Cell-free DNA (cfDNA) profiling enables minimally invasive cancer detection and monitoring. We present SIMMA, a low-input single-molecule sequencing approach that enables multimodal whole-genome and high-depth targeted sequencing of the same cfDNA sample for both tumour-agnostic and tumour-informed liquid biopsy analysis. Across 792 plasma and cerebrospinal fluid cfDNA samples from 277 paediatric patients with diverse brain and extracranial tumours, SIMMA enabled tumour diagnosis, detection of driver mutations, and identification of extrachromosomal DNA (ecDNA) months before clinical relapse. Using conformal prediction trained on genome-wide fragmentomics, genomic and epigenomic data, SIMMA models disease burden as a continuous variable and provides well-calibrated uncertainty estimates for each sample, achieving a limit of detection of ~100 ppm from low-pass whole-genome sequencing data. In summary, SIMMA establishes the clinical utility of multimodal cfDNA profiling with uncertainty quantification for individual patients and unlocks the potential of ecDNA as a liquid biopsy biomarker for disease detection and monitoring across diverse aggressive malignancies.

Proportion of patients with genetic aberrations.

Central nervous system (CNS) tumours are the deadliest cancer for children and currently present limited treatment options. Chimeric antigen receptor (CAR)-T cell therapies have emerged as an innovative approach supported by encouraging clinical results. Current clinical trials using CAR-T cells in the treatment of paediatric CNS cancers differ in a number of variables, including the CAR-T cell route of delivery, presence of lymphodepletion, identified target antigen, and CAR engineering features. Considering early learnings across these areas is an essential step to developing more effective treatment options, especially given the challenges of immunosuppressive tumour microenvironments, various toxicities, CAR-T cell exhaustion, and tumour antigen heterogeneity. In sum, while there is a need for continued innovation, CAR-T cells represent a promising treatment approach for this devastating category of diseases.

Primary vs. relapse copy number profiles.

Top relapse-specific copy number events.

Relapse Vs. primary copy number variants.

Abstract Group 3 medulloblastomas with MYC-amplification (MYC-MBG3) do not respond to standard-of-care regimens and represent one of the worst prognosis childhood brain tumours. MYC is not directly targetable with small molecule inhibitors. The systematic identification of compounds which instead target complementary MYC-dependencies represents a promising approach to rationally re-design effective therapeutic strategies. We derived three MYC-regulable cell avatars from cellular models of MYC-MBG3 (iD425, iD283, iHD-MB03). Transcriptomic analysis, alongside a primary patient tumour cohort (n = 321), identified a common MYC-dependent signature shared between patients and avatars, enriched for proliferative and DNA damage repair processes. Parallel high-throughput drug screens (>500 small molecule inhibitors) revealed differential MYC-dependent sensitivities, consistent across cell avatars, to 80 small molecules spanning 25 drug classes. Bespoke chemoinformatics (predictive compound selectivity, blood-brain-barrier penetration, clinical development stage) integrated with MYC-dependent transcriptomics, identified seven proceedable target dependencies, including CHEK1, AURKA and PLK1. Pharmacological and genetic validation short-listed therapeutic leads and associated small molecules, which were advanced to single agent efficacy trials in a spontaneous in vivo model of MYC-MBG3 (GTML). Inhibition of AURKA (with alisertib) and CHEK1 (with prexasertib) was found to significantly extend survival and reduce tumour burden. To anticipate and circumvent the development of cellular resistance, alisertib and prexasertib were next trialled in combination. In a GTML ex vivo 3D model, the alisertib/prexasertib combination demonstrated strong efficacy, additivity and striking MYC(N)-dependency. In a Myc-dependent in vivo allograft model (Myc-DNp53), alisertib/prexasertib combination therapy significantly increased survival. Pharmacokinetic studies confirmed alisertib/prexasertib central nervous system penetration. Our findings demonstrate dual-AURKA/CHEK1 inhibition exerts a robust anti-tumour effect in MYC-MBG3 that warrants clinical evaluation. Importantly, we present an integrative screening pipeline which encompasses human tumour analysis and avatar-based screening to uncover actionable therapeutic vulnerabilities in MYC-MBG3, which is readily adaptable to other high-risk medulloblastoma disease features.

