Outcome of Hematopoietic Stem Cell Transplantation in 813 Pediatric Patients with Fanconi Anemia.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only established curative option for Fanconi anemia (FA) associated bone marrow failure (BMF)/aplastic anemia (AA) and hematological malignancy. We performed a retrospective multicenter study on 813 FA children undergoing first HSCT between 2010 and 2018. Median duration of follow-up was 3.7 years (interquartile range, 3.4-4.0). Median age at transplant was 8.8 years (6.5-18.1). Overall survival (OS), event-free survival (EFS) and GvHD-free, relapse-free survival (GRFS) at 5 years were 83% (80-86%), 78% (75-81%) and 70% (67-74%) respectively. OS was comparable between matched family donor (MFD, n=441, 88%) and matched unrelated donor (MUD, n=162, 86%) and was superior to that of mismatched family or unrelated donor (MMFD/MMUD, n=144, 72%) and haploidentical donor (HID) (n=66, 70%, p<0.001). In multivariable analysis, a transplant indication of acute myeloid leukaemia/myelodysplastic syndrome compared to AA/BMF, use of MMFD/MMUD and HID compared to MFD, Fludarabine-Cyclophosphamide (FluCy) + other conditioning compared to FluCy independently predicted inferior OS, while alemtuzumab compared to ATG was associated with better OS. Age  10 years was associated with worse EFS and GRFS. Cumulative incidences (CIN) of primary and secondary graft failure were 2% (1-3%) and 3% (2-4%) respectively. CIN of grade II-IV acute GvHD, grade III-IV acute GvHD and chronic GvHD were 23% (20-26%), 12% (10-15%) and 8% (6-10%) respectively. The 5-year CIN of secondary malignancy was 2% (1-3%). These data suggest that HSCT should be offered to Fanconi Anemia patients with AA/BMF at a younger age in the presence of a well-matched donor.

High-dose individualized antithymocyte globulin with therapeutic drug monitoring in high-risk cord blood transplant.

BACKGROUND: 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. OBJECTIVE: 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. STUDY DESIGN: 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. RESULTS: 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%). CONCLUSION: 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.

Ribosomal protein gene RPL9 variants can differentially impair ribosome function and cellular metabolism.

Variants in ribosomal protein (RP) genes drive Diamond-Blackfan anemia (DBA), a bone marrow failure syndrome that can also predispose individuals to cancer. Inherited and sporadic RP gene variants are also linked to a variety of phenotypes, including malignancy, in individuals with no anemia. Here we report an individual diagnosed with DBA carrying a variant in the 5'UTR of RPL9 (uL6). Additionally, we report two individuals from a family with multiple cancer incidences carrying a RPL9 missense variant. Analysis of cells from these individuals reveals that despite the variants both driving pre-rRNA processing defects and 80S monosome reduction, the downstream effects are remarkably different. Cells carrying the 5'UTR variant stabilize TP53 and impair the growth and differentiation of erythroid cells. In contrast, ribosomes incorporating the missense variant erroneously read through UAG and UGA stop codons of mRNAs. Metabolic profiles of cells carrying the 5'UTR variant reveal an increased metabolism of amino acids and a switch from glycolysis to gluconeogenesis while those of cells carrying the missense variant reveal a depletion of nucleotide pools. These findings indicate that variants in the same RP gene can drive similar ribosome biogenesis defects yet still have markedly different downstream consequences and clinical impacts.

Evaluation of the pharmacokinetics of prednisolone in paediatric patients with acute lymphoblastic leukaemia treated according to Dutch Childhood Oncology Group protocols and its relation to treatment response.

