Persistent fetal gene expression in childhood neoplasms is usually explained by a maturation block originating in the prenatal phase. In contrast, reactivation of fetal genes in adult malignancies is considered a consequence of oncofetal reprogramming (OFR) and is associated with aggressive disease. By reconstructing epigenetic ontogeny in juvenile myelomonocytic leukemia (JMML), we identified a postnatal maturation state of JMML stem cells with high transcriptional plasticity indicative of OFR in high-risk disease. Similarly, postnatal activation of oncogenic signaling by inducible Ptpn11E76K mutation in mice triggered molecular plasticity and reactivation of fetal gene expression. Integrative multi-omics analysis revealed aberrant CD52 expression as a feature of high-risk JMML stem cells. Anti-CD52 treatment depleted JMML stem cells and blocked disease propagation in xenograft models. Our results challenge the prevailing maturation block model of pediatric leukemogenesis and establish RAS-associated stem cell plasticity as a determinant of OFR and potential therapeutic vulnerabilities in high-risk JMML. SIGNIFICANCE:Persistent fetal gene expression in pediatric malignancies is considered a consequence of prenatal maturation blockade. In this study, we demonstrate that oncogenic PTPN11 mutations enhance cellular plasticity. This leads to partial restoration of fetal molecular programs, creating new therapeutically exploitable vulnerabilities. See related commentary by Miao and Xu, pp. 168.

Background:Pediatric acute leukemia (AL) typically presents with symptoms of cytopenias, organomegaly, and fever. In some instances, however, musculoskeletal pain may be the predominant-occasionally the sole-initial symptom, leading to misdiagnoses such as osteomyelitis. Methods:We conducted a retrospective, multicenter, observational, cohort study including children and adolescents (<18 years) diagnosed with acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) between 2017 and 2024 who initially presented with osteomyelitis-like clinical and/or radiological features. Clinical presentation, laboratory findings, MRI characteristics, and time to leukemia diagnosis were systematically analyzed and compared with the available literature. Results:Six patients were included (four ALL, one ALL relapse, and one AML). All patients presented with musculoskeletal pain; at first evaluation, fever was present in 50% of the individuals. Initial peripheral blood counts were normal in four and only mildly abnormal in two cases. Inflammatory markers and lactate dehydrogenase were elevated in five and four patients, respectively. MRI demonstrated multifocal or diffuse bone marrow signal alterations in all but one case. The median interval from first presentation to leukemia diagnosis was 17 days (range, 1-53). Features observed in patients with delayed diagnosis included transient clinical response to antibiotic therapy, lack of hematologic abnormalities, and multifocal MRI involvement. Conclusion:Pediatric AL can mimic osteomyelitis clinically, hematologically, and radiologically. Awareness of clinical red flags and suspicious MRI patterns may prompt earlier consideration of an underlying hematologic malignancy and reduce diagnostic delay.

BACKGROUND:B-cell lymphoma 2 (BCL-2) is overexpressed in certain solid tumors (including neuroblastoma), representing a promising target. Venetoclax is a first-in-class, oral, highly selective BCL-2 inhibitor. We report safety, pharmacokinetics, and efficacy of venetoclax in children and young adults with relapsed/refractory solid tumors. PROCEDURE:M13-833 (NCT03236857) was a Phase 1, open-label, global, two-part study. Patients received age-/weight-adjusted venetoclax (400 or 800 mg/day adult equivalent dose [AED]) continuously or intermittently (Days 1-10 of 21-day cycles) as monotherapy or with cyclophosphamide and topotecan (Cy-Topo; Cy 250 mg/m2/day + Topo 0.75 mg/m2/day; intravenously Days 1-5) and myeloid growth factor support. Primary objectives included safety and pharmacokinetic assessments of venetoclax; secondary objectives included efficacy. RESULTS:Fifty-nine patients in the neuroblastoma (n = 36; median age: 8 years [range: 1-17]) and solid tumor cohorts (n = 23; median age: 16 years [range: 3-24]) were assessed. Grade ≥3 treatment-emergent adverse events (TEAEs) occurred in 35/36 (97%) and 21/23 (91%) neuroblastoma and solid tumor patients, respectively; febrile neutropenia was the most common serious TEAE (neuroblastoma 69%; solid tumors 57%). TEAEs leading to discontinuation of venetoclax occurred in 17% (neuroblastoma) and 9% (solid tumors) of patients. No events of tumor lysis syndrome were observed. Peak venetoclax concentrations (Tmax) occurred 4-6 h post-dose; exposure was comparable across age/weight subgroups. Objective response rates were 31% (neuroblastoma) and 22% (solid tumors). CONCLUSIONS:Recommended Phase 2 dose of venetoclax was 800 mg/day AED (Days 1-10 of 21-day cycles). Venetoclax combined with Cy-Topo chemotherapy had a predictable safety profile and showed modest clinical activity in these cohorts.