BACKGROUND:Reversible electroporation involves the use of pulsed electric fields to temporarily disrupt and permeabilise cell membranes. Electroporation combined with chemotherapy - electrochemotherapy (ECT) - increases tumour cell permeability to chemotherapeutic drugs and enhances their effect. We investigated the efficacy of electroporation and cisplatin as a treatment for neuroblastoma in vitro, and explored whether inhibition of DNA repair mechanisms with olaparib potentiates its effects. MATERIALS AND METHODS:Three immortalised neuroblastoma cell lines were exposed to eight 1 ms square wave pulses of 0-0.93 kV/cm electric fields in the presence of propidium iodide and reversible electroporation was identified by detecting live propidium iodide positive cells by flow cytometry. Intracellular cisplatin and olaparib levels after electroporation were investigated by measuring intracellular platinum via inductively coupled plasma mass spectrometry and by quantifying a fluorescent olaparib with flow cytometry. Cells were exposed to electroporation in the presence of cisplatin and olaparib, and cell death was quantified by flow cytometry. Presence of DNA damage was evaluated by quantifying γ-H2AX immunofluorescence. RESULTS:Reversible electroporation (0.74 kV/cm) increased intracellular cisplatin and olaparib levels. Electroporation with cisplatin (1-100 μM) significantly reduced cell viability compared to cisplatin treatment in all cell lines. The addition of olaparib (5 μM) modestly potentiated the effects of cisplatin and electroporation. DNA damage was significantly higher following treatment with a combination of electroporation, cisplatin and olaparib, compared to each treatment alone. CONCLUSIONS:Electroporation with cisplatin appears effective against neuroblastoma in vitro. Further work will investigate the efficacy of electroporation with cisplatin using clinical ECT devices.

Abstract Glypican-2 (GPC2) is highly expressed across paediatric malignancies, including high-risk neuroblastomas and medulloblastomas, and GPC2-directed CART-cells have now entered phase 1 trials (NCT05650749, NCT07087002). Yet durable responses remain limited due to heterogeneous antigen expression, treatment-driven antigen loss, and suboptimal CART persistence. Because B7-H3 is broadly expressed in GPC2+medulloblastoma, it represents a compelling partner target for a dual GPC2.B7H3 CAR approach. To enable dual targeting, we generated seven bicistronic GPC2/B7-H3 CAR constructs incorporating CD28, 4-1BB, or OX40 costimulatory domains and either two (dual, d1–3) or one (parallel, p1–4) CD3-ζ signalling domain. Among these, constructs d1 and p2 were prioritised for further study. Both induced strong activation of Jurkat NFAT-GFP CART-cells when co-cultured with isogenic NALM-6 cells expressing either or both antigens and produced higher cytokine secretion than singleantigen B7-H3 (B) or GPC2 (G) CARs. In medulloblastoma in vitro models, repeated tumourstimulation assays consistently showed superior activity for d1 and p2. These findings translated in vivo. In a GPC2-low medulloblastoma PDX, dual CARs matched the efficacy of B7H3-2G-CARs and outperformed GPC2-2G-CARs. In an aggressive group3 medulloblastoma PDX where all GPC2 CAR–treated mice relapsed, the p2-CAR achieved the strongest therapeutic effect, yielding 16week cure rates of 100% versus 71.4% for d1 and 60% for the single B7H3-CAR, demonstrating the advantage of dual GPC2.B7H3 targeting and suggesting a benefit of parallel CD3ζ–sharing designs. Dual-targeting CART-cells also drove potent regression of COG-N-453x neuroblastoma PDXs, with 16-week cure rates of 83.3%, 83.3%, and 100%, exceeding single-antigen CARs. Long-term persistence of human CART-cells in bone marrow and spleen correlated with durable remissions. Upon tumour rechallenge, only d1 and p2 prevented recurrence, with sustained CART-cell persistence through 36-weeks. Overall, GPC2.B7-H3 bicistronic CARs generated durable and often curative responses across neuroblastoma and medulloblastoma models, supporting this strategy as a next-generation GPC2-directed therapy for clinical evaluation.

Fraction of genome altered by disease group.

Paediatric cancer specific cfDNA panel (ctPC Panel).

Concordance between ctDNA and tissue copy number profiles.

Cohort-wide oncoplot for tissue vs cfDNA variants.

