CD371-positive pediatric B-cell acute lymphoblastic leukemia: propensity to lineage switch and slow early response to treatment.

ABSTRACT: In the effort to improve immunophenotyping and minimal residual disease (MRD) assessment in acute lymphoblastic leukemia (ALL), the international Berlin-Frankfurt-Münster (iBFM) Flow Network introduced the myelomonocytic marker CD371 for a large prospective characterization with a long follow-up. In the present study, we aimed to investigate the clinical and biological features of CD371-positive (CD371pos) pediatric B-cell precursor ALL (BCP-ALL). From June 2014 to February 2017, 1812 pediatric patients with newly diagnosed BCP-ALLs enrolled in trial AIEOP-BFM ALL 2009 were evaluated as part of either a screening (n = 843, Italian centers) or validation cohort (n = 969, other iBFM centers). Laboratory assessment at diagnosis consisted of morphological, immunophenotypic, and genetic analysis. Response assessment relied on morphology, multiparametric flow cytometry (MFC), and polymerase chain reaction (PCR)-MRD. At diagnosis, 160 of 1812 (8.8%) BCP-ALLs were CD371pos. This correlated with older age, lower ETV6::RUNX1 frequency, immunophenotypic immaturity (all P < .001), and strong expression of CD34 and of CD45 (P < .05). During induction therapy, CD371pos BCP-ALLs showed a transient myelomonocytic switch (mm-SW: up to 65.4% of samples at day 15) and an inferior response to chemotherapy (slow early response, P < .001). However, the 5-year event-free survival was 88.3%. Among 420 patients from the validation cohort, 27 of 28 (96.4%) cases positive for DUX4-fusions were CD371pos. In conclusion, in the largest pediatric cohort, CD371 is the most sensitive marker of transient mm-SW, whose recognition is essential for proper MFC MRD assessment. CD371pos is associated to poor early treatment response, although a good outcome can be reached after MRD-based ALL-related therapies.

Outcomes of HLA-mismatched HSCT with TCRαß/CD19 depletion or post-HSCT cyclophosphamide for inborn errors of immunity.

HLA-mismatched transplants with either in vitro depletion of CD3+TCRαß/CD19 (TCRαß) cells or in vivo T-cell depletion using post-transplant cyclophosphamide (PTCY) have been increasingly used for patients with inborn errors of immunity (IEI). We performed a retrospective multicenter study via the EBMT registry on 306 children with IEI undergoing first transplant between 2010-2019 from an HLA-mismatched donor using TCRαß (n=167) or PTCY (n=139). Median age at HSCT was 1.2 years (range, 0.03-19.6 years). The 3-year overall survival (OS) was 78% (95% confidence interval (CI), 71-84%) after TCRαß and 66% (57-74%) after PTCY (p=0.013). Pre-HSCT morbidity score (hazard ratio (HR) 2.27, 1.07-4.80, p=0.032) and non-Busulfan/Treosulfan conditioning (HR 3.12, 1.98-4.92, p<0.001) were the only independent predictors of unfavorable OS. The 3-year event-free survival (EFS) was 58% (50-66%) after TCRαß and 57% (48-66%) after PTCY (p=0.804). Cumulative incidence of severe acute GvHD was higher after PTCY (15%, 9-21%) than TCRαß (6%, 2-9%, p=0.007), with no difference in chronic GvHD (PTCY, 11%, 6-17%; TCRαß, 7%, 3-11%, p=0.173). The 3-year GvHD-free EFS was 53% (44-61%) after TCRαß and 41% (32-50%) after PTCY (p=0.080). PTCY had significantly higher rates of veno-occlusive disease (14.4% versus TCRαß 4.9%, p=0.009), acute kidney injury (12.7% versus 4.6%, p=0.032) and pulmonary complications (38.2% versus 24.1%, p=0.017). Adenoviraemia (18.3% versus PTCY 8.0%, p=0.015), primary graft failure (10%, versus 5%, p=0.048), and second HSCT (17.4% versus 7.9%, p=0.023) were significantly higher in TCRαß. In conclusion, this study demonstrates that both approaches are suitable options in IEI patients, although characterized by different advantages and outcomes.

