Treatment personalization via tumor molecular testing holds promise for improving outcomes for patients with pediatric low-grade glioma (PLGG). We evaluate the health economic impact of employing tumor molecular testing to guide treatment for patients diagnosed with PLGG, particularly the avoidance of radiation therapy (RT) for patients with BRAF-fusion.

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Cancers arising from germline DNA mismatch repair deficiency or polymerase proofreading deficiency (MMRD and PPD) in children harbour the highest mutational and microsatellite insertion-deletion (MS-indel) burden in humans. MMRD and PPD cancers are commonly lethal due to the inherent resistance to chemo-irradiation. Although immune checkpoint inhibitors (ICIs) have failed to benefit children in previous studies, we hypothesized that hypermutation caused by MMRD and PPD will improve outcomes following ICI treatment in these patients. Using an international consortium registry study, we report on the ICI treatment of 45 progressive or recurrent tumors from 38 patients. Durable objective responses were observed in most patients, culminating in a 3 year survival of 41.4%. High mutation burden predicted response for ultra-hypermutant cancers (>100 mutations per Mb) enriched for combined MMRD + PPD, while MS-indels predicted response in MMRD tumors with lower mutation burden (10-100 mutations per Mb). Furthermore, both mechanisms were associated with increased immune infiltration even in 'immunologically cold' tumors such as gliomas, contributing to the favorable response. Pseudo-progression (flare) was common and was associated with immune activation in the tumor microenvironment and systemically. Furthermore, patients with flare who continued ICI treatment achieved durable responses. This study demonstrates improved survival for patients with tumors not previously known to respond to ICI treatment, including central nervous system and synchronous cancers, and identifies the dual roles of mutation burden and MS-indels in predicting sustained response to immunotherapy.

In children, higher general intelligence corresponds with better processing speed ability. However, the relationship between structural brain connectivity and processing speed in the context of intelligence is unclear. Furthermore, the impact of brain injury on this relationship is also unknown. Structural networks were constructed for 36 brain tumor patients (mean age: 13.45 ± 2.73, 58% males) and 35 typically developing children (13.30 ± 2.86, 51% males). Processing speed and general intelligence scores were acquired using standard batteries. The relationship between network properties, processing speed, and intelligence was assessed using a partial least squares analysis. Results indicated that structural networks in brain-injured children were less integrated (β = -.38, p = 0.001) and more segregated (β = 0.4, p = 0.0005) compared to typically developing children. There was an indirect effect of network segregation on general intelligence via processing speed, where greater network segregation predicted slower processing speed which in turn predicted worse general intelligence (GoF = 0.37). These findings provide the first evidence of relations between structural connectivity, processing speed, and intelligence in children. Injury-related disruption to the structural network may result in worse intelligence through impacts on information processing. Our findings are discussed in the context of a network approach to understanding brain-behavior relationships.

Molecular groups of medulloblastoma (MB) are well established. Novel risk stratification parameters include Group 3/4 (non-WNT/non-SHH) methylation subgroups I-VIII or whole-chromosomal aberration (WCA) phenotypes. This study investigates the integration of clinical and molecular parameters to improve risk stratification of non-WNT/non-SHH MB. Non-WNT/non-SHH MB from the HIT2000 study and the HIT-MED registries were selected based on availability of DNA-methylation profiling data. MYC or MYCN amplification and WCA of chromosomes 7, 8, and 11 were inferred from methylation array-based copy number profiles. In total, 403 non-WNT/non-SHH MB were identified, 346/403 (86%) had a methylation class family Group 3/4 methylation score (classifier v11b6) ≥ 0.9, and 294/346 (73%) were included in the risk stratification modeling based on Group 3 or 4 score (v11b6) ≥ 0.8 and subgroup I-VIII score (mb_g34) ≥ 0.8. Group 3 MB (5y-PFS, survival estimation ± standard deviation: 41.4 ± 4.6%; 5y-OS: 48.8 ± 5.0%) showed poorer survival compared to Group 4 (5y-PFS: 68.2 ± 3.7%; 5y-OS: 84.8 ± 2.8%). Subgroups II (5y-PFS: 27.6 ± 8.2%) and III (5y-PFS: 37.5 ± 7.9%) showed the poorest and subgroup VI (5y-PFS: 76.6 ± 7.9%), VII (5y-PFS: 75.9 ± 7.2%), and VIII (5y-PFS: 66.6 ± 5.8%) the best survival. Multivariate analysis revealed subgroup in combination with WCA phenotype to best predict risk of progression and death. The integration of clinical (age, M and R status) and molecular (MYC/N, subgroup, WCA phenotype) variables identified a low-risk stratum with a 5y-PFS of 94 ± 5.7 and a very high-risk stratum with a 5y-PFS of 29 ± 6.1%. Validation in an international MB cohort confirmed the combined stratification scheme with 82.1 ± 6.0% 5y-PFS in the low and 47.5 ± 4.1% in very high-risk groups, and outperformed the clinical model. These newly identified clinico-molecular low-risk and very high-risk strata, accounting for 6%, and 21% of non-WNT/non-SHH MB patients, respectively, may improve future treatment stratification.

