Abstract Background Paediatric-type diffuse high-grade glioma (PDHGG) carries a dismal prognosis despite multimodal therapy. Profound intra- and intertumoral heterogeneity,an immunosuppressivemicroenvironment, and adaptive resistance drive treatment failure and rapid progression. Improved understanding of tumor-intrinsic and therapy-induced molecular alterations is thereforerequiredto enable rational, personalizedcombinationstrategies integrating immune-based therapies. Methods A clinical study in adult and children with high-grade glioma was conducted in a swedish cohort with autologous tumor-lysate-pulsed DC vaccinations. Primary outcomes weretoxicityand secondary outcomes included progression-free survival (PFS) and overall survival (OS), alongsideimmuno-monitoringof peripheral T-cell and regulatory T-cell (Treg) activation.Toidentifyrelapse-associated pathways and therapeutic targets, the therapy-associated molecular changes were assessed in bulk and single-cell RNA sequencing ofmatched primary–relapse tumors from one pediatric patient. Results The vaccinations were feasible and well tolerated. Immunomonitoringrevealed heterogeneous CD4+, CD8+, and T-reg responses. From longitudinal transcriptomic profiling and single-cell analysis we could show thatthe majority oftumor cells displayed an oligodendrocyte precursor-like (OPC-like) phenotype andat relapse,multipleradiation- andtherapy-responsesignatureswereidentifiedaspotential driversinrelapsebiology,whereOPC-likecellsdisplayed upregulation ofIGFBP2,as well as loss ofCDKN2Aexpressionsuggesting a complex SV signature including increasing MYC enhancer mutations among other oncogenes. The plasticity of these tumors, their aggressivnessand the evolution of treatmentresistance through time must be takenintoconsiderationwhenidentifyingtargetsfordrugrepurposing. Conclusion DC vaccination patients showed immunologicallyactivitybutshowedlimitedclinicalbenefit. Atrelapsetherapy-drivenreprogrammingsupportedintegratedimmunogenomicstrategiesandrationalcombinationtherapiesforrecurrentPDHGGs.

We report the de novo discovery of a cyclic peptide inhibitor of ROR1/2 from the RaPID (random nonstandard peptides integrated discovery) system. Multiple independent affinity selection campaigns converged on a highly conserved family of sequences exhibiting single-digit nanomolar binding affinities to ROR1. One peptide from this family containing three nonproteinogenic residues, including a key cyclic β-amino acid, displayed pronounced activity in cellular assays. Strikingly, this peptide induced potent growth inhibition across multiple Diffuse Intrinsic Pontine Glioma (DIPG) cell lines, whereas other family members had no measurable cellular effects. Mechanistic analysis revealed that the active peptide uniquely competes with Wnt5a, the endogenous ligand of ROR1, for receptor binding. To our knowledge, this report provides the first evidence implicating ROR1/2 as a viable therapeutic target in DIPG and establishes macrocyclic peptides as a promising modality for its pharmacological inhibition.

Abstract A major challenge in cancer care is predicting a patient’s risk of relapse after primary treatment. Diffuse Midline Gliomas (DMGs) are intrinsically resistant to standard chemoradiotherapies, and high-throughput genomic profiling of primary and relapse tumors integrated with functional precision medicine are used here to target relapse-initiating cells early. We have established patient-derived xenografts (PDXs) from matched primary–relapse pairs of pediatric diffuse high-grade glioma to study tumor evolution within individual patients using integrated analyses of patient tissue, PDXs, three-dimensional assembloids, and xenograft-derived cell models (XDCLs). Tumoral single cell suspensions from matched primary-relapse pairs were injected orthotopically in immunodeficient NOG mice. We established in vitro XDCLs in stem cell conditions from the first generation of animals transplanted. Co-culture of these cells in 3D assembloids were performed using 180-250 days old cortical organoids. Single-cell RNA sequencing and bulk RNA sequencing (RNA-seq) on patient biopsies, their corresponding PDXs from first generation of mice, and XDCLs, was performed to track potential changes in vivo and in vitro and to further validate primary-relapse specific evolution. Multi-omics profiling across models revealed conserved yet distinct transcriptional programs between primary and relapse, with identification of persistent ACVR1 mutation in both primary and relapse. Phenotypic characterizations across different models through density mapping and invasion analyses confirmed proliferative-driven growth mode in relapse tumor models. Relapse assembloid cultures displayed reduced sensitivity to radiation treatment which highlighted the emergence of resistance in the relapse induced through increased mTOR signaling and reduced TP53 pathway. DMG primary-relapse in vitro XDCLs / 3D assembloid models together with PDXs is a robust approach to study different aspects of relapse-specific tumor biology. Our findings reveal a phenotypic transition from an infiltrative, astrocytic-like, low-proliferative state in primary tumor to a bulky, high proliferative, OPC-like, TP53 mutated, mTOR-associated growth state in relapse. While certain PI3K/mTOR inhibitors showcased attenuated growth in relapse derived models, incomplete responses suggested compensatory signaling pathways, motivating our ongoing evaluation on combination strategies targeting both mTOR and ACVR1 pathways. Our work underlines the importance of modelling disease evolution, showcasing distinct therapeutic vulnerabilities in relapse compared to primary counterparts.

