BackgroundSevere leukocyte adhesion deficiency-I (LAD-I) results from biallelic deleterious ITGB2 variants leading to deficient/defective CD18 leukocyte expression and impaired endothelial adhesion and extravasation.Children with <2% of normal CD18 neutrophil expression experience recurrent, life-threatening bacterial and fungal infections, and extensive mortality. Allogeneic hematopoietic stem cell transplantation (alloHSCT) is potentially curative but limited by donor availability, graft-versus-host disease (GvHD), and graft failure (GF).AimsTo evaluate the long-term safety and efficacy of RP-L201 (marnetegragene autotemcel), an autologous CD34+ hematopoietic stem cell gene therapy utilizing the Chim-CD18-WPRE lentiviral vector carrying ITGB2, including restoration of peripheral blood (PB) polymorphonuclear cell (PMN) CD18 and CD11 expression.MethodsPatients ≥3 months old with severe LAD-I enrolled in the pivotal phase I/II study (NCT03812263), underwent G-CSF/plerixafor mobilization, apheresis, ex vivo transduction of CD34+ cells with RP-L201, and myeloablative busulfan (cAUC 71.6mg/L*h) conditioning, including therapeutic drug monitoring prior to infusion. Assessments included alloHSCT-free survival, PB PMN CD18 expression, PB vector copy number (VCN) and integration site analysis (ISA), leukocytosis normalization, and infection-related hospitalization annualized events. All patients subsequently entered the long-term follow-up (LTFU) study (NCT06282432).ResultsAs of June 18, 2025, nine patients (age at infusion 9.8–117.4 months) were treated and followed for a median (range) of 50.92 (42.6–67.9) months. AlloHSCT-free survival is 100% with no GF. VCN was 0.42–2.4 at 3 months post-infusion and remained durable thereafter with a mean VCN of 1.73 at M12, sustained through M24 and beyond with concomitant sustained PMN CD18 expression. There were no new skin or oral lesions from the end of the parent study to the data-cut date in the LTFU study.Significant infections (either requiring IV antimicrobials or hospitalization) were markedly (90.7%) reduced from pre-infusion through the following 91 days post-engraftment up to the end of the study. RP-L201 was well-tolerated with no discontinuations or RP-L201-related adverse events. ISA demonstrated highly polyclonal integration patterns without evidence of predominant clones.Summary/ConclusionTreatment with RP-L201 results in durable phenotypic correction across all relevant clinical and laboratory parameters of severe LAD-I patients. The long-term benefit-risk profile for RP-L201 remains favorable, with no RP-L201-related adverse events and 100% alloHSCT-free survival.

Introduction Pathogenic variants of Plakophilin-2 (PKP2) are a significant cause of arrhythmogenic cardiomyopathy (ACM), a disorder characterized by increased risk of ventricular tachyarrhythmias, progressive heart failure, and sudden cardiac death. PKP2 variants account for up to 45% of ACM cases. Administration of AAVrh.74-PKP2a (RP-A601), a recombinant adeno-associated viral (AAV) vector in a clinically affected PKP2-ACM mouse model (typical survival ≤50 days post disease onset) after disease onset extended survival for >5 months, improved right ventricular function, decreased cardiac dilation and myocardial fibrosis, and reduced arrhythmia burden. Additional studies in rodents and non-human primates demonstrated RP-A601 safety. Hypothesis RP-A601 will be associated with acceptable safety and will ameliorate aspects of disease pathophysiology in high-risk adult patients with PKP2-ACM, based on preclinical data. Methods NCT05885412 is a multicenter, open-label, dose escalation ongoing Phase 1 trial that will evaluate the safety and tolerability of a single intravenous RP-A601 infusion in adult patients (≥18 yrs) with a PKP2-ACM and an implanted ICD. Preliminary efficacy will be evaluated by assessing change in myocardial PKP2a protein expression and clinical markers of cardiomyopathy and arrhythmia burden. Patients with severe right ventricular dysfunction, left ventricular ejection fraction ≤50% (echocardiogram or cardiac MRI) and/or NYHA Class IV are excluded. Results As of March 11, 2025, three patients with PKP2-ACM have received RP-A601 at a low dose of 8 × 1013 GC/kg (cohort 1). All patients discontinued immunomodulatory therapies at a median of 4.1 months (range: 3.3-4.8) following RP-A601 infusion. All treatment emergent adverse events (TEAEs) experienced by these patients were not serious and mild/moderate in severity. The most common non-serious TEAEs were transaminase elevations and other laboratory abnormalities, the majority of which resolved to normal range within 2 weeks following RP-A601 infusion. One patient experienced treatment-emergent serious adverse events (SAEs) of transaminase elevation, pancreatitis, sepsis and bacteremia, while receiving immunomodulatory therapy. All events fully resolved without sequalae. No arrhythmia SAEs were identified during the initial 5-11 months of post-treatment follow-up. Transient complement activation and Grade 2 thrombocytopenia were observed without evidence of thrombotic microangiopathy. Cardiac transduction and increased myocardial PKP2a protein expression were evident at 3-6 months following RP-A601 infusion via Western Blot and Immunofluorescence. Conclusions To date, RP-A601 has been well-tolerated and led to increased myocardial PKP2a protein expression in patients with this inherited ACM which predisposes to progressive cardiac failure and serious ventricular arrhythmias including sudden cardiac death.

