The clinical significance of sub-total surgical resection in childhood medulloblastoma: a multi-cohort analysis of 1100 patients.

Background: Medulloblastoma patients with a sub-total surgical resection (STR; >1.5 cm2 primary tumour residuum post-surgery) typically receive intensified treatment. However, the association of STR with poor outcomes has not been observed consistently, questioning the validity of STR as a high-risk disease feature. Methods: We collected extent of resection (EOR) data from 1110 patients (from UK CCLG centres (n = 416, collected between September 1990 and July 2014) and published (n = 694) cohorts), the largest cohort of molecularly and clinically annotated tumours assembled to specifically assess the significance of EOR. We performed association and univariable/multivariable survival analyses, assessing overall survival (OS) cohort-wide and with reference to the four consensus medulloblastoma molecular groups and clinical features. Findings: STR was reported in 20% (226/1110) of patients. Non-WNT (p = 0.047), children <5 years at diagnosis (p = 0.021) and metastatic patients (p < 0.0001) were significantly more likely to have a STR. In cohort-wide analysis, STR was associated with worse survival in univariable analysis (p < 0.0001). Examination of specific disease contexts showed that STR was prognostic in univariate analysis for patients receiving cranio-spinal irradiation (CSI) and chemotherapy (p = 0.016) and for patients with Group 3 tumours receiving CSI (p = 0.039). STR was not independently prognostic in multivariable analyses; outcomes for patients who have STR as their only risk-feature are as per standard-risk disease. Specifically, STR was not prognostic in non-metastatic patients that received upfront CSI. Interpretation: In a cohort of 1100 molecularly characterised medulloblastoma patients, STR (n = 226) predicted significantly lower OS in univariable analysis, but was not an independent prognostic factor. Our data suggest that maximal safe resection can continue to be carried out for patients with medulloblastoma and suggest STR should not inform patient management when observed as a sole, isolated risk-feature. Funding: Cancer Research UK, Newcastle Hospitals Charity, Children's Cancer North, British Division of the International Academy of Pathology.

Metabolite profiles of medulloblastoma for rapid and non-invasive detection of molecular disease groups.

BACKGROUND: The malignant childhood brain tumour, medulloblastoma, is classified clinically into molecular groups which guide therapy. DNA-methylation profiling is the current classification 'gold-standard', typically delivered 3-4 weeks post-surgery. Pre-surgery non-invasive diagnostics thus offer significant potential to improve early diagnosis and clinical management. Here, we determine tumour metabolite profiles of the four medulloblastoma groups, assess their diagnostic utility using tumour tissue and potential for non-invasive diagnosis using in vivo magnetic resonance spectroscopy (MRS). METHODS: Metabolite profiles were acquired by high-resolution magic-angle spinning NMR spectroscopy (MAS) from 86 medulloblastomas (from 59 male and 27 female patients), previously classified by DNA-methylation array (WNT (n = 9), SHH (n = 22), Group3 (n = 21), Group4 (n = 34)); RNA-seq data was available for sixty. Unsupervised class-discovery was performed and a support vector machine (SVM) constructed to assess diagnostic performance. The SVM classifier was adapted to use only metabolites (n = 10) routinely quantified from in vivo MRS data, and re-tested. Glutamate was assessed as a predictor of overall survival. FINDINGS: Group-specific metabolite profiles were identified; tumours clustered with good concordance to their reference molecular group (93%). GABA was only detected in WNT, taurine was low in SHH and lipids were high in Group3. The tissue-based metabolite SVM classifier had a cross-validated accuracy of 89% (100% for WNT) and, adapted to use metabolites routinely quantified in vivo, gave a combined classification accuracy of 90% for SHH, Group3 and Group4. Glutamate predicted survival after incorporating known risk-factors (HR = 3.39, 95% CI 1.4-8.1, p = 0.025). INTERPRETATION: Tissue metabolite profiles characterise medulloblastoma molecular groups. Their combination with machine learning can aid rapid diagnosis from tissue and potentially in vivo. Specific metabolites provide important information; GABA identifying WNT and glutamate conferring poor prognosis. FUNDING: Children with Cancer UK, Cancer Research UK, Children's Cancer North and a Newcastle University PhD studentship.