Glucocorticoids form the backbone of paediatric acute lymphoblastic leukaemia (ALL) treatment. Many studies have been performed on steroid resistance; however, few studies have addressed the relationship between dose, concentration and clinical response. The aim of the present study was to evaluate the pharmacokinetics of prednisolone in the treatment of paediatric ALL and the correlation with clinical parameters. A total of 1028 bound and unbound prednisolone plasma concentrations were available from 124 children (aged 0-18 years) with newly diagnosed ALL enrolled in the Dutch Childhood Oncology Group studies. A population pharmacokinetic model was developed and post hoc area under the curve (AUC) was tested against treatment outcome parameters. The pharmacokinetics of unbound prednisolone in plasma was best described with allometric scaling and saturable binding to proteins. Plasma protein binding decreased with age. The AUC of unbound prednisolone was not associated with any of the disease parameters or treatment outcomes. Unbound prednisolone plasma concentrations correlated with age. No effect of exposure on clinical treatment outcome parameters was observed and does not substantiate individualised dosing. Poor responders, high-risk and relapsed patients showed a trend towards lower exposure compared to good responders. However, the group of poor responders was small and requires further research.

Parental experiences in end-of-life decision-making in allogeneic pediatric stem cell transplantation: "Have I been a good parent?"

BACKGROUND: In pediatric hematopoietic stem cell transplantation (HSCT), the end-of-life (EOL) phase and the loss of the child is often characterized by a sudden deterioration of the child following a period of intensive curative treatment. This demands a fast transition for parents. Therefore, an understanding of the parents' perspective on decision-making in such a complex situation is needed. This study aims to gain insight in parental experiences in EOL decision-making in allogeneic pediatric HSCT. METHODS: A qualitative descriptive study was performed among parents of eight families. Data were thematically analyzed. RESULTS: All parents were aware of their child's deterioration. Six families were confronted with a rapid deterioration, while two families experienced a gradual realization that their child would not survive. Parental EOL decision-making in pediatric HSCT shows a reflective perspective on the meaning of parenthood in EOL decision-making. Two central themes were identified: "survival-oriented decision-making" and "struggling with doubts in hindsight." Six subthemes within the first theme described the parents' goal of doing everything to achieve survival. DISCUSSION: Parents experienced EOL decision-making mainly as a process guided by health care professionals (HCPs) based on the child's condition and treatment possibilities. The decision-making is characterized by following opportunities and focusing on hope for cure. In hindsight parents experienced doubts about treatment steps and their child's suffering. HCPs can strengthen the parental role by an early integration of palliative care, providing timely support to parents in the process of imminent loss. Advance care planning can be used to support communication processes, defining preferences for future care.

Autophagy inhibition as a potential future targeted therapy for ETV6-RUNX1-driven B-cell precursor acute lymphoblastic leukemia.

Translocation t(12;21), resulting in the ETV6-RUNX1 (or TEL-AML1) fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia and is considered a first hit in leukemogenesis. A targeted therapy approach is not available for children with this subtype of leukemia. To identify the molecular mechanisms underlying ETV6-RUNX1-driven leukemia, we performed gene expression profiling of healthy hematopoietic progenitors in which we ectopically expressed ETV6-RUNX1. We reveal an ETV6-RUNX1-driven transcriptional network that induces proliferation, survival and cellular homeostasis. In addition, Vps34, an important regulator of autophagy, was found to be induced by ETV6-RUNX1 and up-regulated in ETV6-RUNX1-positive leukemic patient cells. We show that induction of Vps34 was transcriptionally regulated by ETV6-RUNX1 and correlated with high levels of autophagy. Knockdown of Vps34 in ETV6-RUNX1-positive cell lines severely reduced proliferation and survival. Inhibition of autophagy by hydroxychloroquine, a well-tolerated autophagy inhibitor, reduced cell viability in both ETV6-RUNX1-positive cell lines and primary acute lymphoblastic leukemia samples, and selectively sensitized primary ETV6-RUNX1-positive leukemia samples to L asparaginase. These findings reveal a causal relationship between ETV6-RUNX1 and autophagy, and provide pre-clinical evidence for the efficacy of autophagy inhibitors in ETV6-RUNX1-driven leukemia.