An empowered, clinically viable hematopoietic stem cell gene therapy for the treatment of multisystemic mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is a rare X-linked recessive lysosomal storage disorder due to a mutation in the lysosomal enzyme iduronate-2-sulfatase (IDS) gene. IDS deficiency leads to a progressive, multisystem accumulation of glycosaminoglycans (GAGs) and results in central nervous system (CNS) manifestations in the severe form. We developed up to clinical readiness a new hematopoietic stem cell (HSC) gene therapy approach for MPS II that benefits from a novel highly effective transduction protocol. We first provided proof of concept of efficacy of our approach aimed at enhanced IDS enzyme delivery to the CNS in a murine study of immediate translational value, employing a lentiviral vector (LV) encoding a codon-optimized human IDS cDNA. Then the therapeutic LV was tested for its ability to efficiently and safely transduce bona fide human HSCs in clinically relevant conditions according to a standard vs. a novel protocol that demonstrated superior ability to transduce bona fide long-term repopulating HSCs. Overall, these results provide strong proof of concept for the clinical translation of this approach for the treatment of Hunter syndrome.

Immunological Aspects of Kabuki Syndrome: A Retrospective Multicenter Study of the Italian Primary Immunodeficiency Network (IPINet).

Kabuki Syndrome (KS) is a multisystemic genetic disorder. A portion of patients has immunological manifestations characterized by increased susceptibility to infections and autoimmunity. Aiming to describe the clinical and laboratory immunological aspects of KS, we conducted a retrospective multicenter observational study on patients with KS treated in centers affiliated to the Italian Primary Immunodeficiency Network.Thirty-nine patients were enrolled, with a median age at evaluation of 10 years (range: 3 m-21y). All individuals had organ malformations of variable severity. Congenital heart defect (CHD) was present in 19/39 patients (49%) and required surgical correction in 9/39 (23%), with associated thymectomy in 7/39 (18%). Autoimmune cytopenia occurred in 6/39 patients (15%) and was significantly correlated with thymectomy (p < 0.002), but not CHD. Individuals with cytopenia treated with mycophenolate as long-term immunomodulatory treatment (n = 4) showed complete response. Increased susceptibility to infections was observed in 22/32 patients (69%). IgG, IgA, and IgM were low in 13/29 (45%), 13/30 (43%) and 4/29 (14%) patients, respectively. Immunoglobulin substitution was required in three patients. Lymphocyte subsets were normal in all patients except for reduced naïve T-cells in 3/15 patients (20%) and reduced memory switched B-cells in 3/17 patients (18%). Elevated CD3 + TCRαß + CD4-CD8-T-cells were present in 5/17 individuals (23%) and were correlated with hematological and overall autoimmunity (p < 0.05).In conclusion, immunological manifestations of KS in our cohort include susceptibility to infections, antibody deficiency, and autoimmunity. Autoimmune cytopenia is correlated with thymectomy and elevated CD3 + TCRαß + CD4-CD8-T-cells, and benefits from treatment with mycophenolate.

Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease.