Deriving a continuum score for group 3 and 4 medulloblastoma tumor samples analyzed via RNA-sequencing or DNA methylation microarray.

Here, we present a protocol for deriving a continuum score for group 3 and 4 medulloblastoma tumor samples analyzed via RNA-sequencing or DNA methylation microarray. We describe steps for utilizing NMF-defined group 3/group 4 metagenes to calculate a continuum score between 0 and 1 that can be projected onto new sample data analyzed via RNA-sequencing. We then detail procedures for reverse engineering a continuum score for samples analyzed via DNA methylation microarray using a random forest classifier.

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.

Timing is everything: A connection between medulloblastoma prognosis and foetal cerebellar development.

The childhood brain tumour medulloblastoma is typically classified into multiple discrete molecular subgroups with characteristic DNA methylation and expression patterns. Several of these subgroups are used as, or proposed to be, an effective basis for treatment stratification. Here, we highlight the close connection between the findings described in a recent series of studies which, together, strongly imply a continuous association between survival outcome, the transcriptional profile of a Group3/Group4 (i.e. non-WNT/non-SHH) medulloblastoma and the specific point during early foetal cerebellar development at which initial pathogenic disruption took place. This has important implications for future efforts to model the disease by incorporating driving molecular features into their specific developmental context. This further suggests that instead of relying upon discrete DNA methylation subgroups, using expression biomarkers as the basis of a continuous risk predictor may produce a more effective risk stratification of patients with Group3/Group4 medulloblastoma.

Molecular characterization and clinical outcome of B-cell precursor acute lymphoblastic leukemia with IG-MYC rearrangement.

Rarely, immunophenotypically immature B-cell precursor acute lymphoblastic leukemia (BCP-ALL) carries an immunoglobulin- MYC rearrangement (IG-MYC-r). This can result in diagnostic confusion with Burkitt lymphoma/leukemia and use of individualized treatment schedules of unproven efficacy. Here we compare the molecular characteristics of these conditions and investigate historic clinical outcome data. We identified 90 cases registered in a national BCP-ALL clinical trial/registry. When present, diagnostic material underwent cytogenetic, exome, methylome and transcriptome analyses. The outcomes analyzed were 3-year event-free survival and overall survival. IG-MYC-r was identified in diverse cytogenetic backgrounds, co-existing with either established BCP-ALL-specific abnormalities (high hyperdiploidy, n=3; KMT2A-rearrangement, n=6; iAMP21, n=1; BCR-ABL1, n=1); BCL2/BCL6-rearrangements (n=15); or, most commonly, as the only defining feature (n=64). Within this final group, precursor-like V(D)J breakpoints predominated (8/9) and KRAS mutations were common (5/11). DNA methylation identified a cluster of V(D)J-rearranged cases, clearly distinct from Burkitt leukemia/lymphoma. Children with IG-MYC-r within that subgroup had a 3-year event-free survival of 47% and overall survival of 60%, representing a high-risk BCP-ALL. To develop effective management strategies this group of patients must be allowed access to contemporary, minimal residual disease-adapted, prospective clinical trial protocols.

Genome wide DNA methylation analysis identifies novel molecular subgroups and predicts survival in neuroblastoma.