Excellent T-cell reconstitution and survival depend on low ATG exposure after pediatric cord blood transplantation.

Successful immune reconstitution (IR) is associated with improved outcomes following pediatric cord blood transplantation (CBT). Usage and timing of anti-thymocyte globulin (ATG), introduced to the conditioning to prevent graft-versus-host disease and graft failure, negatively influences T-cell IR. We studied the relationships among ATG exposure, IR, and clinical outcomes. All pediatric patients receiving a first CBT between 2004 and 2015 at the University Medical Center Utrecht were included. ATG-exposure measures were determined with a validated pharmacokinetics model. Main outcome of interest was early CD4+ IR, defined as CD4+ T-cell counts >50 × 106/L twice within 100 days after CBT. Other outcomes of interest included event-free survival (EFS). Cox proportional-hazard and Fine-Gray competing-risk models were used. A total of 137 patients, with a median age of 7.4 years (range, 0.2-22.7), were included, of whom 82% received ATG. Area under the curve (AUC) of ATG after infusion of the cord blood transplant predicted successful CD4+ IR. Adjusted probability on CD4+ IR was reduced by 26% for every 10-point increase in AUC after CBT (hazard ratio [HR], 0.974; P < .0001). The chance of EFS was higher in patients with successful CD4+ IR (HR, 0.26; P < .0001) and lower ATG exposure after CBT (HR, 1.005; P = .0071). This study stresses the importance of early CD4+ IR after CBT, which can be achieved by reducing the exposure to ATG after CBT. Individualized dosing of ATG to reach optimal exposure or, in selected patients, omission of ATG may contribute to improved outcomes in pediatric CBT.

Effects of a combined physical and psychosocial training for children with cancer: a randomized controlled trial.

BACKGROUND: Physical fitness and psychosocial function is often reduced in children during or shortly after cancer treatment. This study evaluates the effect of a combined physical exercise and psychosocial intervention on cardiorespiratory fitness, muscle strength, body composition, psychosocial function and health-related quality of life (HrQoL). In addition, intervention mediators, applicability and adherence were examined. METHODS: This multicenter randomized controlled trial included 68 children with cancer [mean age 13.2 (SD: 3.1) years; 54% male] during treatment or within 12-months post-treatment. The 12-week intervention consisted of 24 individual physical exercise sessions supervised by a physiotherapist, and 6 psychosocial training sessions for children and 2 for parents. Physical fitness and psychosocial function were assessed at baseline, directly post-intervention and at 12 months' post-baseline. Generalized estimating equations were used to simultaneously assess intervention effects at short and long-term. Additionally, we evaluated within-group differences over time. Potential physical and psychosocial mediators in the intervention effect on HrQoL were examined using the product-of-coefficient test. Applicability and adherence were assessed by trainer-report. RESULTS: This study was able to compare 26 children who received the study intervention, with 33 children who received usual care. No significant differences in the effects of the intervention were found on physical fitness and psychosocial function at short-term. At 12-months follow-up, significantly larger improvements in lower body muscle strength (ß = 56.5 Newton; 95% CI: 8.5; 104.5) were found in the intervention group when compared to the control group. Within-group changes showed significant improvements over time in HrQoL and bone density in both groups. Intervention effects on HrQoL were not significantly mediated by physical fitness and psychological function. Intervention applicability was satisfactory with an average session attendance of 67% and 22% dropout (mainly due to disease recurrence). CONCLUSIONS: This 12-week physical exercise and psychosocial training intervention for children with cancer was applicable and showed satisfactory adherence. We found no significant between-group differences in effect, except for a significant improvement in lower body muscle strength at long-term in the intervention group compared to the control group. Yet, both the intervention and the control group showed improvements in bone mineral density and HrQoL over time. TRIAL REGISTRATION: The trial was registered at the Dutch Trial Registry ( NTR1531 ). Registered 12 November 2008.