We conducted integrative somatic-germline analyses by deeply sequencing 864 cancer-associated genes, complete genomes and transcriptomes for 300 mostly previously treated children and adolescents/young adults with cancer of poor prognosis or with rare tumors enrolled in the SickKids Cancer Sequencing (KiCS) program. Clinically actionable variants were identified in 56% of patients. Improved diagnostic accuracy led to modified management in a subset. Therapeutically targetable variants (54% of patients) were of unanticipated timing and type, with over 20% derived from the germline. Corroborating mutational signatures (SBS3/BRCAness) in patients with germline homologous recombination defects demonstrates the potential utility of PARP inhibitors. Mutational burden was significantly elevated in 9% of patients. Sequential sampling identified changes in therapeutically targetable drivers in over one-third of patients, suggesting benefit from rebiopsy for genomic analysis at the time of relapse. Comprehensive cancer genomic profiling is useful at multiple points in the care trajectory for children and adolescents/young adults with cancer, supporting its integration into early clinical management.

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications.

Two distinct genetically defined entities of ependymoma arising in the supratentorial compartment are characterized by the presence of either a C11orf95-RELA or a YAP-MAMLD1 fusion, respectively. There is growing evidence that supratentorial ependymomas without these genetic features exist. In this study, we report on 18 pediatric non-RELA/non-YAP supratentorial ependymomas that were systematically characterized by means of their histology, immunophenotype, genetics, and epigenomics. Comprehensive molecular analyses included high-resolution copy number analysis, methylation profiling, analysis of fusion transcripts by Nanostring technology, and RNA sequencing. Based upon histological and immunohistochemical features two main patterns were identified-RELA-like (n = 9) and tanycytic ependymomas (n = 6). In the RELA-like group histologically assigned to WHO grade III and resembling RELA-fused ependymomas, tumors lacked nuclear expression of p65-RelA as a surrogate marker for a pathological activation of the NF-κB pathway. Three tumors showed alternative C11orf95 fusions to MAML2 or NCOA1. A methylation-based brain tumor classifier assigned two RELA-like tumors to the methylation class "EP, RELA-fusion"; the others demonstrated no significant similarity score. Of the tanycytic group, 5/6 tumors were assigned a WHO grade II. No gene fusions were detected. Methylation profiling did not show any association with an established methylation class. We additionally identified two astroblastoma-like tumors that both presented with chromothripsis of chromosome 22 but lacked MN1 breaks according to FISH analysis. They revealed novel fusion events involving genes in chromosome 22. One further tumor with polyploid cytogenetics was interpreted as PFB ependymoma by the brain tumor methylation classifier but had no relation to the posterior fossa. Clinical follow-up was available for 16/18 patients. Patients with tanycytic and astroblastoma-like tumors had no relapse, while 2 patients with RELA-like ependymomas died. Our data indicate that in addition to ependymomas discovered so far, at least two more supratentorial ependymoma types (RELA-like and tanycytic) exist.

