Rebecca Hill

Subgroup-Specific Prognostic Implications of TP53 Mutation in Medulloblastoma

PURPOSE Reports detailing the prognostic impact of TP53 mutations in medulloblastoma offer conflicting conclusions. We resolve this issue through the inclusion of molecular subgroup profiles. PATIENTS AND METHODS We determined subgroup affiliation, TP53 mutation status, and clinical outcome in a discovery cohort of 397 medulloblastomas. We subsequently validated our results on an independent cohort of 156 medulloblastomas. RESULTS TP53 mutations are enriched in wingless (WNT; 16%) and sonic hedgehog (SHH; 21%) medulloblastomas and are virtually absent in subgroups 3 and 4 tumors (P < .001). Patients with SHH/TP53 mutant tumors are almost exclusively between ages 5 and 18 years, dramatically different from the general SHH distribution (P < .001). Children with SHH/TP53 mutant tumors harbor 56% germline TP53 mutations, which are not observed in children with WNT/TP53 mutant tumors. Five-year overall survival (OS; ± SE) was 41% ± 9% and 81% ± 5% for patients with SHH medulloblastomas with and without TP53 mutations, respectively (P < .001). Furthermore, TP53 mutations accounted for 72% of deaths in children older than 5 years with SHH medulloblastomas. In contrast, 5-year OS rates were 90% ± 9% and 97% ± 3% for patients with WNT tumors with and without TP53 mutations (P = .21). Multivariate analysis revealed that TP53 status was the most important risk factor for SHH medulloblastoma. Survival rates in the validation cohort mimicked the discovery results, revealing that poor survival of TP53 mutations is restricted to patients with SHH medulloblastomas (P = .012) and not WNT tumors. CONCLUSION Subgroup-specific analysis reconciles prior conflicting publications and confirms that TP53 mutations are enriched among SHH medulloblastomas, in which they portend poor outcome and account for a large proportion of treatment failures in these patients.

Combined MYC and P53 defects emerge at medulloblastoma relapse and define rapidly progressive, therapeutically targetable disease

Summary We undertook a comprehensive clinical and biological investigation of serial medulloblastoma biopsies obtained at diagnosis and relapse. Combined MYC family amplifications and P53 pathway defects commonly emerged at relapse, and all patients in this group died of rapidly progressive disease postrelapse. To study this interaction, we investigated a transgenic model of MYCN-driven medulloblastoma and found spontaneous development of Trp53 inactivating mutations. Abrogation of p53 function in this model produced aggressive tumors that mimicked characteristics of relapsed human tumors with combined P53-MYC dysfunction. Restoration of p53 activity and genetic and therapeutic suppression of MYCN all reduced tumor growth and prolonged survival. Our findings identify P53-MYC interactions at medulloblastoma relapse as biomarkers of clinically aggressive disease that may be targeted therapeutically.

Novel molecular subgroups for clinical classification and outcome prediction in childhood medulloblastoma: a cohort study