BACKGROUND: Neuroblastoma is the most common malignancy in infancy, accounting for 15% of childhood cancer deaths. Outcome for the high-risk disease remains poor. DNA-methylation patterns are significantly altered in all cancer types and can be utilised for disease stratification. METHODS: Genome-wide DNA methylation (n = 223), gene expression (n = 130), genetic/clinical data (n = 213), whole-exome sequencing (n = 130) was derived from the TARGET study. Methylation data were derived from HumanMethylation450 BeadChip arrays. t-SNE was used for the segregation of molecular subgroups. A separate validation cohort of 105 cases was studied. RESULTS: Five distinct neuroblastoma molecular subgroups were identified, based on genome-wide DNA-methylation patterns, with unique features in each, including three subgroups associated with known prognostic features and two novel subgroups. As expected, Cluster-4 (infant diagnosis) had significantly better 5-year progression-free survival (PFS) than the four other clusters. However, in addition, the molecular subgrouping identified multiple patient subsets with highly increased risk, most notably infant patients that do not map to Cluster-4 (PFS 50% vs 80% for Cluster-4 infants, P = 0.005), and allowed identification of subgroup-specific methylation differences that may reflect important biological differences within neuroblastoma. CONCLUSIONS: Methylation-based clustering of neuroblastoma reveals novel molecular subgroups, with distinct molecular/clinical characteristics and identifies a subgroup of higher-risk infant patients.

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.

Single-cell DNA sequencing identifies risk-associated clonal complexity and evolutionary trajectories in childhood medulloblastoma development.

We reconstructed the natural history and temporal evolution of the most common childhood brain malignancy, medulloblastoma, by single-cell whole-genome sequencing (sc-WGS) of tumours representing its major molecular sub-classes and clinical risk groups. Favourable-risk disease sub-types assessed (MBWNT and infant desmoplastic/nodular MBSHH) typically comprised a single clone with no evidence of further evolution. In contrast, highest risk sub-classes (MYC-amplified MBGroup3 and TP53-mutated MBSHH) were most clonally diverse and displayed gradual evolutionary trajectories. Clinically adopted biomarkers (e.g. chromosome 6/17 aberrations; CTNNB1/TP53 mutations) were typically early-clonal/initiating events, exploitable as targets for early-disease detection; in analyses of spatially distinct tumour regions, a single biopsy was sufficient to assess their status. Importantly, sc-WGS revealed novel events which arise later and/or sub-clonally and more commonly display spatial diversity; their clinical significance and role in disease evolution post-diagnosis now require establishment. These findings reveal diverse modes of tumour initiation and evolution in the major medulloblastoma sub-classes, with pathogenic relevance and clinical potential.

Emergence and maintenance of actionable genetic drivers at medulloblastoma relapse.

BACKGROUND: Less than 5% of medulloblastoma (MB) patients survive following failure of contemporary radiation-based therapies. Understanding the molecular drivers of medulloblastoma relapse (rMB) will be essential to improve outcomes. Initial genome-wide investigations have suggested significant genetic divergence of the relapsed disease. METHODS: We undertook large-scale integrated characterization of the molecular features of rMB-molecular subgroup, novel subtypes, copy number variation (CNV), and driver gene mutation. 119 rMBs were assessed in comparison with their paired diagnostic samples (n = 107), alongside an independent reference cohort sampled at diagnosis (n = 282). rMB events were investigated for association with outcome post-relapse in clinically annotated patients (n = 54). RESULTS: Significant genetic evolution occurred over disease-course; 40% of putative rMB drivers emerged at relapse and differed significantly between molecular subgroups. Non-infant MBSHH displayed significantly more chromosomal CNVs at relapse (TP53 mutation-associated). Relapsed MBGroup4 demonstrated the greatest genetic divergence, enriched for targetable (eg, CDK amplifications) and novel (eg, USH2A mutations) events. Importantly, many hallmark features of MB were stable over time; novel subtypes (>90% of tumors) and established genetic drivers (eg, SHH/WNT/P53 mutations; 60% of rMB events) were maintained from diagnosis. Critically, acquired and maintained rMB events converged on targetable pathways which were significantly enriched at relapse (eg, DNA damage signaling) and specific events (eg, 3p loss) predicted survival post-relapse. CONCLUSIONS: rMB is characterised by the emergence of novel events and pathways, in concert with selective maintenance of established genetic drivers. Together, these define the actionable genetic landscape of rMB and provide a basis for improved clinical management and development of stratified therapeutics, across disease-course.