File containing Supplementary Figures S1-S15

ABSTRACT Molecular testing in hematology requires different assays for disease subgroup identification, risk stratification and selection of appropriate treatment regimens. Yet, molecular tests are not necessarily standardized between diagnostic laboratories, resulting in varying turnaround times and potentially divergent results. To resolve this issue and enable single-assay molecular testing, we have developed a hierarchical classification framework that combines epigenetic and genetic data from whole genome nanopore sequencing (WGNS) with machine learning to determine disease entities, epigenetic subgroups (epitypes) and genetic aberrations in hematopoietic neoplasms. We curated DNA methylation data from 5,420 samples and trained a classifier allowing entity-level diagnostics featuring 21 conditions, including healthy controls, acute and chronic myeloid and lymphoid neoplasms. This classifier was subsequently combined with entity-specific epitype classifiers predicting 44 therapeutically or prognostically relevant states, followed by integration of genetic data. Benchmarking of the combined (epi-)genetic testing strategy using WGNS confirmed high accuracy in the detection of diagnostic groups and risk stratification, and identified diagnosis-defining molecular alterations that were not reported by standard-of-care work-up.

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myelodysplastic/myeloproliferative neoplasm characterized by distinct epigenetic signatures that facilitate molecular classification. This study aimed to evaluate the diagnostic utility of locus-specific DNA methylation in the bone morphogenetic protein 4 (BMP4) gene as a single predictor of disease outcomes in a cohort of 111 children diagnosed with JMML, alongside 9 healthy controls. Methylation levels of BMP4, assessed through targeted bisulfite next-generation sequencing (bs-NGS), were heterogeneous within the JMML cohort and were significantly associated with clinical risk factors, such as patient age, and fetal hemoglobin (HbF) levels. A comparative analysis of BMP4 bs-NGS and genome-wide methylation array data revealed a strong positive correlation (p < 0.001). The sensitivity and specificity of BMP4 bs-NGS for classifying high-methylation cases were 0.612 and 0.887, respectively. For PTPN11-mutant patients (N = 40), the sensitivity was 0.667 and the specificity was 0.842. Survival analysis indicated that patients with high BMP4 methylation (BMP4h) had lower 5-year disease-free survival (DFS) rates than those with normal BMP4 methylation (BMP4n). Specifically, the 20% of patients with highest BMP4 methylation had a 5-year DFS of 0.38, in contrast to 0.62 for the lowest 20% (p = 0.007). These findings highlight the potential of BMP4 methylation analysis as a complementary biomarker for JMML risk stratification, mirroring genome-wide methylation profiles known to associate with prognostic subgroups.

PURPOSE:Modern ALL therapy aims to reduce toxicity, while maintaining and improving the current high cure rates. Acute and late sequelae of anthracyclines are of major concern. The AIEOP-BFM ALL 2009 trial aimed to clarify the need for anthracyclines in low-risk patients. PATIENTS AND METHODS:After 2 weeks of induction therapy, which included two daunorubicin (DNR) doses once weekly (30 mg/m2 each) as part of a 4-drug therapy, patients age 1-17 years with newly diagnosed non-high-risk B-ALL either positive for ETV6::RUNX1 or with rapid treatment response, as assessed by induction day-15 evaluation, were randomly assigned to receive either two additional doses of DNR (control arm [CA]) or no further DNR during induction (experimental arm [EA]). Patients treated as randomly assigned were included in the primary analysis on noninferiority in event-free survival (EFS). Adverse reactions of special interest (ARSI) were analyzed in the as-treated population. RESULTS:Of 6,136 patients enrolled in AIEOP-BFM ALL 2009, 2,514 patients (41.0%) were eligible for this random assignment, with 82.7% actually randomly assigned (EA: n = 1,040 and CA: n = 1,039). The 5-year EFS was 92.5% (SE 0.8%) in CA and 92.2% (SE 0.9%) in EA. Accordingly, cumulative incidence of relapse was 5.8% (SE 0.7%) and 5.7% (SE 0.7%), and overall survival was 97.6% (SE 0.5%) and 97.4% (SE 0.5%) in CA and EA, respectively. Life-threatening and fatal ARSI were similar in the two arms, but there was a three times lower incidence of invasive fungal infections in the EA (0.5% v 1.5%, P = .02). CONCLUSION:A reduced DNR dose during induction did not compromise the outcome of patients with favorable prognostic factors but did diminish infectious toxicity indicated by the lower rate of invasive fungal infections.

Background: Febrile neutropenia (FN) is a common and potentially life-threatening complication in pediatric oncology. Rapid initiation of empiric antibiotics is critical to improving prognosis. This study evaluated the impact of simple changes to a standard operating procedure (SOP) for FN treatment on the time-to-antibiotic (TTA) in pediatric cancer patients, as well as potential clinical effects. Methods: In this retrospective, single-center, cohort study, children with cancer presenting with FN at the emergency room (pedER) or oncology outpatient department (OD) were included over two one-year periods—before and after SOP adaption. The revised SOP defined a target TTA of ≤30 min. The primary endpoint was to compare median TTA and the proportion of FN episodes meeting target TTA. Secondary endpoints comprised adverse events (AEs) (e.g., ICU admission, need for respiratory or circulatory support, sepsis criteria). Results: After SOP adaption, 32.9% of episodes met target TTA, up from 5.9% before. Median TTA was significantly reduced (44 min vs. 93 min). The improvement persisted during the study period. AE rates showed no significant change. Conclusions: Simple procedural adjustments may significantly improve quality indicators of care, e.g., reducing TTA in pediatric FN patients. These adjustments may be transferable to other pediatric oncology settings.

Neurofibromatosis 1 (NF1) is the most common cancer predisposition syndrome and is characterized by a wide range of clinical manifestations, including increased risk of developing various types of malignancies. We report nine patients with NF1 who presented with concurrent diagnoses of juvenile myelomonocytic leukemia and low-grade glioma, a rare combination. One notable feature of this cohort is that eight patients (89%) harbored nonsense germline NF1 variants. This case series highlights the emergence of synchronous or metachronous malignancies in children with NF1 and provides a rationale for heightened awareness of this phenomenon.

