91; p = 0.024). No effect was seen in D+R+ pairs (n = 82; SHR, 0.65; p = 0.241); D+R- (n = 28) had too few events to model. A donor Tel-AA/recipient Tel-B+ mismatch was independently associated with a higher reactivation risk (adjusted HR, 2.41; 95% CI, 1.33-4.37; p = 0.004). The overall survival was unaffected in either stratum. Conclusions: A high donor KIR B-content score protects against CMV reactivation in D-R+, but not D+R+, HSCT recipients, consistent with NK dominance when CMV-specific donor T-cells are sparse. A specific donor-recipient telomeric mismatch independently modifies the risk. Donor KIR profiling warrants prospective evaluation in donor-selection algorithms.

ABSTRACTThis study characterized the micro‐/defect‐structure of laser powder bed fused (L‐PBF) Scalmalloy and investigated its mechanical behavior across a wide temperature range. Hot isostatic pressing effectively reduced defect content while maintaining similar microstructure and room temperature tensile properties to stress relief condition; thus, it was applied to all specimens. Tensile and strain‐controlled fatigue (fully reversed at 0.005, 0.003, 0.002, and 0.001 mm/mm amplitudes) tests were conducted from cryogenic (−195°C) to elevated temperatures (up to 400°C for tensile and 200°C for fatigue). Tensile strength decreased gradually until 100°C and then sharply at 200°C due to excessive grain boundary sliding. Fatigue resistance was consistent across temperatures at the highest strain amplitude (0.005 mm/mm) but reduced significantly at 200°C and intermediate amplitudes of 0.003 and 0.002 mm/mm. At the lowest amplitude (0.001 mm/mm), fatigue life depended more on crack‐initiating defect size than temperature.

Antithymocyte globulin (ATG; Thymoglobulin) infusion may result in infusional side effects (ISEs) resembling cytokine release syndrome. This study aimed to identify cytokines associated with ISEs, factors predicting ISEs, and the impact of ISEs on hematopoietic cell transplantation (HCT) outcomes. We studied 211 allogeneic HCT recipients who received 3 infusions of ATG, on days -2, -1, and 0. The focus was on the first infusion. ISE was defined as a maximum temperature ≥38°C, maximum heart rate >125/minute, minimum systolic blood pressure <90 mmHg, or supplemental oxygen use between the start of the first infusion and the start of the second infusion. In 158 of the 211 patients, we determined the post-first infusion serum levels of 34 cytokines using Luminex and compared the levels in patients with ISEs and patients without ISEs using a signed-rank test with Bonferroni correction for multiple comparisons. In all 211 patients, we compared overall survival (OS), relapse-free survival (RFS), and moderate to severe chronic graft-versus-host disease (cGVHD)- and relapse-free survival (GRFS) between patients with ISEs and those without ISEs using Cox regression, as well as in the incidences of acute GVHD (aGVHD), chronic GVHD (cGVHD), relapse, and nonrelapse death using Fine-Gray regression. At least 1 ISE occurred in 93 patients (44%). Median levels of the following cytokines were significantly higher in the patients with ISEs: interleukin-1 receptor antagonist (IL1-RA) (30,166 pg/mL versus 6394 pg/mL; P < .001), interleukin-6 (IL-6) (188 pg/mL versus 49 pg/mL; P < .001), and interferon gamma-induced protein-10 (IP-10) (106 pg/mL versus 70 pg/mL; P = .004). Patients with ISEs and those without ISEs did not differ in terms of weight; body mass index; day -2 leukocyte, neutrophil, monocyte, or lymphocyte count; post-first infusion ATG level; or ATG area under the time-concentration curve. There were no significant between-group differences in OS (hazard ratio [HR], 0.83; P = .43), RFS (HR, 0.85; P = .38), or GRFS (HR, 1.01; P = .98). There also were no significant differences in the rates of grade II-IV aGVHD, grade III-IV aGVHD, moderate to severe cGVHD, relapse, or nonrelapse death. IL-6, IP-10, and IL1-RA appear to be involved in the pathogenesis of ISEs; however, ISEs appear to have no significant impact on HCT outcomes. © 2025 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Rituximab is commonly used as prevention, preemption, or therapeutically for post-transplant lymphoproliferative disorder (PTLD) after hematopoietic cell transplantation (HCT). Although it is generally assumed that rituximab toxicity (ie, infections resulting from hypogammaglobulinemia and neutropenia) is negligible in relation to mortality due to PTLD, limited evidence supports the validity of this assumption. We sought to determine the impact of rituximab on immunoglobulin levels, neutrophil count, infection density, and mortality outcomes. This study retrospectively analyzed 349 HCT recipients, 289 of whomdid not receive rituximab and 60 of whomreceived rituximab preemptively or therapeutically at a median of 55 days post-transplantation. IgM, IgG, and IgA levels at 6 months and 12 months post-transplantation were lower in patients who received rituximab compared with those who did not (significant at P <.05 for IgM and IgA at 6 months and for IgM and IgG at 12 months). Rituximab recipients also had a higher incidence of severe neutropenia (<.5/nl) between 3 and 24 months (subhazard ratio [SHR], 2.3; P =.020). Regarding nonEpstein-Barr viral infections/PTLD, the rituximab group had a higher infection density between 3 and 24 months compared with the no-rituximab group (3.8 versus 1.6 infections per 365 days at risk; incidence rate ratio, 2.2; P <.001). The rituximab group also had a higher incidence of fatal infections (SHR, 3.1; P =.026), higher nonrelapse mortality (SHR, 2.4; P =.006), and higher overall mortality (hazard ratio, 1.7; P =.033). There were no significant between-group differences in the incidence of clinically significant graft-versus-host disease, graft failure, or relapse. Based on this study, rituximab given for PTLD is associated with substantial morbidity and mortality. Whether the benefit of preemptive rituximab outweighs the risk remains to be determined. (C) 2022 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. (c) 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.