Integration of genome-level data to allow identification of subtype-specific vulnerability genes as novel therapeutic targets.

The identification of cancer-specific vulnerability genes is one of the most promising approaches for developing more effective and less toxic cancer treatments. Cancer genomes exhibit thousands of changes in DNA methylation and gene expression, with the vast majority likely to be passenger changes. We hypothesised that, through integration of genome-wide DNA methylation/expression data, we could exploit this inherent variability to identify cancer subtype-specific vulnerability genes that would represent novel therapeutic targets that could allow cancer-specific cell killing. We developed a bioinformatics pipeline integrating genome-wide DNA methylation/gene expression data to identify candidate subtype-specific vulnerability partner genes for the genetic drivers of individual genetic/molecular subtypes. Using acute lymphoblastic leukaemia as an initial model, 21 candidate subtype-specific vulnerability genes were identified across the five common genetic subtypes, with at least one per subtype. To confirm the approach was applicable across cancer types, we also assessed medulloblastoma, identifying 15 candidate subtype-specific vulnerability genes across three of four established subtypes. Almost all identified genes had not previously been implicated in these diseases. Functional analysis of seven candidate subtype-specific vulnerability genes across the two tumour types confirmed that siRNA-mediated knockdown induced significant inhibition of proliferation/induction of apoptosis, which was specific to the cancer subtype in which the gene was predicted to be specifically lethal. Thus, we present a novel approach that integrates genome-wide DNA methylation/expression data to identify cancer subtype-specific vulnerability genes as novel therapeutic targets. We demonstrate this approach is applicable to multiple cancer types and identifies true functional subtype-specific vulnerability genes with high efficiency.

Advanced molecular pathology for rare tumours: A national feasibility study and model for centralised medulloblastoma diagnostics.

AIMS: Application of advanced molecular pathology in rare tumours is hindered by low sample numbers, access to specialised expertise/technologies and tissue/assay QC and rapid reporting requirements. We assessed the feasibility of co-ordinated real-time centralised pathology review (CPR), encompassing molecular diagnostics and contemporary genomics (RNA-seq/DNA methylation-array). METHODS: This nationwide trial in medulloblastoma (<80 UK diagnoses/year) introduced a national reference centre (NRC) and assessed its performance and reporting to World Health Organisation standards. Paired frozen/formalin-fixed, paraffin-embedded tumour material were co-submitted from 135 patients (16 referral centres). RESULTS: Complete CPR diagnostics were successful for 88% (120/135). Inadequate sampling was the most common cause of failure; biomaterials were typically suitable for methylation-array (129/135, 94%), but frozen tissues commonly fell below RNA-seq QC requirements (53/135, 39%). Late reporting was most often due to delayed submission. CPR assigned or altered histological variant (vs local diagnosis) for 40/135 tumours (30%). Benchmarking/QC of specific biomarker assays impacted test results; fluorescent in-situ hybridisation most accurately identified high-risk MYC/MYCN amplification (20/135, 15%), while combined methods (CTNNB1/chr6 status, methylation-array subgrouping) best defined favourable-risk WNT tumours (14/135; 10%). Engagement of a specialist pathologist panel was essential for consensus assessment of histological variants and immunohistochemistry. Overall, CPR altered clinical risk-status for 29% of patients. CONCLUSION: National real-time CPR is feasible, delivering robust diagnostics to WHO criteria and assignment of clinical risk-status, significantly altering clinical management. Recommendations and experience from our study are applicable to advanced molecular diagnostics systems, both local and centralised, across rare tumour types, enabling their application in biomarker-driven routine diagnostics and clinical/research studies.

Inter and intra-tumoral heterogeneity as a platform for personalized therapies in medulloblastoma.