Summary Background International consensus recognises four medulloblastoma molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGrp3), and group 4 (MBGrp4), each defined by their characteristic genome-wide transcriptomic and DNA methylomic profiles. These subgroups have distinct clinicopathological and molecular features, and underpin current disease subclassification and initial subgroup-directed therapies that are underway in clinical trials. However, substantial biological heterogeneity and differences in survival are apparent within each subgroup, which remain to be resolved. We aimed to investigate whether additional molecular subgroups exist within childhood medulloblastoma and whether these could be used to improve disease subclassification and prognosis predictions. Methods In this retrospective cohort study, we assessed 428 primary medulloblastoma samples collected from UK Childrens Cancer and Leukaemia Group (CCLG) treatment centres (UK), collaborating European institutions, and the UKCCSG-SIOP-PNET3 European clinical trial. An independent validation cohort (n=276) of archival tumour samples was also analysed. We analysed samples from patients with childhood medulloblastoma who were aged 0–16 years at diagnosis, and had central review of pathology and comprehensive clinical data. We did comprehensive molecular profiling, including DNA methylation microarray analysis, and did unsupervised class discovery of test and validation cohorts to identify consensus primary molecular subgroups and characterise their clinical and biological significance. We modelled survival of patients aged 3–16 years in patients (n=215) who had craniospinal irradiation and had been treated with a curative intent. Findings Seven robust and reproducible primary molecular subgroups of childhood medulloblastoma were identified. MBWNT remained unchanged and each remaining consensus subgroup was split in two. MBSHH was split into age-dependent subgroups corresponding to infant (<4·3 years; MBSHH-Infant; n=65) and childhood patients (≥4·3 years; MBSHH-Child; n=38). MBGrp3 and MBGrp4 were each split into high-risk (MBGrp3-HR [n=65] and MBGrp4-HR [n=85]) and low-risk (MBGrp3-LR [n=50] and MBGrp4-LR [n=73]) subgroups. These biological subgroups were validated in the independent cohort. We identified features of the seven subgroups that were predictive of outcome. Cross-validated subgroup-dependent survival models, incorporating these novel subgroups along with secondary clinicopathological and molecular features and established disease risk-factors, outperformed existing disease risk-stratification schemes. These subgroup-dependent models stratified patients into four clinical risk groups for 5-year progression-free survival: favourable risk (54 [25%] of 215 patients; 91% survival [95% CI 82–100]); standard risk (50 [23%] patients; 81% survival [70–94]); high-risk (82 [38%] patients; 42% survival [31–56]); and very high-risk (29 [13%] patients; 28% survival [14–56]). Interpretation The discovery of seven novel, clinically significant subgroups improves disease risk-stratification and could inform treatment decisions. These data provide a new foundation for future research and clinical investigations. Funding Cancer Research UK, The Tom Grahame Trust, Star for Harris, Action Medical Research, SPARKS, The JGW Patterson Foundation, The INSTINCT network (co-funded by The Brain Tumour Charity, Great Ormond Street Childrens Charity, and Children with Cancer UK).

Histologically-defined central nervous system primitive neuro-ectodermal tumours (CNS-PNETs) display heterogeneous DNA methylation profiles and show relationships to other paediatric brain tumour types