The study identifies potential carcinogenic health risk-zone of Chattogram city for the occurrence of trihalomethanes (THMs) at its water distribution network. The EPANET-THMs simulation model along with an empirical model have been adopted in the study to predict THMs content of supply water of the distribution network of the city's Karnaphuli service area. The empirical model has estimated THMs level of supply water based on influential water quality parameters, and few of these have been used as pre-set values for subsequent EPANET simulation. The simulation (R2= 0.7) shows that THMs' concentrations throughout the network vary from 33 to 486 & mu;g/L. Around 60% of total junctions showed THMs concentrations above 150 & mu;g/L, while that is above 50 & mu;g/L for most (99%) of the junctions. Residual Free chlorine, one of the precursors for the THMs formation in distribution line, has also been simulated by EPANET considering varying applied chlorine dose at the water purification unit and wall (Kw) and bulk (Kb) decay constants. The simulated free residual chlorine peaks are found to be closer to the actual values with chlorine dose of 2 mg/L, and decay constants, Kw = 1 d-1 and Kb = 1 d-1. A mean lifetime total risk of cancer due to the presence of THMs has been found to be very high. Spatial distribution of carcinogenic risk shows that the central zone of the service area is the most vulnerable zone, followed by the western and northern zone. The first ever zone wise risk identification could be used as baseline data for operational and regulatory purposes and may raise awareness among the city's inhabitants. Furthermore, the application of EPANET in combination with an empirical model could be an effective tool for predicting THMs' concentration in water distribution networks in developing countries like Bangladesh to minimize the expenses of measuring THMs.

The paper aims at assessing the effect of heat treatment on the fatigue behavior of a novel laser-powder bed fused (L-PBF) Al-Cu-Mg-Ag-TiB2 composite, otherwise known as A20X alloy. Heat treatments, (i) stress relieving, and (ii) T7 over-aging and stabilizing, were performed on L-PBF A20X materials, followed by advanced microstructural characterizations, mechanical property measurements (utilizing tensile and depth-sensing indentation testing), and force-controlled fatigue testing. Microstructural assessments revealed the ultra-fine and fine equiaxed grain structure of the L-PBF stress-relieved and aged A20X materials. Mechanical properties (yield stress, ultimate strength, and nano-hardness quantities) of the aged material were recorded as higher than the stress-relieved counter materials. By the same token, the aged material showed better low-cycle and high-cycle fatigue performance and higher fatigue strength relative to the stress-relieved conditions. Based on detailed fractography assessments, the life-limiting fatigue failures of the studied materials (stress relived and T7 aged) were primarily governed by sub-surface L-PBF-induced volumetric defects such as pores and/or lack of fusion. The findings of this study present useful information on the role/mechanism of inevitable process-induced interior defects on fatigue behavior of the Al-Cu-Mg-Ag-TiB2 composites and the effect of post-processing (i.e., aging in this research) on fatigue behavior, which is of great significance for the high-demanding and critical applications of lightweight high-strength L-PBF Al-Cu-Mg-Ag-TiB2 composites in the space and aviation sectors.