The second Paediatric Therapeutic Development Workshop focused on medulloblastoma. Between 60-70% of patients with medulloblastoma survive, but survivors have significant long-term side effects, and the highest-risk groups have a probability of survival <10%. Thus, the unmet need is to develop therapeutics targeting specific vulnerabilities in medulloblastoma including poor prognosis disease groups (SHH-medulloblastoma, MYCN amplified or TP53 mutated; and Group 3 medulloblastoma, c-MYC amplified) and developing less-toxic therapies for good prognosis disease (WNT-medulloblastoma). The Workshop concluded that (i) targeting SRC by a degrader is a high priority, (ii) inhibition of c-MYC and MYCN tumour-relevant functions for poor prognosis groups is a priority, (iii) targeting WNT-medulloblastoma via a radiolabelled theranostic antibody is an innovative approach for good prognosis tumours to further reduce toxicity, and (iv) B7-H3 has many advantages for CAR T-cell and ADC-based approaches. Based on currently available evidence, combinations of central nervous system penetrant selective PARP-1, CHK1/2 or CDK9 inhibitors with an ATR inhibitor could potentially be evaluated in early-phase trials for high-risk patients; however, these combinations require robust evaluation in pre-clinical models first. Early-phase clinical studies should be international, have novel designs to address small patient numbers and based on an understanding of biology with correlative biological studies. Both developing therapeutics targeting specific vulnerabilities in medulloblastoma and evaluating combinations of existing medicinal products are required to improve outcome and reduce long term sequalae.

ABSTRACT Ovarian tissue cryopreservation enables fertility preservation in females undergoing gonadotoxic therapies, restoring fertility in adults. Although offered even before puberty, the childhood ovary and its vulnerability to therapy remain poorly characterized. Here, ovarian tissue from 16 patients undergoing fertility preservation (aged 1-16 years) and 11 adult controls (aged 22-32 years) was analyzed using single-cell RNA sequencing, spatial transcriptomics, and multiplex immunostaining. In chemotherapy-naïve samples, 13 somatic cell populations underwent extracellular matrix remodeling, vascular, neural, and stromal maturation during puberty, whereas changes in germline related to chromatin remodeling. Spatial transcriptomics resolved 23 clusters across, revealing distinct tissue organization and follicular niche composition between children and adults. Chemotherapy exposure depleted perifollicular and vascular cells, suppressed intercellular signaling, and dysregulated over half of puberty-associated genes, converging on stress responses and extracellular matrix remodeling, with SEPTIN7 as a potential biomarker. These findings uncover critical developmental vulnerabilities of the pediatric ovary relevant to fertility preservation.

We report the de novo discovery of a cyclic peptide inhibitor of ROR1 from the RaPID (Random nonstandard Peptides Integrated Discovery) system. Multiple independent affinity selection campaigns converged on a highly conserved family of sequences exhibiting single digit nanomolar binding affinities to ROR1. One peptide from this family containing three non-proteinogenic residues, including a key cyclic β-amino acid, displayed pronounced activity in cellular assays. Strikingly, this peptide induced potent growth inhibition across multiple Diffuse Intrinsic Pontine Glioma (DIPG) cell lines, whereas other family members had no measurable cellular effects. Mechanistic analysis revealed that the active peptide uniquely competes with Wnt5a, the endogenous ligand of ROR1, for receptor binding. To our knowledge, this report provides the first evidence implicating ROR1 as a visible therapeutic target in DIPG and establishes macrocyclic peptides as a promising modality for its pharmacological inh

Results of differential gene expression analysis between GTML tumours and GTS tumours.

Results of differential gene expression analysis between GTML3 tumor cells treated for 48 hours with DMSO or Dox.

Glioblastoma (GBM) is an aggressive and incurable brain tumor, with treatment resistance and recurrence posing persistent clinical challenges. The transcription factor SOX9 has recently emerged as a key regulator of therapy resistance. Still, no specific SOX9 drugs exist, likely due to the inherent difficulty of targeting transcription factors. Here, we delineate a critical role for SOX9 in driving resistance to standard-of-care radiation and temozolomide (TMZ) therapy. Using a panel of 35 patient-derived GBM cell lines of varying subgroups and MGMT status we defined TMZ sensitivities and demonstrated that SOX9 is selectively activated in resistant lines following cytotoxic stress. Elevated SOX9 expression correlated with poor patient survival by attenuating TMZ-induced DNA damage. CRISPR-based depletion of SOX9 sensitized GBM cells to radiation and TMZ, underscoring its functional importance in mediating resistance. To therapeutically exploit this vulnerability, we developed a SOX9-responsive suicide gene therapy. This system couples the Herpes Simplex Virus Thymidine Kinase (HSV-TK) gene to a SOX9-inducible enhancer, enabling selective ablation of SOX9-positive GBM cells upon ganciclovir (GCV) treatment. Delivery via AAV2 or AAV9 vectors effectively eliminated resistant tumors and significantly extended survival in both immunodeficient and immunocompetent orthotopic GBM mouse models. SOX9-directed gene therapy synergized with radiation to enhance DNA damage and even led to long term survival due to a reprogrammed tumor immune microenvironment. We establish SOX9 as a central driver of therapeutic resistance in GBM and present a first-in-class strategy to selectively target resistant tumor populations. Clinical translation of this strategy holds promise for improving outcomes in patients with SOX9-driven tumor relapse.

The transcription factor SOX9 is associated with poor prognosis and therapy resistance in medulloblastoma, one of the most common malignant pediatric brain tumors. While SOX9 knockdown inhibits tumor recurrence in animal models, no direct SOX9 inhibitors are currently available for clinical use. SOX9 functions as a potent activator of a minimal COL2A1 enhancer that can work as a SOX9-specific response element. In this study, we aimed to engineer this response element to identify therapy-resistant tumor cells and further use it as a gene therapy to selectively target SOX9-expressing medulloblastoma. We employed lentiviral and adeno-associated virus (AAV) vectors, flow cytometry, immunohistochemistry, immunoblotting, in vitro cell models, and in vivo orthotopic mouse models to track and eliminate therapy-resistant medulloblastoma cells. We demonstrated that SOX9-positive cells can be efficiently labeled using a reporter construct containing four tandem SOX9-specific response elements coupled to a minimal promoter (S9RE) driving expression of enhanced green fluorescent protein and luciferase. Furthermore, S9RE was used in an AAV vector to drive expression of herpes simplex virus thymidine kinase (TK), enabling a suicide gene therapy approach wherein ganciclovir selectively kills dividing SOX9-positive tumor cells. This AAV-based gene therapy, when combined with fractionated radiation, synergized with standard therapy to effectively eliminate SOX9-positive medulloblastoma cells in vitro and in vivo. The findings support the potential of SOX9-driven gene therapy as a targeted strategy to overcome therapy resistance in malignant pediatric brain tumors.

Paediatric high-grade gliomas (pedHGGs) are highly invasive brain tumours accounting for approximately 15 % of all central nervous system (CNS) tumours in children and adolescents. The outcome for these tumours is generally poor with 5-year survival rates of less than 20 %. Despite improved biological insights into pedHGGs and the promise of more effective therapies, little progress has been made in the effective treatment and the outcome of these tumours over the last four decades. Much of the evidence for the use of chemotherapy in pedHGGs is extrapolated from adult data, and the evidence for its use in the paediatric population is still weak. This guideline was written by members of the SIOPE HGG Working Group as part of the European Standard Clinical Practice (ESCP) Project. The guideline aims to integrate available evidence-based and expert opinion-based information to assist healthcare professionals in the management of pedHGGs and in an attempt to provide equity in healthcare reflecting the varying resources of each European country.

The transcription factor SOX9 is a marker of poor prognosis and therapy resistance in medulloblastoma, one of the most common malignant brain tumor types in children. Knockdown or downregulation of SOX9 inhibits medulloblastoma recurrence in animal models but there are currently no direct SOX9 inhibitors available for clinical use. SOX9 acts as a selective and potent activator of a minimal COL2A1 enhancer. Here, we show that cells with SOX9 activity can be efficiently labeled when four copies of this response element are coupled to a minimal promoter (S9RE-minP) driving enhanced green fluorescent protein and luciferase. We further show that S9RE-minP can be used in an adeno-associated virus (AAV) to drive herpes simplex virus thymidine kinase in a suicide gene therapy where ganciclovir can be converted to cytotoxically target mitotic SOX9-positive cancer cells. This AAV gene therapy could be safely combined with fractionated radiation and worked efficiently together with standard therapy to eradicate SOX9-positive medulloblastoma cells in vitro and in vivo. We suggest this treatment as an attractive approach to target therapy-resistant malignant pediatric brain tumors.

Results of differential gene expression analysis between pre-GTS1 tumor cells treated for 48 hours with DMSO or Dox.

List of genes that are part of a quiescent cell signature and display regions of differentially opened chromatin.

Medulloblastomas comprise a molecularly diverse set of malignant pediatric brain tumors in which patients are stratified according to different prognostic risk groups that span from very good to very poor. Metastasis at diagnosis is most often a marker of poor prognosis and the relapse incidence is higher in these children. Medulloblastoma relapse is almost always fatal and recurring cells have, apart from resistance to standard of care, acquired genetic and epigenetic changes that correlate with an increased dormancy state, cell state reprogramming and immune escape. Here, we review means to carefully study metastasis and relapse in preclinical models, in light of recently described molecular subgroups. We will exemplify how therapy resistance develops at the cellular level, in a specific niche or from therapy-induced secondary mutations. We further describe underlying molecular mechanisms on how tumors acquire the ability to promote leptomeningeal dissemination and discuss how they can establish therapy-resistant cell clones. Finally, we describe some of the ongoing clinical trials of high-risk medulloblastoma and suggest or discuss more individualized treatments that could be of benefit to specific subgroups.

