Molecular Profiling Reveals Biologically Discrete Subsets and Pathways of Progression in Diffuse Glioma.

Therapy development for adult diffuse glioma is hindered by incomplete knowledge of somatic glioma driving alterations and suboptimal disease classification. We defined the complete set of genes associated with 1,122 diffuse grade II-III-IV gliomas from The Cancer Genome Atlas and used molecular profiles to improve disease classification, identify molecular correlations, and provide insights into the progression from low- to high-grade disease. Whole-genome sequencing data analysis determined that ATRX but not TERT promoter mutations are associated with increased telomere length. Recent advances in glioma classification based on IDH mutation and 1p/19q co-deletion status were recapitulated through analysis of DNA methylation profiles, which identified clinically relevant molecular subsets. A subtype of IDH mutant glioma was associated with DNA demethylation and poor outcome; a group of IDH-wild-type diffuse glioma showed molecular similarity to pilocytic astrocytoma and relatively favorable survival. Understanding of cohesive disease groups may aid improved clinical outcomes.

Cryopreservation of mouse thymus depletes thymocytes but supports immune reconstitution on transplantation.

Cryopreservation of mouse thymus depletes donor thymocytes but preserves thymus function when transplanted after thawing into athymic mice. No differences in immune reconstitution were observed between fresh and frozen/thawed transplants suggesting that donor thymocyte depletion does not affect outcome. Thus, cryopreservation of thymus may improve outcomes in thymus transplant patients.

The pioneer transcription factors Foxa1 and Foxa2 regulate alternative RNA splicing during thymocyte positive selection.

During positive selection at the transition from CD4+CD8+ double-positive (DP) to single-positive (SP) thymocyte, TCR signalling results in appropriate MHC restriction and signals for survival and progression. We show that the pioneer transcription factors Foxa1 and Foxa2 are required to regulate RNA splicing during positive selection of mouse T cells and that Foxa1 and Foxa2 have overlapping/compensatory roles. Conditional deletion of both Foxa1 and Foxa2 from DP thymocytes reduced positive selection and development of CD4SP, CD8SP and peripheral naïve CD4+ T cells. Foxa1 and Foxa2 regulated the expression of many genes encoding splicing factors and regulators, including Mbnl1, H1f0, Sf3b1, Hnrnpa1, Rnpc3, Prpf4b, Prpf40b and Snrpd3. Within the positively selecting CD69+DP cells, alternative RNA splicing was dysregulated in the double Foxa1/Foxa2 conditional knockout, leading to >850 differentially used exons. Many genes important for this stage of T-cell development (Ikzf1-3, Ptprc, Stat5a, Stat5b, Cd28, Tcf7) and splicing factors (Hnrnpab, Hnrnpa2b1, Hnrnpu, Hnrnpul1, Prpf8) showed multiple differentially used exons. Thus, Foxa1 and Foxa2 are required during positive selection to regulate alternative splicing of genes essential for T-cell development, and, by also regulating splicing of splicing factors, they exert widespread control of alternative splicing.

Pan-cancer genome and transcriptome analyses of 1,699 paediatric leukaemias and solid tumours.

Analysis of molecular aberrations across multiple cancer types, known as pan-cancer analysis, identifies commonalities and differences in key biological processes that are dysregulated in cancer cells from diverse lineages. Pan-cancer analyses have been performed for adult but not paediatric cancers, which commonly occur in developing mesodermic rather than adult epithelial tissues. Here we present a pan-cancer study of somatic alterations, including single nucleotide variants, small insertions or deletions, structural variations, copy number alterations, gene fusions and internal tandem duplications in 1,699 paediatric leukaemias and solid tumours across six histotypes, with whole-genome, whole-exome and transcriptome sequencing data processed under a uniform analytical framework. We report 142 driver genes in paediatric cancers, of which only 45% match those found in adult pan-cancer studies; copy number alterations and structural variants constituted the majority (62%) of events. Eleven genome-wide mutational signatures were identified, including one attributed to ultraviolet-light exposure in eight aneuploid leukaemias. Transcription of the mutant allele was detectable for 34% of protein-coding mutations, and 20% exhibited allele-specific expression. These data provide a comprehensive genomic architecture for paediatric cancers and emphasize the need for paediatric cancer-specific development of precision therapies.

