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Metachromatic leukodystrophy (MLD) is a rare autosomal recessive lysosomal storage disorder resulting from arylsulfatase A enzyme deficiency, leading to toxic sulfatide accumulation. As a result affected individuals exhibit progressive neurodegeneration. Treatments such as hematopoietic stem cell transplantation (HSCT) and gene therapy are effective when administered pre-symptomatically. Newborn screening (NBS) for MLD has recently been shown to be technically feasible and is indicated because of available treatment options. However, there is a lack of guidance on how to monitor and manage identified cases. This study aims to establish consensus among international experts in MLD and patient advocates on clinical management for NBS-identified MLD cases. A real-time Delphi procedure using eDELPHI software with 22 experts in MLD was performed. Questions, based on a literature review and workshops, were answered during a seven-week period. Three levels of consensus were defined: A) 100%, B) 75–99%, and C) 50–74% or >75% but >25% neutral votes. Recommendations were categorized by agreement level, from strongly recommended to suggested. Patient advocates participated in discussions and were involved in the final consensus. The study presents 57 statements guiding clinical management of NBS-identified MLD patients. Key recommendations include timely communication by MLD experts with identified families, treating early-onset MLD with gene therapy and late-onset MLD with HSCT, as well as pre-treatment monitoring schemes. Specific knowledge gaps were identified, urging prioritized research for future evidence-based guidelines. Consensus-based recommendations for NBS in MLD will enhance harmonized management and facilitate integration in national screening programs. Structured data collection and monitoring of screening programs are crucial for evidence generation and future guideline development. Involving patient representatives in the development of recommendations seems essential for NBS programs. • Newborn screening for metachromatic leukodystrophy aligns with established newborn screening criteria. • Prediction of symptom onset should be based on family history, genotype and blood ARSA enzyme activity. • Recommendations for pre-symptomatic treatment depend on the predicted MLD subtype. • Consensus-based recommendations for NBS in MLD will enhance harmonized management and facilitate integration in national screening programs. [ABSTRACT FROM AUTHOR]

CD4 immune reconstitution (IR) after allogeneic hematopoietic cell transplant (allo-HCT) correlates with lower non-relapse mortality (NRM), but its impact on leukemia relapse remains less clear, especially in children. We studied the correlation between IR of lymphocyte subsets and HCT outcomes in a large cohort of children/young adults with hematological malignancies. We retrospectively analyzed CD4, CD8, B-cell and natural killer (NK) cell reconstitution in patients after first allo-HCT for a hematological malignancy at three large academic institutions (n = 503; period 2008–2019). We used Cox proportional hazard and Fine–Gray competing risk models, martingale residual plots and maximally selected log-rank statistics to assess the impact of IR on outcomes. Achieving CD4 >50 and/or B cells >25 cells/μL before day 100 after allo-HCT was a predictor of lower NRM (CD4 IR: hazard ratio [HR] 0.26, 95% confidence interval [CI] 0.11–0.62, P = 0.002; CD4 and B cell IR: HR 0.06, 95% CI 0.03–0.16, P < 0.001), acute graft-versus-host disease (GVHD) (CD4 and B cell IR: HR 0.02, 95% CI 0.01–0.04, P < 0.001) and chronic GVHD (CD4 and B cell IR: HR 0.16, 95% CI 0.05–0.49, P = 0.001) in the full cohort, and of lower risk of relapse (CD4 and B cell IR: HR 0.24, 95% CI 0.06–0.92, P = 0.038) in the acute myeloid leukemia subgroup. No correlation between CD8 and NK-cell IR and relapse or NRM was found. CD4 and B-cell IR was associated with clinically significant lower NRM, GVHD and, in patients with acute myeloid leukemia, disease relapse. CD8 and NK-cell IR was neither associated with relapse nor NRM. If confirmed in other cohorts, these results can be easily implemented for risk stratification and clinical decision making. [Display omitted] [ABSTRACT FROM AUTHOR]

Poor graft function (PGF) after allogeneic hematopoietic stem cell transplantation (HCT) is a serious complication with high morbidity and mortality. The reported incidence of PGF, its risk factors and outcome vary substantially between studies. This variability may be explained by heterogeneity in patient cohorts and HCT strategies, differences in the underlying causes of cytopenia, as well as by differences in PGF definition. In this systematic review and meta-analysis, we provide an overview of the various PGF definitions used and determined the impact of this variability on the reported incidence and outcome. We searched MEDLINE, EMBASE and Web of Science up to July 2022, for any study on PGF in HCT recipients. We performed random-effect meta-analyses for incidence and outcome and subgroup analyses based on different PGF criteria. Among 69 included studies (14.265 HCT recipients), we found 63 different PGF definitions, using various combinations of 11 common criteria. The median incidence of PGF was 7% (IQR: 5–11%, 22 cohorts). The pooled survival of PGF patients was 53% (95% CI: 45–61%, 23 cohorts). The most commonly reported risk factors associated with PGF were history of cytomegalovirus infection and prior graft-versus-host disease. Incidence was lower in studies with strict cytopenic cutoffs, while survival was lower for primary compared to secondary PGF. This work indicates that a standardized, quantitative definition of PGF is needed to facilitate clinical guideline development and to advance scientific progress. • Definitions of poor graft function (PGF) are heterogeneous across studies. • Definition heterogeneity affects the reported incidence and outcome of PGF. • A standardized, quantitative and evidence-based definition of PGF is required. • A standardized definition facilitates progress in understanding and treating PGF. [ABSTRACT FROM AUTHOR]

