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Edward C Schwalbe,
Daniel Williamson,
Janet C Lindsey,
Dolores Hamilton,
Sarra L Ryan,
Hisham Megahed,
Miklós Garami,
Peter Hauser,
Bożena Dembowska-Baginska,
Danuta Perek,
Paul A Northcott,
Michael D Taylor,
Roger E Taylor,
David W Ellison, Simon Bailey,
Steven C Clifford
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ABSTRACT: Molecular subclassification is rapidly informing the clinical management of medulloblastoma. However, the disease remains associated with poor outcomes and therapy-associated late effects, and the majority of patients are not characterized by a validated prognostic biomarker. Here, we investigated the potential of epigenetic DNA methylation for disease subclassification, particularly in formalin-fixed biopsies, and to identify biomarkers for improved therapeutic individualization. Tumor DNA methylation profiles were assessed, alongside molecular and clinical disease features, in 230 patients primarily from the SIOP-UKCCSG PNET3 clinical trial. We demonstrate by cross-validation in frozen training and formalin-fixed test sets that medulloblastoma comprises four robust DNA methylation subgroups (termed WNT, SHH, G3 and G4), highly related to their transcriptomic counterparts, and which display distinct molecular, clinical and pathological disease characteristics. WNT patients displayed an expected favorable prognosis, while outcomes for SHH, G3 and G4 were equivalent in our cohort. MXI1 and IL8 methylation were identified as novel independent high-risk biomarkers in cross-validated survival models of non-WNT patients, and were validated using non-array methods. Incorporation of MXI1 and IL8 into current survival models significantly improved the assignment of disease risk; 46 % of patients could be classified as 'favorable risk' (>90 % survival) compared to 13 % using current models, while the high-risk group was reduced from 30 to 16 %. DNA methylation profiling enables the robust subclassification of four disease subgroups in frozen and routinely collected/archival formalin-fixed biopsy material, and the incorporation of DNA methylation biomarkers can significantly improve disease-risk stratification. These findings have important implications for future risk-adapted clinical disease management.
Acta Neuropathologica 01/2013; · 9.32 Impact Factor
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David W. Ellison,
James Dalton,
Mehmet Kocak,
Sarah Leigh Nicholson,
Charles Fraga,
Geoff Neale,
Anna M. Kenney,
Dan J. Brat,
Arie Perry,
William H. Yong,
Roger E. Taylor, Simon Bailey,
Steven C. Clifford,
Richard J. Gilbertson
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ABSTRACT: Medulloblastoma is heterogeneous, being characterized by molecular subgroups that demonstrate distinct gene expression profiles.
Activation of the WNT or SHH signaling pathway characterizes two of these molecular subgroups, the former associated with
low-risk disease and the latter potentially targeted by novel SHH pathway inhibitors. This manuscript reports the validation
of a novel diagnostic immunohistochemical method to distinguish SHH, WNT, and non-SHH/WNT tumors and details their associations
with clinical, pathological and cytogenetic variables. A cohort (n=235) of medulloblastomas from patients aged 0.4–52years was studied for expression of four immunohistochemical markers:
GAB1, β-catenin, filamin A, and YAP1. Immunoreactivity (IR) for GAB1 characterizes only SHH tumors and nuclear IR for β-catenin
only WNT tumors. IRs for filamin A and YAP1 identify SHH and WNT tumors. SHH, WNT, and non-SHH/WNT tumors contributed 31,
14, and 55% to the series. All desmoplastic/nodular (D/N) medulloblastomas were SHH tumors, while most WNT tumors (94%) had
a classic phenotype. Monosomy 6 was strongly associated with WNT tumors, while PTCH1 loss occurred almost exclusively among SHH tumors. MYC or MYCN amplification and chromosome 17 imbalance occurred predominantly among non-SHH/WNT tumors. Among patients aged 3–16years
and entered onto the SIOP PNET3 trial, outcome was significantly better for children with WNT tumors, when compared to SHH
or non-SHH/WNT tumors, which showed similar survival curves. However, high-risk factors (M+ disease, LC/A pathology, MYC amplification) significantly influenced survival in both SHH and non-SHH/WNT groups. We describe a robust method for detecting
SHH, WNT, and non-SHH/WNT molecular subgroups in formalin-fixed medulloblastoma samples. In corroborating other studies that
indicate the value of combining clinical, pathological, and molecular variables in therapeutic stratification schemes for
medulloblastoma, we also provide the first outcome data based on a clinical trial cohort and novel data on how molecular subgroups
are distributed across the range of disease.