BACKGROUND: Sickle cell disease is characterized by hemolytic anemia, pain, and progressive organ damage. A high level of erythrocyte fetal hemoglobin (HbF) comprising α- and γ-globins may ameliorate these manifestations by mitigating sickle hemoglobin polymerization and erythrocyte sickling. BCL11A is a repressor of γ-globin expression and HbF production in adult erythrocytes. Its down-regulation is a promising therapeutic strategy for induction of HbF. METHODS: We enrolled patients with sickle cell disease in a single-center, open-label pilot study. The investigational therapy involved infusion of autologous CD34+ cells transduced with the BCH-BB694 lentiviral vector, which encodes a short hairpin RNA (shRNA) targeting BCL11A mRNA embedded in a microRNA (shmiR), allowing erythroid lineage-specific knockdown. Patients were assessed for primary end points of engraftment and safety and for hematologic and clinical responses to treatment. RESULTS: As of October 2020, six patients had been followed for at least 6 months after receiving BCH-BB694 gene therapy; median follow-up was 18 months (range, 7 to 29). All patients had engraftment, and adverse events were consistent with effects of the preparative chemotherapy. All the patients who could be fully evaluated achieved robust and stable HbF induction (percentage HbF/(F+S) at most recent follow-up, 20.4 to 41.3%), with HbF broadly distributed in red cells (F-cells 58.9 to 93.6% of untransfused red cells) and HbF per F-cell of 9.0 to 18.6 pg per cell. Clinical manifestations of sickle cell disease were reduced or absent during the follow-up period. CONCLUSIONS: This study validates BCL11A inhibition as an effective target for HbF induction and provides preliminary evidence that shmiR-based gene knockdown offers a favorable risk-benefit profile in sickle cell disease. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT03282656).

TP53 DNA binding domain mutational status and rituximab-based treatment are independent prognostic factors for pediatric Burkitt lymphoma patients stratification.

Not available.

Matched unrelated donor transplantation versus haploidentical transplantation with post-transplant cyclophosphamide in children with acute myeloid leukemia: a PDWP-EBMT study.

In children with acute myeloid leukemia (AML) who lack a human leukocyte antigen (HLA) identical sibling, the donor can be replaced with an HLA-matched unrelated donor (MUD) or a haploidentical donor (haplo). We compared outcomes of patients <18 years with AML in first and second complete remission (CR1 and CR2) undergoing a hematopoietic stem cell transplantation (HCT) either with a MUD with anti-thymocyte globulin (ATG) (N=420) or a haplo HCT with post-transplant cyclophosphamide (PT-CY) (N=96) after a myeloablative conditioning regimen (MAC) between 2011 and 2021, reported to the European Society for Blood and Marrow Transplantation. A matched pair analysis was performed to adjust for differences among groups. The final analysis was performed on 253 MUD and 95 haplo-HCT. In the matched cohort, median age at HCT was 11.2 and 10 years and median year of HCT was 2017 and 2018, in MUD and haplo-HCT recipients, respectively. The risk of grade III-IV acute graft-versus-host disease (aGVHD) was significantly higher in the haplo group (hazard ratio [HR]=2.33, 95% confidence interval [CI]: 1.18-4.58; P=0.01). No significant differences were found in 2 years overall survival (OS; 78.4% vs. 71.5%; HR=1.39, 95% CI: 0.84-2.31; P=0.19), leukemia-free survival (LFS; 72.7% vs. 69.5%; HR=1.22, 95% CI: 0.76-1.95; P=0.41), CI of relapse (RI; 19.3% vs. 19.5%; HR=1.14, 95% CI: 0.62-2.08; P=0.68) non-relapse-mortality (NRM; 8% vs. 11%; HR=1.39, 95% CI: 0.66-2.93; P=0.39) and graft-versus-host free relapse-free survival (GRFS; 60.7% vs. 54.5%, HR=1.38, 95% CI: 0.95-2.02; P=0.09) after MUD and haplo-HCT respectively. Our study suggests that haplo-HCT with PT-CY is a suitable option to transplant children with AML lacking a matched related donor.

Application of the Weighted-Incidence Syndromic Combination Antibiogram (WISCA) to guide the empiric antibiotic treatment of febrile neutropenia in oncological paediatric patients: experience from two paediatric hospitals in Northern Italy.