Background: Anti-thymocyte globulin (ATG) serotherapy for graft-versus-host disease (GVHD) prophylaxis in allogeneic hematopoietic cell transplantation (HCT) is frequently associated with infusional side-effects (ISEs) including fever, tachycardia, respiratory distress, and hypotension/shock. Although this constellation closely resembles cytokine release syndrome, the mechanism behind ATG ISEs is poorly understood. The aim of this study was to characterize the frequency and severity of ATG ISEs, and determine the association of ISEs with cytokine levels and clinical outcomes. Methods: This study included 118 adult HCT recipients receiving myeloablative conditioning including fludarabine, busulfan, low-dose total body irradiation, and 4.5mg/kg ATG (0.5mg/kg on day -2 and 2mg/kg on days -1 and 0). Patient electronic medical records were retrospectively reviewed for clinical data collection. ISE were classified as thermal (fever/rigors), respiratory (tachypnea/hypoxemia/increased O 2 supplementation), tachycardia, or circulatory (hypotension/fluid resuscitation requirement). ISEs were further characterized into mild or severe (e.g., mild tachycardia = 105-120bpm, severe ≥120bpm). Serum levels of 31 cytokines were determined pre- and post-1 st ATG infusion in all patients (and up to 13 timepoints in select patients) using Luminex. Cox and Fine-Gray regression were used to determine the association of ISEs with clinical outcomes including grade 2-4 acute GVHD (aGVHD), moderate-severe chronic GVHD (cGVHD), cumulative incidence of relapse (CIR), and overall survival (OS). Results: At least one ISE occurred in 74% (87/118) of patients, with a maximum severity of mild in 56 (47%) and severe in 31 (26%). Patients experienced an average of 1.4 ISEs (median 1), most commonly tachycardia (57%) and thermal (50%), while respiratory (26%) and circulatory (7%) ISEs were less frequent. ISE frequency was 62%, 60%, and 35% after the 1 st-3 rd ATG infusions, respectively, with the median time from infusion to first ISE being ~4.5 hours. Compared to pre-infusion levels, there was a >1-fold change in 11/34 cytokines (all P<0.001) [Table 1]. Of greatest interest was IL-6, which demonstrated a progressive increase in median levels between patients with no vs. mild vs. severe ISEs (P<0.05). Severe ISEs were associated with a lower incidence of grade 2-4 aGVHD (SHR=0.12, P=0.032), but not with any other outcomes. Patient age, sex, primary disease, or pre-infusion peripheral blood cell counts (leukocytes, lymphocytes, neutrophils, and monocytes) did not appear to influence ISE frequency or severity. Conclusion: ISEs occur in the majority of ATG recipients, with one-fourth experiencing severe ISEs. The concomitant rise in serum cytokine levels (particularly IL-6) suggests that anti-cytokine antibodies (e.g., tocilizumab) may assist in the prevention or treatment of ISEs, however, the impact this may have on outcomes is unclear and warrants investigation in upcoming clinical studies.