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 Group 3 medulloblastoma (G3 MB) is an aggressive poor prognosis paediatric brain tumour. Chimeric Antigen Receptor (CAR) T-cell therapy targeting B7H3, an immune checkpoint molecule overexpressed in medulloblastoma and minimally expressed in healthy brain tissues, represents a promising therapeutic. However, the immunosuppressive tumour microenvironment (TME), often mediated by transforming growth factor (TGFβ), limits CAR T-cell efficacy and persistence. We hypothesised that inhibition of TGFβ signalling through blockade of TGFβ receptor I (TGFBRI) could preserve CAR T-cell function and enhance anti-tumour activity in G3 MB. TGFβ pathway activation was assessed in both immunocompetent and immunodeficient MB mouse models, as well as in patient tumour samples using immunohistochemical detection of phosphorylated SMAD3 (pSMAD3). Anti-B7H3 CAR T-cells were generated from healthy donor PBMCs using gamma-retroviral transduction, either alone or co-expressing a dominant negative TGFBRII (DNR). CAR T-cells functionality was evaluated in in vitro co-culture assays with G3 MB cell lines in the presence or absence of exogenous TGFβ (10ng/ml). Pharmacological inhibition of TGFβ signalling was tested using the selective TGFBRI inhibitors Vactosertib and LY3200882. Robust TGFβ pathway activation was observed across preclinical models and patient samples, with significantly higher pSMAD3 levels in Group 3 tumours compared with other medulloblastoma subgroups (SHH n = 71, Group 3 n = 63, Group 4 n = 71, WNT n = 15). Exogenous TGFβ markedly impaired CAR T-cell proliferation, phenotype and cytokine release; these effects were reversed by DNR expression. Both TGFBRI inhibitors were well tolerated, preserved CAR T-cell expansion and phenotype, and restored effector function in TGFβ-rich conditions. Drug treatment protected CAR-T effector function (cytokine release and proliferation) upon repeated tumour and TGFβ challenge. In vivo studies are evaluating efficacy, safety, feasibility, and pharmacokinetic and pharmacodynamic profiles. These findings provide an initial preclinical rationale for combining TGFβ-pathway inhibition with anti-B7H3 CAR T-cell therapy in G3 medulloblastoma.

Background Relapsed medulloblastoma remains a significant therapeutic challenge as it is near universally fatal. The tumor microenvironment of medulloblastoma plays a critical role in tumor progression, influencing tumor growth, immune evasion, and therapeutic resistance. We hypothesized that defining tumor-immune interactions in diagnostic and relapsed medulloblastoma may uncover mechanisms of immune evasion and identify novel therapeutic targets.Methods We analyzed paired primary and recurrent RNA-sequencing data from 140 medulloblastoma patients to profile immune cell composition and validate spatial relationships within the TME. To identify key tumor-immune interactions, we developed a novel algorithm to detect receptor-ligand pairs using single-cell RNA-sequencing data. These interactions were validated across RNA and proteomic datasets. Their functional significance was empirically demonstrated in newly developed immunocompetent models of recurrent medulloblastoma that closely recapitulate the human disease.Results We observed a shift toward a heightened immunosuppressive TME at relapse. Using our algorithm, we identified biologically significant receptor-ligand interactions, most notably MIF-CD74, constitutively expressed at RNA and protein levels across medulloblastoma subgroups, at diagnosis and relapse. Disrupting MIF-CD74 interactions led to significant alterations in the tumor microenvironment, highlighting its functional significance.Conclusions Our multifaceted approach identified key tumor-immune interactions in medulloblastoma. Among these, MIF-CD74 was validated as a targetable interaction, demonstrating the utility of our integrative approach for identifying novel therapeutic targets across multiple tumor types.

Relapse vs. primary somatic variants.

Abstract CAR-T cell therapies are effective in haematological malignancies, yet responses in solid tumours remain limited by tonic signalling, exhaustion, and poor persistence. B7-H3 is expressed most high-risk paediatric brain tumours, making it an attractive target for CAR-T cell immunotherapy in CNS malignancies. Switchable CAR-T cell technologies enable pharmacologic control of CAR expression using drug-inducible degron tags. The iTAG2 degron permits reversible downregulation of CAR expression both during manufacture—limiting tonic signalling and T-cell exhaustion prior to infusion—and following administration, offering a potential strategy for reversing tumour-induced T cell exhaustion. Immunomodulatory drugs (IMiDs) act as molecular glues that induce cereblon-mediated proteasomal degradation of degron-tagged proteins. We evaluated the effects of IMiDs, including Iberdomide and Pomalidomide, on anti–B7-H3 TE9-28z-iTAG2 CAR-T cells to achieve reversible control of CAR expression either during manufacture or post-manufacture. Functional activity of CAR-T cells, generated in GMP-compatible conditions, was assessed in vitro using solid tumour cell lines, including the Med8A medulloblastoma brain tumour model, and in vivo using NSG mouse models of medulloblastoma and neuroblastoma. Compared with CAR-T cells manufactured in the absence of IMiDs, iTAG2 CAR-T cells exposed to IMiDs during manufacture demonstrated enhanced proliferation, increased cytotoxicity, and elevated cytokine production in vitro upon tumour challenge. In vivo, IMiD-manufactured iTAG2 CAR-T cells showed superior tumour control and increased proliferative capacity. Post-manufacture regulation of CAR expression using IMiD scheduling was also evaluated. In long-term tumour challenge assays, IMiD-induced CAR downregulation prolonged CAR-T cell functional persistence and was associated with reduced markers of T-cell exhaustion. Incorporation of IMiDs either during manufacture or following infusion enhances the functional activity and therapeutic potential of iTAG2-regulated anti–B7-H3 CAR T cells. TE9-28z-iTAG2 CAR T cells are being evaluated in a Phase I study of repeated intracerebroventricular administration in children and young adults with recurrent or refractory high-grade CNS tumours.