Impact of environment on pediatric and adult brain tumors: The 2023 Brain Tumor Epidemiology Consortium meeting report.

The Brain Tumor Epidemiology Consortium (BTEC) is an international organization with membership of individuals from the scientific community with interests related to brain tumor epidemiology including surveillance, classification, methodology, etiology, and factors associated with morbidity and mortality. The 2023 annual BTEC meeting entitled "Impact of Environment on Pediatric and Adult Brain Tumors" was held in Lexington, KY, USA on May 22 - 24, 2023. The meeting gathered scientists from the United States, Canada, Australia, and Europe and included four keynote sessions covering genomic, epigenomic, and metabolomic considerations in brain tumor epidemiology, cancer clusters, environmental risk factors, and new approaches to cancer investigation. The meeting also included three abstract sessions and a brainstorming session. A summary of the meeting content is included in this report.

The transcriptional repressor Bcl6 promotes pre-TCR-induced thymocyte differentiation and attenuates Notch1 activation.

Pre-T-cell receptor (TCR) signal transduction is required for developing thymocytes to differentiate from CD4-CD8- double-negative (DN) cell to CD4+CD8+ double-positive (DP) cell. Notch signalling is required for T-cell fate specification and must be maintained throughout ß-selection, but inappropriate Notch activation in DN4 and DP cells is oncogenic. Here, we show that pre-TCR signalling leads to increased expression of the transcriptional repressor Bcl6 and that Bcl6 is required for differentiation to DP. Conditional deletion of Bcl6 from thymocytes reduced pre-TCR-induced differentiation to DP cells, disrupted expansion and enrichment of intracellular TCRß+ cells within the DN population and increased DN4 cell death. Deletion also increased Notch1 activation and Notch-mediated transcription in the DP population. Thus, Bcl6 is required in thymocyte development for efficient differentiation from DN3 to DP and to attenuate Notch1 activation in DP cells. Given the importance of inappropriate NOTCH1 signalling in T-cell acute lymphoblastic leukaemia (T-ALL), and the involvement of BCL6 in other types of leukaemia, this study is important to our understanding of T-ALL.

Pediatric brain tumors: Origins, epidemiology, and classification - The 2022 Brain Tumor Epidemiology Consortium meeting report.

The Brain Tumor Epidemiology Consortium (BTEC) is an international organization that fosters collaboration among scientists focused on understanding the epidemiology of brain tumors with interests ranging from the etiology of brain tumor development and outcomes to the control of morbidity and mortality. The 2022 annual BTEC meeting with the theme "Pediatric Brain Tumors: Origins, Epidemiology, and Classification" was held in Lyon, France on June 20 - 22, 2022. Scientists from North America and Europe presented recent research and progress in the field. The meeting content is summarized in this report.

The Role of FAS Receptor Methylation in Osteosarcoma Metastasis.

Osteosarcoma is the most frequent primary malignant bone tumor with an annual incidence of about 400 cases in the United States. Osteosarcoma primarily metastasizes to the lungs, where FAS ligand (FASL) is constitutively expressed. The interaction of FASL and its cell surface receptor, FAS, triggers apoptosis in normal cells; however, this function is altered in cancer cells. DNA methylation has previously been explored as a mechanism for altering FAS expression, but no variability was identified in the CpG island (CGI) overlapping the promoter. Analysis of an expanded region, including CGI shores and shelves, revealed high variability in the methylation of certain CpG sites that correlated significantly with FAS mRNA expression in a negative manner. Bisulfite sequencing revealed additional CpG sites, which were highly methylated in the metastatic LM7 cell line but unmethylated in its parental non-metastatic SaOS-2 cell line. Treatment with the demethylating agent, 5-azacytidine, resulted in a loss of methylation in CpG sites located within the FAS promoter and restored FAS protein expression in LM7 cells, resulting in reduced migration. Orthotopic implantation of 5-azacytidine treated LM7 cells into severe combined immunodeficient mice led to decreased lung metastases. These results suggest that DNA methylation of CGI shore sites may regulate FAS expression and constitute a potential target for osteosarcoma therapy, utilizing demethylating agents currently approved for the treatment of other cancers.