Pediatric Diamond-Blackfan anemia in the Netherlands: An overview of clinical characteristics and underlying molecular defects.

INTRODUCTION: Diamond-Blackfan anemia (DBA) is characterized by hypoplastic anemia, congenital anomalies, and a predisposition for malignancies. Most of our understanding of this disorder stems from molecular studies combined with extensive data input from international patient registries. OBJECTIVES: To create an overview of the pediatric DBA population in the Netherlands. METHODS: Forty-three patients diagnosed with DBA from all Dutch university pediatric hospitals were included in this study, and their clinical and genetic characteristics were collected from patient records. RESULTS: Congenital malformations were present in 24 of 43 patients (55.8%). An underlying genetic defect was identified in 26 of 43 patients (60.5%), the majority of which were found in the RPS19 gene (12 of 43, 27.9%) with 1 patient carrying a mutation in a novel DBA candidate gene, RPL9. In 31 of 35 (88.6%) patients, an initial response to glucocorticoid treatment was observed. Six patients (14.0%) underwent hematopoietic stem cell transplantation, and eleven patients (11 of 43, 25.6%) became treatment-independent spontaneously. CONCLUSION: In agreement with previous reports, the Dutch pediatric DBA population is both clinically and genetically heterogeneous. National and international registries, together with more extensive genetic testing, are crucial to increase our understanding of genotype and phenotype correlations of this intriguing disorder.

Viral reactivations and associated outcomes in the context of immune reconstitution after pediatric hematopoietic cell transplantation.

BACKGROUND: Viral reactivations (VRs) after hematopoietic cell transplantation (HCT) contribute to significant morbidity and mortality. Timely immune reconstitution (IR) is suggested to prevent VR. OBJECTIVES: We studied the relation between IR (as a continuous predictor over time) and VR (as a time-varying predictor) and the relation between VR and other clinical outcomes. METHODS: In this retrospective analysis all patients receiving a first HCT between January 2004 and September 2014 were included. IR (CD3/CD4/CD8 T, natural killer, and B cells) was measured biweekly until 12 weeks and monthly thereafter. Main outcomes of interest were VR of adenovirus, EBV, human herpesvirus 6 (HHV6), cytomegalovirus (CMV), and BK virus screened weekly. Clinical outcomes included overall survival (OS), event-free-survival, nonrelapse mortality (NRM), and graft-versus-host disease. Cox proportional hazard and Fine and Gray competing risk models were used. RESULTS: Two hundred seventy-three patients (age, 0.1-22.7 years; median follow-up, 58 months) were included. Delayed CD4 reconstitution predicted reactivation of adenovirus (hazard ratio [HR], 0.995; P = .022), EBV (HR, 0.994; P = .029), and HHV6 (HR, 0.991; P = .012) but not CMV (P = .31) and BK virus (P = .27). Duration of adenovirus reactivation was shorter with timely CD4 reconstitution, which was defined as 50 × 106 cells/L or greater within 100 days. Adenovirus reactivation predicted lower OS (HR, 2.17; P = .0039) and higher NRM (HR, 2.96; P = .0008). Concomitant CD4 reconstitution abolished this negative effect of adenovirus reactivation (OS, P = .67; NRM, P = .64). EBV and HHV6 reactivations were predictors for the occurrence of graft-versus-host disease, whereas CMV and BK virus reactivation did not predict clinical outcomes. CONCLUSION: These results stress the importance of timely CD4 reconstitution. Strategies to improve CD4 reconstitution can improve HCT outcomes, including survival, and reduce the need for toxic antiviral therapies.

Acetylation of C/EBPε is a prerequisite for terminal neutrophil differentiation.