Introduction: Pharmacokinetically (PK)-targeting intravenous busulfan improves outcomes following allogeneic hematopoietic cell transplantation (HCT). However, the optimal busulfan area under the curve (AUC) likely differs based on the specific conditioning regimen, and the target busulfan exposure is unknown in combination with fludarabine and low-dose total body irradiation (TBI). Methods: This study included 1019 adult HCT recipients that received myeloablative conditioning including fludarabine, busulfan, anti-thymocyte globulin, and low-dose (4cGy) TBI. Busulfan was administered as a total dose of ~3.2mg/kg given equally from days -5 to -2 pre-transplant. Total AUC was estimated using measurements of serial serum samples. Multivariate Cox and Fine-Gray regression were used for comparison of AUC subgroups. The primary outcomes of interest were relapse-free survival (RFS) and overall survival (OS). Results: Median AUC was 62.3 mg∙hr/L (range: 39.4-128.0 mg∙hr/L). Total AUC exposure of 49.3-57.5 mg∙hr/L was associated with greater RFS (67% vs. 47%, HR=1.82, P=0.014) and OS (71% vs. 46%, HR=1.99, P=0.008) compared to patients with higher AUCs of 57.5-73.9 mg∙hr/L. Although very low (<49.3 mg∙hr/L) or very high (>73.9 mg∙hr/L) AUCs trended towards worse RFS and OS compared to 49.3-57.5 mg∙hr/L, this analysis was limited by the small number of patients with extreme AUCs and did not reach statistical significance. Except potentially for patients with a high/very high HCT disease risk index, 49.3-57.5 mg∙hr/L appeared to be the optimal AUC regardless of patient sex, age, or primary disease. Conclusion: Within the evaluated AUC range, 49.3-57.5 mg∙h/L appeared to be associated with the most favourable survival. Pharmacokinetic-targeting to this range may improve outcomes.

Rituximab-based chemoimmunotherapy (R-CHOP) is the standard of care in most B-cell lymphomas, however there is marked interpatient heterogeneity in treatment response. An important mechanism of Rituximab (anti-CD20) action is through natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), but the factors influencing this are poorly understood. Here, we present findings from a preclinical study investigating the role of NK cell receptor polymorphisms and HLA allelic variation on ADCC against aggressive B-cell lymphoma cell lines. Genotyping was conducted using Luminex-based SSO and Sanger sequencing. Multicolor flow cytometry was used to assess the response of activated NK cells from healthy donors against 8 lymphoma cell lines. Individuals carrying the GG or GT allele in CD16a were found to have a 3-8 fold greater NK cell response compared to individuals with a TT allele and rituximab-mediated ADCC was significantly greater in individuals with a KIR-3DL1 allele when tested against B-cell lymphomas lacking the HLA-Bw4 allele. An additive effect was observed when considering both CD16a and KIR-HLA together. As expected, CD20 expression was a strong correlate of NK cell response. In summary, NK cell-mediated ADCC in response to ADCC appears to be influenced by the KIR-HLA-CD16a genotype. These findings suggest a potential role for immunogenotyping in B-cell lymphomas treated with Rituximab. Additionally, the presence of the shared F c receptor domain opens the possibility of applying this framework towards predicting NK cell responses to antibody therapies in other diseases.

Additive manufacturing (AM), an enabler of Industry 4.0, recently opened limitless possibilities in various sectors covering personal, industrial, medical, aviation and even extra-terrestrial applications. Although significant research thrust is prevalent on this topic, a detailed review covering the impact, status, and prospects of artificial intelligence (AI) in the manufacturing sector has been ignored in the literature. Therefore, this review provides comprehensive information on smart mechanisms and systems emphasizing additive, subtractive and/or hybrid manufacturing processes in a collaborative, predictive, decisive, and intelligent environment. Relevant electronic databases were searched, and 248 articles were selected for qualitative synthesis. Our review suggests that significant improvements are required in connectivity, data sensing, and collection to enhance both subtractive and additive technologies, though the pervasive use of AI by machines and software helps to automate processes. An intelligent system is highly recommended in both conventional and non-conventional subtractive manufacturing (SM) methods to monitor and inspect the workpiece conditions for defect detection and to control the machining strategies in response to instantaneous output. Similarly, AM product quality can be improved through the online monitoring of melt pool and defect formation using suitable sensing devices followed by process control using machine learning (ML) algorithms. Challenges in implementing intelligent additive and subtractive manufacturing systems are also discussed in the article. The challenges comprise difficulty in self-optimizing CNC systems considering real-time material property and tool condition, defect detections by in-situ AM process monitoring, issues of overfitting and underfitting data in ML models and expensive and complicated set-ups in hybrid manufacturing processes.