BACKGROUND: Guidelines about febrile neutropenia in paediatric patients are not homogeneous; the best empiric treatment of this condition should be driven by local epidemiology. The Weighted-Incidence Syndromic Combination Antibiogram (WISCA) addresses the need for disease-specific local susceptibility evidence that could guide empiric antibiotic prescriptions based on outcome estimates of treatment regimens obtained as a weighted average of pathogen susceptibilities. This study developed a WISCA model to inform empirical antibiotic regimen selection for febrile neutropenia (FN) episodes in onco-haematological paediatric patients treated at two Italian paediatric tertiary centres. METHODS: We included blood cultures from patients with a bloodstream infection and neutropenia admitted to the Paediatric Haematology-Oncology wards in Padua and Genoa Hospitals from 2016 to 2021. WISCAs were developed by estimating the coverage of 20 antibiotics as monotherapy and of 21 combined regimens with a Bayesian probability distribution. RESULTS: We collected 350 blood cultures, including 196 g-negative and 154 g-positive bacteria. Considering the most used antibiotic combinations, such as piperacillin-tazobactam plus amikacin, the median coverage for the pool of bacteria collected in the study was 78%. When adding a glycopeptide, the median coverage increased to 89%, while the replacement of piperacillin-tazobactam with meropenem did not provide benefits. The developed WISCAs showed that no monotherapy offered an adequate coverage rate for the identified pathogens. CONCLUSIONS: The application of WISCA offers the possibility of maximizing the clinical utility of microbiological surveillance data derived from large hospitals to inform the choice of the best empiric treatment while contributing to spare broad-spectrum antibiotics.

A Multi-Omics Approach Reveals Enrichment in Metabolites Involved in the Regulation of the Glutathione Pathway in LIN28B-Dependent Cancer Cells.

The RNA-binding protein LIN28B, identified as an independent risk factor in high-risk neuroblastoma patients, is implicated in adverse treatment outcomes linked to metastasis and chemoresistance. Despite its clinical significance, the impact of LIN28B on neuroblastoma cell metabolism remains unexplored. This study employs a multi-omics approach, integrating transcriptome and metabolome data, to elucidate the global metabolic program associated with varying LIN28B expression levels over time. Our findings reveal that escalating LIN28B expression induces a significant metabolic rewiring in neuroblastoma cells. Specifically, LIN28B prompts a time-dependent increase in the release rate of metabolites related to the glutathione and aminoacyl-tRNA biosynthetic pathways, concomitant with a reduction in glucose uptake. These results underscore the pivotal role of LIN28B in governing neuroblastoma cell metabolism and suggest a potential disruption in the redox balance of LIN28B-bearing cells. This study offers valuable insights into the molecular mechanisms underlying LIN28B-associated adverse outcomes in neuroblastoma, paving the way for targeted therapeutic interventions.

A novel mutation in MECOM affects MPL regulation in vitro and results in thrombocytopenia and bone marrow failure.

MECOM-associated syndrome (MECOM-AS) is a rare disease characterized by amegakaryocytic thrombocytopenia, progressive bone marrow failure, pancytopenia and radioulnar synostosis with high penetrance. The clinical phenotype may also include finger malformations, cardiac and renal alterations, hearing loss, B-cell deficiency and predisposition to infections. The syndrome, usually diagnosed in the neonatal period because of severe thrombocytopenia, is caused by mutations in the MECOM gene, encoding for the transcription factor EVI1. The mechanism linking the alteration of EVI1 function and thrombocytopenia is poorly understood. In a paediatric patient affected by severe thrombocytopenia, we identified a novel variant of the MECOM gene (p.P634L), whose effect was tested on pAP-1 enhancer element and promoters of targeted genes showing that the mutation impairs the repressive activity of the transcription factor. Moreover, we demonstrated that EVI1 controls the transcriptional regulation of MPL, a gene whose mutations are responsible for congenital amegakaryocytic thrombocytopenia (CAMT), potentially explaining the partial overlap between MECOM-AS and CAMT.

Lentiviral haematopoietic stem-cell gene therapy for early-onset metachromatic leukodystrophy: long-term results from a non-randomised, open-label, phase 1/2 trial and expanded access.