Creating a Vaccine-like Supplement against Respiratory Infection Using Recombinant Bacillus subtilis Spores Expressing SARS-CoV-2 Spike Protein with Natural Products.

Vaccination is the most effective method of combating COVID-19 infection, but people with a psychological fear of needles and side effects are hesitant to receive the current vaccination, and alternative delivery methods may help. Bacillus subtilis, a harmless intestinal commensal, has recently earned a strong reputation as a vaccine production host and delivery vector, with advantages such as low cost, safety for human consumption, and straightforward oral administration. In this study, we have succeeded generating "S spores" by engineering B. subtilis with spore coat proteins resembling the spike (S) protein of the ancestral SARS-CoV-2 coronavirus. With the addition of two immunostimulating natural products as adjuvants, namely Astragalus membranaceus (Fisch.) Bge (AM) and Coriolus versicolor (CV), oral administration of S spores could elicit mild immune responses against COVID-19 infection without toxicity. Mucosal IgA against the S protein was enhanced by co-feeding with AM and CV in an S spores-inoculated mouse model. Faster and stronger IgG responses against the S protein were observed when the mice were fed with S spores prior to vaccination with the commercial COVID-19 vaccine CoronaVac. In vitro studies demonstrated that AM, CV, and B. subtilis spores could dose-dependently activate both macrophages and dendritic cells by secreting innate immunity-related IL-1ß, IL-6, and TNF-α, and some other proinflammatory chemokines and cytokines. In conclusion, the combination of S spores with AM and CV may be helpful in developing a vaccine-like supplement against respiratory infection.

Early experience with targeted therapy as a first-line adjuvant treatment for pediatric low-grade glioma.

OBJECTIVE: Pediatric low-grade gliomas (pLGGs) frequently exhibit dysregulation of the mitogen-activated protein kinase (MAPK) pathway. Targeted therapies, including mutant BRAF inhibitors (dabrafenib) and MEK inhibitors (trametinib), have shown promise in patients in whom conventional chemotherapy has failed. However, few studies have investigated the use of targeted therapy as a first-line treatment for pLGG. Here, the authors reviewed their institutional experience with using a personalized medicine approach to patients with newly diagnosed pLGGs. METHODS: All pediatric patients at the authors' institution who had been treated with dabrafenib or trametinib for pLGG without first receiving conventional chemotherapy or radiation were retrospectively reviewed. Demographic, clinical, and radiological data were collected. RESULTS: Eight patients underwent targeted therapy as a first-line treatment for pLGG. Five patients had a BRAF alteration (1 with a BRAFV600E mutation, 4 with a KIAA1549:BRAF fusion), and 3 patients had an NF1 mutation. One of the 8 patients was initially treated with dabrafenib, and trametinib was added later. Seven patients were initially treated with trametinib; of these, 2 later transitioned to dual therapy, whereas 5 continued with trametinib monotherapy. Six patients (75%) demonstrated a partial response to therapy during their treatment course, whereas stable disease was identified in the remaining 2 patients (25%). One patient experienced mild disease progression after completing a course of trametinib monotherapy, but ultimately stabilized after a period of close observation. Another patient experienced tumor progression while on dabrafenib, but subsequently responded to dual therapy with dabrafenib and trametinib. The most common adverse reactions to targeted therapy were cutaneous toxicity (100%) and diarrhea (50%). CONCLUSIONS: Targeted therapies have the potential to become a standard treatment option for pLGG due to their favorable toxicity profile and oral route of administration. This case series provides preliminary evidence that targeted therapies can induce an early disease response as a first-line adjuvant treatment; however, large-scale studies are required to assess long-term durability and safety.

Integrated DNA Copy Number and Expression Profiling Identifies IGF1R as a Prognostic Biomarker in Pediatric Osteosarcoma.

Osteosarcoma is a primary malignant bone tumor arising from bone-forming mesenchymal cells in children and adolescents. Despite efforts to understand the biology of the disease and identify novel therapeutics, the survival of osteosarcoma patients remains dismal. We have concurrently profiled the copy number and gene expression of 226 osteosarcoma samples as part of the Strategic Partnering to Evaluate Cancer Signatures (SPECS) initiative. Our results demonstrate the heterogeneous landscape of osteosarcoma in younger populations by showing the presence of genome-wide copy number abnormalities occurring both recurrently among samples and in a high frequency. Insulin growth factor receptor 1 (IGF1R) is a receptor tyrosine kinase which binds IGF1 and IGF2 to activate downstream pathways involved in cell apoptosis and proliferation. We identify prevalent amplification of IGF1R corresponding with increased gene expression in patients with poor survival outcomes. Our results substantiate previously tenuously associated copy number abnormalities identified in smaller datasets (13q34+, 20p13+, 4q35-, 20q13.33-), and indicate the significance of high fibroblast growth factor receptor 2 (FGFR2) expression in distinguishing patients with poor prognosis. FGFR2 is involved in cellular proliferation processes such as division, growth and angiogenesis. In summary, our findings demonstrate the prognostic significance of several genes associated with osteosarcoma pathogenesis.

A functional variant on 20q13.33 related to glioma risk alters enhancer activity and modulates expression of multiple genes.

Genome-wide association studies (GWAS) have identified single-nucleotide polymorphisms (SNPs) associated with glioma risk on 20q13.33, but the biological mechanisms underlying this association are unknown. We tested the hypothesis that a functional SNP on 20q13.33 impacted the activity of an enhancer, leading to an altered expression of nearby genes. To identify candidate functional SNPs, we identified all SNPs in linkage disequilibrium with the risk-associated SNP rs2297440 that mapped to putative enhancers. Putative enhancers containing candidate functional SNPs were tested for allele-specific effects in luciferase enhancer activity assays against glioblastoma multiforme (GBM) cell lines. An enhancer containing SNP rs3761124 exhibited allele-specific effects on activity. Deletion of this enhancer by CRISPR-Cas9 editing in GBM cell lines correlated with an altered expression of multiple genes, including STMN3, RTEL1, RTEL1-TNFRSF6B, GMEB2, and SRMS. Expression quantitative trait loci (eQTL) analyses using nondiseased brain samples, isocitrate dehydrogenase 1 (IDH1) wild-type glioma, and neurodevelopmental tissues showed STMN3 to be a consistent significant eQTL with rs3761124. RTEL1 and GMEB2 were also significant eQTLs in the context of early CNS development and/or in IDH1 wild-type glioma. We provide evidence that rs3761124 is a functional variant on 20q13.33 related to glioma/GBM risk that modulates the expression of STMN3 and potentially other genes across diverse cellular contexts.

Bi-allelic Mutations in Phe-tRNA Synthetase Associated with a Multi-system Pulmonary Disease Support Non-translational Function.

The tRNA synthetases catalyze the first step of protein synthesis and have increasingly been studied for their nuclear and extra-cellular ex-translational activities. Human genetic conditions such as Charcot-Marie-Tooth have been attributed to dominant gain-of-function mutations in some tRNA synthetases. Unlike dominantly inherited gain-of-function mutations, recessive loss-of-function mutations can potentially elucidate ex-translational activities. We present here five individuals from four families with a multi-system disease associated with bi-allelic mutations in FARSB that encodes the beta chain of the alpha2beta2 phenylalanine-tRNA synthetase (FARS). Collectively, the mutant alleles encompass a 5'-splice junction non-coding variant (SJV) and six missense variants, one of which is shared by unrelated individuals. The clinical condition is characterized by interstitial lung disease, cerebral aneurysms and brain calcifications, and cirrhosis. For the SJV, we confirmed exon skipping leading to a frameshift associated with noncatalytic activity. While the bi-allelic combination of the SJV with a p.Arg305Gln missense mutation in two individuals led to severe disease, cells from neither the asymptomatic heterozygous carriers nor the compound heterozygous affected individual had any defect in protein synthesis. These results support a disease mechanism independent of tRNA synthetase activities in protein translation and suggest that this FARS activity is essential for normal function in multiple organs.

Gli3 in fetal thymic epithelial cells promotes thymocyte positive selection and differentiation by repression of Shh.

Gli3 is a Hedgehog (Hh)-responsive transcription factor that can function as a transcriptional repressor or activator. We show that Gli3 activity in mouse thymic epithelial cells (TECs) promotes positive selection and differentiation from CD4+ CD8+ to CD4+ CD8- single-positive (SP4) cells in the fetal thymus and that Gli3 represses Shh Constitutive deletion of Gli3, and conditional deletion of Gli3 from TECs, reduced differentiation to SP4, whereas conditional deletion of Gli3 from thymocytes did not. Conditional deletion of Shh from TECs increased differentiation to SP4, and expression of Shh was upregulated in the Gli3-deficient thymus. Use of a transgenic Hh reporter showed that the Hh pathway was active in thymocytes, and increased in the Gli3-deficient fetal thymus. Neutralisation of endogenous Hh proteins in the Gli3-/- thymus restored SP4 differentiation, indicating that Gli3 in TECs promotes SP4 differentiation by repression of Shh Transcriptome analysis showed that Hh-mediated transcription was increased whereas TCR-mediated transcription was decreased in Gli3-/- thymocytes compared with wild type.

T cell phenotype in paediatric heart transplant recipients.

Paediatric heart transplantation recipients suffer an increased incidence of infectious, autoimmune and allergic problems. The relative roles of thymus excision and immunosuppressive treatments in contributing to these sequelae are not clear. We compared the immunological phenotypes of 25 heart transplant recipients (Tx), 10 children who underwent thymus excision during non-transplantation cardiac surgery (TE) and 25 age range-matched controls, in two age bands: 1-9 and 10-16 years. Significant differences from controls were seen mainly in the younger age band with Tx showing lower CD3 and CD4 cell counts whilst TE showed lower CD8 cell counts. Naïve T cell and recent thymic emigrant proportions and counts were significantly lower than controls in both groups in the lower age band. T cell recombination excision circle (TREC) levels were lower than controls in both groups in both age bands. There were no differences in regulatory T cells, but in those undergoing thymus excision in infancy, their proportions were higher in TE than Tx, a possible direct effect of immunosuppression. T cell receptor V beta spectratyping showed fewer peaks in both groups than in controls (predominantly in the older age band). Thymus excision in infancy was associated with lower CD8 cell counts and higher proportions of Tregs in TE compared to Tx. These data are consistent with thymus excision, particularly in infancy, being the most important influence on immunological phenotype after heart transplantation.

Association of heated tobacco product use with smoking cessation in Chinese cigarette smokers in Hong Kong: a prospective study.

INTRODUCTION: Heated tobacco products (HTPs) are increasingly popular worldwide, but whether they aid or undermine cigarette abstinence remains uncertain. We examined the predictors of HTP initiation and the prospective association of HTP use with cigarette abstinence in community-based smokers in Hong Kong. DESIGN: Secondary analysis of a randomised clinical trial aimed to evaluate the effectiveness of brief advice and referral for smoking cessation. The interventions were not related to HTP use. PARTICIPANTS AND SETTINGS: 1213 carbon monoxide-verified daily cigarette smokers with intentions to quit or reduce smoking proactively recruited from community sites throughout Hong Kong MAIN EXPOSURE: Current (past 7 day) use of HTP at baseline. MAIN OUTCOME: Self-reported 7-day point-prevalence cigarette abstinence at 6 months (exclusive use of HTP permitted). RESULTS: At baseline, 201 (16.6%) and 60 (4.9%) were ever and current HTP users, respectively. During the 6-month follow-up period, 110 of 1012 (10.9%) never users at baseline initiated HTPs. Younger age and higher education significantly predicted initiation. After adjusting for sociodemographic, smoking-related and quitting-related factors, current HTP use at baseline was not associated with cigarette abstinence at 6 months (adjusted prevalence ratio (aPR) 1.08, 95% CI 0.63 to 1.85). The results were similar in persistent users from baseline to 1-month/3-month follow-up (vs non-users; aPR 1.14, 95% CI 0.57 to 2.29). Use of smoking cessation service between baseline and 3-month follow-up significantly predicted cigarette abstinence (aPR 1.70, 95% CI 1.26 to 2.30). CONCLUSION: HTP use was not associated with cigarette abstinence at 6 months in a community-based cohort of smokers with intentions to quit or reduce smoking. Trial registration details ClinicalTrials.gov, NCT03565796.

