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.
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New digital droplet PCR assays measure copy number of human centromeresLarge variation in copy number and individual-specific fingerprints existCentromere array copy number is stable in cultured human cellsAnalysis of primary human cancer samples suggest copy number can change in cancer
de Lima et al. develop a PCR method to estimate human centromere size based on a measurement of copy number in centromere arrays. The authors use this method to analyze normal tissue samples across populations, finding that the size of centromeres is highly variable among chromosomes as well as among individuals. They also demonstrate centromeric copy number changes in cancer samples, suggesting a new category of genome instability.
Abstract
INTRODUCTION: International consensus recognises four molecular subgroups of medulloblastoma, each with distinct molecular features and clinical outcomes. Assigning molecular subgroup is ...typically achieved via the Illumina DNA methylation microarray. Given the rapidly-expanding WGS capacity in healthcare institutions, there is an unmet need to develop platform-independent, sequence-based subgrouping assays. Whole genome bisulfite sequencing (WGBS) enables the assessment of genome-wide methylation status at single-base resolution. To date, its routine application for subgroup assignment has been limited, due to high economic cost and sample input requirements. Currently, no optimised pipeline exists that is tailored to handle samples sequenced at low-pass (i.e., <10x depth). METHODOLOGY: Two datasets were utilised; 36 newly sequenced low-depth (10x) and 42 publicly available high-depth (30x) WGBS medulloblastoma samples (n=34), alongside cerebellar control samples (n=8), all with matched DNA methylation microarray data. We applied imputation to low-pass WGBS data, assessed inter-platform correlation and identified molecular subgroups by directly integrating WGBS sample data with pre-existing array-trained models. We developed machine learning WGBS-based classifiers and compared performance against microarray. We optimised reference-free aneuploidy detection with low-pass WGBS and assessed concordance with microarray-derived aneuploidy calls. RESULTS: We optimised a pipeline for processing, QC, and analysis of low-pass WGBS data, suitable for routine molecular subgrouping and reference-free aneuploidy assessment that achieves 96% sensitivity compared to microarray approaches. A pilot study of the suitability of FFPE was promising, and we demonstrate that WGBS data can be integrated into existing array-trained models with high assignment probabilities. Also, WGBS-derived classifier performance measures exceeded microarray-derived classifiers. CONCLUSION: We describe a platform-independent WGBS assay for molecular subgrouping of medulloblastoma. It performs equivalently to array-based methods at increasingly comparable cost ($400 vs $580) and provides a proof-of-concept for routine clinical adoption using standard WGS technology. Finally, the full methylome enabled elucidation of additional biological heterogeneity that has hitherto been inaccessible.
Abstract
Introduction
International consensus recognises four molecular subgroups of medulloblastoma, each with distinct molecular features and clinical outcomes. The current gold-standard for ...subgroup assignment is DNA methylation microarray. There is an unmet need to develop platform-independent subgrouping assays which are both non-proprietary and compatible with rapidly-expanding WGS capacity in healthcare. Whole Genome Bisulfite Sequencing (WGBS) enables the assessment of genome-wide methylation status at single-base resolution. Previously, WGBS adoption has been limited by cost and sample quality/quantity requirements. Its application for routine detection of medulloblastoma subgroups has not previously been reported.
Methodology
Two datasets were utilised; 36 newly-sequenced low-depth (10x coverage) and 34 publicly-available high-depth (30x) WGBS medulloblastomas, all with matched DNA methylation microarray data. We compared platform concordance and identified molecular subgroups. Machine-learning WGBS-based subgroup classifiers were optimised and compared between platforms. Aneuploidy and mutation detection using WGBS was optimised and compared to microarray-derived estimates where possible. Finally, comprehensive subgroup-specific DNA methylation signatures were identified.
Results
We optimised a pipeline for processing, quality control and analysis of low-depth WGBS data, suitable for routine molecular subgrouping and aneuploidy assessment. We demonstrated the suitability of fresh-frozen and FFPE DNA for WGBS, and, using downsampling, showed that subgroup calling is robust at coverages as low as 2x. We identified differentially methylated regions that, due to poor representation, could not be detected using methylation microarrays. Molecular subgroups of medulloblastoma assigned using WGBS were concordant with array-based definitions, and WGBS-derived classifier performance measures exceeded microarray-derived classifiers.