Primary gliomas arising within midline structures of the central nervous system are associated with a worse prognosis compared with hemispheric gliomas. In adults, compared to their pediatric counterparts, adult midline gliomas are not as clearly characterized on the clinical behavior, prognostic factors, and treatment approaches for these diseases. This retrospective cohort assessed all adult (≥ 18 years) patients from our institution with diffuse gliomas arising from midline structures at time of diagnosis (2014–2020). Molecular features characterized using immunohistochemistry, targeted next-generation sequencing, and chromosomal microarray analysis were collected. Patient characteristics were compared across groups using analysis of variance, Kruskal–Wallis, and the chi-square test as appropriate. Cumulative progression-free survival (PFS) and overall survival (OS) probabilities were estimated using the Kaplan–Meier method. Comparisons across groups were made using the log rank test. 79 patients were included in analysis, with a median follow-up of 22.5 months (range, 0.6–123). The mean age at diagnosis was 44.5 years (range, 19.4–76.4), and 51

GSEA of genes associated with positive or negative correlation with radiographic response to ONC201 treatment.

Eltrombopag stimulates hematopoiesis in aplastic anemia, and preclinical studies suggest it promotes DNA repair in Fanconi Anemia (FA) hematopoietic stem cells. We conducted a clinical trial to explore its safety and efficacy in bone marrow failure due to FA. Eltrombopag was administered to 8 pediatric patients with one or more significant cytopenias. After 6 months, those who achieved at least a partial response in peripheral blood continued treatment for 6 additional months. Median age was 7 years (4-12), one patient had somatic mosaicism in bone marrow, and two had been treated by gene therapy (GT). At 6 months, three patients (37.5%) achieved a response, persisting in one at 12 months. The three patients with either somatic mosaicism or with GT-corrected cells showed an enhanced increase of corrected cells. One patient required dose modifications due to gastrointestinal intolerance and two because of hepatobiliary laboratory toxicity. No clonal evolution was observed by conventional cytogenetics, but a patient developed a transient somatic variant in RUNX1. In conclusion, eltrombopag did not mediate clinically relevant responses, although a possible impact of dose reductions and treatment duration must be considered. The increase of gene-corrected cells suggests it promoted a proliferative advantage over uncorrected ones. Www.clinicaltrials.gov #NCT06045052.

GSEA analysis of genes with significantly lower chromatin accessibility at promoters and enhancers and reduced gene expression in DIPG007 cells treated with ONC201 versus vehicle.

Fanconi anemia (FA) constitutes the most common of the inherited bone marrow failure syndromes, a group of rare heterogeneous disorders characterized by cytopenia, predisposition to hematologic and solid malignancies and diverse clinical features. Currently, the only available hematopoietic curative treatment for bone marrow failure is an allogeneic hematopoietic stem cell transplantation (HSCT), although gene therapy has demonstrated evidence of efficacy and substantially reduced toxicity. It has been demonstrated that eltrombopag stimulates trilineage hematopoiesis in aplastic anemia, and preclinical studies suggest it promotes DNA repair in FA hematopoietic stem cells (HSCs). Herein, we report the experience with eltrombopag in a patient misdiagnosed with aplastic anemia and subsequently determined to have FA mosaicism and in two FA patients who previously received gene therapy but who were infused with very low numbers of gene-corrected HSCs. Strikingly, the patient with somatic mosaicism achieved transfusion independence and averted HSCT, and the gene-therapy patients showed a marked increase of corrected cells during treatment.

VST-transformed gene expression values for all samples and protein-coding genes.

BACKGROUND:Danon disease is a rare, X-linked, monogenic cardiomyopathy caused by mutations in the lysosomal-associated membrane 2 gene (LAMP2), which encodes the LAMP2 protein. In male patients, the predominant phenotype is progressive cardiac hypertrophy, cardiac dysfunction, and early death. There are no directed therapies for the disease. METHODS:In this phase 1 study, we evaluated the safety and efficacy of a single infusion of RP-A501, a recombinant adeno-associated virus serotype 9 containing the transgene LAMP2B, which encodes an isoform of LAMP2. The primary outcomes were the safety and toxic effects of RP-A501, myocardial LAMP2 transduction and protein expression, stabilization of or reduction in heart-failure symptoms, and stabilization of or improvement in cardiac structure and function. Key secondary outcomes were sustained reduction in or stabilization of symptoms, immunologic response to RP-A501, end-stage heart failure, and overall survival. Exploratory outcomes included improvement in serologic markers of cardiac disease, patient-reported outcomes, and quality-of-life assessments. RESULTS:RP-A501 infusion was administered to seven male patients with Danon disease: five who were 15 years of age or older and two who were between 11 and 14 years of age. All the patients received a transient immunomodulatory regimen of prednisone, tacrolimus or sirolimus, and rituximab. Phase 1 data over 24 to 54 months, including interim data from a long-term follow-up study, are reported here. One patient had complement-mediated thrombotic microangiopathy (grade 4) with thrombocytopenia and acute kidney injury. Three patients had glucocorticoid-related exacerbation (grade 3) of Danon disease-related skeletal myopathy. One patient with left ventricular systolic dysfunction at baseline had progressive heart failure and underwent transplantation 5 months after infusion. In the six patients with normal left ventricular ejection fraction at baseline, we observed cardiac LAMP2 protein expression and a reduction from baseline in or stabilization of the left ventricular mass index, preservation of left ventricular ejection fraction, and reduction in or stabilization of the levels of cardiac troponin I and N-terminal pro-B-type natriuretic peptide. At 24 to 54 months, all the patients were alive, with complete resolution of side effects. CONCLUSIONS:A single infusion of RP-A501 appeared to be safe and was associated with cardiac LAMP2 expression and evidence of clinical improvement over a period of 24 to 54 months. (Funded by Rocket Pharmaceuticals; ClinicalTrials.gov number, NCT03882437.).