Acta Neuropathologica 04/2012; 121(3):381-396. · 9.32 Impact Factor
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ABSTRACT: We report the case of a 13-year old with a radiological diagnosis of relapsed medulloblastoma. Eighteen months after the last course of palliative chemotherapy MRI showed radiological resolution. The lesions were retrospectively assumed to be a radiation effect, highlighting the potential benefits of biopsy to confirm presumed radiological recurrence.
British Journal of Neurosurgery 12/2011; 26(4):542-4. · 0.88 Impact Factor
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Sarra L Ryan,
Ed C Schwalbe,
Michael Cole,
Yuan Lu,
Meryl E Lusher,
Hisham Megahed,
Kieran O'Toole,
Sarah Leigh Nicholson,
Laszlo Bognar,
Miklos Garami,
Peter Hauser,
Andrey Korshunov,
Stefan M Pfister,
Daniel Williamson,
Roger E Taylor,
David W Ellison, Simon Bailey,
Steven C Clifford
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ABSTRACT: The MYC oncogenes are the most commonly amplified loci in medulloblastoma, and have previously been proposed as biomarkers of adverse disease prognosis by us and others. Here, we report focussed and comprehensive investigations of MYCC, MYCN and MYCL in an extensive medulloblastoma cohort (n = 292), aimed to define more precisely their biological significance and optimal clinical application to direct improved disease risk-stratification and individualisation of therapy. MYCC and MYCN expression elevations were multifactorial, associated with high-risk (gene amplification, large-cell/anaplastic pathology (LCA)) and favourable-risk (WNT/SHH molecular subgroups) disease features. Highly variable cellular gene amplification patterns underlay overall MYC copy number elevations observed in tumour biopsies; we used these alternative measures together to define quantitative methodologies and thresholds for amplification detection in routinely collected tumour material. MYCC and MYCN amplification, but not gain, each had independent prognostic significance in non-infants (≥3.0-16.0 years), but MYCC conferred a greater hazard to survival than MYCN when considered across this treatment group. MYCN's weaker group-wide survival relationship may be explained by its pleiotropic behaviour between clinical disease-risk groups; MYCN predicted poor prognosis in clinical high-risk (metastatic (M+) or LCA), but not standard-risk, patients. Extending these findings, survival decreased in proportion to the total number of independently significant high-risk features present (LCA, M+ or MYCC/MYCN amplification). This cumulative-risk model defines a patient group characterised by ≥2 independent risk-factors and an extremely poor prognosis (<15% survival), which can be identified straightforwardly using the reported MYC amplification detection methodologies alongside clinical assessments, enabling targeting for novel/intensified therapies in future clinical studies.
Acta Neuropathologica 12/2011; 123(4):501-13. · 9.32 Impact Factor
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Janet C Lindsey,
Rebecca M Hill,
Hisham Megahed,
Meryl E Lusher,
Ed C Schwalbe,
Michael Cole,
Twala L Hogg,
Richard J Gilbertson,
David W Ellison, Simon Bailey,
Steven C Clifford
Journal of Clinical Oncology 02/2011; 29(12):e344-6; author reply e347-8. · 18.37 Impact Factor
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Ed C Schwalbe,
Janet C Lindsey,
Debbie Straughton,
Twala L Hogg,
Michael Cole,
Hisham Megahed,
Sarra L Ryan,
Meryl E Lusher,
Michael D Taylor,
Richard J Gilbertson,
David W Ellison, Simon Bailey,
Steven C Clifford
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ABSTRACT: Microarray studies indicate medulloblastoma comprises distinct molecular disease subgroups, which offer potential for improved clinical management.
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.
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.
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.