BACKGROUND: Effective treatment for metachromatic leukodystrophy (MLD) remains a substantial unmet medical need. In this study we investigated the safety and efficacy of atidarsagene autotemcel (arsa-cel) in patients with MLD. METHODS: This study is an integrated analysis of results from a prospective, non-randomised, phase 1/2 clinical study and expanded-access frameworks. 29 paediatric patients with pre-symptomatic or early-symptomatic early-onset MLD with biochemical and molecular confirmation of diagnosis were treated with arsa-cel, a gene therapy containing an autologous haematopoietic stem and progenitor cell (HSPC) population transduced ex vivo with a lentiviral vector encoding human arylsulfatase A (ARSA) cDNA, and compared with an untreated natural history (NHx) cohort of 31 patients with early-onset MLD, matched by age and disease subtype. Patients were treated and followed up at Ospedale San Raffaele, Milan, Italy. The coprimary efficacy endpoints were an improvement of more than 10% in total gross motor function measure score at 2 years after treatment in treated patients compared with controls, and change from baseline of total peripheral blood mononuclear cell (PBMC) ARSA activity at 2 years after treatment compared with values before treatment. This phase 1/2 study is registered with ClinicalTrials.gov, NCT01560182. FINDINGS: At the time of analyses, 26 patients treated with arsa-cel were alive with median follow-up of 3·16 years (range 0·64-7·51). Two patients died due to disease progression and one due to a sudden event deemed unlikely to be related to treatment. After busulfan conditioning, all arsa-cel treated patients showed sustained multilineage engraftment of genetically modified HSPCs. ARSA activity in PBMCs was significantly increased above baseline 2 years after treatment by a mean 18·7-fold (95% CI 8·3-42·2; p<0·0001) in patients with the late-infantile variant and 5·7-fold (2·6-12·4; p<0·0001) in patients with the early-juvenile variant. Mean differences in total scores for gross motor function measure between treated patients and age-matched and disease subtype-matched NHx patients 2 years after treatment were significant for both patients with late-infantile MLD (66% [95% CI 48·9-82·3]) and early-juvenile MLD (42% [12·3-71·8]). Most treated patients progressively acquired motor skills within the predicted range of healthy children or had stabilised motor performance (maintaining the ability to walk). Further, most displayed normal cognitive development and prevention or delay of central and peripheral demyelination and brain atrophy throughout follow-up; treatment benefits were particularly apparent in patients treated before symptom onset. The infusion was well tolerated and there was no evidence of abnormal clonal proliferation or replication-competent lentivirus. All patients had at least one grade 3 or higher adverse event; most were related to conditioning or to background disease. The only adverse event related to arsa-cel was the transient development of anti-ARSA antibodies in four patients, which did not affect clinical outcomes. INTERPRETATION: Treatment with arsa-cel resulted in sustained, clinically relevant benefits in children with early-onset MLD by preserving cognitive function and motor development in most patients, and slowing demyelination and brain atrophy. FUNDING: Orchard Therapeutics, Fondazione Telethon, and GlaxoSmithKline.

CD72 is a pan-tumor antigen associated to pediatric acute leukemia.

In the development of novel immunotherapeutic approaches, the step of target identification is a challenging process, because it aims at identifying robust tumor-associated antigens (TAAs) specific for the pathological population and causing no off-target effects. Here we propose CD72 as a novel and robust TAA for pediatric acute leukemias. We provided an outline of CD72 expression assessed by flow cytometry on a variety of cancer cell lines and primary samples, including normal bone marrow (BM) samples and hematopoietic stem and progenitor cells. We analyzed CD 72 expression on a cohort of 495 pathological pediatric BM aspirates, including: 215 B-cell precursor acute lymphoblastic leukemias (BCP-ALL), 156 acute myeloid leukemias (AMLs), 88 T-lineage ALLs or lymphoblastic lymphomas with BM infiltration, 13 B-lineage lymphoblastic lymphomas with BM infiltration, 9 myelodysplastic syndromes with increased blasts (5%-9% blasts on BM: MDS-IB1) and 14 non-hematopoietic solid tumors infiltrating BM. Results showed that CD72 is highly expressed in almost all BCP-ALL and the majority of AML at diagnosis, including BCP-ALL cases characterized by CD19 loss. These findings support a potential role for advanced diagnostics and novel immunotherapy approaches, providing a pan-ALL and AML target.