C/EBPε, a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, is exclusively expressed in myeloid cells and regulates transition from the promyelocytic stage to the myelocytic stage of neutrophil development, being indispensable for secondary and tertiary granule formation. Knowledge concerning the functional role of C/EBPε posttranslational modifications is limited to studies concerning phosphorylation and sumoylation. In the current study, using ectopic expression and ex vivo differentiation of CD34(+) hematopoietic progenitor cells, we demonstrate that C/EBPε is acetylated, which was confirmed by mass spectrometry analysis, identifying 4 acetylated lysines in 3 distinct functional domains. Regulation of C/EBPε acetylation levels by the p300 acetyltransferase and the sirtuin 1 deacetylase controls transcriptional activity, which can at least in part be explained by modulation of DNA binding. During neutrophil development, acetylation of lysines 121 and 198 were found to be crucial for terminal neutrophil differentiation and the expression of neutrophil-specific granule proteins, including lactoferrin and collagenase. Taken together, our data illustrate a critical role for acetylation in the functional regulation of C/EBPε activity during terminal neutrophil development.

ICL-induced miR139-3p and miR199a-3p have opposite roles in hematopoietic cell expansion and leukemic transformation.

Interstrand crosslinks (ICLs) are toxic DNA lesions that cause severe genomic damage during replication, especially in Fanconi anemia pathway-deficient cells. This results in progressive bone marrow failure and predisposes to acute myeloid leukemia (AML). The molecular mechanisms responsible for these defects are largely unknown. Using Ercc1-deficient mice, we show that Trp53 is responsible for ICL-induced bone marrow failure and that loss of Trp53 is leukemogenic in this model. In addition, Ercc1-deficient myeloid progenitors gain elevated levels of miR-139-3p and miR-199a-3p with age. These microRNAs exert opposite effects on hematopoiesis. Ectopic expression of miR-139-3p strongly inhibited proliferation of myeloid progenitors, whereas inhibition of miR-139-3p activity restored defective proliferation of Ercc1-deficient progenitors. Conversely, the inhibition of miR-199a-3p functions aggravated the myeloid proliferation defect in the Ercc1-deficient model, whereas its enforced expression enhanced proliferation of progenitors. Importantly, miR-199a-3p caused AML in a pre-leukemic mouse model, supporting its role as an onco-microRNA. Target genes include HuR for miR-139-3p and Prdx6, Runx1, and Suz12 for miR-199a-3p. The latter genes have previously been implicated as tumor suppressors in de novo and secondary AML. These findings show that, in addition to TRP53-controlled mechanisms, miR-139-3p and miR-199a-3p are involved in the defective hematopoietic function of ICL-repair deficient myeloid progenitors.

Population pharmacokinetics of intravenous Erwinia asparaginase in pediatric acute lymphoblastic leukemia patients.

Erwinia asparaginase is an important component in the treatment of pediatric acute lymphoblastic leukemia. A large variability in serum concentrations has been observed after intravenous Erwinia asparaginase. Currently, Dutch Childhood Oncology Group protocols dose alterations are based on trough concentrations to ensure adequate asparaginase activity (≥100 IU/L). The aim of this study was to describe the population pharmacokinetics of intravenous Erwinia asparaginase to quantify and gather insight into inter-individual and inter-occasion variability. The starting dose was evaluated on the basis of the derived population pharmacokinetic parameters. In a multicenter prospective observational study, a total of 714 blood samples were collected from 51 children (age 1-17 years) with acute lymphoblastic leukemia. The starting dose was 20,000 IU/m2 three times a week and adjusted according to trough levels from week three onwards. A population pharmacokinetic model was developed using NONMEM® A 2-compartment linear model with allometric scaling best described the data. Inter-individual and inter-occasion variability of clearance were 33% and 13%, respectively. Clearance in the first month of treatment was 14% higher (P<0.01). Monte Carlo simulations with our pharmacokinetic model demonstrated that patients with a low weight might require higher doses to achieve similar concentrations compared to patients with high weight. The current starting dose of 20,000 IU/m2 might result in inadequate concentrations, especially for smaller, lower weight patients, hence dose adjustments based on individual clearance are recommended. The protocols were approved by the institutional review boards. (Registered at NTR 3379 Dutch Trial Register; www.trialregister.nl).