Medulloblastoma is the most common malignant CNS tumor of childhood, affecting ~350 patients/year in the USA. In 2020, most children are cured of their disease, however, survivors are left with life-long late-effects as a consequence of intensive surgery, and application of chemo- and radio-therapy to the developing brain. A major contributor to improvements in patient survival has been the development of risk-stratified treatments derived from a better understanding of the prognostic value of disease biomarkers. The characterization and validation of these biomarkers has engendered a comprehensive understanding of the extensive heterogeneity that exists within the disease, which can occur both between and within tumors (inter- and intra-tumoral heterogeneity, respectively). In this review, we discuss inter-tumoral heterogeneity, describing the early characterization of clinical and histopathological disease heterogeneity, the more recent elucidation of molecular disease subgroups, and the potential for novel therapies based on specific molecular defects. We reflect on the limitations of current approaches when applied to a rare disease. We then review early investigations of intra-tumoral heterogeneity using FISH and immunohistochemical approaches, and focus on the application of next generation sequencing on bulk tumors to elucidate intra-tumoral heterogeneity. Finally, we critically appraise the applications of single-cell sequencing approaches and discuss their potential to drive next biological insights, and for routine clinical application.

Time, pattern, and outcome of medulloblastoma relapse and their association with tumour biology at diagnosis and therapy: a multicentre cohort study.

BACKGROUND: Disease relapse occurs in around 30% of children with medulloblastoma, and is almost universally fatal. We aimed to establish whether the clinical and molecular characteristics of the disease at diagnosis are associated with the nature of relapse and subsequent disease course, and whether these associations could inform clinical management. METHODS: In this multicentre cohort study we comprehensively surveyed the clinical features of medulloblastoma relapse (time to relapse, pattern of relapse, time from relapse to death, and overall outcome) in centrally reviewed patients who relapsed following standard upfront therapies, from 16 UK Children's Cancer and Leukaemia Group institutions and four collaborating centres. We compared these relapse-associated features with clinical and molecular features at diagnosis, including established and recently described molecular features, prognostic factors, and treatment at diagnosis and relapse. FINDINGS: 247 patients (175 [71%] boys and 72 [29%] girls) with medulloblastoma relapse (median year of diagnosis 2000 [IQR 1995-2006]) were included in this study. 17 patients were later excluded from further analyses because they did not meet the age and treatment criteria for inclusion. Patients who received upfront craniospinal irradiation (irradiated group; 178 [72%] patients) had a more prolonged time to relapse compared with patients who did not receive upfront craniospinal irradiation (non-irradiated group; 52 [21%] patients; p<0·0001). In the non-irradiated group, craniospinal irradiation at relapse (hazard ratio [HR] 0·27, 95% CI 0·11-0·68) and desmoplastic/nodular histology (0·23, 0·07-0·77) were associated with prolonged time to death after relapse, MYC amplification was associated with a reduced overall survival (23·52, 4·85-114·05), and re-resection at relapse was associated with longer overall survival (0·17, 0·05-0·57). In the irradiated group, patients with MBGroup3 tumours relapsed significantly more quickly than did patients with MBGroup4 tumours (median 1·34 [0·99-1·89] years vs 2·04 [1·39-3·42 years; p=0·0043). Distant disease was prevalent in patients with MBGroup3 (23 [92%] of 25 patients) and MBGroup4 (56 [90%] of 62 patients) tumour relapses. Patients with distantly-relapsed MBGroup3 and MBGroup4 displayed both nodular and diffuse patterns of disease whereas isolated nodular relapses were rare in distantly-relapsed MBSHH (1 [8%] of 12 distantly-relapsed MBSHH were nodular alone compared with 26 [34%] of 77 distantly-relapsed MBGroup3 and MBGroup4). In MBGroup3 and MBGroup4, nodular disease was associated with a prolonged survival after relapse (HR 0·42, 0·21-0·81). Investigation of second-generation MBGroup3 and MBGroup4 molecular subtypes refined our understanding of heterogeneous relapse characteristics. Subtype VIII had prolonged time to relapse and subtype II had a rapid time from relapse to death. Subtypes II, III, and VIII developed a significantly higher incidence of distant disease at relapse whereas subtypes V and VII did not (equivalent rates to diagnosis). INTERPRETATION: This study suggests that the nature and outcome of medulloblastoma relapse are biology and therapy-dependent, providing translational opportunities for improved disease management through biology-directed disease surveillance, post-relapse prognostication, and risk-stratified selection of second-line treatment strategies. FUNDING: Cancer Research UK, Action Medical Research, The Tom Grahame Trust, The JGW Patterson Foundation, Star for Harris, The Institute of Child Health - Newcastle University - Institute of Child Health High-Risk Childhood Brain Tumour Network (co-funded by The Brain Tumour Charity, Great Ormond Street Children's Charity, and Children with Cancer UK).

Pediatric pan-central nervous system tumor analysis of immune-cell infiltration identifies correlates of antitumor immunity.

Immune-therapy is an attractive alternative therapeutic approach for targeting central nervous system (CNS) tumors and the constituency of the Tumor Immune Microenvironment (TIME) likely to predict patient response. Here, we describe the TIME of >6000 primarily pediatric CNS tumors using a deconvolution approach (methylCIBERSORT). We produce and validate a custom reference signature defining 11 non-cancer cell types to estimate relative proportions of infiltration in a panCNS tumor cohort spanning 80 subtypes. We group patients into three broad immune clusters associated with CNS tumor types/subtypes. In cohorts of medulloblastomas (n = 2325), malignant rhabdoid tumors (n = 229) and pediatric high-grade gliomas (n = 401), we show significant associations with molecular subgroups/subtypes, mutations, and prognosis. We further identify tumor-specific immune clusters with phenotypic characteristics relevant to immunotherapy response (i.e. Cytolytic score, PDL1 expression). Our analysis provides an indication of the potential future therapeutic and prognostic possibilities of immuno-methylomic profiling in pediatric CNS tumor patients that may ultimately inform approach to immune-therapy.

Second-generation molecular subgrouping of medulloblastoma: an international meta-analysis of Group 3 and Group 4 subtypes.

In 2012, an international consensus paper reported that medulloblastoma comprises four molecular subgroups (WNT, SHH, Group 3, and Group 4), each associated with distinct genomic features and clinical behavior. Independently, multiple recent reports have defined further intra-subgroup heterogeneity in the form of biologically and clinically relevant subtypes. However, owing to differences in patient cohorts and analytical methods, estimates of subtype number and definition have been inconsistent, especially within Group 3 and Group 4. Herein, we aimed to reconcile the definition of Group 3/Group 4 MB subtypes through the analysis of a series of 1501 medulloblastomas with DNA-methylation profiling data, including 852 with matched transcriptome data. Using multiple complementary bioinformatic approaches, we compared the concordance of subtype calls between published cohorts and analytical methods, including assessments of class-definition confidence and reproducibility. While the lowest complexity solutions continued to support the original consensus subgroups of Group 3 and Group 4, our analysis most strongly supported a definition comprising eight robust Group 3/Group 4 subtypes (types I-VIII). Subtype II was consistently identified across all component studies, while all others were supported by multiple class-definition methods. Regardless of analytical technique, increasing cohort size did not further increase the number of identified Group 3/Group 4 subtypes. Summarizing the molecular and clinico-pathological features of these eight subtypes indicated enrichment of specific driver gene alterations and cytogenetic events amongst subtypes, and identified highly disparate survival outcomes, further supporting their biological and clinical relevance. Collectively, this study provides continued support for consensus Groups 3 and 4 while enabling robust derivation of, and categorical accounting for, the extensive intertumoral heterogeneity within Groups 3 and 4, revealed by recent high-resolution subclassification approaches. Furthermore, these findings provide a basis for application of emerging methods (e.g., proteomics/single-cell approaches) which may additionally inform medulloblastoma subclassification. Outputs from this study will help shape definition of the next generation of medulloblastoma clinical protocols and facilitate the application of enhanced molecularly guided risk stratification to improve outcomes and quality of life for patients and their families.