Relationship between hemoglobin, hemolysis, and transcranial Doppler velocities in children with sickle cell disease: Results from a long-term natural history study in Italy in the era of multimodal therapy.

BACKGROUND: Stroke and cerebral vasculopathy are leading causes of morbidity and mortality in patients with sickle cell disease (SCD). Transcranial Doppler (TCD) is a reliable and validated predictor of stroke risk. Children with conditional or abnormal TCD are at an increased risk for stroke, which can be mitigated by red blood cell transfusion or hydroxyurea. Elucidating the relationship between cerebral hemodynamics and hemolytic anemia can help identify novel therapeutic approaches to reduce stroke risk and transfusion dependence. METHODS: This long-term, real-world study was designed to evaluate the prevalence of TCD imaging (TCDi)-assessed flow velocities in children and to interrogate their relationship with markers of anemia and hemolysis. RESULTS: In total, 155 children (median follow-up 79.8 months, 1358.44 patient-years) had 583 evaluable TCDi results. Only patients with HbSS or HbSß0 had abnormal (1.6%) or conditional (10.9%) TCDi. Children with abnormal or conditional TCDi had lower hemoglobin (Hb) and higher hemolysis markers. A linear correlation was detected between TCD velocity and Hb: an Hb increase of 1 g/dL corresponded to decreases in velocity in the internal carotid and middle cerebral arteries (6.137 cm/s and 7.243 cm/s). Moreover, patients with Hb >9 g/dL presented a lower risk of TCDi-associated events. CONCLUSION: These results support the need to optimize disease-modifying treatments that increase Hb and reduce hemolysis for stroke prevention in young children with SCD.

Hypogammaglobulinemia in Children After Hematopoietic Stem Cell Transplantation and Rituximab Treatment: Relevance of B Cell Subsets.

Rituximab (RTX) is widely employed to treat Epstein-Barr virus reactivation in children undergoing Hematopoietic Cell Transplantation (HCT). The resulting loss of B cells may cause persistent hypogammaglobulinemia. This retrospective cross-sectional study aims to identify flow cytometry biomarkers associated with persistent hypogammaglobulinemia in patients receiving RTX after HCT. We analyzed 5 patients (cases group) requiring immunoglobulin substitution due to low level of IgG (IgG <5 g/L) detected after RTX treatment and 5 patients (controls group) not requiring long-term immunoglobulin (Ig) substitution. We investigated the B cell reconstitution, and in patients group we observed a significantly lower count in B total, IgD+CD27+ marginal B cells and IgD-CD27+ switched-memory B cells, after a median of 5 years from HCT, compared with the control group. Despite the importance limits of our study and the heterogeneity of our data (age of included patients, time of evaluation, interval between RTX dose and assessment) we conclude that RTX given early after HCT might cause a deranged B cell maturation, contributing to the delation in B cell recovery following HCT, and switched memory and marginal zone B cell counts could be a promising biomarker to identify patients requiring long-term Ig substitution.

Evaluating high power laser therapy (HPLT) as treatment for chemotherapy-induced oral mucositis in paediatric patients with oncohematological diseases.