Abstract Background Post Transcatheter aortic valve implantation (TAVI) coronary re-access is predicted to become more common as TAVI expands into lower risk cohorts. The use of a self-expanding valve (SEV) has been recognised as an independent predictor for unsuccessful coronary cannulation. Other predictors include reduced transcatheter heart valve (THV) to coronary distance and small sino-tubular junction. Despite this however a patient cohort remains in which the use of a SEV is preferable due to anatomical complexity or extensive calcification. Purpose Using patient-specific computer modelling, we aimed to assess how the deployment of different sized THVs and at different depths affected the distance from valve to coronary and valve to sino-tubular junction in bicuspid aortic valve cases. Methods In this modelling study we have used pre-procedural, CT-derived, patient-specific finite element analysis computer models to predict the eventual valve result. We studied two anatomical measures for both right and left coronary sinuses. The distance from coronary ostium to nearest structure, either THV or displaced native leaflet – Sinus of Valsalva (SoV) free space. The second distance was from the STJ to THV – STJ free space. THVs were modelled at both high and medium implant positions. THV size was based upon annular dimensions. Where the annular dimensions fell near an anatomical grey-zone, a second size THV was modelled. For each patient and measure, a mean of all distances was calculated, in addition to both minimum and maximum distances. Results The free space SOV left main coronary artery (LMCA) mean was 6.39 mm (4.87-7.91 mm). The difference between maximum and minimum was 0.9461 mm (0.54–1.36 mm), p<0.01. The largest patient-specific difference between minimum and maximum was 2.41 mm, using the same THV size but at different depths. The free space SOV right coronary artery (RCA) mean was 5.73 mm (4.17-7.30 mm). The difference between maximum and minimum was 0.789 mm (0.49-1.09 mm), p<0.01. The STJ free space LMCA mean was 4.3 mm (2.35–6.27 mm), The difference between maximum and minimum was 0.99 mm (0.63-1.35 mm), p<0.01. The STJ free space RCA mean was 3.76 mm (2.2-5.32 mm).The difference between maximum and minimum was 1.18 mm (0.9-1.56 mm), p<0.01. The largest patient-specific difference between minimum and maximum was 2.02 mm, using the same valve size but at different depths. Data presented as mean (95% confidence intervals), n=16 patients/48 models. Conclusions This modelling study has indicated a significant difference in all four measured parameters when different THVs are modelled at different depths. Note some of the largest differences are seen with the same size THV but at a different depth. We would highlight the small mean effect size seen across the results, however in cases where the difference is >2mm, this could have an effect on coronary re-access and warrants further investigation.

Hematopoietic cell transplant (HCT) is the only established cure for sickle cell disease (SCD). Engraftment (chimerism) of donor red blood cells (RBCs) determines HCT success but current methods measure only white blood cell chimerism and may be inaccurate or fail to indicate graft rejection in a timely manner. Measurement of differentially methylated regions (DMRs) in nucleated red blood cells (nRBCs) to distinguish individuals with sickle cell anemia (genotype HbSS) nRBCs from HbAA and HbAS nRBCs to quantify RBC chimerism could improve post-HCT monitoring by increasing sensitivity to detect rejection and allow for early intervention. Cell-free DNA derived from nRBCs represents a potential method to specifically measure RBC chimerism. Methylation of cytosine nucleotides that are followed by a guanine (methylated CpG sites) is a major means of cell-specific gene expression, and measuring regions of multiple differentially methylated cytosines (DMRs) enables the identification of cell-of-origin of DNA fragments. Cell-free DNA is released into the circulation from all body tissues and cells, including nRBCs, and retains their cell-specific methylation signatures (figure 1). We hypothesized that DMRs unique to healthy and/or SCD nRBC DNA could be identified and later quantified to assess RBC donor chimerism. We generated genome-wide methylomes of nRBC DNA from pediatric SCD (n=2) and non-SCD control (n=3) patients using enzymatic methyl-sequencing (figure 2). From these 5 novel nRBC methylomes and publicly available methylomes and hydroxymethylomes from 35 different tissues and hematopoietic cells we computationally identified 105 DMRs uniquely found in nRBCs compared to other tissues and cells. There were 34 DMRs that specifically distinguished control nRBCs from SCD nRBCs. The 105 DMRs unique to nRBCs were all relatively hypomethylated. These DMRs were intergenic and not associated with CpG islands or shores. DMRs that distinguished SCD nRBCs from control nRBCs were predominantly hypermethylated (76%) in SCD nRBCs and were commonly (21%-24%) located at promoter, exon or intron sequences while 47% were intergenic. 24% were in CpG islands and 5% were in CpG shores. The most significant gene ontology terms in SCD-specific nRBC DMRs were cardiac-related, including heart contraction and conduction. Other highly significant terms were related to cell signaling, transmembrane transporters, and ion channels. Enriched pathway analysis identified terms related to cell signaling, though these were associated with neuronal and immune signaling pathways. To validate our in silico findings, we amplified 8 DMRs from genomic DNA isolated from 23 different cells and tissues and compared methylation patterns. Four DMRs were relatively hypomethylated in control nRBCs compared to SCD nRBCs and other tissues and cells. Amplification of these DMRs from cell-free DNA in patient plasma before and after HCT will validate them as potential unique markers of successful RBC chimerism. Quantification in recipients with HbAA and HbAS donors will be performed. The successful identification of nRBC-specific DMRs in plasma cell-free DNA will enable novel methodology for the quantification of RBC chimerism after HCT for SCD that can be compared to conventional methods of chimerism measurement in future clinical trials.