Conclusion
We describe a platform-independent assay for molecular subgrouping of medulloblastoma using WGBS. It performs equivalently to current array-based methods at comparable cost ($405 vs $596) and provides a proof-of-concept for its routine clinical adoption using standard WGS technology. Finally, the full methylome enabled elucidation of additional biological heterogeneity that has hitherto been inaccessible.
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.
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•Group 3 and group 4 medulloblastoma exist along a transcriptomic continuum•Position on the continuum is prognostic and reflects key molecular aberrations•Intratumoral transcriptional heterogeneity is limited according to subtype•This continuum maps to early fetal development, implicating cells of origin
The childhood brain tumor medulloblastoma is classified into multiple DNA methylation-based subtypes. Using RNA-seq, Williamson et al. show that group 3 and group 4 tumors manifest as intermediates on a transcriptomic continuum. Position on the continuum is associated with molecular pathology and disease course. The continuum mirrors early cerebellar development, implicating cells of origin.
Abstract
Determinants of survivorship outcomes are emerging from limited studies of medulloblastoma (MB) survivors. We undertook an integrated analysis of biological (tumour group, host genetics) and ...clinico-demographic features in patients treated on the SIOP-UKCCSG-PNET3 and HIT-SIOP-PNET4 clinical trials with available quality of survival (QoS) data (n=218), to determine key correlates of survivorship, and their clinical potential. Treatment/demographic factors and molecular subgroup (MBWNT, MBSHH, MBGrp3, MBGrp4) were assessed against health status, behavioural functioning, and health-related quality of life (HrQoL). In DNA from HIT-SIOP-PNET4 (n=74), 39 candidate SNPs with known modifying effects on neurocognitive outcomes (e.g., involved in oxidative stress/inflammation) were genotyped and assessed against Wechsler Intelligence Scale (WISC) scores. As expected, MBSHH was associated with improved HrQoL, but subgroup did not associate further with QoS outcomes. SIOP-UKCCSG-PNET3 patients receiving chemotherapy before craniospinal irradiation (CSI) had significantly lower health status (p=0.021) and behavioural functioning (p<0.016) compared to patients treated with CSI alone, and those treated on both arms (maintenance chemotherapy and hyperfractionated (36Gy) or standard (23.4Gy) CSI) of HIT-SIOP-PNET4. SIOP-UKCCSG-PNET3 patients receiving CSI-only had better HrQoL scores than those who received pre-CSI chemotherapy and both HIT-SIOP-PNET4 arms (p=0.004). Females reported worse HrQoL/behavioural functioning across both trials (p<0.04). In HIT-SIOP-PNET4, longer intervals from diagnosis to CSI predicted worse HrQoL/health status (p<0.05). Neither molecular group nor clinico-demographic features tested were associated with neurocognition. In contrast, 6 SNPs significantly associated with ≥1 WISC domain; 4/6 showed multiple associations and were independently prognostic; further associations were apparent at the gene/pathway level. This large, integrated and multi-disciplinary analysis of two independent trials cohorts has revealed multiple factors predictive of medulloblastoma survivorship including treatment (chemotherapy, time to CSI), tumour (molecular group) and host genetic factors. Assessment in further prospective series are required to determine their potential as a basis for modifications to disease management.
Group 4 tumours (MB
Grp4
) represent the majority of non-WNT/non-SHH medulloblastomas. Their clinical course is poorly predicted by current risk-factors. MB
Grp4
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 MB
Grp4
molecular landscape and determined its utility to improve clinical management. A clinically-annotated discovery cohort (n = 362 MB
Grp4
) 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 MB
Grp4
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 MB
Grp4
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 MB
Grp4
disease. Novel validated survival models, integrating clinical features, methylation and WCA groups, improve outcome prediction and re-define risk-status for ~ 80% of MB
Grp4
. Our MB
Grp4
favourable-risk group has MB
WNT
-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.
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