(Abstracted from N Engl J Med 2025;392:1698-1709) Leukocyte adhesion deficiency type I (LAD-I) is an autosomal recessive immune disorder marked by recurrent, life-threatening infections, with 25% to 39% mortality before age 2 years without curative therapy. Common symptoms include umbilical-cord complications, poorly healing cutaneous lesions, persistent leukocytosis, and periodontitis.

Persistent hypoxemia in children with an underlying diagnosis of cancer presents a clinical challenge for physicians. Hypoxemia may arise directly from the malignancy, the subsequent treatment the child is receiving, or from complications associated with curative intent. The complex interplay of these factors necessitates a multidisciplinary approach to diagnosis and management, ensuring that each potential etiology is sufficiently examined and addressed. This report seeks to explore the case of persistent hypoxemia in a child with medulloblastoma following bone marrow transplant, emphasizing the importance of a systematic and comprehensive evaluation to guide both the diagnosis and the treatment. By better understanding the potential etiologies of hypoxemia, clinicians can improve patient care and meet the unique needs of this specific population. A 3-year-old boy with disseminated Group 4 medulloblastoma status postmidline craniotomy, emergent, and abbreviated course of spinal radiation followed by induction chemotherapy (vincristine, methotrexate, etoposide, cyclophosphamide, and cisplatin) developed new-onset hypoxemia on Day +4 following his third and final autologous hematopoietic stem cell transplant (HSCT) conditioned with carboplatin and thiotepa. A chest X-ray (CXR) revealed bilateral hazy pulmonary opacities and a chest computed tomography (CT) revealed diffuse bilateral peripheral ground glass opacities and central bronchial wall thickening. The heart size was noted to be normal. He underwent a bronchoscopy with lavage the following day which revealed mild visible evidence of inflammatory airway disease consistent with diffuse bronchitis, but absence of inflammation on bronchoalveolar lavage (BAL). A central venous catheter blood culture and BAL revealed pansensitive Serratia marcescens and he completed a course of cefepime for 10 days for bacteremia and possible pneumonia. Despite the 10-day cefepime course for bacteremia and possible pneumonia, he remained intermittently hypoxemic with an escalation in oxygen requirement (2 L nasal cannula [NC] 100% FiO2) on Day +17 with new-onset tachypnea and dry cough. A repeat CXR noted improved hazy bilateral opacities but new cardiac enlargement. An echocardiogram (ECHO) revealed a severely dilated right ventricle with moderately decreased systolic function with markedly increased estimated right ventricular (RV) systolic pressure (67 mmHg). A CT angiogram was negative for pulmonary embolism (PE) but did demonstrate new circumferential pericardial effusion. Focal dilation of left lower lobe pulmonary arteries with ground glass opacities was suspicious for regional venous obstruction though infection required exclusion (Figure 1). He continued to receive scheduled diuretic therapy to maintain a slight net negative balance with good effect. Cardiology was consulted and recommended maintaining oxygen saturation levels above 92% to prevent worsening pulmonary resistance from his previous pneumonia and to promote pulmonary vasodilator effect. A baseline N-terminal pro-brain natriuretic peptide was obtained which was elevated at 13,775 (normal 23–289 pg/mL). A pulmonary hypertension specialist was consulted, and the prevailing concern was for pulmonary arterial hypertension due to an acute pulmonary process. Despite these measures, the child continued to have an oxygen need (maximum 10 L via high-flow NC 100% FiO2) and underwent a repeat bronchoscopy with BAL on Day +22, which revealed essentially normal findings with no visible evidence of inflammatory lower airway disease or hemorrhage. Infectious disease studies were negative including bacterial culture, legionella polymerase chain reaction (PCR), aspergillus antigen, adenovirus PCR, beta-d-glucan, pneumocystis PCR, fungal culture, acid fast smear for mycobacteria, nocardia stain, and respiratory viral pathogen panel. A venous blood gas revealed normal CO2 (41–51 mmHg) and HCO3 (18-26 mmol/L). During routine anesthesia for the bronchoscopy, the child experienced sustained hypotension despite normal oxygen saturation of 94% requiring vasopressin suggesting a pulmonary hypertensive crisis. A repeat ECHO the following day continued to demonstrate a severely dilated right ventricle with severely decreased systolic function with further increasing estimated RV systolic pressure (79 mmHg). He was subsequently initiated on sildenafil 5 mg (0.33 mg/kg) three times a day and escalated to 10 mg (0.67 mg/kg) three times per day as tolerated based on recommendations from cardiology. Cardiac catheterization was considered but deferred at that time due to initiation of sildenafil. Challenge point: A 3-year-old boy presents with increasing oxygen requirements on Day +17 after third and final autologous stem cell transplant. CT demonstrates pericardial effusion. ECHO reveals severely dilated right ventricle with decreased systolic function. Infectious disease workup is negative. Learner reflection: What is your leading and differential diagnosis? What do you recommend next and why? Listen to podcast 1 to hear the consortium's decision-making progress. In conjunction with a pulmonary hypertension specialist and pulmonologist, the differential was broadened to include pulmonary veno-occlusive disease (PVOD), PE (however CT angiogram negative although could not exclude thrombotic microangiopathy) and pneumonia (however repeat negative BAL culture and absence of inflammation). Due to the child's persistent hypoxemia and tendency towards pulmonary edema with diuretic responsiveness, his oxygen needs were ultimately felt to be characteristic of PVOD. Thus, the child began defibrotide (6.25 mg/kg q6h) on Day +29 posttransplant due to the possibility of PVOD and pulmonary thrombi contributing to his oxygen needs. Anticoagulation was deferred due to risk of alveolar hemorrhage. Though considered, a diagnostic lung biopsy was likewise deferred given anesthesia risk. A repeat ECHO 5 days later revealed a moderate-severely dilated right ventricle with slightly improved but severely decreased systolic function with estimated RV systolic pressure (59 mmHg). Catheterization was discussed at length with an anesthesiologist, an intensivist, a cardiologist, and a pulmonary hypertension expert. Ultimately, and because he had begun empiric treatment for PVOD, the decision was made to forgo cardiac catheterization as the child demonstrated significant right ventricle dysfunction raising the concern for a postanesthesia pulmonary hypertensive crisis and hemodynamic collapse requiring intubation, which would have been detrimental for this child. For him, the risks outweighed the benefits. As he was not a candidate for a cardiac catheterization to accurately measure his RV systolic pressure, he was transferred to the pediatric intensive care unit for right ventricle optimization with milrinone (0.25 mcg/kg/min) and epinephrine (0.01–0.02 mcg/kg/min) until repeat ECHO and CT showed improvement in systolic function. A 7-day course of methylprednisolone (2 mg/kg) was initiated a week later to improve lung function. During this time, the child was gradually able to tolerate oxygen wean to 0.25–0.50 L oxygen supplementation and continued to receive diuretic therapy. A repeat ECHO a week later revealed RV size and function improvement, decrease in tricuspid regurgitation, and decreased pericardial effusion (Figure 2). A repeat chest CT demonstrated decreased patchy ground glass opacities in both lungs and resolved pericardial effusion. The child completed a 14-day course of defibrotide. There were no adverse events such as bleeding during this time. The diagnosis of PVOD was supported by resolution of cough and alveolar opacities with steroids and defibrotide, resolution of