To the editors: Central nervous system primitive neuro-ectodermal tumours (CNS-PNETs) are a group of rare childhood embryonal brain tumours associated with a poor prognosis (approximately 50% overall survival) and defined by a common histology according to the current consensus World Health Organisation (WHO) classification [7]. CNS-PNETs occur supratentorially and are defined by histological features shared with cerebellar PNETs (termed medulloblastomas), however the histological classification of CNS-PNET can be challenging. Individual CNS-PNETs are often reclassified as other paediatric supratentorial tumour groups, including anaplastic astrocytoma, atypical teratoid rhabdoid tumour (ATRT), anaplastic oligodendroglioma and anaplastic ependymoma, following central immunophenotypic and histological review [3, 12]. Initial studies have shown that substantial molecular heterogeneity exists within CNS-PNETs; molecular features characteristic of other cerebral brain tumour types (e.g. IDH1 mutation, CDKN2A deletion) have been detected in subsets, but unifying genomic defects have not yet been reported [4, 8, 10, 11]. The recent definition of embryonal tumours with abundant neuropil and true rosettes (ETANTR) as a discrete tumour entity occurring in very young children within the CNS-PNET group - characterised by focal amplification of 19q13.42 and dismal outcome [5, 6, 9] - suggests the existence of currently unrecognised molecular pathological variants, and a refined understanding of CNS-PNET biology could lead to their improved subclassification and the subsequent development of directed therapies. We and others have recently demonstrated the utility of DNA methylation profiling for the discovery and distinction of clinical and molecular sub-classes of brain tumour types including medulloblastomas, gliomas and ependymomas [13-15]. To investigate the potential of DNA methylation profiles to enhance the molecular classification of CNS-PNETs, we assessed 1505 CpG residues across 807 genes in a series of 29 archival CNS-PNETs using established methods [14], alongside assessment of clinical and molecular characteristics (Figure 1h). All biopsies underwent central neuropathological review according to WHO criteria [7] by a three pathologist panel (TSJ, KR and JL). Tumours representing ETANTRs, CNS-PNETs with significant glial (GFAP) or neuronal (synaptophysin) differentiation, and SMARCB1/INI1-negative tumours (by immunohistochemistry (IHC)), were excluded and not assessed, thus defining a study population of morphologically homogeneous CNS-PNETs for analysis (Figure 1a). Finally, DNA methylation profiles from 136 further paediatric brain tumours were generated contemporaneously and assessed in comparison. These included medulloblastomas of defined molecular subgroup (n=60; 15 representative examples each from the WNT (MBWNT), SHH (MBSHH), Group 3 (MBGroup3) and Group 4 (MBGroup4) [14]), alongside ependymomas (n=61; 45 posterior fossa (16 anaplastic, 29 classic; median age at diagnosis, 2.8 years), 16 supratentorial (9 anaplastic, 7 classic; median age, 6.9 years)) and cerebral high-grade gliomas (pHGG; n=15; 12 glioblastoma multiforme, 3 anaplastic astrocytoma; median age 7.1 years) with histology confirmed by central histological review (by WHO criteria [7]). Figure 1 Consensus clustering of CNS-PNETs with other molecularly and histologically-defined paediatric brain tumours does not identify a discrete CNS-PNET tumour subgroup We first undertook unsupervised clustering of the CNS-PNET tumour group based on their DNA methylation patterns using non-negative matrix factorisation (NMF [2, 14]). Three sub-groups produced the most consistent consensus clustering; the majority of tumours clustered confidently into a single large group (21/29), while the two smaller remaining groups (n≤5) were less well defined (Figure 1b). Next, we sought to compare the DNA methylation patterns observed for CNS-PNETs with those of the seven other paediatric brain tumour groups with available data. Prior to the addition of CNS-PNETs into our analysis, these tumours formed seven groups as expected (Figure 1c,d,f), representing discrete confidently-defined (average silhouette width, 0.82) groups of MBWNT, MBSHH, MBGroup3 and MBGroup4, posterior-fossa ependymomas and pHGG tumours, and a mixed tumour group containing all (n=16) supratentorial ependymomas alongside some posterior fossa ependymomas (n=9) and pHGGs (n=3). Whilst the inclusion of CNS-PNETs in the analysis yielded 8 optimal clusters (Figure 1c,e,f), the overall quality of these clusters was reduced (average silhouette width, 0.69) and CNS-PNETs did not form a single discrete group; indeed, CNS-PNETs clustered into six of the different tumour groups observed (Figure 1e,f), showing closer similarities to the other clinically and molecularly-defined paediatric brain tumour groups investigated than to each other. Finally, we made an initial assessment of relationships between the clustered CNS-PNETs and other clinical and molecular disease features (Figure 1g,h). Although numbers were limited, TP53 nuclear stabilisation was common and detected in most clusters, while TP53 mutation and MYCN amplification were rare. Most notably, both IDH1 mutations were exclusively detected in pHGG-like CNS-PNETs arising in adults [4], although no pHGG-characteristic HIST1H3B or H3F3A hotspot mutations were observed [15]. The single WNT pathway-activating CTNNB1 mutation was detected in a MBWNT-like CNS-PNET tumour. No relationships to clinical or pathological disease features were observed in this cohort (Figure 1h). In summary, our data show that despite a defining histological homogeneity using current diagnostic criteria, CNS-PNETs display highly heterogeneous DNA methylation patterns which are more commonly related to other paediatric brain tumour types than to each other. These initial findings raise important issues in the classification of CNS-PNETs and indicate their current clinical definition and grouping by common ‘PNET’ histology [7], and treatment using uniform therapeutic approaches, does not adequately address their underlying biological and clinical complexity. Moreover, our data suggest the potential of refined molecular sub-classification for the improved diagnosis and discrimination of CNS-PNET molecular variants, and to support molecularly-directed clinical trials across tumour types defined currently by clinical and pathological criteria. Despite the modest resolution of our platform, robust discrimination of recognised non-CNS-PNET tumour groups was achieved, both supporting these conclusions and highlighting the potential benefits of higher-resolution molecular investigations in expanded cohorts to validate and extend our findings. The variable DNA methylation patterns observed in CNS-PNETs are likely to represent complex factors, including cellular and developmental origins and ‘driver’ events in tumourigenesis [1]. The collection of snap-frozen tumour cohorts will now be essential to support comprehensive integrated genomic/epigenomic investigations, and comparison with transcriptomic features [11], which were not tractable in our current archival cohort. Finally, understanding the biological significance of epigenetic events in CNS-PNET and related tumour types could lead to the development of novel and/or targeted approaches for the improved therapy of these tumours.

Development of a targeted sequencing approach to identify prognostic, predictive and diagnostic markers in paediatric solid tumours

The implementation of personalised medicine in childhood cancers has been limited by a lack of clinically validated multi-target sequencing approaches specific for paediatric solid tumours. In order to support innovative clinical trials in high-risk patients with unmet need, we have developed a clinically relevant targeted sequencing panel spanning 311 kb and comprising 78 genes involved in childhood cancers. A total of 132 samples were used for the validation of the panel, including Horizon Discovery cell blends (n=4), cell lines (n=15), formalin-fixed paraffin embedded (FFPE, n=83) and fresh frozen tissue (FF, n=30) patient samples. Cell blends containing known single nucleotide variants (SNVs, n=528) and small insertion-deletions (indels n=108) were used to define panel sensitivities of ≥98% for SNVs and ≥83% for indels [95% CI] and panel specificity of ≥98% [95% CI] for SNVs. FFPE samples performed comparably to FF samples (n=15 paired). Of 95 well-characterised genetic abnormalities in 33 clinical specimens and 13 cell lines (including SNVs, indels, amplifications, rearrangements and chromosome losses), 94 (98.9%) were detected by our approach. We have validated a robust and practical methodology to guide clinical management of children with solid tumours based on their molecular profiles. Our work demonstrates the value of targeted gene sequencing in the development of precision medicine strategies in paediatric oncology.

MYC and TP53 defects interact at medulloblastoma relapse to define rapidly progressive disease and can be targeted therapeutically

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Disease recurrence following multi-modal therapy is the single most adverse event in medulloblastoma (MB). Currently >90% of relapsing patients die, accounting for ∼10% of childhood cancer deaths. MB is heterogeneous at diagnosis, comprising four molecular subgroups with distinct clinicopathological and molecular features and outcomes. The relevance of these features at relapse is unknown, making characterisation, modelling and targeted therapy of relapse biology essential to improve outcomes. However, relapsed MBs are not routinely biopsied in clinical practice. We undertook a first comprehensive investigation of the molecular, clinical and pathological features of 29 relapsed MBs and paired tumour samples taken at diagnosis, including the assessment of features with established significance at diagnosis (e.g. chromosome 17 and TP53 pathway status, MYC family ( MYC, MYCN ) gene amplification, polyploidy, C TNNB1 mutation and molecular subgroup status). Molecular subgroup was concordant at diagnosis and relapse, however evidence of alteration of all other features examined was found in relapsed tumours, with the majority of changes (30/44) representing acquired high-risk events. Most notably, MYC family gene amplifications and TP53 pathway defects commonly emerged in combination at relapse following conventional multimodal treatment ( P =0.02, 7/22, 32%) and predicted rapid progression to death ( P =0.016). These observations suggested aberrant activation of MYC family genes synergizes with TP53 inactivation in the genesis of biologically aggressive MB. To investigate any such relationship, we examined Trp53 status in our transgenic mouse model of spontaneously-arising MYCN-driven MB (GTML; Glt1-tTA/TRE-MYCN-Luc ). Somatic Trp53 mutations were found in 83% of tumors (n=10/12). Direct modelling of this interaction in GTML/ Trp53 KI/KI mice dramatically enhanced MB formation with 100% penetrance (43/43, median survival 47 days) in GTML/ Trp53 KI/KI versus 6% (3/50) in GTML; P <0.0001), faithfully mimicked clinicopathological characteristics of TP53-MYC family gene-associated relapsed human tumors, and validated the essential role of TP53 in potentiating the growth of MYCN-driven MB. Finally, therapeutic inhibition of Aurora-A kinase using MLN8237 in these tumours, and in derived neurospheres in vitro , promoted degradation of MYCN, reduced tumor growth and prolonged survival. In summary, while subgroup status remains stable, MBs display altered molecular, pathological and clinical features at relapse, and the emergence of combined TP53-MYC family gene defects is common following conventional therapy. Their association with rapid demise, coupled with their biological validation as driving and therapeutically exploitable events in a novel mouse MB model, strongly support further investigation and routine biopsy of relapse disease to drive future individualised therapeutic strategies. Citation Format: Rebecca M. Hill, Sanne Kuijper, Janet Lindsey, Ed C. Schwalbe, Karen Barker, Jessica Boult, Daniel Williamson, Zai Ahmad, Albert Hallsworth, Sarra Ryan, Evon Poon, Simon Robinson, Ruth Ruddle, Florence Raynaud, Louise Howell, Colin Kwok, Abhijit Joshi, Sarah Nicholson, Stephen Crosier, Stephen Wharton, Tom Jacques, Keith Robson, Antony Michalski, Darren Hargrave, Barry Pizer, Simon Bailey, Fredrik J. Swartling, Kevin Petrie, William A. Weiss, Louis Chesler, Steve Clifford. MYC and TP53 defects interact at medulloblastoma relapse to define rapidly progressive disease and can be targeted therapeutically. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-201. doi:10.1158/1538-7445.AM2014-LB-201

Abstract 3442: TP53 mutations in favorable risk WNT subtype medulloblastomas

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Although overall cure rates of ∼70% are now achieved, advances in our biological understanding of the disease will be essential for future improvements in outcome, through improved disease risk stratification and molecular therapeutic strategies. Mutations of TP53 are reported in ∼10% of MBs and have recently been reported to be associated with universally poor disease outcome (Tabori et al. (2010). J Clin Oncol 28: 1345-50), however, this relationship now requires validation in further cohorts. Additionally, there is now strong evidence that MB comprises a series of distinct molecular sub-types with characteristic molecular defects, transcriptional profiles and disease behaviour. WNT subgroup tumors (15% of cases) display activation of the WNT pathway and are associated with a favorable prognosis (>90% survival), while sonic hedgehog signalling pathway (SHH) activated tumors (∼25% of cases) also form a distinct subgroup. We therefore sought to analyse the frequency of TP53 mutations in these major MB molecular subgroups and investigate their impact on disease prognosis. A cohort of 50 MBs was selected to include representatives of all major established clinicopathological groups. WNT subgroup cases (n=15) were selected for inclusion by the presence of CTNNB1 mutations and chromosome 6 defects, while SHH subgroup cases (n=12) were identified by their characteristic transcriptional profiles. TP53 mutations were investigated by PCR-based direct DNA sequence analysis of exons 4-9. TP53 missense mutations were observed in 5 cases (10%); all were in non-infant, non-metastatic cases, classified clinically as ‘standard risk’. On review, one was identified as a germline mutation (Li-Fraumeni syndrome: LFS), whilst all others were somatic mutations in sporadic tumors. All 4 cases with a somatic TP53 mutation achieved a durable remission of between 5.6-11.1 years, while the LFS case relapsed and died within 1.1 years. Evidence of TP53 mutations were found in all MB subgroups, but mutations appeared enriched in the WNT subgroup (3/15 (20%) WNT tumors; 1/12 (8%) SHH tumors (LFS case); 1/23 (4%) non-WNT/SHH tumors). These data suggest that the prognostic significance of TP53 mutations in MB is dependent on the molecular and clinical context in which they arise. Whilst a poor outcome was observed for the TP53 mutation-associated LFS case, our findings demonstrate that TP53 mutation is not associated with a universally poor prognosis, and indicate TP53 mutations are enriched in the WNT subgroup, where subgroup membership remains the overriding favorable prognostic determinant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3442. doi:10.1158/1538-7445.AM2011-3442