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Hematopoietic stem cell transplantation (HSCT) is often the only curative treatment option for patients suffering from various types of malignant diseases and some non-cancerous conditions. Nevertheless, it is associated with a high risk of complications leading to transplant-related mortality and long-term morbidity. An increasing number of therapeutic and prevention strategies have been developed over the last few years to tackle the complications arising in patients receiving an HSCT. These strategies have been mainly carried out in adults and some are now being translated into children. In this manuscript, we review the recent advancements in the development and implementation of treatment options for post-HSCT non-infectious complications in pediatric patients with leukemia and other non-malignant conditions, with a special attention on the new agents available within clinical trials. We focused on the following conditions: graft failure, prevention of relapse and early interventions after detection of minimal residual disease positivity following HSCT in acute lymphoblastic and myeloid leukemia, chronic graft versus host disease, non-infectious pulmonary complications, and complications of endothelial origin. [ABSTRACT FROM AUTHOR]

The outcome of allogeneic hematopoietic cell transplantation (allo-HCT) largely depends on the development and management of graft-versus-host disease (GvHD), infections, and the occurrence of relapse of malignancies. Recent studies showed a lower incidence of chronic GvHD and severe acute GvHD in patients receiving naive T cell depleted grafts compared to patients receiving complete T cell depleted grafts. On the other hand, the incidence of acute GvHD in patients receiving cord blood grafts containing only naive T cells is rather low, while potent graft-versus-leukemia (GvL) responses have been observed. These data suggest the significance of naive T cells as both drivers and regulators of allogeneic reactions. The naive T cell pool was previously thought to be a quiescent, homogenous pool of antigen-inexperienced cells. However, recent studies showed important differences in phenotype, differentiation status, location, and function within the naive T cell population. Therefore, the adequate recovery of these seemingly innocent T cells might be relevant in the imminent allogeneic reactions after allo-HCT. Here, an extensive review on naive T cells and their contribution to the development of GvHD and GvL responses after allo-HCT is provided. In addition, strategies specifically directed to stimulate adequate reconstitution of naive T cells while reducing the risk of GvHD are discussed. A better understanding of the relation between naive T cells and alloreactivity after allo-HCT could provide opportunities to improve GvHD prevention, while maintaining GvL effects to lower relapse risk. [ABSTRACT FROM AUTHOR]

Pediatric allogeneic hematopoietic stem cell transplantation (HSCT) patients often suffer from gastro-intestinal (GI) disease caused by viruses, Graft-versus-Host Disease (GVHD) or a combination of the two. Currently, the GI eukaryotic virome of HSCT recipients remains relatively understudied, which complicates the understanding of its role in GVHD pathogenicity. As decisions regarding immunosuppressive therapy in the treatment of virus infection or GVHD, respectively, can be completely contradicting, it is crucial to better understand the prevalence and relevance of viruses in the GI tract in the HSCT setting. A real time PCR panel for a set of specific viruses widely used to diagnose the most common causes of GI viral gastroenteritis is possibly insufficient to grasp the full extent of viruses present. Therefore, we applied the targeted sequence capture method ViroCap to residual fecal samples of 11 pediatric allogeneic HSCT recipients with GI symptoms and a suspicion of GVHD, to enrich for nucleic acids of viruses that are known to infect vertebrate hosts. After enrichment, NGS was applied to broadly detect viral sequences. Using ViroCap, we were able to detect viruses such as norovirus and adenovirus (ADV), that had been previously detected using clinical diagnostic PCR on the same sample. In addition, multiple, some of which clinically relevant viruses were detected, including ADV, human rhinovirus (HRV) and BK polyomavirus (BKV). Interestingly, in samples in which specific PCR testing for regular viral GI pathogens did not result in a diagnosis, the ViroCap pipeline led to the detection of viral sequences of human herpesvirus (HHV)-7, BKV, HRV, KI polyomavirus and astrovirus. The latter was an only recently described variant and showed extensive sequence mismatches with the applied real time PCR primers and would therefore not have been detected if tested. Our results indicate that target enrichment of viral nucleic acids through ViroCap leads to sensitive and broad possibly clinically relevant virus detection, including the detection of newer variants in clinical HSCT recipient samples. As such, ViroCap could be a useful detection tool clinically, but also in studying the associations between viral presence and GVHD. [ABSTRACT FROM AUTHOR]

The article discusses the use of RIG-I/MAVS and STING signaling, citing the molecular pathways that regulate the tissue repair function of type I interferon (IFN-I) in tissue injury. It cites on the development graft-versus-host disease (GVHD) in mice used in laboratory research of allogeneic hematopoietic stem cell transplantation. The article also suggests that activation of either RIG-I/MAVS or STING pathways during the acute intestinal tissue injury.

Graft-versus-host disease (GvHD) is a major complication of allogeneic hematopoietic (stem) cell transplantation (HCT). Clinically, GvHD is associated with severe and long-lasting hematopoietic dysfunction, which may contribute to the high mortality of GvHD after HCT. During GvHD, excessive immune activation damages both hematopoietic stem and progenitor cells and their surrounding bone marrow niche, leading to a reduction in cell number and functionality of both compartments. Hematopoietic dysfunction can be further aggravated by the occurrence—and treatment—of HCT-associated complications. These include immune suppressive therapy, coinciding infections and their treatment, and changes in the microbiome. In this review, we provide a structured overview of GvHD-mediated hematopoietic dysfunction, including the targets in the bone marrow, the mechanisms of action and the effect of GvHD-related complications and their treatment. This information may aid in the identification of treatment options to improve hematopoietic function in patients, during and after GvHD. [ABSTRACT FROM AUTHOR]

• 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]

The article presents the author's views on an article on graft-versus-host disease (GVHD) by researchers A. Mensen and colleagues published within the issue. The researchers provided an evaluation of B-cell development in adult patients who had allogeneic hematopoietic stem cell transplantation. It is stated that the researchers made a significant step in understanding of B-cell development after transplantation as well as its impairment in GVHD.

• 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]

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]