pulmonary edema, and marked improvement in RV size and function. The child was discharged home with 0.25–0.5 L oxygen supplementation, to wean as tolerated with a goal of 94% oxygen saturation. The child is currently 12 months from completion of therapy, doing well and remains in room air without oxygen support. ECHO performed at 8 months after completion of therapy revealed normal heart size and function with no evidence of pulmonary hypertension. Challenge point: A 3-year-old boy with presumed PVOD is treated with defibrotide and steroids along with milrinone and epinephrine. The child experienced significant improvement in oxygen saturation. Repeat ECHO and CT studies show improvement as well. Learner reflection: How do these treatments address the underlying pathophysiology of PVOD? What are the advantages and disadvantages of this form of treatment? Listen to podcast 2 to hear the consortium's decision-making process. This child's unclear pulmonary presentation and subsequent directed management of suspected PVOD with improvement highlights the importance of maintaining a high suspicion for PVOD in children who undergo multiple autologous or other high risk HSCTs. PVOD is a progressive occlusion of the pulmonary venous system characterized by occlusion of the small pulmonary veins by fibrous intimal thickening in addition to patchy capillary proliferation.1 PVOD can progress to increased pulmonary vascular resistance leading to pulmonary vascular congestion, progressive dyspnea, and RV heart failure.2 Clinical presentation of PVOD is characterized by fatigue and shortness of breath.3 Other findings include decreased exercise tolerance, chest pain with exertion, dizziness, and cough.3 Additionally, the difficulty of diagnosis is exacerbated by the fact that PVOD often presents with vague symptoms, nonspecific findings on chest radiographs, and atypical cardiopulmonary hemodynamics.2 Definite diagnosis is obtained via lung biopsy; however, this carries substantial risks for children with pulmonary hypertension and after individual assessment of risks and benefits, biopsy is often deferred.3 In pediatrics, the actual incidence of PVOD is unknown due to the complicated nature of diagnosis—many are misdiagnosed as idiopathic pulmonary hypertension.3 The annual incidence is estimated to be 0.1–0.2 cases per million. A broad literature search identified 68 cases of pediatric PVOD.3 In the post-HSCT setting, less than 20 pediatric PVOD cases were identified.4 Diagnosis of PVOD is particularly challenging as many findings are nonspecific.5 However, findings on radiographic imaging and on clinical presentation should prompt consideration for PVOD post-HSCT.6 Noninvasive diagnostics include clinical presentation, thin-slice chest CT with contrast, and Doppler ECHO. Findings on CT suggestive of PVOD include mediastinal lymph node involvement, centrilobular ground glass opacities, smooth thickening of the interlobular septa, and pleural effusion.3 Although the child did not have mediastinal lymph node involvement or interlobular septa thickening, he did have an original CT that revealed diffuse bilateral peripheral ground glass opacities and central bronchial wall thickening which correlates with these PVOD findings. Furthermore, pulmonary arteries were regionally dilated in areas of ground glass opacities suggestive of patchy pulmonary edema due to pulmonary venous obstruction. In addition, a subsequent CT demonstrated a new circumferential pericardial effusion which is also often seen in children with PVOD; although, the mechanism is unclear.3 Findings on Doppler ECHO suggestive of PVOD include elevated tricuspid regurgitation velocity with or without right sided chamber dysfunction.1 This child developed acute pulmonary hypertensive changes with severely dilated right atrium and ventricle with severe RV dysfunction secondary to near-systemic RV systolic pressures, which is similar to other PVOD case report findings.7 The fact that the child had so much right heart dysfunction in the absence of RV hypertrophy is likely indicative of the acuity of onset. The combined findings on CT and ECHO are suggestive of a PVOD diagnosis for the child. Diagnosis of PVOD was aided by the rule-out criteria of negative BAL cultures for pneumonia and negative CT angiogram for PE. Response to treatment is also supportive. Challenge point: The diagnosis of PVOD is often confirmed with biopsy. In this case, biopsy was not possible. Diagnosis was made utilizing imaging such as CT and ECHO. Findings here were consistent with PVOD. Rule-out criteria also contributed to the diagnosis of PVOD. Learner reflection: How do these noninvasive diagnostic tools aid in identifying PVOD? Which findings were crucial in the diagnosis of PVOD? Why are rule-out criteria also important in this case? Listen to podcast 3 to hear the consortium's decision-making process. One of the greatest risk factors for developing PVOD is endothelial injury from cytotoxic chemotherapy and irradiation.6 Recent research is also suggestive of infection playing a role in development of PVOD.8 Before the child's episode of hypoxemia leading to the PVOD diagnosis and treatment with defibrotide, the child had received spinal radiation along with chemotherapy which utilized the drugs carboplatin and thiotepa for his disseminated medulloblastoma. The use of cytotoxic drugs in this case report is consistent with other reported cases of PVOD post-HSCT.2, 8 It is possible that the spinal radiation and cytotoxic chemotherapy treatment caused extensive endothelial injury which increased the child's risk factors for developing PVOD. Additionally, infection has been shown to cause reduction in tissue plasminogen activator.9 Subsequently, this reduction may cause thrombus formation leading to intimal fibrosis of the small veins and venules. The child recovered from a Serratia marcescens infection before the decline of his hypoxemia and it can be reasonably hypothesized that this infection further predisposed the child to acquiring PVOD. The onset of PVOD after transplant typically occurs within weeks to months, with a few case reports of PVOD manifesting within days.2 This timeline is similar to the child's presentation with new oxygen requirements on Day +17 posttransplant. Challenge point: Endothelial injury contributes to the underlying pathophysiology of PVOD. Injury to the pulmonary endothelium can come from different sources such as cytotoxic injury or infection. In this case, the child underwent chemotherapy, radiation, and had a previous infection. Learner reflection: How might these risk factors contribute to endothelial injury and subsequently increase the likelihood of developing PVOD? Listen to podcast 4 to hear the consortium's decision-making process. Other noninvasive diagnostics for PVOD include resting hypoxemia, severe desaturation upon exercise, and occult alveolar hemorrhage on BAL.1 This child did not have alveolar hemorrhage, as PVOD was suspected early and rapidly treated with empiric high dose steroids. Pulmonary hypertension is often treated with pulmonary vasodilators; however, in the setting of pulmonary venous obstruction, the use of vasodilators can increase congestion in the pulmonary capillary bed, resulting in progressive pulmonary edema. Therefore, vasodilators are often contraindicated in treatment of PVOD.8 The child did receive sildenafil, but fortunately did not worsen as his PVOD was likely patchy and regional. One of the main challenges of this case was to maintain the child's oxygen at an adequate level. The child had resting hypoxemia and thus required supplemental oxygen to maintain adequate saturations. The hypoxemia combined with the tendency of the child towards pulmonary edema with diuretic responsiveness contributed to the PVOD diagnosis. Lung biopsy is often used as confirmation for a diagnosis of PVOD.5 Pathology of lung tissue in a person with PVOD characteristically contains primary venous-based lesions without plexiform lesions. Other findings include secondary arteriolar medial hypertrophy, intimal proliferation, and alveolar hemorrhage.5 