In haploidentical (haplo)-cord blood (CB) transplantations, early haplo donor engraftment serves as a myeloid bridge to sustainable CB engraftment and is associated with early neutrophil recovery. The conditioning regimens as published for haplo-cord protocols usually contain serotherapy, such as rabbit antithymocyte globulin (ATG) (Thymoglobulin, Genzyme, Cambridge, MA). However, reducing or omitting serotherapy is an important strategy to improve early immune reconstitution after transplantation. The need for serotherapy in successful haplo-cord transplantation, defined as having a haplo-derived myeloid bridge to CB engraftment, has not been investigated before. Two consecutive cohorts of patients underwent transplantation with haplo-CB. The first group underwent transplantation with haplo-CB for active infection and/or an underlying condition with expected difficult engraftment without a conventional donor available. They received a single unit (s) CB and haplo donor cells (CD34 + selected, 5 × 10 6 CD34 + /kg). The second cohort included patients with poor-risk malignancies, not eligible for other treatment protocols. They received a sCB and haplo donor cells (CD19/αβTCR-depleted; 5 × 10 6 CD34 + /kg). Retrospectively in both cohorts, active ATG (Thymoglobulin) levels were measured and post–hematopoietic cell transplantation area under the curve (AUC) was calculated. The influence of ATG exposure for having a successful haplo-myeloid bridge (early haplo donor engraftment before CB engraftment and no secondary neutropenia) and transplantation-related mortality (TRM) were analyzed as primary endpoints. Twenty patients were included (16 in the first cohort and 4 in the second cohort). In 58% of evaluable patients, there was no successful haplo-derived myeloid bridge to CB engraftment, for which a low post-transplantation ATG exposure appeared to be a predictor ( P < . 001). TRM in the unsuccessful haplo-bridge group was 70% ± 16% versus 12% ± 12% in the successful haplo-bridge group ( P = . 012). In conclusion, sufficient in vivo T depletion with ATG is required for a successful haplo-myeloid bridge to CB engraftment. [ABSTRACT FROM AUTHOR]

Cord blood is increasingly recognized for its excellent stem cell potential, lenient matching criteria, instant availability and clinical behavior in transplants when cell dose criteria can be met. However with 1-2 log fewer total (stem cell) numbers in the graft compared with other cell sources, the infused cell dose per kilogram is critical for engraftment and outcome, creating the need for development of stem cell support platforms. The co-transplant platforms of haplo cord and double unit cord blood (DUCB) transplantation are aimed toward increasing stem cell dose. Together with the optimization of reducedintensity protocols, long-term sustained engraftment using cord blood has become available to most patients, including elderly patients. Haplo cord has a low incidence of both acute and chronic graft-versus-host disease but may require antithymocyte globulin ATG for effective neutrophil recovery. DUCB can be performed without anti-thymocyte globulin with excellent immune reconstitution and disease-free survival, but engraftment is considerably slower, and graft-versus-host disease incidence significant. Both haplo-cord and DUCB transplantation appear to both be valid alternatives to matched unrelated donors in adults. [ABSTRACT FROM AUTHOR]

Background: Eosinophils isolated from the blood of patients with allergic asthma exhibit enhanced responsiveness to multiple stimuli compared with cells from normal controls, a phenomenon generally referred to as priming. This priming response is essential for optimal activation with augmented responses including chemotaxis, cytotoxicity, respiratory burst, and the release of proinflammatory lipid mediators. Objective: To monitor the kinetics of priming of eosinophils in the peripheral blood and in the bronchoalveolar lavage fluid of patients with allergic asthma before and after allergen challenge. Methods: Priming of blood eosinophils obtained from patients with allergy and donors without allergy was measured by labeling with monoclonal phage antibodies A17 and A27 recognizing priming-associated epitopes on phagocytes. In addition, blood and bronchoalveolar lavage fluid eosinophils from subjects with allergy after segmental and whole lung allergen challenge were similarly analyzed. Results: A dose-dependent cytokine-induced upregulation of priming-associated epitopes on blood eosinophils was found. Patients with allergic asthma exhibited an in vivo partially primed eosinophil phenotype, which is further primed in vitro after cytokine or chemokine incubation. Priming was increased in peripheral blood 6 hours after whole lung challenge as well as after segmental allergen challenge. Interestingly, eosinophils obtained from the bronchoalveolar lavage fluid 48 hours after segmental allergen challenge exhibited a higher primed phenotype. Conclusion: These data are consistent with a model in which local allergic inflammatory reactions induce partial systemic eosinophil priming in the peripheral blood. Eosinophils found in the airway are highly primed, consistent with the markedly upregulated inflammatory capacity observed in these cells. [Copyright &y& Elsevier]

