Ed Schwalbe

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.

Rapid diagnosis of medulloblastoma molecular subgroups.

Purpose: Microarray studies indicate medulloblastoma comprises distinct molecular disease subgroups, which offer potential for improved clinical management. Experimental Design: Minimal mRNA expression signatures diagnostic for the Wnt/Wingless (WNT) and Sonic Hedgehog (SHH) subgroups were developed, validated, and used to assign subgroup affiliation in 173 tumors from four independent cohorts, alongside a systematic investigation of subgroup clinical and molecular characteristics. Results: WNT tumors [12% (21/173)] were diagnosed >5 years of age (peak, 10 years), displayed classic histology, CTNNB1 mutation (19/20), and associated chromosome 6 loss, and have previously been associated with favorable prognosis. SHH cases [24% (42/173)] predominated in infants (<3 years) and showed an age-dependent relationship to desmoplastic/nodular pathology; all infant desmoplastic/nodular cases (previously associated with a good outcome) were SHH-positive, but these relationships broke down in noninfants. PTCH1 mutations were common [34% (11/32)], but PTCH1 exon1c hypermethylation, chromosome 9q and REN (KCTD11) genetic loss were not SHH associated, and SMO or SUFU mutation, PTCH1 exon1a or SUFU hypermethylation did not play a role, indicating novel activating mechanisms in the majority of SHH cases. SHH tumors were associated with an absence of COL1A2 methylation. WNT/SHH-independent medulloblastomas [64% (110/173)] showed all histologies, peaked at 3 and 6 years, and were exclusively associated with chromosome 17p loss. Conclusions: Medulloblastoma subgroups are characterized by distinct genomic, epigenomic and clinicopathologic features, and clinical outcomes. Validated array-independent gene expression assays for the rapid assessment of subgroup affiliation in small biopsies provide a basis for their routine clinical application, in strategies including molecular disease-risk stratification and delivery of targeted therapeutics. Clin Cancer Res; 17(7); 1883–94. ©2011 AACR.

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).

Minimal methylation classifier (MIMIC): A novel method for derivation and rapid diagnostic detection of disease-associated DNA methylation signatures

Rapid and reliable detection of disease-associated DNA methylation patterns has major potential to advance molecular diagnostics and underpin research investigations. We describe the development and validation of minimal methylation classifier (MIMIC), combining CpG signature design from genome-wide datasets, multiplex-PCR and detection by single-base extension and MALDI-TOF mass spectrometry, in a novel method to assess multi-locus DNA methylation profiles within routine clinically-applicable assays. We illustrate the application of MIMIC to successfully identify the methylation-dependent diagnostic molecular subgroups of medulloblastoma (the most common malignant childhood brain tumour), using scant/low-quality samples remaining from the most recently completed pan-European medulloblastoma clinical trial, refractory to analysis by conventional genome-wide DNA methylation analysis. Using this approach, we identify critical DNA methylation patterns from previously inaccessible cohorts, and reveal novel survival differences between the medulloblastoma disease subgroups with significant potential for clinical exploitation.

Supratentorial and spinal pediatric ependymomas display a hypermethylated phenotype which includes the loss of tumor suppressor genes involved in the control of cell growth and death

Epigenetic alterations, including methylation, have been shown to be an important mechanism of gene silencing in cancer. Ependymoma has been well characterized at the DNA copy number and mRNA expression levels. However little is known about DNA methylation changes. To gain a more global view of the methylation profile of ependymoma we conducted an array-based analysis. Our data demonstrated tumors to segregate according to their location in the CNS, which was associated with a difference in the global level of methylation. Supratentorial and spinal tumors displayed significantly more hypermethylated genes than posterior fossa tumors, similar to the ‘CpG island methylator phenotype’ (CIMP) identified in glioma and colon carcinoma. This hypermethylated profile was associated with an increase in expression of genes encoding for proteins involved in methylating DNA, suggesting an underlying mechanism. An integrated analysis of methylation and mRNA expression array data allowed us to identify methylation-induced expression changes. Most notably genes involved in the control of cell growth and death and the immune system were identified, including members of the JNK pathway and PPARG. In conclusion, we have generated a global view of the methylation profile of ependymoma. The data suggests epigenetic silencing of tumor suppressor genes is an important mechanism in the pathogenesis of supratentorial and spinal, but not posterior fossa ependymomas. Hypermethylation correlated with a decrease in expression of a number of tumor suppressor genes and pathways that could be playing an important role in tumor pathogenesis.