Chimeric antigen receptor (CAR) T cell therapy is an increasingly prominent form of next-generation immunotherapy in both hematologic and solid tumor malignancies. Multiple CAR T products have received regulatory approval for use as treatment in relapsed and refractory leukemias and lymphomas. However, approximately half of these patients do not respond to therapy and production of CAR T cells is a difficult, expensive, and time-consuming process. Although much has been learned about the fundamental biology of CAR T cells over the past decade, we are still unable to accurately predict which patients will respond to therapy. In particular, few studies have specifically examined biological characteristics of manufactured CAR T cells and correlated these to relevant clinical outcomes. In this study, we analyzed the lymphocyte populations and CAR T cells from patients enrolled in ACIT001/EXC002, a Canadian multi-centre phase Ib/II single-arm clinical trial of decentralized production of second-generation CD19/41BB/CD3z CAR T cells for treatment of multiply relapsed/refractory non-Hodgkin lymphoma (NHL) and acute lymphoblastic leukemia (ALL). CD3-positive T cells and CAR T products were comprehensively immunophenotyped by flow cytometry. Thecapacity for manufactured CAR T cells to kill tumor cells was assessed using an ex vivo cytotoxicity assay. CAR T cell immunophenotype and cytotoxic potential were correlated with clinical outcomes including response to therapy, progress-free survival (PFS) and incidence of serious adverse events. To date, 23 patients have been enrolled, of which 16 had an aggressive NHL while the remaining 7 had been diagnosed with B cell ALL. Mean age of participants at time of enrollment was 57 (range: 26-77) and 65.2% were male. CAR T product was manufactured using lentiviral transduction with a median transduction efficiency of 32.1% (range: 9.1% to 55.5%). A majority of patients achieved a complete response as evaluated by PET scan on day 28 post-CAR T cell infusion with only 3 patients showing disease progression, 1 patient achieving only partial response, and 3 patients being unevaluable. We were able to evaluate PFS at 6 months in 15 out of 23 patients, with 46.7% of patients experiencing progression and/or mortality within this period. Surprisingly, while CAR T cell cytotoxicity ex vivo trended higher in patients that achieved a complete metabolic response compared to those with partial response or disease progression, this did not reach statistical significance (p = 0.280). However, expression of T cell exhaustion markers such as PD-1 CAR T products was found to be significantly increased in non-responders (p = 0.010). Higher expression of exhaustion markers was also found to be associated with decreased CAR T persistence in vivo as measured by circulating CAR T cell levels at day 14 post-infusion. In our study cohort, CAR T products had either similar or significantly greater proportions of CD4+ as compared to CD8+ T cells. CAR T cells were found to express high levels of HLA-DR and CD127, however this did not correlate with cytotoxicity, response to therapy or PFS. Patient CAR T cells, regardless of the level of exhaustion marker expression, tended to be highly skewed towards the T effector memory subset, with almost no populations of naïve or memory T cells identified. In this analysis of the impact of biological characteristics of CAR T cell products on patient outcomes, we found that CAR T cell exhaustion rather than cytotoxic activity is correlated with response to treatment and higher PFS at 6 months. Interestingly, the degree of patient T cell exhaustion had an impact on transduction efficiency but not the cytotoxic potential of the manufactured CAR T cell product. Taken together, our data indicate that CAR T cell exhaustion and persistence following infusion rather than cytotoxic potential is a key predictor for patient response to treatment. While our conclusions are currently limited by small sample size, more patients are presently being accrued to allow for more robust analysis in the future.