BACKGROUND: Oral mucositis (OM) is one of the most debilitating side effects of chemotherapy. Among the current methods used in OM management, the feasibility of high power laser therapy (HPLT) has been proposed but not fully evaluated. AIM: The aim of this study was to evaluate the efficacy of HPLT in the treatment of OM in paediatric patients. DESIGN: Fourteen oncohematological paediatric patients treated with chemotherapy and/or hematopoietic stem cell transplantation and affected by OM at different grades were enrolled in this study. All patients were treated with a class IV laser device in four sessions for four consecutive days, OM was evaluated with the WHO-OTS scale. The perception of pain was assessed by the visual analog scale. Photographs of and information about lesions and patient questionnaires were taken and collected, respectively. Descriptive analyses and the Wilcoxon signed-rank test (nonparametric test) were used, with a statistical significance of α = .05. RESULTS: After a week into the treatment, 57% of patients were completely healed, whereas the whole cohort experienced a drastic decrease in pain, from an average value of 5.8-1.1 (p = .0016). The average number of injuries per patient decreased from 7.4 to 3.1 (p = .008). CONCLUSION: High power laser therapy appears to be a safe and efficacious method for managing OM. It reduces pain and severity of oral cavity injuries. Further studies are needed to determine the optimal parameters useful in OM treatment and to evaluate the relative effectiveness of HPLT to other treatments like LLLT.

Neonatal Manifestations of Chronic Granulomatous Disease: MAS/HLH and Necrotizing Pneumonia as Unusual Phenotypes and Review of the Literature.

Chronic granulomatous disease (CGD) is a rare inborn error of immunity (IEI), characterized by a deficient phagocyte killing due to the inability of NADPH oxidase to produce reactive oxygen species in the phagosome. Patients with CGD suffer from severe and recurrent infections and chronic inflammatory disorders. Onset of CGD has been rarely reported in neonates and only as single case reports or small case series. We report here the cases of three newborns from two different kindreds, presenting with novel infectious and inflammatory phenotypes associated with CGD. A girl with CYBA deficiency presented with necrotizing pneumonia, requiring a prolonged antibiotic treatment and resulting in fibrotic pulmonary changes. From the second kindred, the first of two brothers developed a fatal Burkholderia multivorans sepsis and died at 24 days of life. His younger brother had a diagnosis of CYBB deficiency and presented with Macrophage Activation Syndrome/Hemophagocytic Lympho-Histiocytosis (MAS/HLH) without any infection, that could be controlled with steroids. We further report the findings of a review of the literature and show that the spectrum of microorganisms causing infections in neonates with CGD is similar to that of older patients, but the clinical manifestations are more diverse, especially those related to the inflammatory syndromes. Our findings extend the spectrum of the clinical presentation of CGD to include unusual neonatal phenotypes. The recognition of the very early, potentially life-threatening manifestations of CGD is crucial for a prompt diagnosis, improvement of survival and reduction of the risk of long-term sequelae.

Patient-Customized Oligonucleotide Therapy for a Rare Genetic Disease.

Genome sequencing is often pivotal in the diagnosis of rare diseases, but many of these conditions lack specific treatments. We describe how molecular diagnosis of a rare, fatal neurodegenerative condition led to the rational design, testing, and manufacture of milasen, a splice-modulating antisense oligonucleotide drug tailored to a particular patient. Proof-of-concept experiments in cell lines from the patient served as the basis for launching an "N-of-1" study of milasen within 1 year after first contact with the patient. There were no serious adverse events, and treatment was associated with objective reduction in seizures (determined by electroencephalography and parental reporting). This study offers a possible template for the rapid development of patient-customized treatments. (Funded by Mila's Miracle Foundation and others.).