Next-generation systematics: An innovative approach to resolve the structure of complex prokaryotic taxa.

Prokaryotic systematics provides the fundamental framework for microbiological research but remains a discipline that relies on a labour- and time-intensive polyphasic taxonomic approach, including DNA-DNA hybridization, variation in 16S rRNA gene sequence and phenotypic characteristics. These techniques suffer from poor resolution in distinguishing between closely related species and often result in misclassification and misidentification of strains. Moreover, guidelines are unclear for the delineation of bacterial genera. Here, we have applied an innovative phylogenetic and taxogenomic approach to a heterogeneous actinobacterial taxon, Rhodococcus, to identify boundaries for intrageneric and supraspecific classification. Seven species-groups were identified within the genus Rhodococcus that are as distantly related to one another as they are to representatives of other mycolic acid containing actinobacteria and can thus be equated with the rank of genus. It was also evident that strains assigned to rhodococcal species-groups are underspeciated with many misclassified using conventional taxonomic criteria. The phylogenetic and taxogenomic methods used in this study provide data of theoretical value for the circumscription of generic and species boundaries and are also of practical significance as they provide a robust basis for the classification and identification of rhodococci of agricultural, industrial and medical/veterinary significance.

TERT promoter mutation and aberrant hypermethylation are associated with elevated expression in medulloblastoma and characterise the majority of non-infant SHH subgroup tumours.