The increase of the global donor inventory is of limited benefit to patients of non-Northwestern European descent.

Between 2001 and 2012, the number of unrelated donors registered worldwide increased from 7 to 21 million, and the number of public cord blood units increased to over 500,000. We addressed the question of whether this expansion resulted in higher percentages of patients reaching transplantation. Unrelated donor searches were evaluated for 3,124 eligible patients in the Netherlands in two cohorts (2001-2006, n=995; 2007-2012, n=2129), comparing results for patients of Northwestern European and non-Northwestern European origin. Endpoints were 'donor found' and 'transplantation reached'. The substantial growth of the donor inventory over the period studied did not increase the median number of potential unrelated donors (n=7) for non-Northwestern European patients, but almost doubled the number for Northwestern European patients from 42 to 71. Before and after 2007, an unrelated donor or cord blood was identified for 91% and 95%, respectively, of Northwestern European patients and for 65% and 82% of non-Northwestern European patients (P<0.0001). Non-Northwestern European patients more often needed a cord blood transplant. The degree of HLA matching was significantly lower for non-Northwestern European patients (P<0.0006). The time needed to identify a donor decreased for both populations. The percentage of Northwestern European patients reaching transplantation increased from 77% to 83% and for non-Northwestern European patients from 57% to 72% (P=0.0003). The increase of the global inventory resulted in more transplants for patients lacking a family donor, although the quality and quantity of (potential) haematopoietic cell grafts for patients of a non-Northwestern European descent remained inferior, indicating the need for adaptation of recruitment.

Eating behavior during dexamethasone treatment in children with acute lymphoblastic leukemia.

BACKGROUND AND AIM: Large prospective studies on dexamethasone-induced changes in eating behavior, energy, and nutrient intake are lacking in pediatric acute lymphoblastic leukemia (ALL). We prospectively studied eating behavior, energy, nutrient intake, and the effect on leptin and adiponectin levels during dexamethasone administration in children with ALL. PATIENTS: Parents of patients with ALL (3-16 years) completed a dietary diary for their child during 4 days of dexamethasone (6 mg/m2 ) administration. Energy intake and nutrient intake (energy percentage = E%) were assessed and compared with the recommended intake. The Dutch Eating Behavior Questionnaire for Children was completed before start and after 4 days of dexamethasone administration by patients of 7-12 years of age. Fasting leptin and adiponectin levels were also measured before start and after 4 days of dexamethasone administration. RESULTS: Energy intake per day(kcal) (N = 44) increased significantly during dexamethasone (median day 1: 1,103 (717-1,572) versus day 4: 1,482 (1,176-1,822), P < 0.01), including an increase in total protein, fat, saturated fat, carbohydrate, and sodium intake. Intake of saturated fat (median day 4: 12 E%) and salt (median day 4: 1.9 g/day) exceeded the healthy range for age and gender. With respect to eating behavior, dexamethasone significantly decreased restrained eating (P = 0.04). Leptin levels as well as adiponectin levels increased significantly during the dexamethasone course. CONCLUSIONS: Four days of dexamethasone treatment significantly increased energy intake, including excessive saturated fat and salt intake, and changed eating behavior in children with ALL. Nutritional and behavioral interventions during dexamethasone treatment are recommended to stimulate a healthy lifestyle.

Ribosomal protein mutations induce autophagy through S6 kinase inhibition of the insulin pathway.

