Keith Robson

Hyperfractionated Versus Conventional Radiotherapy Followed by Chemotherapy in Standard-Risk Medulloblastoma: Results From the Randomized Multicenter HIT-SIOP PNET 4 Trial

PURPOSE To compare event-free survival (EFS), overall survival (OS), pattern of relapse, and hearing loss in children with standard-risk medulloblastoma treated by postoperative hyperfractionated or conventionally fractionated radiotherapy followed by maintenance chemotherapy. PATIENTS AND METHODS In all, 340 children age 4 to 21 years from 122 European centers were postoperatively staged and randomly assigned to treatment with hyperfractionated radiotherapy (HFRT) or standard (conventional) fractionated radiotherapy (STRT) followed by a common chemotherapy regimen consisting of eight cycles of cisplatin, lomustine, and vincristine. RESULTS After a median follow-up of 4.8 years (range, 0.1 to 8.3 years), survival rates were not significantly different between the two treatment arms: 5-year EFS was 77% ± 4% in the STRT group and 78% ± 4% in the HFRT group; corresponding 5-year OS was 87% ± 3% and 85% ± 3%, respectively. A postoperative residual tumor of more than 1.5 cm(2) was the strongest negative prognostic factor. EFS of children with all reference assessments and no large residual tumor was 82% ± 2% at 5 years. Patients with a delay of more than 7 weeks to the start of RT had a worse prognosis. Severe hearing loss was not significantly different for the two treatment arms at follow-up. CONCLUSION In this large randomized European study, which enrolled patients with standard-risk medulloblastoma from more than 100 centers, excellent survival rates were achieved in patients without a large postoperative residual tumor and without RT treatment delays. EFS and OS for HFRT was not superior to STRT, which therefore remains standard of care in this disease.

Homozygous loss of ADAM3A revealed by genome-wide analysis of pediatric high-grade glioma and diffuse intrinsic pontine gliomas

Overall, pediatric high-grade glioma (pHGG) has a poor prognosis, in part due to the lack of understanding of the underlying biology. High-resolution 244 K oligo array comparative genomic hybridization (CGH) was used to analyze DNA from 38 formalin-fixed paraffin-embedded predominantly pretreatment pHGG samples, including 13 diffuse intrinsic pontine gliomas (DIPGs). The patterns of gains and losses were distinct from those seen in HGG arising in adults. In particular, we found 1q gain in up to 27% of our cohort compared with 9% reported in adults. A total of 13% had a balanced genetic profile with no large-scale copy number alterations. Homozygous loss at 8p12 was seen in 6 of 38 (16%) cases of pHGG. This novel deletion, which includes the ADAM3A gene, was confirmed by quantitative real-time PCR (qPCR). Loss of CDKN2A/CDKN2B in 4 of 38 (10%) samples by oligo array CGH was confirmed by fluorescent in situ hybridization on tissue microarrays and was restricted to supratentorial tumors. Only ∼50% of supratentorial tumors were positive for CDKN2B expression by immunohistochemistry (IHC), while ∼75% of infratentorial tumors were positive for CDKN2B expression (P = 0.03). Amplification of the 4q11-13 region was detected in 8% of cases and included PDGFRA and KIT, and subsequent qPCR analysis was consistent with the amplification of PDGFRA. MYCN amplification was seen in 5% of samples being significantly associated with anaplastic astrocytomas (P= 0.03). Overall, DIPG shared similar spectrum of changes to supratentorial HGG with some notable differences, including high-frequency loss of 17p and 14q and lack of CDKN2A/CDKN2B deletion. Informative genetic data providing insight into the underlying biology and potential therapeutic possibilities can be generated from archival tissue and typically small biopsies from DIPG. Our findings highlight the importance of obtaining pretreatment samples.

Multifactorial analysis of predictors of outcome in pediatric intracranial ependymoma.