To the editor: The childhood brain tumour medulloblastoma comprises four molecular disease subgroups (MBWNT, MBSHH, MBGroup3 and MBGroup4). However, large-scale whole-exome sequencing investigations have not identified defining genetic lesions for the non-MBWNT subgroups [8,11]. Recent studies reported in this journal and others [1,3,6,7,9] have identified frequent TERT promoter mutations and aberrant DNA methylation in CNS malignancies, suggesting an important mechanism in tumour development (Figure 1a). In medulloblastoma, Castelo-Branco et al.[3] reported a high frequency of TERT promoter methylation, while Killela et al.[6] described TERT promoter mutations which Koelsche et al.[7] and Remke et al.[9] subsequently reported were most frequent in adult MBSHH, but rarer in childhood tumours. However, while TERT mutations have been associated with elevated expression in other cancers [1, 5], and account for a proportion of MBSHH, the relative contribution of TERT methylation alterations has not yet been investigated alongside mutational analysis. Moreover, relationships between TERT promoter methylation and gene expression are unclear; the positive association reported across multiple malignancies by Castelo-Branco et al.[3] is contradicted by the inverse association described by Arita et al.[1] in TERT wild-type adult gliomas. Figure 1 TERT non-coding mutations, aberrant DNA hypermethylation, and expression in medulloblastoma We therefore sought to clarify the role of TERT alterations in medulloblastoma, by assessing the frequency of TERT promoter hot-spot mutations [1,6,7,9], aberrant methylation of the critical cg11625005 TERT promoter CpG residue [3], and TERT expression, in our tumour series. We show a common, subgroup-specific, involvement of TERT mutations in MBSHH alongside a wider involvement of TERT methylation across the MB subgroups, both associated with elevated TERT expression. Notably, in the non-infant MBSHH patient group aged 4 and over at diagnosis within our cohort, we show these genetic and epigenetic aberrations occur in both childhood and adult tumours, and in a mutually exclusive fashion, representing a defining molecular alteration in >75% of this patient group. TERT promoter mutations occurred at high frequency in both childhood (14/41 (34%)) and adult (8/11 (73%)) MBSHH (Figure 1b) in our cohort, more common than any coding mutation reported in these groups to date (TP53, 30%; PTCH1, 27%; DDX3X, 18%; all other genes, <6% (n=33, data from cancer.sanger.ac.uk). The age distributions of mutated (4.7-15.5 years) and non-mutated (5.2-15.4 years) childhood patients did not differ significantly (p=0.27; Mann-Whitney U test). TERT mutations were tumour-specific where germline DNA was available for comparison (n=4) and exclusive to non-infant MBSHH in our investigations. Mutations were not found in MBWNT (n=16; age range, 4.7-16.8 years), MBGroup3 (n=16; 1.5-16.1 years) or MBGroup4 (n=20; 2.4-15.8 years) from infants and children, or in tumours from infant MBSHH (<4.0 at diagnosis; n=17; 0.2-3.5 years), consistent with the rarity of mutations in these subgroups reported by Remke et al. [9] Aberrant TERT promoter methylation at cg11625005 was a feature of all medulloblastoma molecular subgroups, but varied significantly in level and incidence between tumour groups (p=9×10−6, ANOVA); aberrant hypermethylation (with respect to normal cerebellar levels; n=17, foetal to 67 years) was observed in 63% (10/16) MBWNT, 69% (11/16) MBGroup3 and in 10% (2/20) MBGroup4, while MBSHH tumours (36%; 16/44 hypermethylated) showed greatest variation (Figure 1c). Notably, TERT hypermethylation showed significant age-dependent associations within the MBSHH group (0% (0/6) >16 years; 52% (11/21) 4-16 years; 29% (5/17) <4 years; p=0.05, χ2 test). Moreover, TERT promoter mutation and aberrant methylation at cg11625005 were mutually exclusive in non-infant MBSHH within our cohort (Figure 1d) suggesting methylation alterations contribute significantly to TERT alteration in this group, and possible common mechanistic effects. Aberrant hypermethylation was detected in 11/17 non-mutated vs. 0/10 mutated non-infant MBSHH tumours assessed (p=0.001; Fisher’s exact test (Figure 1d)). To assess the potential mechanistic contributions of promoter mutation and methylation to TERT gene expression, we next assessed their association within our cohort using expression data generated by RNA-seq. TERT methylation and expression showed a significant positive and linear relationship (p=0.001; Pearson’s correlation test) in TERT wild-type tumours across all medulloblastoma subgroups, while all TERT mutant tumours (all MBSHH > 4 years (Figure 1b)) displayed high TERT expression in the absence of hypermethylation (Figure 1e). Non-coding TERT promoter alterations, encompassing mutually-exclusive mutation and aberrant hypermethylation, both associated with elevated TERT expression, are therefore a defining feature for the majority (>75%) of non-infant MBSHH in our cohort, indicating a key mechanism in their molecular pathogenesis. Moreover TERT hypermethylation and deregulation, in the absence of mutation, suggests a wider involvement across the other medulloblastoma molecular subtypes, notably frequent in MBWNT and MBGroup3, but less so in MBGroup4, which now mandates further investigation. Finally, our findings raise the potential importance of additional non-coding and/or epigenetic regulatory alterations in medulloblastoma, which have hitherto been overlooked by exome sequencing studies [8]. Despite current nomenclature, MBSHH is not solely defined by SHH pathway activation and SHH is likely to contribute alongside other frequently disrupted pathways, with TERT alterations representing the most common identified to date. We believe these findings have important implications for future diagnosis, research and targeted therapy of a significant proportion of medulloblastoma patients.

Glycerol and acetaminophen as adjuvant therapy did not affect the outcome of bacterial meningitis in Malawian children.

We investigated the benefit of 2 candidate adjunctive therapies in bacterial meningitis: glycerol, which has shown promise in earlier studies, and acetaminophen, which is reportedly beneficial in adult septicemia. In a hospital in Blantyre, Malawi, we enrolled 360 children aged ≥ 2 months with proven bacterial meningitis (36% HIV infected) in a double-blind, randomized, placebo-controlled trial of glycerol and acetaminophen in a 2 × 2 factorial design. Of 4 groups, first group received oral glycerol, second received rectal acetaminophen, third received both therapies and the fourth received placebos only. Adjuvant therapies were given for the first 48 hours of antibiotic therapy. Endpoints were mortality and neurological sequelae. Baseline findings were similar across all groups, except that many children had prior antibiotics in the acetaminophen group and many were anemic in the acetaminophen and glycerol group. Outcomes were similar for all groups. We found no benefit from oral glycerol or rectal acetaminophen in, mostly pneumococcal, meningitis in Malawian children.

Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance

Background:Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes.Methods:A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays.Results:Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a ‘methylator’ phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype.Conclusion:Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy.