Molecular characterisation defines clinically-actionable heterogeneity within Group 4 medulloblastoma and improves disease risk-stratification.

Group 4 tumours (MBGrp4) represent the majority of non-WNT/non-SHH medulloblastomas. Their clinical course is poorly predicted by current risk-factors. MBGrp4 molecular substructures have been identified (e.g. subgroups/cytogenetics/mutations), however their inter-relationships and potential to improve clinical sub-classification and risk-stratification remain undefined. We comprehensively characterised the paediatric MBGrp4 molecular landscape and determined its utility to improve clinical management. A clinically-annotated discovery cohort (n = 362 MBGrp4) was assembled from UK-CCLG institutions and SIOP-UKCCSG-PNET3, HIT-SIOP-PNET4 and PNET HR + 5 clinical trials. Molecular profiling was undertaken, integrating driver mutations, second-generation non-WNT/non-SHH subgroups (1-8) and whole-chromosome aberrations (WCAs). Survival models were derived for patients ≥ 3 years of age who received contemporary multi-modal therapies (n = 323). We first independently derived and validated a favourable-risk WCA group (WCA-FR) characterised by ≥ 2 features from chromosome 7 gain, 8 loss, and 11 loss. Remaining patients were high-risk (WCA-HR). Subgroups 6 and 7 were enriched for WCA-FR (p < 0·0001) and aneuploidy. Subgroup 8 was defined by predominantly balanced genomes with isolated isochromosome 17q (p < 0·0001). While no mutations were associated with outcome and overall mutational burden was low, WCA-HR harboured recurrent chromatin remodelling mutations (p = 0·007). Integration of methylation and WCA groups improved risk-stratification models and outperformed established prognostication schemes. Our MBGrp4 risk-stratification scheme defines: favourable-risk (non-metastatic disease and (i) subgroup 7 or (ii) WCA-FR (21% of patients, 5-year PFS 97%)), very-high-risk (metastatic disease with WCA-HR (36%, 5-year PFS 49%)) and high-risk (remaining patients; 43%, 5-year PFS 67%). These findings validated in an independent MBGrp4 cohort (n = 668). Importantly, our findings demonstrate that previously established disease-wide risk-features (i.e. LCA histology and MYC(N) amplification) have little prognostic relevance in MBGrp4 disease. Novel validated survival models, integrating clinical features, methylation and WCA groups, improve outcome prediction and re-define risk-status for ~ 80% of MBGrp4. Our MBGrp4 favourable-risk group has MBWNT-like excellent outcomes, thereby doubling the proportion of medulloblastoma patients who could benefit from therapy de-escalation approaches, aimed at reducing treatment induced late-effects while sustaining survival outcomes. Novel approaches are urgently required for the very-high-risk patients.

In vivo modeling recapitulates radiotherapy delivery and late-effect profile for childhood medulloblastoma.

Background: Medulloblastoma (MB) is the most common malignant pediatric brain tumor, with 5-year survival rates > 70%. Cranial radiotherapy (CRT) to the whole brain, with posterior fossa boost (PFB), underpins treatment for non-infants; however, radiotherapeutic insult to the normal brain has deleterious consequences to neurocognitive and physical functioning, and causes accelerated aging/frailty. Approaches to ameliorate radiotherapy-induced late-effects are lacking and a paucity of appropriate model systems hinders their development. Methods: We have developed a clinically relevant in vivo model system that recapitulates the radiotherapy dose, targeting, and developmental stage of childhood medulloblastoma. Consistent with human regimens, age-equivalent (postnatal days 35-37) male C57Bl/6J mice received computerized tomography image-guided CRT (human-equivalent 37.5 Gy EQD2, n = 12) ±â€…PFB (human-equivalent 48.7 Gy EQD2, n = 12), via the small animal radiation research platform and were longitudinally assessed for > 12 months. Results: CRT was well tolerated, independent of PFB receipt. Compared to a sham-irradiated group (n = 12), irradiated mice were significantly frailer following irradiation (frailty index; P = .0002) and had reduced physical functioning; time to fall from a rotating rod (rotarod; P = .026) and grip strength (P = .006) were significantly lower. Neurocognitive deficits were consistent with childhood MB survivors; irradiated mice displayed significantly worse working memory (Y-maze; P = .009) and exhibited spatial memory deficits (Barnes maze; P = .029). Receipt of PFB did not induce a more severe late-effect profile. Conclusions: Our in vivo model mirrored childhood MB radiotherapy and recapitulated features observed in the late-effect profile of MB survivors. Our clinically relevant model will facilitate both the elucidation of novel/target mechanisms underpinning MB late effects and the development of novel interventions for their amelioration.

Medulloblastoma group 3 and 4 tumors comprise a clinically and biologically significant expression continuum reflecting human cerebellar development.

Medulloblastoma is currently subclassified into distinct DNA methylation subgroups/subtypes with particular clinico-molecular features. Using RNA sequencing (RNA-seq) in large, well-annotated cohorts of medulloblastoma, we show that transcriptionally group 3 and group 4 medulloblastomas exist as intermediates on a bipolar continuum between archetypal group 3 and group 4 entities. Continuum position is prognostic, reflecting a propensity for specific DNA copy-number changes, and specific switches in isoform/enhancer usage and RNA editing. Examining single-cell RNA-seq (scRNA-seq) profiles, we show that intratumoral transcriptional heterogeneity along the continuum is limited in a subtype-dependent manner. By integrating with a human scRNA-seq reference atlas, we show that this continuum is mirrored by an equivalent continuum of transcriptional cell types in early fetal cerebellar development. We identify distinct developmental niches for all four major subgroups and link each to a common developmental antecedent. Our findings show a transcriptional continuum arising from oncogenic disruption of highly specific fetal cerebellar cell types, linked to almost every aspect of group 3/group 4 molecular biology and clinico-pathology.

PCR amplicons identify widespread copy number variation in human centromeric arrays and instability in cancer.

Centromeric α-satellite repeats represent ~6% of the human genome, but their length and repetitive nature make sequencing and analysis of those regions challenging. However, centromeres are essential for the stable propagation of chromosomes, so tools are urgently needed to monitor centromere copy number and how it influences chromosome transmission and genome stability. We developed and benchmarked droplet digital PCR (ddPCR) assays that measure copy number for five human centromeric arrays. We applied them to characterize natural variation in centromeric array size, analyzing normal tissue from 37 individuals from China and 39 individuals from the US and UK. Each chromosome-specific array varies in size up to 10-fold across individuals and up to 50-fold across chromosomes, indicating a unique complement of arrays in each individual. We also used the ddPCR assays to analyze centromere copy number in 76 matched tumor-normal samples across four cancer types, representing the most-comprehensive quantitative analysis of centromeric array stability in cancer to date. In contrast to stable transmission in cultured cells, centromeric arrays show gain and loss events in each of the cancer types, suggesting centromeric α-satellite DNA represents a new category of genome instability in cancer. Our methodology for measuring human centromeric-array copy number will advance research on centromeres and genome integrity in normal and disease states.