Background Autoimmune hepatitis (AIH) is a substantial UK health burden, but there is variation in care, facilities and in opinion regarding management. We conducted an audit of service provision and care of patients with AIH in 28 UK hospitals. Methods Centres provided information about staffing, infrastructure and patient management (measured against predefined guideline-based standards) via a web-based data collection tool. Results Hospitals (14 university hospitals (UHs), 14 district general hospitals (DGHs)) had median (range) of 8 (3-23) gastroenterologists; including 3 (0-10) hepatologists. Eight hospitals (29%, all DGHs) had no hepatologist. In individual hospital departments, there were 50% (18-100) of all consultants managing AIH: in DGH's 92% (20-100) vs 46% (17-100) in UHs. Specialist nurses managed AIH in only 18%. Seventeen (61%) hospitals had a histopathologist with a liver interest, these were more likely to find rosettes than those without (172/795 vs 50/368; p<0.001). Of 999 steroid-treated patients with >= 12 months follow-up, 25% received steroids for <12 months. After 1 year of treatment, 82% of patients achieved normal serum alanine aminotransaminase (ALT); this was higher in UHs than DGHs. Three-monthly liver blood tests were inadequately recorded in 26%. Of potentially eligible patients with liver decompensation, transplantation was apparently not considered in 5% (n=7). The same standards were attained in different types of hospital. Conclusion Management of AIH in UK hospitals is often shared between most gastroenterologists. Blood test monitoring and treatment duration are not always in line with recommendations. Some eligible patients with decompensation are not discussed with transplant teams. Care might be improved by expanding specialist input and management by fewer designated consultants.

Background aims: The value of routine chimerism determination after myeloablative hematopoietic cell transplantation (HCT) is unclear, particularly in the setting of anti-thymocyte globulin (ATG)-based graft -ver-sus-host disease (GVHD) prophylaxis. Methods: Blood samples were collected at 3 months post-HCT from 558 patients who received myeloablative conditioning and ATG-based GVHD prophylaxis. Chimerism was assessed using multiplex polymerase chain reaction of short tandem repeats in sorted T cells (CD3+) and leukemia lineage cells (CD13+CD33+ for mye-loid malignancies and CD19+ for B-lymphoid malignancies). ATG exposure was determined using a flow cytometry-based assay. The primary outcomes of interest were relapse and chronic GVHD (cGVHD).Results: Incomplete (<95%) T-cell chimerism and leukemia lineage chimerism were present in 17% and 4% of patients, respectively. Patients with incomplete T-cell chimerism had a significantly greater incidence of relapse (36% versus 22%, subhazard ratio [SHR] = 2.03, P = 0.001) and lower incidence of cGVHD (8% versus 25%, SHR = 0.29, P < 0.001) compared with patients with complete chimerism. In multivariate modeling, patients with high post-transplant ATG area under the curve and any cytomegalovirus (CMV) serostatus other than donor/recipient seropositivity (non-D+R+) had an increased likelihood of incomplete T-cell chimerism. Patients with incomplete leukemia lineage chimerism had a significantly greater incidence of relapse (50% versus 23%, SHR = 2.70, P = 0.011) and, surprisingly, a greater incidence of cGVHD (45% versus 20%, SHR = 2.64, P = 0.003).Conclusions: High post-transplant ATG exposure and non-D+R+ CMV serostatus predispose patients to incomplete T-cell chimerism, which is associated with an increased risk of relapse. The increased risk of cGVHD with incomplete B-cell/myeloid chimerism is a novel finding that suggests an important role for recipient antigen-presenting cells in cGVHD pathogenesis.(c) 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.

Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 is next generation immunotherapy that has curative potential in relapsed ALL and aggressive NHL. Though effective, about half of the patients do not respond to the therapy. In addition, the CAR T production process is difficult, time consuming, expensive and these costs are subsequently passed down to health care systems. Therefore, predicting the clinical efficacy and resistance to the CAR T cell therapy before going through all CAR T cell production process to rule out the non-responders is needed to save both time and cost. Herein we describe a holistic, correlative approach to identifying responders and non-responders on the phase 1b portion of ACIT001/EXC002, a clinical trial of decentralized production of anti-CD19/41BB/CD3z in multiply relapsed/refractory NHL and acute lymphoblastic leukemia (ALL) patients. The outcomes were correlated with the clinical response. The median transduction efficiency was 27% (range: 7.35% to 54.57%). Among 10 patients enrolled in phase I clinical trial, 3 patients never responded (2 primary refractory and 1 unevaluable), and 1 showed partial response. All 10 products were tested for cytotoxicity ex vivo in increasing effector to target ratios against a CD19+ cell line (Raji) after 4 and 24 hours in culture. Except one (A02), all of the complete responders exhibited effective CD19 positive cells killing while 2 out of 3 non-responders exhibited poor response in ex vivo cytotoxic assay. Although one of the non-responders expressed high level of PD1, expression was not statistically different between responders and non-responders. Flow cytometry analysis of CAR T products demonstrated that naïve T cell subset was negligible in all samples. Central memory subset was low in all CAR T samples except CAR T from one patient (A06). In general, proportion of effector memory subset was higher than other T cell subsets in CAR T from all patients and highest proportion was observed in the one of the CAR T from non-response group (A03). Gene expression analysis using nCounter CAR T characterization panel from Nanostring revealed upregulation and downregulation of gene sets in CAR T from non-response group as compared to the CAR T from complete response. Glucose-6-phosphate isomerase (GPI), Aldolase (ALDOC), Myc were the top downregulated genes and interferon-gamma-inducible-protein 30 (IFI30), chemokine receptor CX3CR1, FCGR3A/B were the top upregulated gene in CAR T from non-response group. Advanced analysis showed a trend in higher pathway score for apoptosis and exhaustion pathway and a trend in lower score for activation, glycolysis and JAK-STAT pathway in CAR T from non-response as compared to the complete response group. Since there was a substantial cross patient heterogeneity in gene expression of CAR T, more samples in each group are needed to validate significance of differences in gene expression. Interestingly, expression of top genes in CAR T from one the non-response group (A07) was different than other group although it showed good response in ex vivo cytotoxicity. While the three individual assays by themselves would not be sufficient to predict for response, taken together, the multiple criteria from ex vivo cytotoxicity assay, T cell phenotypic analysis and gene expression analysis is capable of identifying response to this second generation CAR T product. This study will continue to analyze the CAR T from upcoming phase II clinical trial and perform robust corelative analysis to predict the clinical outcomes based on multiple characteristics of CAR T products while striving to identify statistical weight behind these assays to develop a more reliable prediction model through use of machine learning.

Background aims: The internal tandem duplication of FLT3 (FLT3(ITD)) and NPM1 mutations (NPM1(mut)) are well-established prognostic factors in cytogenetically intermediate-risk acute myeloid leukemia (AML) when treated with chemotherapy alone. However, their prognostic value in the setting of allogeneic hematopoietic cell transplantation (HCT) is controversial. Methods: FLT3 and NPM1 mutational status was determined at diagnosis using single-gene polymerase chain reaction or next-generation sequencing in 247 adult patients with cytogenetically intermediate-risk AML who underwent myeloablative HCT. Multivariate Fine-Gray and Cox regression was used to analyze the cumulative incidence of relapse (CIR), relapse-free survival (RFS) and overall survival (OS). Results: FLT3(ITD) and NPM1(mut) were present in 74 of 247 (30%) and 79 of 247 (32%) patients, respectively. There was no significant difference between patients without a FLT3(ITD) or NPM1(mut) (FLT3(NONITD)/NPM1(WT)) and patients with a FLT3(ITD) mutation alone (FLT3(ITD)/NPM1(WT)) with regard to CIR (P = 0.60), RFS (P = 0.91) or OS (P = 0.66). Similarly, there was no significant difference between FLT3(NONITD)/NPM1(WT) and FLT3(NONITD)/NPM1(mut) patients with regard to CIR (P = 0.70), RFS (P = 0.75) or OS (P = 0.95). The presence of a concurrent mutation in NPM1 did not appear to modify the impact of having a FLT3(ITD) mutation. Conclusions: In contrast to chemotherapy-only treatment, FLT3 and NPM1 mutational status does not appear to predict outcomes in patients with cytogenetically intermediate-risk AML following HCT. These results suggest that HCT may ameliorate the poor prognostic effect of FLT3(ITD) mutation and that HCT should be considered over chemotherapy-only treatment in FLT3(ITD)-mutated AML. (C) 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.