BACKGROUND:Invasive fungal diseases (IFD) pose significant challenges in paediatric oncology. Their management is complicated by limited paediatric-specific evidence, lack of standardised protocols and variability in resources across centres. This study assessed current practices and addressed the challenges in the prevention, diagnosis and treatment of IFDs in paediatric oncology centres across Germany, Austria and Switzerland. METHODS:A questionnaire was distributed to senior paediatric oncologists in 70 paediatric oncology centres across Germany, Austria and Switzerland, gathering data on centre infrastructure, infectious disease (ID) expertise, annual cumulative IFD incidence in 2023, diagnostic tools, antifungal prophylaxis, treatment and follow-up practices for IFD. Responses were analysed descriptively. RESULTS:Sixty-two centres responded, with a median of 56 (IQR 40-75) new oncological diagnoses per centre; 54.8% of centres managed allogeneic HCT patients. IFDs were reported in 88.7% of centres, with a median cumulative IFD incidence of 4.6% (IQR 3.0%-5.9%). No significant association was found between cumulative IFD incidence and the number of transplants, antifungal prophylaxis protocols and availability of ID consultation services. ID consultation was available in 58.1% of centres, with 24/7 support provided in 41.7% of these centres. Larger centres more frequently had paediatric ID specialists, ID consultation services and access to therapeutic drug monitoring. CONCLUSIONS:The observed heterogeneity in mycology expertise and IFD management strategies across centres reflects the inherent complexity of IFDs and the diagnostic and therapeutic uncertainties amid limited evidence. Strengthening oncology-ID networks and implementing digital consultation platforms may promote high-quality, equitable care, particularly for those with fewer in-house resources.

Juvenile myelomonocytic leukemia (JMML) is a myelodysplastic/myeloproliferative neoplasm of early childhood driven by RAS pathway mutations. Allogeneic hematopoietic stem cell transplantation (HSCT) is the therapy of choice for most patients. However, relapse rate is high, in patients with adverse features, frequently noted in PTPN11 -mutated JMML, or in patients without evidence of graft-versus-host disease (GvHD). Here we set out to understand the mechanisms associated with oncogenic PTPN11 immune escape. Analyzing primary PTPN11 -mutated JMML samples and MxCre;Ptpn11D61Y/+ mice, we observed elevated expression of immune checkpoint molecules, including ectonucleotidases CD39 and CD73 - key mediators of the adenosine pathway - on monocytic and granulocytic leukemic cells. Stimulation with GM-CSF, a central mediator of JMML pathogenesis, induced ectonucleotidases expression on granulocytes and monocytes. In contrast, MEK inhibition downstream of Ptpn11D61Y/ + reduced ectonucleotidases expression. Functionally, Ptpn11D61Y/ +-mutated myeloid cells suppressed activation and proliferation of wild-type (WT) T lymphocytes, an effect recapitulated by adenosine and reversed by pharmacological CD39 inhibition with POM-1. In vivo , POM-1 treatment of MxCre ; Ptpn11D61Y/+ mice presenting with myeloproliferation reduced spleen size and partially restored immune responsiveness. Moreover, POM-1 induced apoptosis in murine Ptpn11D61Y/+ myeloid cells, highlighting a dual therapeutic benefit of CD39 inhibition in JMML. Together, these findings suggest that targeting the adenosine pathway may represent an immunomodulatory approach to enhance T cell-mediated control of JMML, particularly in the context of HSCT and relapse prevention. ### Competing Interest Statement The authors have declared no competing interest. Collaborative Research Center (CRC) 1479 Oncoescape, 441891347

EWOG-MESRAT (European Working Group-Methylation Signatures and Response to Azacitidine Therapy; DRKS00007185) is an investigator-initiated trial that studied EPIC array-based DNA methylation patterns and next generation sequencing (NGS)-based variant allele frequencies (VAFs) of driver mutations in peripheral blood (PB) and bone marrow (BM) of 11 patients with newly diagnosed juvenile myelomonocytic leukaemia (JMML) during therapy with azacitidine. We demonstrate that the pharmacodynamic activity of azacitidine can efficiently be monitored in PB and BM. DNA methylation subgroup classification was linked to clinical response after three cycles of azacitidine and found to be conserved between PB and BM in all patients. In contrast, neither changes in VAFs nor changes in DNA methylation patterns during the course of therapy correlated with therapy outcome among the 11 study patients. This work thus supports the value of DNA methylation subgroup classification from PB samples for response prediction of single-agent azacitidine in patients with JMML.