• High-dose individualized ATG with TDM is safe. • Actual exposures of ATG can be well predicted. • The protocol is effective to prevent GvHD and rejection in this high-risk population. Graft-versus-host disease (GvHD) and rejection are main limitations of cord blood transplantation (CBT), more so in patients with severe inflammation or previous rejections. While rigorous T-cell depletion with antithymocyte globulin (ATG) is needed to prevent GvHD and rejection, overexposure to ATG leads to slow T-cell recovery after transplantation, especially in CBT. To evaluate high-dose, upfront ATG with individualized dosing and therapeutic drug monitoring (TDM) in pediatric CBT for patients at high risk for GvHD and rejection. Heavily inflamed patients and patients with a recent history of rejection were eligible for individualized high-dose ATG with real-time TDM. The ATG dosing scheme was adjusted to target a post-CBT exposure of <10 AU*day/mL, while achieving a pre-CBT exposure of 60–120 AU*day/mL; exposure levels previously defined for optimal efficacy and safety in terms of reduced GvHD and rejection, respectively. Main outcomes of interest included efficacy (target exposure attainment) and safety (incidence of GvHD and rejection). Other outcomes of interest included T-cell recovery and survival. Twenty-one patients were included ranging from 2 months to 18 years old, receiving an actual median cumulative dose of ATG of 13.3 mg/kg (range 6–30 mg/kg) starting at a median 15 days (range 12–17) prior to CBT. Dosing was adjusted in 14 patients (increased in 3 and decreased in 11 patients). Eighteen (86%) and 19 (91%) patients reached the target pre-CBT and post-CBT exposure, respectively. Cumulative incidence for acute GvHD was 34% (95% CI 23–45) and 5% (95% CI 0–10%) for grade 2–4 and grade 3–4, respectively; cumulative incidence of rejection was 9% (95% CI 2–16%). Overall survival was 75% (95% CI 65–85%). Individualized high-dose ATG with TDM is feasible and safe for patients with hyperinflammation in a CBT setting. We observe high target ATG exposure attainment, good immune reconstitution (despite very high doses of ATG) and acceptable rates of GvHD and rejection. [ABSTRACT FROM AUTHOR]

Introduction Pulmonary complications (PCs) after pediatric hematopoietic cell transplant (HCT) are heterogeneous in nature and are strongly associated with transplant-related mortality. Microbial pathogens play a role in the evolution of many post-HCT PCs and may contribute to or develop in the setting of an altered inflammatory microenvironment in the lungs. Objectives To improve characterization of the pulmonary microbiome and transcriptome in pediatric HCT patients with PCs. Methods Bronchoalveolar lavage (BAL) was performed at the University Medical Center in Utrecht between April 2004 and November 2016 for all children <18 years of age prior to undergoing HCT and again in select patients after HCT. In this study, n=257 BALs (n=210 obtained pre-HCT and n=47 obtained post-HCT) underwent metatranscriptomic sequencing to a median depth of 41.5 million read-pairs per sample followed by alignment to human and microbial genomes. Pathogenic bacteria and fungi were identified if RNA was quantified ≥2 standard deviations above the pre-HCT cohort mean (Z-score ≥2). Pathogenic and non-pathogenic viruses were noted when quantified ≥10 and ≥1 reads per million total reads, respectively. Diversity of the bacterial microbiome was considered abnormal if the Simpson's Diversity Index was ≤0.6. Differential gene expression was calculated using DEseq2 with FDR-adjusted p-values <0.05, followed by Gene Ontology analysis. Results Relative to pre-HCT patients, post-HCT patients were more likely to have abnormal quantities of RNA aligning to pathogenic microbes (59.6% vs. 40.5%, p=0.017), including RNA aligning to pathogenic bacteria (23.4% vs. 11.0%, p=0.023) and pathogenic viruses (48.9% vs 29.0%, p=0.009), but not pathogenic fungi (6.4% vs 7.1%, p=0.854). In addition, post-HCT patients were also more likely to have depressed bacterial diversity (23.4% vs. 10.0%, p=0.012) and to harbor non-pathogenic viruses (27.7% vs. 7.1%, p<0.001). Relative to pre-HCT patients without an identified pathogen, post-HCT patients with a pathogen demonstrated at least 396 differentially expressed genes in the biological processes of immunity (leukocyte transendothelial migration; neutrophil degranulation; T-cell receptor activation, IL-1, IL-10 and TNF/NF-κβ mediated signaling; regulation of type 1 IFN production) and cellular response to stress (regulation of HIF-1, response to hypoxia, and regulation of apoptosis and programmed cell death). Conclusions As detection of pathogenic pulmonary microbes is exceedingly common pre-HCT and increases after HCT, incorporation of host gene expression profiles along with standard diagnostic criteria for pulmonary complications may improve classification accuracy and facilitate patient-targeted therapies. Further investigation into the microbiologic and human transcriptional heterogeneity within these high-risk patients is required. [ABSTRACT FROM AUTHOR]

Background Common variable immunodeficiency (CVID) is usually well controlled with immunoglobulin substitution and immunomodulatory drugs. A subgroup of patients has a complicated disease course with high mortality. For these patients, investigation of more invasive, potentially curative treatments, such as allogeneic hematopoietic stem cell transplantation (HSCT), is warranted. Objective We sought to define the outcomes of HSCT for patients with CVID. Methods Retrospective data were collected from 14 centers worldwide on patients with CVID receiving HSCT between 1993 and 2012. Results Twenty-five patients with CVID, which was defined according to international criteria, aged 8 to 50 years at the time of transplantation were included in the study. The indication for HSCT was immunologic dysregulation in the majority of patients. The overall survival rate was 48%, and the survival rate for patients undergoing transplantation for lymphoma was 83%. The major causes of death were treatment-refractory graft-versus-host disease accompanied by poor immune reconstitution and infectious complications. Immunoglobulin substitution was stopped in 50% of surviving patients. In 92% of surviving patients, the condition constituting the indication for HSCT resolved. Conclusion This multicenter study demonstrated that HSCT in patients with CVID was beneficial in most surviving patients; however, there was a high mortality associated with the procedure. Therefore this therapeutic approach should only be considered in carefully selected patients in whom there has been extensive characterization of the immunologic and/or genetic defect underlying the CVID diagnosis. Criteria for patient selection, refinement of the transplantation protocol, and timing are needed for an improved outcome. [ABSTRACT FROM AUTHOR]