IFITM proteins drive type 2 T helper cell differentiation and exacerbate allergic airway inflammation.

The interferon-inducible transmembrane (Ifitm/Fragilis) genes encode homologous proteins that are induced by IFNs. Here, we show that IFITM proteins regulate murine CD4+ Th cell differentiation. Ifitm2 and Ifitm3 are expressed in wild-type (WT) CD4+ T cells. On activation, Ifitm3 was downregulated and Ifitm2 was upregulated. Resting Ifitm-family-deficient CD4+ T cells had higher expression of Th1-associated genes than WT and purified naive Ifitm-family-deficient CD4+ T cells differentiated more efficiently to Th1, whereas Th2 differentiation was inhibited. Ifitm-family-deficient mice, but not Ifitm3-deficient mice, were less susceptible than WT to induction of allergic airways disease, with a weaker Th2 response and less severe disease and lower Il4 but higher Ifng expression and IL-27 secretion. Thus, the Ifitm family is important in adaptive immunity, influencing Th1/Th2 polarization, and Th2 immunopathology.

C11orf95-RELA reprograms 3D epigenome in supratentorial ependymoma.

Supratentorial ependymoma (ST-EPN) is a type of malignant brain tumor mainly seen in children. Since 2014, it has been known that an intrachromosomal fusion C11orf95-RELA is an oncogenic driver in ST-EPN [Parker et al. Nature 506:451-455 (2014); Pietsch et al. Acta Neuropathol 127:609-611 (2014)] but the molecular mechanisms of oncogenesis are unclear. Here we show that the C11orf95 component of the fusion protein dictates DNA binding activity while the RELA component is required for driving the expression of ependymoma-associated genes. Epigenomic characterizations using ChIP-seq and HiChIP approaches reveal that C11orf95-RELA modulates chromatin states and mediates chromatin interactions, leading to transcriptional reprogramming in ependymoma cells. Our findings provide important characterization of the molecular underpinning of C11orf95-RELA fusion and shed light on potential therapeutic targets for C11orf95-RELA subtype ependymoma.

Correction to: C11orf95-RELA reprograms 3D epigenome in supratentorial ependymoma.

In the original publication, Fig. 1 was incorrectly published with same two histograms at the bottom.

A genome-wide association study on medulloblastoma.

INTRODUCTION: Medulloblastoma is a malignant embryonal tumor of the cerebellum that occurs predominantly in children. To find germline genetic variants associated with medulloblastoma risk, we conducted a genome-wide association study (GWAS) including 244 medulloblastoma cases and 247 control subjects from Sweden and Denmark. METHODS: Genotyping was performed using Illumina BeadChips, and untyped variants were imputed using IMPUTE2. RESULTS: Fifty-nine variants in 11 loci were associated with increased medulloblastoma risk (p < 1 × 10-5), but none were statistically significant after adjusting for multiple testing (p < 5 × 10-8). Thirteen of these variants were genotyped, whereas 46 were imputed. Genotyped variants were further investigated in a validation study comprising 249 medulloblastoma cases and 629 control subjects. In the validation study, rs78021424 (18p11.23, PTPRM) was associated with medulloblastoma risk with OR in the same direction as in the discovery cohort (ORT = 1.59, pvalidation = 0.02). We also selected seven medulloblastoma predisposition genes for investigation using a candidate gene approach: APC, BRCA2, PALB2, PTCH1, SUFU, TP53, and GPR161. The strongest evidence for association was found for rs201458864 (PALB2, ORT = 3.76, p = 3.2 × 10-4) and rs79036813 (PTCH1, ORA = 0.42, p = 2.6 × 10-3). CONCLUSION: The results of this study, including a novel potential medulloblastoma risk loci at 18p11.23, are suggestive but need further validation in independent cohorts.