Study of the origin and development of cerebellar tumours has been hampered by the complexity and heterogeneity of cerebellar cells that change over the course of development. Here we use single-cell transcriptomics to study more than 60,000 cells from the developing mouse cerebellum and show that different molecular subgroups of childhood cerebellar tumours mirror the transcription of cells from distinct, temporally restricted cerebellar lineages. The Sonic Hedgehog medulloblastoma subgroup transcriptionally mirrors the granule cell hierarchy as expected, while group 3 medulloblastoma resembles Nestin+ stem cells, group 4 medulloblastoma resembles unipolar brush cells, and PFA/PFB ependymoma and cerebellar pilocytic astrocytoma resemble the prenatal gliogenic progenitor cells. Furthermore, single-cell transcriptomics of human childhood cerebellar tumours demonstrates that many bulk tumours contain a mixed population of cells with divergent differentiation. Our data highlight cerebellar tumours as a disorder of early brain development and provide a proximate explanation for the peak incidence of cerebellar tumours in early childhood.

Myxopapillary ependymoma (MPE) is a heterogeneous disease regarding histopathology and outcome. The underlying molecular biology is poorly understood, and markers that reliably predict the patients' clinical course are unknown.

Ependymomas encompass a heterogeneous group of central nervous system (CNS) neoplasms that occur along the entire neuroaxis. In recent years, extensive (epi-)genomic profiling efforts have identified several molecular groups of ependymoma that are characterized by distinct molecular alterations and/or patterns. Based on unsupervised visualization of a large cohort of genome-wide DNA methylation data, we identified a highly distinct group of pediatric-type tumors (n = 40) forming a cluster separate from all established CNS tumor types, of which a high proportion were histopathologically diagnosed as ependymoma. RNA sequencing revealed recurrent fusions involving the pleomorphic adenoma gene-like 1 (PLAGL1) gene in 19 of 20 of the samples analyzed, with the most common fusion being EWSR1:PLAGL1 (n = 13). Five tumors showed a PLAGL1:FOXO1 fusion and one a PLAGL1:EP300 fusion. High transcript levels of PLAGL1 were noted in these tumors, with concurrent overexpression of the imprinted genes H19 and IGF2, which are regulated by PLAGL1. Histopathological review of cases with sufficient material (n = 16) demonstrated a broad morphological spectrum of tumors with predominant ependymoma-like features. Immunohistochemically, tumors were GFAP positive and OLIG2- and SOX10 negative. In 3/16 of the cases, a dot-like positivity for EMA was detected. All tumors in our series were located in the supratentorial compartment. Median age of the patients at the time of diagnosis was 6.2 years. Median progression-free survival was 35 months (for 11 patients with data available). In summary, our findings suggest the existence of a novel group of supratentorial neuroepithelial tumors that are characterized by recurrent PLAGL1 fusions and enriched for pediatric patients.

Approximately 20 TP53 retrogenes exist in the African and Asian elephant genomes (Loxodonta Africana, Elephas Maximus) in addition to a conserved TP53 gene that encodes a full-length protein. Elephant TP53-RETROGENE 9 (TP53-R9) encodes a p53 protein (p53-R9) that is truncated in the middle of the canonical DNA binding domain. This C-terminally truncated p53 retrogene protein lacks the nuclear localization signals and oligomerization domain of its full-length counterpart. When expressed in human osteosarcoma cells (U2OS), p53-R9 binds to Tid1, the chaperone protein responsible for mitochondrial translocation of human p53 in response to cellular stress. Tid1 expression is required for p53-R9-induced apoptosis. At the mitochondria, p53-R9 binds to the pro-apoptotic BCL-2 family member Bax, which leads to caspase activation, cytochrome c release, and cell death. Our data show, for the first time, that expression of this truncated elephant p53 retrogene protein induces apoptosis in human cancer cells. Understanding the molecular mechanism by which the additional elephant TP53 retrogenes function may provide evolutionary insight that can be utilized for the development of therapeutics to treat human cancers.