Immunogenicity to meningococcal serogroup ACWY (MenACWY) conjugate vaccine has not been studied in immunocompromised minors with juvenile idiopathic arthritis (JIA) or inflammatory bowel disease (IBD). We determined immunogenicity of a MenACWY-TT vaccine in JIA and IBD patients at adolescent age and compared results to data from aged-matched healthy controls (HCs). We performed a prospective observational cohort study in JIA and IBD patients (14–18 years old), who received a MenACWY vaccination during a nationwide catch-up campaign (2018–2019) in the Netherlands. Primary aim was to compare MenACWY polysaccharide-specific serum IgG geometric mean concentrations (GMCs) in patients with HCs and secondary between patients with or without anti-TNF therapy. GMCs were determined before and 3–6, 12, and 24 months postvaccination and compared with data from HCs at baseline and 12 months postvaccination. Serum bactericidal antibody (SBA) titers were determined in a subset of patients at 12 months postvaccination. We included 226 JIA and IBD patients (66 % and 34 % respectively). GMCs were lower for MenA and MenW (GMC ratio 0·24 [0·17-0·34] and 0·16 [0·10-0·26] respectively, p < 0·01) in patients compared to HCs at 12 months postvaccination. Anti-TNF users had lower MenACWY GMCs postvaccination compared with those without anti-TNF (p < 0·01). The proportion protected (SBA ≥ 8) for MenW was reduced in anti-TNF users (76 % versus 92 % in non-anti-TNF and 100 % in HCs, p < 0.01). The MenACWY conjugate vaccine was immunogenic in the vast majority of JIA and IBD patients at adolescent age, but seroprotection was lower in patients using anti-TNF agents. Therefore, an extra booster MenACWY vaccination should be considered. [ABSTRACT FROM AUTHOR]

• Chemo-naivety before HCT and serotherapy are predictors for developing AIC. • aGVHD grades II to IV is a predictor for developing AIC. • IgM, IgG, and IgA levels increase before development of AIC. • AIC is a rare but severe complication after HCT. • Recognizing AIC is challenging, and treatment should begin as quickly as possible. Development of autoimmune cytopenia (AIC) after allogeneic hematopoietic cell transplantation (HCT) is a serious complication requiring urgent intensification of immunosuppressive therapy. The pathophysiology and predictors of AIC are not completely understood. In this retrospective cohort analysis of 380 pediatric patients, we evaluated the incidence, outcomes, and related various variables, including immune reconstitution markers to AIC. Three hundred eighty patients (median age, 7.4 years; range,.1 to 22.7) were included, of which 30 patients (7.8%) developed AIC in 1 (n = 6), 2 (n = 6), or 3 (n = 16) cell lineages at a median of 133 days (range, 46 to 445) after HCT. Using multivariate analysis we found that chemo-naivety before HCT, acute graft-versus-host disease (aGVHD) grades II to IV, and serotherapy were associated with the development of AIC. Development of AIC was preceded by increased levels of IgM, IgA, and IgG. Immune profiles of total absolute lymphocytes were very similar between AIC patients and control subjects. However, CD3-CD16+CD56+ natural killer cells, CD3+ T cells, CD3+CD4+ T cell subset, and CD3+CD8+ T cell subset were lower in AIC patients. Overall survival was good, at 83% (similar between AIC patients and control subjects). In conclusion, we identified chemo-naivety before HCT, preceding aGVHD grades II to IV, and serotherapy as predictors for development of AIC. Increasing levels of IgM, IgA, and IgG preceded AIC development. These data provide clues to further study the biology of AIC. [ABSTRACT FROM AUTHOR]

Highlights • Recovery of monocytes, neutrophils, and NK cells predicts CD4+ T cell reconstitution after HCT. • Innate immune cell recovery is higher after cord blood compared with bone marrow transplantation. • Graft immune cell proliferation potential relates to innate immune cell recovery after HCT. Abstract Innate immune cells are the first to recover after allogeneic hematopoietic cell transplantation (HCT). Nevertheless, reports of innate immune cell recovery and their relation to adaptive recovery after HCT are largely lacking. Especially predicting CD4+ T cell reconstitution is of clinical interest, because this parameter directly associates with survival chances after HCT. We evaluated whether innate recovery relates to CD4+ T cell reconstitution probability and investigated differences between innate recovery after cord blood transplantation (CBT) and bone marrow transplantation (BMT). We developed a multivariate, combined nonlinear mixed-effects model for monocytes, neutrophils, and natural killer (NK) cell recovery after transplantation. A total of 205 patients undergoing a first HCT (76 BMT, 129 CBT) between 2007 and 2016 were included. The median age was 7.3years (range,.16 to 23). Innate recovery was highly associated with CD4+ T cell reconstitution probability (P <.001) in multivariate analysis correcting for covariates. Monocyte (P <.001), neutrophil (P <.001), and NK cell (P <.001) recovery reached higher levels during the first 200days after CBT compared with BMT. The higher innate recovery after CBT may be explained by increased proliferation capacity (measured by Ki-67 expression) of innate cells in CB grafts compared with BM grafts (P =.041) and of innate cells in vivo after CBT compared with BMT (P =.048). At an individual level, patients with increased innate recovery after either CBT or BMT had received grafts with higher proliferating innate cells (CB; P =.004, BM; P =.01, respectively). Our findings implicate the use of early innate immune monitoring to predict the chance of CD4+ T cell reconstitution after HCT, with respect to higher innate recovery after CBT compared with BMT. [ABSTRACT FROM AUTHOR]

Acute Graft-versus-Host-Disease (aGvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic (stem) cell transplantation (HCT). Studies evaluating the relation between immune reconstitution (IR) and aGvHD are largely lacking. We previously showed that successful CD4+ IR strongly predicts non-relapse mortality (NRM) and survival after HCT. We hypothesized that adequate CD4+ IR may influence outcomes in patients who develop aGvHD, because (CD4+) T regulatory (Tregs) play a crucial role in tolerance. Therefore, we studied the relation between CD4+ IR after HCT and outcomes (survival, NRM) in patients who developed moderate to severe aGvHD, in 2 separate pediatric / young adult cohorts. Patients receiving their first allogeneic HCT at the UMC Utrecht / Princess Maxima Center (UMC/PMC; 2004 and 2018) and at Memorial Sloan Kettering Cancer Center (MSK; 2008 – 2018), New York were included. No restrictions applied in terms of donor source, indication, conditioning, age. CD4+ IR was measured every 2-4 weeks. The main outcomes of interest were 5-year overall survival (OS) and 5-year NRM, stratified for aGvHD 2-4 and adequate CD4+ IR (twice >50*106 CD4+/uL) prior to aGvHD onset. Fine and Gray competing risk models and cox-proportional hazard models were used. 591 patients (276 UMC/PMC; 315 MSK) were included (table 1); median age at UMC/PMC was 7.06 years (range 0.16-22.74) and at MSK 10.4 years (range 0.1-35.6). At UMC/PMC 73 (26.4%) and at MSK 70 (22.2%) patients developed aGvHD 2-4, while 29 (10.5%) and 32 (10.2%) developed aGvHD 3-4. At UMC/PMC 35% (26/73) developed aGvHD in the presence of CD4+ IR and at MSK 64% (45/70). The 5-yr OS for patients without aGvHD at UMC/PMC and MSK was 75% and 72%, resp. The only multivariate predictor for OS and NRM in patients with aGvHD 2-4 was CD4+ IR. In both cohorts, CD4+ IR before onset of aGvHD 2-4 was associated with higher 5-yrs OS compared to those who developed aGvHD 2-4 without CD4+ IR; 77% vs 48%, p<0.001 at UMC/PMC and 74.2% vs 36.0%, p=0.007 at MSK. The higher OS in 'aGvHD with CD4+ IR' was associated with lower 5-yrs NRM in both cohorts; 11% vs 34% (p=0.006) at UMC/PMC and 5% vs 54% (p<0.0001) at MSK. For grade 3-4 aGvHD the differences were more striking; 5-year-OS 78% vs. 24% (p<0.001), and a 5-yrs NRM of 12% vs 78% (p<0.001) at UMC/PMC and a 5-yrs OS 75% vs 33% (p=0.066) and a 5-yrs NRM of 5% vs 67% (p<0.001) at MSK. In 2 separate cohorts adequate 'CD4+ IR prior to onset aGvHD' was found to be a simple and robust predictor for survival and NRM. These findings provide insight in the importance of adequate IR for HCT patients to survive moderate to severe aGvHD. Finding strategies to better predict CD4+ T cell recovery after HCT may influence survival chances. [ABSTRACT FROM AUTHOR]

Gastro-intestinal (GI) symptoms in pediatric allogeneic hematopoietic stem cell transplantation (HSCT) patients are common and may be caused by viruses, Graft-Versus-Host-Disease (GVHD) or both. Currently, little is known about the GI viriome in SCT patients, hindering the study of the pathogenicity and role in GVHD. We used ViroCap, a viral target enrichment method, in combination with Next Generation Sequencing (NGS) to detect viruses in stool samples of HSCT patients and compared to PCR results. Using ViroCap, nucleic acid of a broad range of RNA and DNA viruses infecting vertebrate hosts was enriched in banked stool samples of HSCT patients. Upon enrichment, samples were sequenced by NGS on an Illumina MiSeq system and detected viral sequences were analyzed using the automated Genome Detective pipeline (GD) and/or directly aligned (DA) to a reference sequence of a specific virus, using Genious software. Where possible, presence of newly detected viral pathogens was confirmed by virus-specific qPCR. Stool samples of 11 patients, age range 1-17, that had undergone HSCT for a variety of indications were selected for ViroCap analysis. All selected patients suffered from GI-symptoms. Ten were diagnosed with grade I-IV GI-GVHD according to consensus guidelines. In 6 samples adenovirus (ADV), norovirus or both had been previously detected by clinical diagnostic qPCRs (Diagnosis D), while in 5 samples no viral GI pathogens were found (No diagnosis ND). In all D samples the presence of the initially found virus could be confirmed upon ViroCap analysis. ADV was most commonly found and Ct values grossly correlated with the number of sequence specific reads upon ViroCap GD and DA analysis. In addition, ViroCap led to the detection of multiple, possibly clinically relevant viral pathogens in D samples, including human herpesvirus-6B (HHV6B) (n=1), BK polyomavirus (BKV) (n=2), ADV (n=1) and human rhinovirus (HRV) (n=1). Presence of BKV and HRV was confirmed by qPCR. Furthermore, in 4/5 ND samples viral pathogens were detected. These included human herpes Virus 7 (HHV7) (n=1), BKV (n=1), HRV (n=2) and Astrovirus (AV) (n=1). HRV presence was confirmed by qPCR, HHV7 was not tested. BKV could not be confirmed, but other clinical samples from the same patient taken closely before/after the specific stool sample were BKV positive. Further analysis of the AV sequences revealed that the routine PCR failed to detect this virus due to extensive sequence mismatches in the primer/probe binding regions. This AV clade was classified as an AV VA3 (JX857868.1). In summary, application of viral target enrichment strategies with limited pathogen detection bias, such as ViroCap, increase the sensitivity of NGS for virus detection and can lead to discovery of novel variants. As such, it might be a useful screening tool to study associations of viral pathogens with GI-symptoms and GVHD. [ABSTRACT FROM AUTHOR]

Rabbit anti-thymocyte globulin (ATG) is used in allogeneic hematopoietic cell transplantation (HCT) to prevent graft-versus-host-disease (GvHD) and graft failure (GF). Its main and unpredictable toxicity includes poor immune reconstitution associated with increased relapse, viral reactivations and subsequently higher mortality. Early (<100 days) CD4+ T-cell reconstitution has been associated with improved overall survival1. Based on pharmacokinetic and pharmacodynamic (PKPD) studies we developed an individualized ATG dosing regimen aiming for optimal CD4+ T-cell recovery while maintaining a strong anti-GvHD effect. We performed an open label, phase II historically controlled non-randomized prospective clinical trial investigating individualized versus fixed dosing of ATG (Thymoglobulin). Individualized dosing was based on the results from a previous validated population PKPD model1,2 with cumulative doses varying between 2 to 10 mg/kg starting based on weight, recipient lymphocyte counts before first dose of ATG and stem cell source, starting day -9. Primary endpoint was successful CD4+ T-cell reconstitution (CD4+ >50/mm3 at 2 consecutive timepoints within 100 days after HCT1) in patients alive without relapse or graft failure <100 days. Secondary endpoints were overall survival, event free survival, GvHD and graft failure. Results were compared to a previously described historical cohort receiving a fixed cumulative ATG dose of 10mg/kg starting day -51. Minimal follow-up was 1 year. Power was based on 20% effect size with α=0.05 and β=0.10. Multivariate Cox proportional hazard and Fine-Gray competing risks were used. The study was registered in the Dutch Trial Registry (NTR4960). We included 58 patients between 2015-2018, and compared to 112 historical controls (table 1). CD4+ reconstitution was significantly better in the individualized dosing group: 83% versus 54% in the fixed dosing group; HR 2.4 (95% CI 1.6-3.6), p<0.0001 (Figure 1a). The individualized group showed a trend towards improved survival (81% vs. 66%; HR 0.54 [95% CI 0.27-1.07], p = 0.078; Figure 1b) and event free survival (79% vs 61%: HR 0.52 [95% CI 0.27-1.01], p = 0.052). No differences were seen in grade 3-4 acute GvHD (7% vs.6%; HR 1.44 [95% CI 0.47-4.44], p = 0.53), and graft failure (5% vs 5%; HR 1.61 [95% CI 0.38-6.83], p = 0.52). Individualized ATG dosing significantly increases the chance of rapid immune reconstitution without affecting the incidence of aGvHD and graft failure. Together, this is an important step towards predictable CD4+ T cell reconstitution and improved survival changes. [ABSTRACT FROM AUTHOR]

Gastro-intestinal (GI) Graft-versus-Host Disease (GvHD) involves a complex interplay of conditioning-related injury and the pathologic immune response at the epithelial level. Enhanced tissue damage is a risk factor for GVHD. We demonstrated that donor T cell-derived IFNγ causes intestinal stem cell damage in GvHD. Recent studies have proposed that donor T cells directly interact with GI epithelium in GvHD in vivo , but the precise mechanisms remain incompletely understood. Here, we evaluate the influence of both cytokine- and chemotherapy-induced intestinal epithelial damage on T cell migration and direct epithelial cell-T cell interactions using GI organoids as a model system. We developed a human intestinal organoid-T cell interaction co-culture system and studied the role of T cells in intestinal damage during GVHD. We observed that polyclonally activated T cells accumulate in close proximity to intestinal organoids (Fig. 1). In this co-culture system T cell-derived IFNg- plays a key role in tissue damage. RNA-seq analyses of IFNg-treated intestinal organoids indicated significant upregulation of T cell chemotaxis associated genes including CXCL9, 10 and 11. Luminex analysis of conditioned media confirmed release of these chemokines in co-culture versus controls. In addition, stimulation of organoids with IFNγ led to upregulation of HLA-I and HLA-II at both the RNA and membrane protein expression level (Fig. 2A). These experiments place activated T cells at the cusp of intestinal damage during GVHD through the production of cytokines, the induction of T cell recruiting chemokines and mechanisms of antigen driven cytotoxicity. Next, we set out to characterize the effect of chemotherapy on epithelium by treating organoids with Busulfan (Bu), Fludarabine (Flu) or Clofarabine (Clo). Epithelial damage was confirmed by the presence of increasing levels of caspase activation (Fig. 3A), a decrease in the number of surviving organoids, a smaller organoid size and decreased proliferation. Bu, Flu, Clo treatment did not affect HLA-I-II mRNA expression, but a variable, organoid donor-dependent upregulation of membrane HLA-I upon exposure to Bu was observed (Fig. 2B). By using a migration assay, we established that medium that we obtained from chemotherapy-treated organoids exerts increased T cell chemotaxis compared to control medium (Fig. 3B). In conclusion, both (T cell derived) IFNγ- and chemotherapy induced intestinal epithelial damage can lead to increased T cell migration ex vivo , resulting in direct contact between allogeneic T cells and co-cultured human intestinal epithelium. Cytokine signaling and conditioning-related injury may contribute to GVHD pathogenesis in vivo by promoting recruitment to intestinal crypt epithelium. [ABSTRACT FROM AUTHOR]