Nevertheless, biopsy was contraindicated in this case given the child's oxygen needs and anesthesia risk. Placing the child under anesthesia might have precipitated a hypertensive crisis and subsequent hemodynamic collapse with concern for prolonged intubation. This decision was made to empirically treat the patient instead, with input from a pulmonary hypertension specialist and pediatric pulmonologist. Currently, there is still no clear treatment for PVOD. Defibrotide, an oligonucleotide with antithrombotic, fibrinolytic, and endothelial reparative properties has emerged as a treatment for hepatic veno-occlusive disease. Clinical studies utilizing defibrotide for people with hepatic veno-occlusive disease have been shown to be successful. A study of 45 pediatric cases with hepatic VOD demonstrated a 76% complete resolution of VOD with defibrotide treatment.10 Other studies show similar favorable results of defibrotide treatment for hepatic VOD.10 Moreover, a phase III clinical trial reported a 23% increase in survival rates at Day +100 post-HSCT in people with hepatic VOD.11 Defibrotide is a drug that selectively increases prostaglandin I2 and E2 levels in addition to increasing tissue plasminogen activator. These increased prostaglandins and tissue plasminogen activators work by increasing the fibrinolytic capacity of endothelial cells while also reducing their procoagulant activity.12 Pulmonary alveolar hemorrhage was the most common adverse effect with a fatal outcome.11 The child, however, did not develop hemorrhage postdefibrotide treatment. Due to the pathophysiologic similarities between the hepatic and pulmonary forms of veno-occlusive disease, it may be hypothesized that defibrotide would have a similar benefit in treating PVOD. Nonetheless, medical literature does not yet contain any cases reporting the use of defibrotide to successfully treat PVOD.13 Challenge point: A 3-year-old boy presented with signs and symptoms characteristic of PVOD. After treatment with defibrotide and steroids, the child completely recovered. The child is now at home without any oxygen requirements. Learner reflection: What are the potential implications of the positive outcomes of this case for future research and treatment approaches for PVOD? Listen to podcast 5 to hear the consortium's decision-making process. This particular case report details the use of defibrotide in a child with PVOD post-HSCT. This child was successfully treated with defibrotide with resolution of PVOD. The child is doing well in the outpatient setting without oxygen support and without any major respiratory concerns. Although the child had findings not consistent with PVOD including no worsening with sildenafil and an absence of alveolar hemorrhage, the case was still thought to be due to PVOD despite the lack of a confirmatory lung biopsy. The diagnosis of PVOD was aided by the child's tendency toward pulmonary edema with hypoxemia, patchy infiltrates visualized on chest CT, and tricuspid regurgitation seen on ECHO. Jordan Holthe: Writing—original draft; conceptualization; investigation; writing—review and editing. Paul Boesch: Writing—review and editing; data curation; supervision. Mira Kohorst: Supervision; writing—review and editing. Jonathan Schwartz: Data curation; supervision; writing—review and editing. Asmaa Ferdjallah: Conceptualization; investigation; writing—original draft; writing—review and editing; supervision; data curation. The authors have no funding to report. The authors declare no conflict of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. At this point in time, a broad differential was considered. A collaborative differential was developed in conjunction with numerous pediatric specialties, including pulmonology, cardiology, and hematology and oncology. One of the things on the differential was pulmonary embolism due to the patient's hypoxemia and possible occurrence of a pulmonary hypertensive crisis. Another thing on the differential was pneumonia. The patient had previously been treated with cefepime for 10 days due to bacteremia and possible pneumonia. Therefore, it was a possibility that the pneumonia had reoccurred or never been treated completely in the first place. The leading diagnosis was pulmonary veno-occlusive disease. The team came to this conclusion based on the fact that infectious workup was negative and CT angiogram was negative, along with the patient's persistent hypoxemia and the tendency towards pulmonary edema with diuretic responsiveness. The team then decided to move forward with treatment. Lung biopsy was ultimately deferred due to the increased risk of anesthesia with the severe right ventricle dysfunction that the patient had. The cardiology and anesthesia teams were concerned about precipitating a hypertensive crisis and hemodynamic collapse leading to a prolonged intubation, and so the risks outweighed the potential benefit of a clear diagnosis and empiric treatment was started. The patient was started on defibrotide due to its known ability to treat VOD. Eventually the patient was additionally started on milrinone, epinephrine, and methylprednisolone. The team ordered repeat ECHOS to monitor for signs of improvement. The pathophysiology of pulmonary VOD is complex and remains incompletely understood. Therefore the treatment for this disease really required both cardiology and bone marrow support. From a cardiology standpoint, maximizing oxygen saturation was key, as was right ventricular optimization and improvement in systolic function with milrinone and epinephrine. The advantages of this approach are to improve organ function and address the core pathophysiology of essentially a weak heart. The disadvantages of this, of course, is that it is not addressing the exact pathophysiology of pulmonary VOD. From a bone marrow transplant standpoint, defibrotide was used as it has a very specific mechanism of action for VOD in general. Anticoagulation was deferred due to the high risk of bleeding and steroids were started to allow for anti-inflammatory properties as well as cytokine inhibition. Due to the high risk of using anesthesia in a patient with significant pulmonary hypertension, a diagnostic lung biopsy was not able to be performed and was deferred. Therefore, the clinical team had to rely on blood lab tests as well as imaging to guide understanding of what was occurring. From a cardiology standpoint, this includes a chest X-ray, ECHO, CT Angiogram, pro-BNP, and pulse ox to assess oxygenation. From a bone marrow transplant standpoint, blood pressure was studied intensely as was other broad blood tests referring to cell counts. For this patient, there were quite significant risk factors that contributed to endothelial injury and subsequently increased the likelihood of developing pulmonary VOD. Any patient who undergoes myeloablative chemotherapy is at risk for VOD. Particularly for this patient as part of his regimen, he underwent 3 back-to-back myeloablative stem cell transplants or rescues. So he was at an even increased risk of developing VOD in general as well as other endothelialopathies. This is always the case when patients undergo myeloablative chemotherapies as chemotherapy can cause significant damage to the microvasculature. This case is important because it shows that pulmonary VOD can actually be treated, and treated without a pulmonary biopsy. Lung biopsies can be very helpful in making a rapid and accurate clinical diagnosis; however, they are also very invasive and not always practical clinically. On paper, physicians want to make a clear diagnosis, however with a patient in front of us, the clinical status of the patient supersedes all else and we need to figure out the best way to pick an 'empiric treatment' without adding significant risk to the patient. Additionally, this case highlights the absolute non-negotiable importance of multidisciplinary care for all patients undergoing stem cell transplant. This patient had caring providers from bone marrow transplant team, cardiology, and pulmonology, who were willing to work together to come up with a unifying diagnosis.