Clinical Cancer Research 02/2011; 17(7):1883-94. · 7.74 Impact Factor
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Ruth G Tatevossian,
Bo Tang,
James Dalton,
Tim Forshew,
Andrew R Lawson,
Jing Ma,
Geoff Neale,
Sheila A Shurtleff, Simon Bailey,
Amar Gajjar,
Suzanne J Baker,
Denise Sheer,
David W Ellison
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ABSTRACT: Recent studies of genetic abnormalities in pediatric low-grade gliomas (LGGs) have focused on activation of the ERK/MAPK pathway by KIAA1549-BRAF gene fusions in the majority of pilocytic astrocytomas (PAs) and by rare mutations in elements of the pathway across histopathologically diverse LGGs. This study reports that MYB, an oncogene not previously implicated in gliomagenesis, is activated in a diverse subset of pediatric LGGs. The study cohort comprised 57 pediatric LGGs and a comparative cohort of 59 pediatric high-grade gliomas (HGGs). The LGG cohort included 34 PAs and 23 diffuse gliomas; fibrillary astrocytomas (n = 14), oligodendroglial tumors (n = 7), and angiocentric gliomas (n = 2). MYB copy number abnormalities were disclosed using Affymetrix 6.0 SNP arrays and confirmed using interphase fluorescence in situ hybridization. Novel MYB amplifications that upregulate MYB RNA and protein expression were demonstrated in 2/14 diffuse astrocytomas. In addition, focal deletion of the terminal region of MYB was seen in 1 of 2 angiocentric gliomas (AGs). Increased expression of MYB was demonstrated by quantitative RT-PCR and immunohistochemistry. MYB upregulation at the protein level was demonstrated in a proportion of diffuse LGGs (60%), pilocytic astrocytomas (41%), and HGGs (19%), but abnormalities at the genomic level were only a feature of diffuse gliomas. Our data suggest that MYB may have a role in a subset of pediatric gliomas, through a variety of mechanisms in addition to MYB amplification and deletion.
Acta Neuropathologica 11/2010; 120(6):731-43. · 9.32 Impact Factor
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David W Ellison,
Mehmet Kocak,
James Dalton,
Hisham Megahed,
Meryl E Lusher,
Sarra L Ryan,
Wei Zhao,
Sarah Leigh Nicholson,
Roger E Taylor, Simon Bailey,
Steven C Clifford
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ABSTRACT: Medulloblastomas are heterogeneous and include relatively good-prognosis tumors characterized by Wnt pathway activation, as well as those that cannot be successfully treated with conventional therapy. Developing a practical therapeutic stratification that allows accurate identification of disease risk offers the potential to individualize adjuvant therapy and to minimize long-term adverse effects in a subgroup of survivors.
Using formalin-fixed paraffin-embedded (FFPE) tissue for immunohistochemistry, fluorescent in situ hybridization, and direct sequencing to identify tumors with a Wnt pathway signature and those harboring copy number abnormalities (CNAs) of potential prognostic significance (MYC/MYCN amplification, CNAs of chromosome 6 and 17), we evaluated clinical, pathologic, and molecular outcome indicators and stratification models in a cohort (n = 207) of patients with medulloblastoma 3 to 16 years of age from the International Society of Pediatric Oncology CNS9102 (PNET3) trial.
Metastatic disease and large-cell/anaplastic (LC/A) phenotype were the clinicopathologic variables associated with poor progression-free survival (PFS). Nuclear immunoreactivity for β-catenin, CTNNB1 mutation, and monosomy 6 all identified a group of good-prognosis patients. MYC amplification was associated with poor outcome, but other CNAs were not. Low-risk medulloblastomas were defined as β-catenin nucleopositive tumors without metastasis at presentation, LC/A phenotype, or MYC amplification. High-risk medulloblastomas were defined as tumors with metastatic disease, LC/A phenotype, or MYC amplification. Low-risk, standard-risk, and high-risk categories of medulloblastoma had significantly (P < .0001) different outcomes.
Integrating assays of molecular biomarkers undertaken on routinely collected diagnostic FFPE tissue into stratification schemes for medulloblastoma alongside clinical and pathologic outcome indicators can refine current definition of disease risk and guide adjuvant therapy.
Journal of Clinical Oncology 10/2010; 29(11):1400-7. · 18.37 Impact Factor
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ABSTRACT: Flow cytometric minimal residual disease (MRD) monitoring could become more powerful if directed towards the disease-maintaining leukemic stem cell (LSC) compartment. Using a cohort of 48 children with B-lineage acute lymphoblastic leukemia (ALL), we sought the newly proposed candidate-LSC population, CD34(+)CD38(low)CD19(+), at presentation and in end of induction bone marrow samples. We identified the candidate LSC population in 60% of diagnostic samples and its presence correlated with expression of CD38, relative to that of normal B-cell progenitors. In addition, the candidate LSC was not detectable in all MRD positive samples. The absence of the population in 40% of diagnostic and 40% of MRD positive samples does not support the use of this phenotype as a generic biomarker to track LSCs and suggests that this phenotype may be an artifact of CD38 underexpression rather than a biologically distinct LSC population. ClinicalTrials.gov Identifier: NCT00222612.