Acute graft-versus-host disease (aGVHD) can be triggered by inflammatory conditions, including infections and mucositis. We investigated the association between PCR positivity for gastrointestinal (GI) viruses in stool before hematopoietic cell transplantation (HCT) and intestinal aGVHD using Cox proportional hazard models. We included 48 consecutive HCT patients (28 with malignancies and 20 with nonmalignancies) without GI symptoms before HCT. Fifteen patients were GI virus positive: 9 adenovirus, 3 norovirus, 2 parechovirus, and 1 astrovirus. Overall survival was 58% ± 8%. The cumulative incidence of aGVHD grade 2 to 4 was 43% ± 8% (n = 18) after a median of 47 days (range, 14 to 140). In univariate analysis, GI virus PCR positivity was the only predictor for aGVHD (P = .008): within the group of GI virus PCR-positive patients, the cumulative incidence of aGVHD 2 to 4 was 70% ± 12% versus 29 ± 8% in the PCR-negative group (P = .004). In conclusion, GI virus PCR positivity before HCT predicted development of intestinal aGVHD. These results may ultimately affect monitoring, aGVHD prophylaxis, and treatment, as well as rescheduling of elective HCTs. [ABSTRACT FROM AUTHOR]

Background Graft-versus-Host-Disease (GvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic (stem) cell transplantation (HCT). Studies evaluating the relation between immune reconstitution (IR) and GvHD are largely lacking. We previously showed that successful CD4+ IR strongly predicts event-free survival chances (JACI 2018, Blood 2018). Therefore, we were interested in relating IR to acute and chronic GvHD after HCT. Methods Pediatric patients receiving their first allogeneic HCT between 2004 and 2018 were included. Blood samples and outcome data were collected and registered prospectively. Our main outcomes of interest were 5-year overall survival (OS) and non-relapse mortality (NRM) probabilities, stratified for aGvHD (grade II-IV) and for the presence or absence of adequate CD4+ IR (twice >50*106 CD4+CD3+ cells/L) prior to aGvHD onset. Other outcomes of interest were IR of T-cells, B-cells, NK-cells, monocytes, neutrophils, reticulocytes, and basophils, in patients with or without grade II-IV aGvHD or cGvHD. We applied a time-to-event multivariate Log-rank test to evaluate aGvHD-related survival. IR data were analyzed as continuous variables using multivariate linear-mixed-effects-regression modelling. HCT-source; cord blood transplantation, and bone marrow or peripheral blood transplantation, was analyzed as covariate. Results 391 pediatric patients were included; 97 (24.8%) had moderate-severe aGvHD (grade II-IV) and 42 (10.7%) had cGvHD (limited-extensive). Adequate early CD4+ IR before diagnosis of aGvHD grade II-IV was associated with reduced mortality compared to no CD4 +IR; OS was 77% versus 48% (p<0.001; Figure 1A) and NRM 17% versus 35% (p=0.005; Figure 2A), respectively. For grade III-IV differences were even more striking; OS was 78% in patients with CD4+ IR prior to grade III-IV aGvHD onset and only 24% in patients without CD4+ IR (p<0.001, Figure 1B), and NRM was 22% versus 74% (p<0.001; Figure 2B), respectively. Innate immune cell reconstitution was similar in patients with or without acute or chronic GvHD. At diagnosis, patients with grade II-IV aGvHD had drastically lower B-cell counts compared to control patients (p<0.001). We also observed delayed recovery of B-cell subsets prior to cGvHD, both with and without (both p=0.001) prior steroid-treated aGvHD, compared to controls (Figure 2). Interpretation Adequate CD4+ IR prior to aGvHD onset protects against aGvHD-related mortality after HCT. Either regulatory T-cell development or a lower risk of infection may be possible explanations. A specific delay in B-cell recovery after HCT provides an interesting clinical indicator for cGvHD, which demands further research. Our findings provide insight in the importance of monitoring the immune system after HCT to better understand the biology and determine strategies to prevent and treat GvHD. [ABSTRACT FROM AUTHOR]