BACKGROUND:Venetoclax is a potent, oral BCL-2 inhibitor approved as combination therapy for the treatment of adults with newly diagnosed acute myeloid leukaemia (AML) who are ineligible for intensive chemotherapy. This study evaluated the safety and preliminary efficacy of venetoclax alone or combined with chemotherapy in paediatric and adolescent/young adult patients with relapsed/refractory AML. PROCEDURE:In this phase 1, open-label, two-part, multicentre study, paediatric and adolescent/young adult patients (<25 years of age) with relapsed/refractory AML were treated with venetoclax alone or in combination with hypomethylating agents or cytarabine. The study is registered with ClinicalTrials.gov, NCT03236857. RESULTS:A total of 37 patients received treatment with either venetoclax as a monotherapy (n = 3) or in combination with decitabine (n = 5), azacitidine (n = 19), low-dose cytarabine (n = 1) or high-dose cytarabine (HDAC; n = 9). Febrile neutropenia (57%), hypokalaemia (38%), and thrombocytopenia (35% [thrombocytopenia, 19%; platelet count decreased, 16%]) were the most common grade 3/4 treatment-emergent adverse events. Across all venetoclax combinations, the overall response rate (ORR) was 24% (9/37), and the median duration of response was 2.6 months (95% CI, 0.5-7.9). Among the combinations, ORR was 44% with venetoclax plus HDAC and 21% with venetoclax plus azacitidine. In biomarker-evaluable patients, responses to venetoclax plus chemotherapy were observed in patients harbouring mutations across a range of functional classifications and heterogeneous BH3 family member dependencies. CONCLUSIONS:Venetoclax alone or combined with chemotherapy was well tolerated in paediatric and adolescent/young adult patients with relapsed/refractory AML, with promising, although transient, responses with venetoclax plus HDAC or azacitidine.

Introduction: Febrile neutropenia (FN) is a common complication in paediatric patients receiving chemotherapy and ranks among the most common causes of death in patients with cancer. The standard of care is the rapid identification of high-risk patients upon presentation in the emergency room (ER) and initiation of appropriate diagnostic and therapeutic measures. The prompt administration of broad-spectrum antibiotics appeared to reduce morbidity and mortality in all cancer patients developing FN. The interval between admission and antibiotics administration, also referred as time-to-antibiotics (TTA), has therefore been used as a quality-of-care benchmark in cancer treatment. Primary objective: To assess the effect of a change of a standard of procedure (SOP) for the treatment of FN on the TTA in pediatric cancer patients receiving chemotherapy. Secondary objective: To assess the clinical impact of the new SOP on the course of FN episodes in pediatric cancer patients. Study design: This retrospective, single-center, cohort study included pediatric cancer patients presenting with FN in the emergency room (ER) or in the outpatient department of the pediatric hematology and oncology clinic (OD) of the Medical Center of the University of Freiburg from May 2019 until May 2021. Median TTA before and after the introduction of the new SOP, i.e. May 2020, was compared, as well as the occurrence of adverse events during FN episodes. A target TTA ≤ 30 minutes was demanded within the new SOP, compared to no target TTA in the previous SOP. The primary endpoint was defined as fulfilment of target TTA in >50% of FN episodes. Secondary endpoints comprised the rate of complications occurring during FN episodes (e.g. admission in ICU, use of vasopressors, need of respiratory support). Results: 227 Episodes of FN were included in this study. The primary endpoint was not met, as target TTA was achieved in 32.9% of cases only. However, a significant reduction of the median TTA was observed (93 min vs. 44 min). The improvement of median TTA persisted during the study period. The presence of tachycardia and fever at presentation were associated with shorter TTA. Higher CRP values related to increased hospital length of stay and higher rate of complications during the FN episodes. Conclusions: This study highlights how simple implementations of a procedural algorithm even in large pediatric cancer centers can have a significant impact on the quality of care, e.g. on TTA in FN. Our SOP appears as an effective approach with longer lasting effects to improve care of FN in children with cancer. However, the retrospective study design is a major limitation that might obscure the relationship between SOP effects, TTA and patient outcome in FN episodes. Prospective evaluations of standardized procedures may further improve guidelines for the treatment of FN.