Abstract BACKGROUND Whole genome sequencing (WGS) is the most informative singular molecular assay in cancer diagnosis. Recent evidence demonstrates that WGS can add diagnostic information and change the management of childhood cancer, and thus is being increasingly employed in clinical settings globally. However, it remains unknown whether WGS can accurately recapitulate existing multi-assay standard-of-care (SOC) genomic testing used in pediatric cancer diagnostics. In this study we evaluate the concordance between WGS and SOC findings from an unselected cohort of children across 8 centres from two healthcare systems (England and Sweden) that offer routine WGS. METHODS We compared WGS and SOC genomic test reports for children under 18 years presenting with new or relapsed cancer between January 2021 and November 2023 across 2 English centres; Cambridge University Hospital and Great Ormond Street Hospital, and 6 Swedish centres; Gothenburg, Karolinska, Linköping, Lund, Umeå and Uppsala. Only WGS findings reported to clinicians were evaluated, without re-analysis of genomes. Tests were described as ‘concordant’ where WGS and SOC were in complete concordance (positive or negative) for all SOC-detected variants. Discordance described occasions where SOC detected findings not identified by WGS. ‘Additional findings’ described cases where WGS provided disease-relevant findings above SOC testing. Only disease-relevant variants were considered in the analysis. RESULTS A cohort of 1032 patients with 1841 SOC molecular tests was included – 436 with haematological malignancies and 596 with solid tumor malignancies (528 from England, and 504 from Sweden). WGS recapitulated 99.3% of SOC tests performed, across all types of genomic alteration (1829/1841). Of the 12 instances of discordance, 3 related to poor WGS sample purity, 5 were gene fusions, 1 low variant allele frequency (0.02) internal tandem duplication, 2 single nucleotide variants and 1 copy-number aberration. WGS provided additional disease relevant findings in 19.7% of cases (203/1032). DISCUSSION Deployment of available SOC genomic testing for cancer diagnostics is highly variable across nations, individual centres and disease entities, and is usually dictated by test availability, cost and likely clinical yield, in a non-agnostic manner. For the first time we demonstrate, across two national systems, that WGS faithfully recapitulates the vast majority of SOC findings irrespective of mutation class, cancer type and variant calling algorithm. Sample quality and intra-tumoral heterogeneity likely account for the few discrepancies observed. Barriers to implementation of routine WGS as the only molecular diagnostic assay for pediatric cancer are cost, analytical expertise, and turnaround time (TAT). Our group is systematically studying the health economic benefits of WGS as a single assay to replace all SOC testing. Finally, an ongoing collaborative project aimed at reducing TAT to under 48 hours using novel technology has demonstrated feasibility in a small number of patients to date. Citation Format: Jonathan Kennedy, Sarah M. Leiter, Angus Hodder, Sheng-Yuan Kan, Jack Bartram, Giuseppe Barone, Michael Gattens, Matthew J. Murray, Sam Behjati, Patrick Tarpey, Matthew Cullen, Antony Ceraulo, Karin Langenberg, Jan Molenaar, Sandra Wessman, Frida Abel, Gustaf Ljungman, Geraldine Giraud, Hakon Anderson Blomstrand, Zdenek Rohan, Anna Staffas, Christina Orsmark-Pietras, Tatjana Pandzic, Irina Golovleva, Linda Fogelstrand, Jonas Abrahamsson, Ulrika Norèn-Nyström, Josefine Palle, Thoas Fioretos, Lucia Cavelier Franco, Gisela Barbany, Nadège Corradini, Gudrun Schleirmacher, Richard Rosenquist, David Gisselsson, Aditi Vedi. Whole genome sequencing can reproduce all standard-of-care diagnostics for childhood cancer: Results from two national systems [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B011.

BackgroundChildhood cancer predisposition (ChiCaP) syndromes are increasingly recognized as contributing factors to childhood cancer development. Yet, due to variable availability of germline testing, many children with ChiCaP might go undetected today. We report results from the nationwide and prospective ChiCaP study that investigated diagnostic yield and clinical impact of integrating germline whole-genome sequencing (gWGS) with tumor sequencing and systematic phenotyping in children with solid tumors.MethodsgWGS was performed in 309 children at diagnosis of CNS (n = 123, 40%) or extracranial (n = 186, 60%) solid tumors and analyzed for disease-causing variants in 189 known cancer predisposing genes. Tumor sequencing data were available for 74% (227/309) of patients. In addition, a standardized clinical assessment for underlying predisposition was performed in 95% (293/309) of patients.FindingsThe prevalence of ChiCaP diagnoses was 11% (35/309), of which 69% (24/35) were unknown at inclusion (diagnostic yield 8%, 24/298). A second-hit and/or relevant mutational signature was observed in 19/21 (90%) tumors with informative data. ChiCaP diagnoses were more prevalent among patients with retinoblastomas (50%, 6/12) and high-grade astrocytomas (37%, 6/16), and in those with non-cancer related features (23%, 20/88), and ≥2 positive ChiCaP criteria (28%, 22/79). ChiCaP diagnoses were autosomal dominant in 80% (28/35) of patients, yet confirmed de novo in 64% (18/28). The 35 ChiCaP findings resulted in tailored surveillance (86%, 30/35) and treatment recommendations (31%, 11/35).InterpretationOverall, our results demonstrate that systematic phenotyping, combined with genomics-based diagnostics of ChiCaP in children with solid tumors is feasible in large-scale clinical practice and critically guides personalized care in a sizable proportion of patients.FundingThe study was supported by the Swedish Childhood Cancer Fund and the Ministry of Health and Social Affairs.