SH2B3 inactivation through CN-LOH 12q is uniquely associated with B-cell precursor ALL with iAMP21 or other chromosome 21 gain.

In more than 30% of B-cell precursor acute lymphoblastic leukaemia (B-ALL), chromosome 21 sequence is overrepresented through aneuploidy or structural rearrangements, exemplified by intrachromosomal amplification of chromosome 21 (iAMP21). Although frequent, the mechanisms by which these abnormalities promote B-ALL remain obscure. Intriguingly, we found copy number neutral loss of heterozygosity (CN-LOH) of 12q was recurrent in iAMP21-ALL, but never observed in B-ALL without some form of chromosome 21 gain. As a consequence of CN-LOH 12q, mutations or deletions of the adaptor protein, SH2B3, were converted to homozygosity. In patients without CN-LOH 12q, bi-allelic abnormalities of SH2B3 occurred, but only in iAMP21-ALL, giving an overall incidence of 18% in this sub-type. Review of published data confirmed a tight association between overrepresentation of chromosome 21 and both CN-LOH 12q and SH2B3 abnormalities in B-ALL. Despite relatively small patient numbers, preliminary analysis linked 12q abnormalities to poor outcome in iAMP21-ALL (p = 0.03). Homology modelling of a leukaemia-associated SH2 domain mutation and in vitro analysis of patient-derived xenograft cells implicated the JAK/STAT pathway as one likely target for SH2B3 tumour suppressor activity in iAMP21-ALL.

Prognostic effect of whole chromosomal aberration signatures in standard-risk, non-WNT/non-SHH medulloblastoma: a retrospective, molecular analysis of the HIT-SIOP PNET 4 trial.