Either IL-7 activation of JAK-STAT or BEZ inhibition of PI3K-AKT-mTOR pathways dominates the single-cell phosphosignature of ex vivo treated pediatric T-cell acute lymphoblastic leukemia cells.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from lymphoblasts of T-cell origin. While TALL accounts for only 15% of childhood and 25% of adult ALL, 30% of patients relapse with a poor outcome. Targeted therapy of resistant and high-risk pediatric T-ALL is therefore urgently needed, together with precision medicine tools allowing the testing of efficacy in patient samples. Furthermore, leukemic cell heterogeneity requires drug response assessment at the single-cell level. Here we used single-cell mass cytometry to study signal transduction pathways such as JAK-STAT, PI3K-AKT-mTOR and MEK-ERK in 16 diagnostic and five relapsed T-ALL primary samples, and investigated the in vitro response of cells to Interleukin-7 (IL-7) and the inhibitor BEZ-235. T-ALL cells showed upregulated activity of the PI3K-AKT-mTOR and MEK-ERK pathways and increased expression of proliferation and translation markers. We found that perturbation induced by the ex vivo administration of either IL-7 or BEZ-235 reveals a high degree of exclusivity with respect to the phospho-protein responsiveness to these agents. Notably, these response signatures were maintained from diagnosis to relapse in individual patients. In conclusion, we demonstrated the power of mass cytometry single-cell profiling of signal transduction pathways in T-ALL. Taking advantage of this advanced approach, we were able to identify distinct clusters with different responsiveness to IL-7 and BEZ-235 that can persist at relapse. Collectively our observations can contribute to a better understanding of the complex signaling network governing T-ALL behavior and its correlation with influence on the response to therapy.

Pain coping strategies in paediatric patients newly diagnosed with leukaemia compared with healthy peers.

OBJECTIVE: Children with leukaemia experience special difficulties adapting to stressful medical procedures and to the adverse effects of chemotherapy, though they can implement their coping strategies. The aims of the study were to assess whether the coping-with-pain strategies could be influenced by a child's personal and illness factors and to render possible comparisons between children with leukaemia and healthy peers. Another aim was to compare parents' and children's reports on coping strategies. METHODS: A total of 125 patients (average age = 6.79 years; SD = 3.40) with acute leukaemia (lymphocytic leukaemia 90.4% and myeloid leukaemia 9.6%) and age-matched healthy children with their parents were enrolled in the study. A socio-demographic questionnaire and the Waldon-Varni Pediatric Pain Coping Inventory, parent and self-report versions, were administered 1 month after diagnosis. Data regarding the therapy's side effects were recorded. RESULTS: The comparison between proxy-reports of the two groups of parents found significant differences in terms of social support, self-cognitive instructions and catastrophising strategies. Children aged 6-10 years relied more heavily on distraction than children of other ages, using more problem-solving and self-cognitive instructions. The results indicated moderate parent-child agreement. CONCLUSION: Health professionals could help paediatric leukaemic patients in adopting more efficiently pain coping strategies applicable for different ages.

IFIH1 loss-of-function variants contribute to very early-onset inflammatory bowel disease.

Genetic defects of innate immunity impairing intestinal bacterial sensing are linked to the development of Inflammatory Bowel Disease (IBD). Although much evidence supports a role of the intestinal virome in gut homeostasis, most studies focus on intestinal viral composition rather than on host intestinal viral sensitivity. To demonstrate the association between the development of Very Early Onset IBD (VEOIBD) and variants in the IFIH1 gene which encodes MDA5, a key cytosolic sensor for viral nucleic acids. Whole exome sequencing (WES) was performed in two independent cohorts of children with VEOIBD enrolled in Italy (n = 18) and USA (n = 24). Luciferase reporter assays were employed to assess MDA5 activity. An enrichment analysis was performed on IFIH1 comparing 42 VEOIBD probands with 1527 unrelated individuals without gastrointestinal or immunological issues. We identified rare, likely loss-of-function (LoF), IFIH1 variants in eight patients with VEOIBD from a combined cohort of 42 children. One subject, carrying a homozygous truncating variant resulting in complete LoF, experienced neonatal-onset, pan-gastrointestinal, IBD-like enteropathy plus multiple infectious episodes. The remaining seven subjects, affected by VEOIBD without immunodeficiency, were carriers of one LoF variant in IFIH1. Among these, two patients also carried a second hypomorphic variant, with partial function apparent when MDA5 was weakly stimulated. Furthermore, IFIH1 variants were significantly enriched in children with VEOIBD as compared to controls (p = 0.007). Complete and partial MDA5 deficiency is associated with VEOIBD with variable penetrance and expressivity, suggesting a role for impaired intestinal viral sensing in IBD pathogenesis.