Pediatric ependymomas are enigmatic tumors, and their clinical management remains one of the more difficult in pediatric oncology. The identification of biological correlates of outcome and therapeutic targets remains a significant challenge in this disease. We therefore analyzed a panel of potential biological markers to determine optimal prognostic markers. We constructed a tissue microarray from 97 intracranial tumors from 74 patients (WHO grade II-III) and analyzed the candidate markers nucleolin, telomerase catalytic subunit (hTERT; antibody clone 44F12), survivin, Ki-67, and members of the receptor tyrosine kinase I (RTK-I) family by immunohistochemistry. Telomerase activity was determined using the in vitro-based telomere repeat amplification protocol assay, and telomere length was measured using the telomere restriction fragment assay. Primary tumors with low versus high nucleolin protein expression had a 5-year event-free survival of 74%+/-13% and 31%+/-7%, respectively. Multivariate analysis identified low nucleolin expression to be independently associated with a more favorable prognosis (hazard ratio=6.25; 95% confidence interval, 1.6-24.2; p=0.008). Ki-67 and survivin correlated with histological grade but not with outcome. Immunohistochemical detection of the RTK-I family did not correlate with grade or outcome. Telomerase activity was evident in 19 of 22 primary tumors, with telomere lengthening and/or maintenance occurring in five of seven recurrent cases. Low nucleolin expression was the single most important biological predictor of outcome in pediatric intracranial ependymoma. Furthermore, telomerase reactivation and maintenance of telomeric repeats appear necessary for childhood ependymoma progression. These findings require corroboration in a clinical trial setting.

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.

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.

Hyperfractionated Accelerated Radiotherapy (HART) with maintenance chemotherapy for metastatic (M1-3) Medulloblastoma - A safety/feasibility study

BACKGROUND AND PURPOSE To evaluate feasibility and toxicity of Hyperfractionated Accelerated Radiotherapy (HART) 1.24Gy b.i.d. followed by chemotherapy for M1-3 Medulloblastoma (MB). The aim of HART was to use hyperfractionation to improve therapeutic ratio combined with acceleration to minimise tumour cell repopulation during radiotherapy (RT). MATERIALS AND METHODS Between February 2002 and May 2008, 34 eligible patients (22 male, 12 female) aged 3-15years (median 7) with metastatic MB (M1-9; M2-3, M3-22) received HART with a craniospinal radiotherapy (CSRT) dose of 39.68Gy followed by 22.32Gy boost to the whole posterior fossa and 9.92Gy metastatic boosts. The 8th and subsequent patients received vincristine (VCR) 1.5mg/m(2) weekly×8 doses over 8weeks starting during the 1st week of RT. Maintenance chemotherapy comprised 8 six-weekly cycles of VCR 1.5mg/m(2) weekly×3, CCNU 75mg/m(2) and cisplatin 70mg/m(2). RESULTS Median duration of HART was 34days (range 31-38). Grade 3-4 toxicities included mucositis (8), nausea (10), anaemia (5), thrombocytopaenia (2), leucopaenia (24). With 4.5-year median follow-up, 3-year EFS and OS were 59% and 71%, respectively. Of 10 relapses, 1 was outside the central nervous system (CNS), 1 posterior fossa alone and 8 leptomeningeal with 3 also associated with posterior fossa. CONCLUSION HART with or without VCR was well tolerated and may have a place in the multi-modality management of high-risk MB.

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

Localized conjunctival extra-nodal marginal zone B cell lymphoma with presumed paraproteinic crystalline keratopathy

Crystalline corneal deposits have been well reported in individual cases of lymphoproliferative disorders associated with hyper-gammaglobulinemia, hence called ‘Crystalline Paraproteinemic Keratopathy’. This is the first report of corneal deposits in a case of localised conjunctival B-cell Lymphoma without paraproteinaemia/hyper-gammaglobulinemia, hence called ‘Presumed Paraproteinic Crystalline Keratopathy’.

A national feasibility study of real-time central pathology review (CPR) and molecular diagnostics for medulloblastoma

13:15 Registration 14:00 Welcome Paul Ince (University of Sheffield, UK) 14:15 Genetic characterisation of the motor neuron disease/frontotemporal dementia spectrum Vivianna van Deerlin (University of Pennsylvania, USA) 15:00 Molecular and clinical pathology of genetic variants in the motor neuron disease/ frontotemporal dementia spectrum Manuela Neumann (DZNE/University of T€ ubingen, Germany) 15:45 Tea break 16:15 Motor neuron differentiation in stem cell research on motor neuron disease Brian Kaspar (Ohio State University, USA) 17:00 Cellular and molecular mechanisms of disease pathogenesis in spinal muscular atrophy Thomas H. Gillingwater (University of Edinburgh, UK) 17:45 Alfred Meyer’s Memorial Lecture Pamela Shaw (University of Sheffield, UK) Prospects for therapy development and CNS delivery in motor neuron diseases