Selumetinib is an FDA-approved targeted therapy for plexiform neurofibromas in neurofibromatosis type 1(NF1) with durable response rates seen in most, but not all patients. In this proof-of-concept study, we demonstrate single-cell RNA sequencing(scRNAseq) as a technique for quantifying drug response to selumetinib at the single cell level. scRNAseq data from neurofibroma biopsies was obtained from a public genomics repository. Schwann cell populations were identified through standard clustering techniques and single-cell selumetinib sensitivity was quantified on a scale of 0(resistant) to 1(sensitive) based on the expression pattern of a 500 gene selumetinib sensitivity signature from the BeyondCell sensitivity library. A total of seven plexiform neurofibromas were included in our final analysis. The median absolute number of Schwann cells across samples was 658 cells (IQR: 1,029 cells, Q1-Q3: 135 cells to 1,163 cells). There was a statistically significant difference in selumetinib sensitivity profiles across samples (p < 0.001). The tumor with the highest median selumetinib sensitivity score had a median selumetinib sensitivity score of 0.64(IQR: 0.14, Q1-Q3: 0.59–0.70, n = 112 cells) and the tumor with the lowest median selumetinib sensitivity score had a median score of 0.37 (IQR: 0.21, Q1-Q3: 0.27–0.48, n = 1,034 cells). scRNAseq of plexiform neurofibroma biopsies reveals differential susceptibilities to selumetinib on a single cell level. These findings may explain the partial responses seen in clinical trials of selumetinib for NF1 and demonstrate the value of collecting scRNAseq data for future NF1 trials.