BACKGROUND: Most children with medulloblastoma fall within the standard-risk clinical disease group defined by absence of high-risk features (metastatic disease, large-cell/anaplastic histology, and MYC amplification), which includes 50-60% of patients and has a 5-year event-free survival of 75-85%. Within standard-risk medulloblastoma, patients in the WNT subgroup are established as having a favourable prognosis; however, outcome prediction for the remaining majority of patients is imprecise. We sought to identify novel prognostic biomarkers to enable improved risk-adapted therapies. METHODS: The HIT-SIOP PNET 4 trial recruited 338 patients aged 4-21 years with medulloblastoma between Jan 1, 2001, and Dec 31, 2006, in 120 treatment institutions in seven European countries to investigate hyperfractionated radiotherapy versus standard radiotherapy. In this retrospective analysis, we assessed the remaining tumour samples from patients in the HIT-SIOP PNET 4 trial (n=136). We assessed the clinical behaviour of the molecularly defined WNT and SHH subgroups, and identified novel independent prognostic markers and models for standard-risk patients with non-WNT/non-SHH disease. Because of the scarcity and low quality of available genomic material, we used a mass spectrometry-minimal methylation classifier assay (MS-MIMIC) to assess methylation subgroup and a molecular inversion probe array to detect genome-wide copy number aberrations. Prognostic biomarkers and models identified were validated in an independent, demographically matched cohort (n=70) of medulloblastoma patients with non-WNT/non-SHH standard-risk disease treated with conventional therapies (maximal surgical resection followed by adjuvant craniospinal irradiation [all patients] and chemotherapy [65 of 70 patients], at UK Children's Cancer and Leukaemia Group and European Society for Paediatric Oncology (SIOPE) associated treatment centres between 1990 and 2014. These samples were analysed by Illumina 450k DNA methylation microarray. HIT-SIOP PNET 4 is registered with ClinicalTrials.gov, number NCT01351870. FINDINGS: We analysed methylation subgroup, genome-wide copy number aberrations, and mutational features in 136 assessable tumour samples from the HIT-SIOP PNET 4 cohort, representing 40% of the 338 patients in the trial cohort. This cohort of 136 samples consisted of 28 (21%) classified as WNT, 17 (13%) as SHH, and 91 (67%) as non-WNT/non-SHH (we considered Group3 and Group4 medulloblastoma together in our analysis because of their similar molecular and clinical features). Favourable outcomes for WNT tumours were confirmed in patients younger than 16 years, and all relapse events in SHH (four [24%] of 17) occurred in patients with TP53 mutation (TP53mut) or chromosome 17p loss. A novel whole chromosomal aberration signature associated with increased ploidy and multiple non-random whole chromosomal aberrations was identified in 38 (42%) of the 91 samples from patients with non-WNT/non-SHH medulloblastoma in the HIT-SIOP PNET 4 cohort. Biomarkers associated with this whole chromosomal aberration signature (at least two of chromosome 7 gain, chromosome 8 loss, and chromosome 11 loss) predicted favourable prognosis. Patients with non-WNT/non-SHH medulloblastoma could be reclassified by these markers as having favourable-risk or high-risk disease. In patients in the HIT-SIOP PNET4 cohort with non-WNT/non-SHH medulloblastoma, with a median follow-up of 6·7 years (IQR 5·8-8·2), 5-year event-free survival was 100% in the favourable-risk group and 68% (95% CI 57·5-82·7; p=0·00014) in the high-risk group. In the validation cohort, with a median follow-up of 5·6 years (IQR 3·1-8·1), 5-year event-free survival was 94·7% (95% CI 85·2-100) in the favourable-risk group and 58·6% (95% CI 45·1-76·1) in the high-risk group (hazard ratio 9·41, 95% CI 1·25-70·57; p=0·029). Our comprehensive molecular investigation identified subgroup-specific risk models which allowed 69 (51%) of 134 accessible patients from the standard-risk medulloblastoma HIT-SIOP PNET 4 cohort to be assigned to a favourable-risk group. INTERPRETATION: We define a whole chromosomal signature that allows the assignment of non-WNT/non-SHH medulloblastoma patients normally classified as standard-risk into favourable-risk and high-risk categories. In addition to patients younger than 16 years with WNT tumours, patients with non-WNT/non-SHH tumours with our defined whole chromosomal aberration signature and patients with SHH-TP53wild-type tumours should be considered for therapy de-escalation in future biomarker-driven, risk-adapted clinical trials. The remaining subgroups of patients with high-risk medulloblastoma might benefit from more intensive therapies. FUNDING: Cancer Research UK, Swedish Childhood Cancer Foundation, French Ministry of Health/French National Cancer Institute, and the German Children's Cancer Foundation.

Analysis of Listeria using exogenous volatile organic compound metabolites and their detection by static headspace-multi-capillary column-gas chromatography-ion mobility spectrometry (SHS-MCC-GC-IMS).

Listeria monocytogenes is a Gram-positive bacterium and an opportunistic food-borne pathogen which poses significant risk to the immune-compromised and pregnant due to the increased likelihood of acquiring infection and potential transmission of infection to the unborn child. Conventional methods of analysis suffer from either long turn-around times or lack the ability to discriminate between Listeria spp. reliably. This paper investigates an alternative method of detecting Listeria spp. using two novel enzyme substrates that liberate exogenous volatile organic compounds in the presence of α-mannosidase and D-alanyl aminopeptidase. The discriminating capabilities of this approach for identifying L. monocytogenes from other species of Listeria are investigated. The liberated volatile organic compounds (VOCs) are detected using an automated analytical technique based on static headspace-multi-capillary column-gas chromatography-ion mobility spectrometry (SHS-MCC-GC-IMS). The results obtained by SHS-MCC-GC-IMS are compared with those obtained by the more conventional analytical technique of headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results found that it was possible to differentiate between L. monocytogenes and L. ivanovii, based on their VOC response from α-mannosidase activity.