Haematologica 11/2009; 95(4):679-83. · 6.42 Impact Factor
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Cell cycle (Georgetown, Tex.) 07/2009; 8(11):1806-7. · 5.36 Impact Factor
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Tim Forshew,
Ruth G Tatevossian,
Andrew R J Lawson,
Jing Ma,
Geoff Neale,
Babatunji W Ogunkolade,
Tania A Jones,
Johan Aarum,
James Dalton, Simon Bailey,
Tracy Chaplin,
Rowena L Carter,
Amar Gajjar,
Alberto Broniscer,
Bryan D Young,
David W Ellison,
Denise Sheer
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ABSTRACT: We report genetic aberrations that activate the ERK/MAP kinase pathway in 100% of posterior fossa pilocytic astrocytomas, with a high frequency of gene fusions between KIAA1549 and BRAF among these tumours. These fusions were identified from analysis of focal copy number gains at 7q34, detected using Affymetrix 250K and 6.0 SNP arrays. PCR and sequencing confirmed the presence of five KIAA1549-BRAF fusion variants, along with a single fusion between SRGAP3 and RAF1. The resulting fusion genes lack the auto-inhibitory domains of BRAF and RAF1, which are replaced in-frame by the beginning of KIAA1549 and SRGAP3, respectively, conferring constitutive kinase activity. An activating mutation of KRAS was identified in the single pilocytic astrocytoma without a BRAF or RAF1 fusion. Further fusions and activating mutations in BRAF were identified in 28% of grade II astrocytomas, highlighting the importance of the ERK/MAP kinase pathway in the development of paediatric low-grade gliomas.
The Journal of Pathology 03/2009; 218(2):172-81. · 6.32 Impact Factor
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ABSTRACT: Medulloblastoma is the most common malignant brain tumour of childhood. The identification of critical genes involved in its pathogenesis will be central to advances in our understanding of its molecular basis, and the development of improved therapeutic approaches.
We performed a SNP-array based genome-wide copy number analysis in medulloblastoma cell lines, to identify regions of genomic amplification and homozygous deletion, which may harbour critical disease genes. A series of novel and established medulloblastoma defects were detected (MYC amplification (n = 4), 17q21.31 high-level gain (n = 1); 9p21.1-p21.3 (n = 1) and 6q23.1 (n = 1) homozygous deletion). Most notably, a novel recurrent region of genomic amplification at 8q24.22-q24.23 was identified (n = 2), and selected for further investigation. Additional analysis by interphase fluorescence in situ hybridisation (iFISH), PCR-based mapping and SNP-array revealed this novel amplification at 8q24.22-q24.23 is independent of MYC amplification at 8q24.21, and is unique to medulloblastoma in over 800 cancer cell lines assessed from different tumour types, suggesting it contains key genes specifically involved in medulloblastoma development. Detailed mapping identified a 3Mb common minimal region of amplification harbouring 3 coding genes (ZFAT1, LOC286094, KHDRBS3) and two genes encoding micro-RNAs (hsa-miR-30b, hsa-miR-30d). Of these, only expression of hsa-miR-30b, hsa-miR-30d and KHDRBS3 correlated with copy number status, and all three of these transcripts also displayed evidence of elevated expression in sub-sets of primary medulloblastomas, measured relative to the normal cerebellum.
These data implicate hsa-miR-30b, hsa-miR-30d and KHDRBS3 as putative oncogenic target(s) of a novel recurrent medulloblastoma amplicon at 8q24.22-q24.23. Our findings suggest critical roles for these genes in medulloblastoma development, and further support the contribution of micro-RNA species to medulloblastoma pathogenesis.
PLoS ONE 02/2009; 4(7):e6159. · 4.09 Impact Factor
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Sarina Sulong,
Anthony V Moorman,
Julie A E Irving,
Jonathan C Strefford,
Zoe J Konn,
Marian C Case,
Lynne Minto,
Kerry E Barber,
Helen Parker,
Sarah L Wright,
Adam R M Stewart, Simon Bailey,
Nick P Bown,
Andrew G Hall,
Christine J Harrison
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ABSTRACT: Inactivation of the tumor suppressor gene, CDKN2A, can occur by deletion, methylation, or mutation. We assessed the principal mode of inactivation in childhood acute lymphoblastic leukemia (ALL) and frequency in biologically relevant subgroups. Mutation or methylation was rare, whereas genomic deletion occurred in 21% of B-cell precursor ALL and 50% of T-ALL patients. Single nucleotide polymorphism arrays revealed copy number neutral (CNN) loss of heterozygosity (LOH) in 8% of patients. Array-based comparative genomic hybridization demonstrated that the mean size of deletions was 14.8 Mb and biallelic deletions composed a large and small deletion (mean sizes, 23.3 Mb and 1.4 Mb). Among 86 patients, only 2 small deletions were below the resolution of detection by fluorescence in situ hybridization. Patients with high hyperdiploidy, ETV6-RUNX1, or 11q23/MLL rearrangements had low rates of deletion (11%, 15%, 13%), whereas patients with t(9;22), t(1;19), TLX3, or TLX1 rearrangements had higher frequencies (61%, 42%, 78%, and 89%). In conclusion, CDKN2A deletion is a significant secondary abnormality in childhood ALL strongly correlated with phenotype and genotype. The variation in the incidence of CDKN2A deletions by cytogenetic subgroup may explain its inconsistent association with outcome. CNN LOH without apparent CDKN2A inactivation suggests the presence of other relevant genes in this region.
Blood 10/2008; 113(1):100-7. · 9.90 Impact Factor
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ABSTRACT: Deregulation of the RAS-RAF-mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK signaling cascade is often caused by somatic mutations in genes encoding proteins which influence the activity of this pathway and include NRAS, KRAS2, FLT3, PTPN11, and BRAF. We report the first comprehensive mutational screen of key exons of these genes in a large cohort of unselected acute lymphoblastic leukemia (ALL) cases at diagnosis (n = 86) and in a more selected cohort at disease recurrence (n = 47) using the sensitive method of denaturing high-performance liquid chromatography. We show that somatic mutations that deregulate the pathway constitute one of the most common genetic aberrations in childhood ALL (cALL), being found in 35% of diagnostic and 25% of relapse samples. In matched presentation/relapse pairs, mutations predominating at relapse could be shown to be present at very low levels at diagnosis using allele-specific PCR, thus implicating the mutated clone in disease progression. Importantly, in primary samples, we show that mutations are associated with activated ERK and differential cytotoxicity to MEK-ERK inhibitors was shown for some patients. Inhibitors of the pathway, which are currently undergoing clinical trial, may be a novel therapeutic option for cALL, particularly at relapse.
Cancer Research 09/2008; 68(16):6803-9. · 7.86 Impact Factor
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ABSTRACT: Candidate gene investigations have indicated a significant role for epigenetic events in the pathogenesis of medulloblastoma, the most common malignant brain tumor of childhood. To assess the medulloblastoma epigenome more comprehensively, we undertook a genomewide investigation to identify genes that display evidence of methylation-dependent regulation. Expression microarray analysis of medulloblastoma cell lines following treatment with a DNA methyltransferase inhibitor revealed deregulation of multiple transcripts (3%-6% of probes per cell line). Eighteen independent genes demonstrated >3-fold reactivation in all cell lines tested. Bisulfite sequence analysis revealed dense CpG island methylation associated with transcriptional silencing for 12 of these genes. Extension of this analysis to primary tumors and the normal cerebellum revealed three major classes of epigenetically regulated genes: (1) normally methylated genes (DAZL, ZNF157, ASN) whose methylation reflects somatic patterns observed in the cerebellum, (2) X-linked genes (MSN, POU3F4, HTR2C) that show disruption of their sex-specific methylation patterns in tumors, and (3) tumor-specific methylated genes (COL1A2, S100A10, S100A6, HTATIP2, CDH1, LXN) that display enhanced methylation levels in tumors compared with the cerebellum. Detailed analysis of COL1A2 supports a key role in medulloblastoma tumorigenesis; dense biallelic methylation associated with transcriptional silencing was observed in 46 of 60 cases (77%). Moreover, COL1A2 status distinguished infant medulloblastomas of the desmoplastic histopathological subtype, indicating that distinct molecular pathogenesis may underlie these tumors and their more favorable prognosis. These data reveal a more diverse and expansive medulloblastoma epi genome than previously understood and provide strong evidence that the methylation status of specific genes may contribute to the biological subclassification of medulloblastoma.