Background: Leukocyte adhesion deficiency type I (LAD-I) is a rare autosomal recessive inborn error of immunity caused by loss of function mutations in ITGB2, which encodes for the β2 common integrin subunit CD18, impairing leukocyte adhesion to inflamed endothelium and migration to sites of infection or injury. Allo-HSCT represents the only therapeutic option to enable survival beyond early childhood but is limited by donor availability, risk of infections, graft-versus-host disease (GvHD) and graft failure (GF). Objective: To characterize outcomes of patients with LAD-I who underwent allo-HSCT over a very recent 10-year period in a large European cohort. Methods: The European Society for Blood and Marrow Transplantation (EBMT) registry allo1 database was queried for patients with LAD-I who received an allo-HSCT using a single stem cell source between 2012 and 2021 and were under 12 years old at the time of transplant. Patient, donor, and transplant characteristics were summarized using descriptive statistics. Ninety-five percent confidence intervals (CI) were calculated for overall survival (OS) and event-free survival (EFS). EFS was defined as survival without GF and/or grade II-IV acute GvHD (aGvHD). Percentages for categories with missing patient data were calculated based on the number of patients with available data. Results: Among 56 patients with LAD-I who met the inclusion criteria, the median age at diagnosis was 2.0 months (IQR: 0.4-2.2) and median time from diagnosis to transplant was 6.8 months (IQR: 2.9-15.5). All patients received a conditioning regimen, with 80.4% receiving myeloablative conditioning. Most patients (80.4%) received serotherapy (mainly with ATG and alemtuzumab); and 98.2% received GvHD immunosuppressive prophylaxis. Among patients with available information, recipient/donor cytomegalovirus (CMV) status mismatch (−/+ and +/-) was present in 19.2% and 5.8%, respectively. Human Leukocyte Antigens (HLA) matched sibling donor (MSD) recipients comprised 39.3% of the cohort, matched unrelated family donors, 30.4%, matched unrelated donors, 12.5%, mismatched donors, 16.1% and unrelated donors, 1.8%. Transplant-related complications, including veno-occlusive disease, transplant-associated microangiopathy and renal insufficiency among others occurred in 31% of patients. Post-transplant infection-related hospitalizations occurred in 45.5% of patients. GF occurred in 16.4% of patients, while 27.3% developed aGvHD (Grade II-IV) and 7.7% developed chronic GvHD. Overall, the mortality rate was 14.3%. OS at 1 and 3 years in the total patient population was 85% (95% CI: 75-95%). Recipient/donor CMV status was significantly associated with OS (p=0.003) with mismatched -/+ and +/- recipient 1 year and 3 year survival of 57% (range: 25-89%) and 67% (range: 13-100%) respectively; 1-3 year OS was 86% (range: 60-100%) and 100% for -/- and +/+ patients (no deaths occurred between year 1 and 3). The 1-year EFS in the total patient population was 58% (range: 45-71%). EFS at 1 year was highest in MSD recipients (86% [range: 71-100%]), whereas it was lowest among patients with HLA mismatched donors (33% [range: 3-64%]). EFS at 3 years was the same as EFS at 1 year in the total patient population and across HLA matched subgroups, as no additional events were recorded after the first year post-transplant in any group. Conclusions: Allo-HSCT is the current standard-of-care for definitive treatment for LAD-I. However, despite a better OS in patients with LAD-I undergoing allo-HSCT, GvHD, GF, and other transplant-related complications remain significant sources of transplant-related morbidity, as shown in this recent cohort. These findings highlight the need for more effective and safer treatment approaches, including possibly lentiviral-based gene therapy.