The large diversity of central nervous system (CNS) tumor types in children and adolescents results in disparate patient outcomes and renders accurate diagnosis challenging. In this study, we prospectively integrated DNA methylation profiling and targeted gene panel sequencing with blinded neuropathological reference diagnostics for a population-based cohort of more than 1,200 newly diagnosed pediatric patients with CNS tumors, to assess their utility in routine neuropathology. We show that the multi-omic integration increased diagnostic accuracy in a substantial proportion of patients through annotation to a refining DNA methylation class (50%), detection of diagnostic or therapeutically relevant genetic alterations (47%) or identification of cancer predisposition syndromes (10%). Discrepant results by neuropathological WHO-based and DNA methylation-based classification (30%) were enriched in histological high-grade gliomas, implicating relevance for current clinical patient management in 5% of all patients. Follow-up (median 2.5 years) suggests improved survival for patients with histological high-grade gliomas displaying lower-grade molecular profiles. These results provide preliminary evidence of the utility of integrating multi-omics in neuropathology for pediatric neuro-oncology.

Posterior fossa type A (PF-EPN-A, PFA) ependymoma are aggressive tumors that mainly affect children and have a poor prognosis. Histopathology shows significant intratumoral heterogeneity, ranging from loose tissue to often sharply demarcated, extremely cell-dense tumor areas. To determine molecular differences in morphologically different areas and to understand their clinical significance, we analyzed 113 PF-EPN-A samples, including 40 corresponding relapse samples. Cell-dense areas ranged from 0 to 100% of the tumor area and displayed a higher proportion of proliferating tumor cells (p < 0.01). Clinically, cell density was associated with poor progression-free and overall survival (pPFS = 0.0026, pOS < 0.01). Molecularly, tumor areas with low and high cell density showed diverging DNA methylation profiles regarding their similarity to distinct previously discovered PF-EPN-A subtypes in 9/21 cases. Prognostically relevant chromosomal changes at 1q and 6q showed spatial heterogeneity within single tumors and were significantly enriched in cell-dense tumor areas as shown by single-cell RNA (scRNA)-sequencing as well as copy number profiling and fluorescence in situ hybridization (FISH) analyses of different tumor areas. Finally, spatial transcriptomics revealed cell-dense areas of different tumors to be more similar than various different areas of the same tumor. High-density areas distinctly overexpressed genes encoding histone proteins, WNT5A, TGFB1, or IGF2. Relapsing tumors displayed a higher proportion of cell-dense areas (p = 0.036), a change in PF-EPN-A methylation subtypes (13/32 patients), and novel chromosome 1q gains and 6q losses (12/32 cases) compared to corresponding primary tumors. Our data suggest that PF-EPN-A ependymomas habor a previously unrecognized intratumoral heterogeneity with clinical implications, which has to be accounted for when selecting diagnostic material, inter alia, by histological evaluation of the proportion of cell-dense areas.

Functional heterogeneity within tumors presents a significant therapeutic challenge. Here we show that quiescent, therapy-resistant Sox2(+) cells propagate sonic hedgehog subgroup medulloblastoma by a mechanism that mirrors a neurogenic program. Rare Sox2(+) cells produce rapidly cycling doublecortin(+) progenitors that, together with their postmitotic progeny expressing NeuN, comprise tumor bulk. Sox2(+) cells are enriched following anti-mitotic chemotherapy and Smoothened inhibition, creating a reservoir for tumor regrowth. Lineage traces from Sox2(+) cells increase following treatment, suggesting that this population is responsible for relapse. Targeting Sox2(+) cells with the antineoplastic mithramycin abrogated tumor growth. Addressing functional heterogeneity and eliminating Sox2(+) cells presents a promising therapeutic paradigm for treatment of sonic hedgehog subgroup medulloblastoma.

Choroid plexus carcinomas (CPCs) are poorly understood and frequently lethal brain tumors with few treatment options. Using a mouse model of the disease and a large cohort of human CPCs, we performed a cross-species, genome-wide search for oncogenes within syntenic regions of chromosome gain. TAF12, NFYC, and RAD54L co-located on human chromosome 1p32-35.3 and mouse chromosome 4qD1-D3 were identified as oncogenes that are gained in tumors in both species and required for disease initiation and progression. TAF12 and NFYC are transcription factors that regulate the epigenome, whereas RAD54L plays a central role in DNA repair. Our data identify a group of concurrently gained oncogenes that cooperate in the formation of CPC and reveal potential avenues for therapy.