Mutations affecting the ribosome lead to several diseases known as ribosomopathies, with phenotypes that include growth defects, cytopenia, and bone marrow failure. Diamond-Blackfan anemia (DBA), for example, is a pure red cell aplasia linked to the mutation of ribosomal protein (RP) genes. Here we show the knock-down of the DBA-linked RPS19 gene induces the cellular self-digestion process of autophagy, a pathway critical for proper hematopoiesis. We also observe an increase of autophagy in cells derived from DBA patients, in CD34+ erythrocyte progenitor cells with RPS19 knock down, in the red blood cells of zebrafish embryos with RP-deficiency, and in cells from patients with Shwachman-Diamond syndrome (SDS). The loss of RPs in all these models results in a marked increase in S6 kinase phosphorylation that we find is triggered by an increase in reactive oxygen species (ROS). We show that this increase in S6 kinase phosphorylation inhibits the insulin pathway and AKT phosphorylation activity through a mechanism reminiscent of insulin resistance. While stimulating RP-deficient cells with insulin reduces autophagy, antioxidant treatment reduces S6 kinase phosphorylation, autophagy, and stabilization of the p53 tumor suppressor. Our data suggest that RP loss promotes the aberrant activation of both S6 kinase and p53 by increasing intracellular ROS levels. The deregulation of these signaling pathways is likely playing a major role in the pathophysiology of ribosomopathies.

Predicted Indirectly ReCognizable HLA Epitopes Class I Promote Antileukemia Responses after Cord Blood Transplantation: Indications for a Potential Novel Donor Selection Tool.

Unrelated cord blood transplantation (UCBT) provides a curative therapy for patients with hematological malignancies. The effect of HLA mismatches in UCBT is currently the subject of debate. HLA-mismatched UCBT may lead to improved leukemia control but also to graft-versus-host disease (GVHD), resulting in nonrelapse mortality (NRM). The aim of this study was to investigate whether indirect recognition of mismatched HLA provides an explanation for the graft-versus-tumor effect and risk of GVHD. The probability of indirect recognition was predicted by the Predicted Indirectly ReCognizable HLA Epitopes (PIRCHE) model. The effect of the numbers of PIRCHE presented on HLA class I and II (PIRCHE-I and -II) was studied in 134 pediatric patients. To study the effects of higher numbers of PIRCHE, patients were divided in 2 equally sized groups, using the median number of PIRCHE as cutoff values. Proportional hazard models and competing risk analyses were performed to study the effect of PIRCHE on the clinical outcomes relapse, acute and chronic GVHD, NRM, and disease-free and overall survival. Above median PIRCHE-I were associated with reduced relapse risk (HR, .26; 95% CI, .07 to .94; P = .04), evaluating the 50 patients transplanted for a malignancy. Both PIRCHE-I and -II were not associated with other clinical outcomes, including GVHD and NRM. These data suggest that high PIRCHE-I may lead to improved graft-versus-tumor effects after UCBT, without an accompanying GVHD risk. Inclusion of PIRCHE in UCB selection criteria may enhance UCBT outcome, which needs to be tested in prospective studies.

Four decades of stem cell transplantation for Fanconi anaemia in the Netherlands.

This article presents the haematopoietic stem cell transplantation (SCT) results of the complete Dutch Fanconi anaemia (FA) patient cohort. Sixty-eight Dutch FA patients have been transplanted since 1972. In total, 63 (93%) patients engrafted, 54 after first SCT and 9 after second SCT. Fludarabine (FLU)-based conditioning was associated with decreased graft failure (odds ratio 0·21, P = 0·01), decreased early mortality (hazard ratio 0·25, P = 0·01) and improved 5-year overall survival (FLU 87·8% [standard error (SE) 5·1%] versus non-FLU 59·3% [SE 9·5%], P = 0·01). Late mortality was mainly caused by squamous cell carcinoma. Twenty-two patients were treated with the current Dutch FA conditioning regimen (FLU 150 mg/m(2) and cyclophosphamide 30 mg/kg ± anti-thymocyte globulin - no irradiation). Stem cell donors were matched related (n = 8) or alternative donors (n = 14). Stable engraftment after first SCT was achieved in 19 (86%) patients. At a median follow-up of 3·9 years 20 (91%) patients are alive. Our study provides a unique overview of a nation-wide SCT cohort illustrating the major improvements in treatment regimen and patient outcome in recent years. It shows that a non-irradiation and busulfan-free conditioning regimen can be used successfully, also in alternative donor SCT. Furthermore, it underlines the importance of late cancer screening and comprehensive care for this complex disorder.