Protocol for NCT03416530, ONC201-014: ONC201 in Newly Diagnosed Diffuse Intrinsic Pontine Glioma and Recurrent/Refractory Pediatric H3K27M Gliomas.

Upregulated and downregulated metabolites in DIPG007 cells treated with ONC201 versus vehicle.

Introduction: Red cell pyruvate kinase deficiency (PKD) is a rare, autosomal recessive, non-spherocytic hemolytic anemia caused by mutations in the pyruvate kinase liver and red blood cell (PKLR) gene, resulting in a glycolytic defect, causing increased red cell destruction, hyperbilirubinemia, splenomegaly, and iron overload. Splenectomy increases hemoglobin (on average 1.6 g/dL) but is associated with increased infection and thromboembolic risk, and 10-15% of patients with PKD will remain transfusion dependent despite splenectomy. Mitapivat, an allosteric PKLR activator, is approved for the treatment of adult patients, however hemoglobin increases of at least 1.5 g/dL were infrequent in splenectomized patients and/or those with more severe anemia. There is a high unmet medical need for patients with severe PKD who are transfusion dependent. RP-L301 is a gene therapy consisting of autologous CD34+ hematopoietic cells transduced with a lentiviral vector (LV) carrying the codon optimized red cell pyruvate kinase (coRPK) gene. As demonstrated in the RP-L301 Phase 1 study, infusion of genetically corrected autologous hematopoietic cells was associated with favorable safety (there were no RP-L301 associated serious adverse events) and conferred sustained hemoglobin increases and eliminated RBC transfusion requirements. Hb improvement and hemolysis reduction were observed in both adult and pediatric patients with PKD; all patients remain transfusion independent following hematopoietic engraftment; 3 of 4 patients demonstrate sustained normal-range Hb levels with up to 3 years follow-up. These results indicate that RP-L301 has the potential to address the significant unmet need in PKD. Study Design and Methods: This is a single-arm, open-label, global (US, Denmark, Netherlands and Spain) Phase 2 (NCT06422351) clinical trial evaluating a single infusion of RP-L301 in splenectomized pediatric and adult patients (ages 8-55 years) with PKD. Key inclusion criteria include confirmed PKLR mutation, significant anemia and/or RBC transfusion requirements, and adequate organ function. Key exclusion criteria include presence of other causes of hemolysis and previous hematopoietic cell transplant. The primary objective is to evaluate efficacy; the primary endpoint is a Hb increase of >1.5 g/dL at 12 months post-infusion compared to baseline levels. Secondary and exploratory objectives include resolution of anemia to normal range, reduction in transfusion requirements including transfusion-independence, improvement in hemolysis parameters, PKD symptoms and patient-reported outcomes, iron overload reduction, genetic correction, safety, tolerability, and durability of efficacy results. Autologous CD34+ hematopoietic cells are mobilized using granulocyte-colony stimulating factor (G-CSF) and plerixafor and then collected via apheresis, followed by CD34+ cell enrichment by immunoselection and transduction with the LV containing coRPK. RP-L301 is infused following myeloablative busulfan conditioning. Efficacy and safety will be assessed through 24 months post-infusion and patients will have the option for long-term follow-up enrollment up to 15 years. Approximately 10 patients will participate. Conclusion: Based on the favorable efficacy and safety data from the Phase 1 study, this registrational Phase 2 study is designed to enable comprehensive evaluation of the potential of RP-L301 autologous gene therapy to substantively reverse the disease phenotype in pediatric and adult patients with PKD.

Upregulated and downregulated genes in DIPG007 cells treated with ONC201 versus vehicle.

Introduction: Optic pathway gliomas (OPG) are generally low-grade lesions arising from the astrocytes of the visual pathway, most commonly of the optic tract and hypothalamus. While OPGs can be locally aggressive, most are slow growing and detected in one of two ways: (1) during serial screening in NF1 patients and (2) when patients who do not have NF1 begin to experience loss of visual acuity (this is a very rare occurrence in the pediatric population). Large series, such as that of Dutton, and population-based work using the Surveillance, Epidemiology, and End Results (SEER) dataset have shown these lesions to disproportionately affect children (median age at diagnosis of 7 years), accounting for 5% of all pediatric central nervous system (CNS) tumors; males and female appear to be equally affected. Histologically the SEER data show the majority (60%) of pediatric lesions to be pilocytic astrocytoma (PA) and 32% to be low-grade glioma astrocytoma; the latter comprise diffuse and pilomyxoid astrocytoma, though the exact makeup is unclear. Unlike other glial tumors, PA are associated with a high 5-year survival rate (94.1%), and because a majority of OPG are PA, OPG similarly exhibit high short- and long-term survival rates. Because of the low histologic grade and slow-growing behavior of most pediatric OPGs (WHO Grade I lesions), management largely consists of close surveillance. In general, surgery is rarely offered due to tumor location coupled with the fact that other effective treatment options exist for patients with NF1.