Diffuse intrinsic pontine glioma is a uniformly lethal malignant tumor of infancy with no effective therapies. A new study reveals that inhibition of JMJD3 has robust antitumor activity in diffuse intrinsic pontine glioma xenografts.

Medulloblastoma is a malignant childhood brain tumour comprising four discrete subgroups. Here, to identify mutations that drive medulloblastoma, we sequenced the entire genomes of 37 tumours and matched normal blood. One-hundred and thirty-six genes harbouring somatic mutations in this discovery set were sequenced in an additional 56 medulloblastomas. Recurrent mutations were detected in 41 genes not yet implicated in medulloblastoma; several target distinct components of the epigenetic machinery in different disease subgroups, such as regulators of H3K27 and H3K4 trimethylation in subgroups 3 and 4 (for example, KDM6A and ZMYM3), and CTNNB1-associated chromatin re-modellers in WNT-subgroup tumours (for example, SMARCA4 and CREBBP). Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumours identified genes that maintain this cell lineage (DDX3X), as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumorigenesis. These data provide important new insights into the pathogenesis of medulloblastoma subgroups and highlight targets for therapeutic development.

Facial emotion recognition (FER) deficits are evident and pervasive across neurodevelopmental, psychiatric, and acquired brain disorders in children, including children treated for brain tumours. Such deficits are thought to perpetuate challenges with social relationships and decrease quality of life. The present study combined eye-tracking, neuroimaging and cognitive assessments to evaluate if visual attention, brain structure, and general cognitive function contribute to FER in children treated for posterior fossa (PF) tumours (patients: n = 36) and typically developing children (controls: n = 18). To assess FER, all participants completed the Diagnostic Analysis of Nonverbal Accuracy (DANVA2), a computerized task that measures FER using photographs, while their eye-movements were recorded. Patients made more FER errors than controls (p < .01). Although we detected subtle deficits in visual attention and general cognitive function in patients, we found no associations with FER. Compared to controls, patients had evidence of white matter (WM) damage, (i.e., lower fractional anisotropy [FA] and higher radial diffusivity [RD]), in multiple regions throughout the brain (all p < .05), but not in specific WM tracts associated with FER. Despite the distributed WM differences between groups, WM predicted FER in controls only. In patients, factors associated with their disease and treatment predicted FER. Our study provides insight into predictors of FER that may be unique to children treated for PF tumours, and highlights a divergence in associations between brain structure and behavioural outcomes in clinical and typically developing populations; a concept that may be broadly applicable to other neurodevelopmental and clinical populations that experience FER deficits.

In cancer, recurrent somatic single-nucleotide variants-which are rare in most paediatric cancers-are confined largely to protein-coding genes1-3. Here we report highly recurrent hotspot mutations (r.3A>G) of U1 spliceosomal small nuclear RNAs (snRNAs) in about 50% of Sonic hedgehog (SHH) medulloblastomas. These mutations were not present across other subgroups of medulloblastoma, and we identified these hotspot mutations in U1 snRNA in only <0.1% of 2,442 cancers, across 36 other tumour types. The mutations occur in 97% of adults (subtype SHHδ) and 25% of adolescents (subtype SHHα) with SHH medulloblastoma, but are largely absent from SHH medulloblastoma in infants. The U1 snRNA mutations occur in the 5' splice-site binding region, and snRNA-mutant tumours have significantly disrupted RNA splicing and an excess of 5' cryptic splicing events. Alternative splicing mediated by mutant U1 snRNA inactivates tumour-suppressor genes (PTCH1) and activates oncogenes (GLI2 and CCND2), and represents a target for therapy. These U1 snRNA mutations provide an example of highly recurrent and tissue-specific mutations of a non-protein-coding gene in cancer.