The combination of Clofarabine + Fludarabine + Busulfan (CloFluBu) was found to have synergistic anti-leukemic activity against ALL and AML blasts in vitro (Andersson et al: BBMT 2011). As TBI induces significant late effects in childhood ALL, and AML patients have high relapse rates, we hypothesized that CloFluBu may be a potential alternative to TBI in ALL, and could add anti-leukemic activity in AML. Within the "Dutch COG HCT Working Group" we prospectively studied the outcomes of a CloFluBu-conditioning regimen for lymphoblastic and myeloid malignancies. Patients from the 2 pediatric HCT programs (LUMC and UMC Utrecht/Princess Máxima Center for Pediatric Oncology) in the Netherlands with a lymphoblastic or myeloid malignancy receiving their first HCT, between August 2011 and April 2019, were included. Over 4 days, Clofarabine 30mg/m2 was given in 1 hour, followed by Fludarabine 10mg/m2 in 1 hour, followed by a 3-hour infusion of once-daily targeted Busulfan (weight-based dosing + therapeutic drug monitoring to a total Bu-exposure of 90mg*h/L). Thymoglobulin was added in unrelated donors (except in AML receiving cord blood). GvHD-prophylaxis was according to standard protocols. Minimal Residual Disease (MRD) negative was defined as < 10e-4. Primary endpoints were Overall Survival (OS) and Leukemia Free Survival (LFS). Secondary endpoints were Non Relapse Mortality (NRM), Relapse, acute and chronic Graft-versus-Host Disease (GvHD), and VOD/SOS. Cox Proportional Hazard and fine and gray competing risk models were used for data analysis. 155 children were included; 66 ALL (38 in CR1, 28 ³ CR2), 69 AML (28 in CR1, 40 in CR2, 1 in active disease) and 20 other malignancies (mostly MDS-EB). Median age was 9.7 (0.5-18.6) years. Most donors were unrelated (119 vs 36 related); 79 Bone Marrow (BM), 66 Cord Blood (CB) and 10 Peripheral Blood Stem Cells. Median follow up was 964 (19-2994) days. Overall the 3-yr estimated OS and LFS was 72 ± 4.5% and 65 ± 5% respectively. Estimated 3-yr LFS for MRD-neg ALL, MRD-pos ALL, AML CR1 and AML CR2 was 74 ± 7%, 40 ± 12%, 64 ± 10%, 65 ± 9% respectively; Fig 1). NRM in whole cohort was 10.3 ± 3.0% (with 25 ± 4.3% for AML CR1 (n=28), and 5.8 ± 2.3% in the rest; Fig 2). Other endpoints: only 2 graft-failures were noted, incidence of aGvHD III-IV at 6 months was 11 ± 3%, extensive chronic GvHD at 3-yr was 5.2 ± 2.2%. Relapse at 3-yr was 25 ± 4.3% (MRD-neg ALL 16.2 ± 3.7%, versus 60 ± 4.9% in MRD-pos ALL, in AML 22.5 ± 4.2%) and no VOD/SOS was noted. CloFluBu in myeloid- and lymphoblastic malignancies, showed very limited toxicity and encouraging LFS in all groups, in particular for MRD negative ALL. More studies, preferably in randomized controlled clinical trials, are needed to draw firm conclusion with regards to the anti-leukemic effect and late effects. [ABSTRACT FROM AUTHOR]

Early treatment with allogeneic hematopoietic stem cell transplantation (allo-HSCT) for cerebral adrenoleukodystrophy (CALD) has a beneficial effect on clinical indices of disease and long-term survival but is associated with immunologic risks. We aim to understand how outcomes of allo-HSCT differ by disease stage, donor cell source, and conditioning regimen. As of February 2019, 47 boys were enrolled in an ongoing observational study of allo-HSCT in CALD. Efficacy was analyzed for advanced (Loes >9 or Neurologic Function Score [NFS] >1; n=10) and two early disease cohorts (ED1, Loes ≤4 and NFS ≤1 [n=21]; ED2, Loes >4-9, and NFS ≤1 [n=9]). Safety was analyzed by donor source and conditioning regimen. There was no statistical difference in survival and neurologic findings between the ED cohorts. Patients with advanced CALD had the worst outcomes. At 24 months, overall survival and survival free of major functional disabilities were 90.5% (95% CI 67.0, 97.5) and 78.9% (53.2, 91.5) for ED1, 85.7% (33.4, 97.9) and 70.0% (22.5, 91.8) for ED2, and 52.5% (16.8, 79.3) and 35.0% (8.5, 64.0) for advanced CALD, respectively. Most patients in both ED cohorts, and fewer of those with advanced CALD, had stable Loes scores and NFS through last follow up. Of patients with baseline gadolinium enhancement (GdE+) and follow-up data, 9/9 in ED1, 4/5 in ED2, and 2/4 in the advanced disease cohort had resolution of GdE+ throughout follow-up. Acute (Grade ≥2) and chronic graft-versus-host disease (GVHD) occurred in 23.5% (8/34) and 27.6% (8/29) of patients, respectively. One-year transplant-related mortality was 12.1% (4/33), and 21.6% (8/37) of patients had engraftment failure. There were no substantial observed differences in transplant-related outcomes by donor cell source, but more patients who received umbilical cord (UC) cells from an unrelated donor (38.9%, 7/18) had engraftment failure compared to patients who received bone marrow (BM) or UC cells from a matched sibling donor (MSD), or BM cells from an unrelated donor (0% for both [0/8]; p=0.0622). Importantly, while transplants performed using cells from a MSD are considered safe, they resulted in chronic GVHD in 28.6% (2/7) of patients. Trade-offs in transplant-related risks with myeloablative conditioning were noted. Compared to busulfan/ fludarabine (Bu/Flu), the use of busulfan/cyclophosphamide increased the risk of acute and chronic GVHD (6.3% [1/16] vs. 42.9% [6/14] [p=0.0309] and 13.3% [p=2/15] vs. 54.5% [6/11] [p=0.0384], respectively), while myeloablative conditioning with Bu/Flu resulted in higher rates of engraftment failure (28.6% [6/21] vs. 0% [0/11], p=0.0711). These data suggest that treatment in early CALD provides better efficacy outcomes irrespective of the stage of early disease, although neuropsychological outcome data are not yet available. Transplant-related risks highlight an unmet need for improved safety outcomes. [ABSTRACT FROM AUTHOR]