Phenotypic variability in patients with ADA2 deficiency due to identical homozygous R169Q mutations.

OBJECTIVE: To determine the genotype-phenotype association in patients with adenosine deaminase-2 (ADA2) deficiency due to identical homozygous R169Q mutations inCECR1 METHODS: We present a case series of nine ADA2-deficient patients with an identical homozygous R169Q mutation. Clinical and diagnostic data were collected and available MRI studies were reviewed. We performed genealogy and haplotype analyses and measured serum ADA2 activity. ADA2 activity values were correlated to clinical symptoms. RESULTS: Age of presentation differed widely between the nine presented patients (range: 0 months to 8 years). The main clinical manifestations were (hepato)splenomegaly (8/9), skin involvement (8/9) and neurological involvement (8/9, of whom 6 encountered stroke). Considerable variation was seen in type, frequency and intensity of other symptoms, which included aplastic anaemia, acute myeloid leukaemia and cutaneous ulcers. Common laboratory abnormalities included cytopenias and hypogammaglobulinaemia. ADA2 enzyme activity in patients was significantly decreased compared with healthy controls. ADA2 activity levels tended to be lower in patients with stroke compared with patients without stroke. Genealogical studies did not identify a common ancestor; however, based on allele frequency, a North-West European founder effect can be noted. Three patients underwent haematopoietic cell transplantation, after which ADA2 activity was restored and clinical symptoms resolved. CONCLUSION: This case series revealed large phenotypic variability in patients with ADA2 deficiency though they were homozygous for the same R169Q mutation inCECR1 Disease modifiers, including epigenetic and environmental factors, thus seem important in determining the phenotype. Furthermore, haematopoietic cell transplantation appears promising for those patients with a severe clinical phenotype.

A prospective study on drug monitoring of PEGasparaginase and Erwinia asparaginase and asparaginase antibodies in pediatric acute lymphoblastic leukemia.

This study prospectively analyzed the efficacy of very prolonged courses of pegylated Escherichia coli asparaginase (PEGasparaginase) and Erwinia asparaginase in pediatric acute lymphoblastic leukemia (ALL) patients. Patients received 15 PEGasparaginase infusions (2500 IU/m(2) every 2 weeks) in intensification after receiving native E coli asparaginase in induction. In case of allergy to or silent inactivation of PEGasparaginase, Erwinia asparaginase (20 000 IU/m(2) 2-3 times weekly) was given. Eighty-nine patients were enrolled in the PEGasparaginase study. Twenty (22%) of the PEGasparaginase-treated patients developed an allergy; 7 (8%) showed silent inactivation. The PEGasparaginase level was 0 in all allergic patients (grade 1-4). Patients without hypersensitivity to PEGasparaginase had serum mean trough levels of 899 U/L. Fifty-nine patients were included in the Erwinia asparaginase study; 2 (3%) developed an allergy and none silent inactivation. Ninety-six percent had at least 1 trough level ≥100 U/L. The serum asparagine level was not always completely depleted with Erwinia asparaginase in contrast to PEGasparaginase. The presence of asparaginase antibodies was related to allergies and silent inactivation, but with low specificity (64%). Use of native E coli asparaginase in induction leads to high hypersensitivity rates to PEGasparaginase in intensification. Therefore, PEGasparaginase should be used upfront in induction, and we suggest that the dose could be lowered. Switching to Erwinia asparaginase leads to effective asparaginase levels in most patients. Therapeutic drug monitoring has been added to our ALL-11 protocol to individualize asparaginase therapy.