Neuro-Oncology 08/2008; 10(6):981-94. · 5.72 Impact Factor
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ABSTRACT: Abnormalities of chromosome 10 are frequently observed in the development of medulloblastoma, the most common malignant brain tumor of childhood. To identify critical genetic loci involved, we performed detailed physical mapping of regions of allelic loss on this chromosome. 18% of cases (5/32 primary tumors, 2/8 cell lines) harbored allelic losses on 10q. Refined mapping identified a 21.7Mb common interval, affecting the region 10q23.3-10q25.3. This region contains three genes, MXI1, SUFU and BTRC, which represent putative medulloblastoma tumor suppressor (TS) genes on the basis of either (i) negative regulation of critical medulloblastoma pathways, or (ii) mutation in other cancer types. We therefore sought evidence of their genetic inactivation in 46 cases, by mutational analysis of their entire coding regions. A MXI1 mutation was identified which abolishes its translation initiation site (A1G; MET1VAL), however no further tumor-specific sequence variations were detected. We next identified and characterised CpG islands associated with 5' regions of the MXI1, SUFU and BTRC genes; analysis of these regions for evidence of DNA hypermethylation, alongside expression analysis of their respective transcripts, revealed no evidence to support epigenetic inactivation of any gene. These findings implicate the inactivation of critical TS loci at 10q23.3-25.3 in medulloblastoma, however comprehensive analysis of SUFU, BTRC and MXI1 indicates they are unlikely to represent major targets of these allelic losses. MXI1 mutation appears to play a role in the pathogenesis of a small subset of cases, and suggests an alternative mechanism to MYC amplification for disruption of the MYC/MAD/MAX network in medulloblastoma.
Cell cycle (Georgetown, Tex.) 11/2006; 5(20):2381-9. · 5.36 Impact Factor
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ABSTRACT: MCJ (DNAJD1) is a recently discovered member of the DNAJ protein family whose expression is controlled epigenetically by methylation of a CpG island located within the 5' transcribed region of its gene. Methylation-dependent transcriptional silencing of MCJ has been observed in ovarian cancers and associated with increased resistance to chemotherapeutic agents; however, its role in other cancer types has not been widely investigated. We examined the status of MCJ in intracranial primitive neuroectodermal tumours [PNETs, comprising cerebellar PNETs (medulloblastomas) and supratentorial PNETs (stPNETs)] and ependymomas, together representing the most common malignant brain tumours of childhood. Evidence of MCJ hypermethylation was found in all 3 tumour types [medulloblastomas, 3/9 (33%) cell lines, 2/28 (7%) primary tumours; stPNETs, 2/2 (100%) cell lines, 3/10 (30%) primary tumours; and ependymomas, 2/20 (10%) primary tumours] but not in nonneoplastic brain tissues (n = 11), indicating that MCJ methylation is a tumour-specific event. In methylated cases, the distribution of methylated CpG sites across the CpG island could be broadly divided into 2 patterns: (i) extensive methylation of the majority of CpG sites across the island or (ii) limited methylation of individual CpG sites concentrated towards the 5' end of the island. Extensive methylation patterns were associated with the methylation-dependent transcriptional silencing of MCJ in medulloblastoma and stPNET cell lines. Further investigations of the mechanism of MCJ inactivation revealed that its loss could occur either through biallelic epigenetic methylation or by methylation in association with genetic loss of its second allele. These data indicate that epigenetic inactivation of MCJ may play a role in the development of a range of paediatric brain tumour types, and its role in disease pathogenesis and chemotherapeutic resistance should now be investigated further.
International Journal of Cancer 02/2006; 118(2):346-52. · 5.44 Impact Factor
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ABSTRACT: Glucocorticoids are pivotal in the treatment of children with acute lymphoblastic leukemia (ALL) and have significant antileukemic effects in the majority of children. However, clinical resistance is a significant problem. Although cell line models implicate somatic mutations and loss of heterozygosity (LOH) of the glucocorticoid receptor (GR) gene as a mechanism of in vitro glucocorticoid resistance, the relevance of this mechanism as a cause of clinical resistance in children with ALL is not known. Mutational screening of all coding exons of the GR gene and LOH analyses were done in a large cohort of relapsed ALL. We show that somatic mutations and LOH of the GR rarely contribute to relapsed disease in children with ALL. However, we report the second case of ALL with a somatic mutation of the GR involving a 29-bp deletion in exon 8 and resulting in a truncated protein with loss of part of the ligand-binding domain. There was no evidence of a remaining wild-type allele. Allele-specific PCR detected the mutated clone at day 28 after presentation, which persisted at a low level throughout the disease course before relapse several years later. We hypothesize that the mutated allele present in a leukemic subclone at initial diagnosis was selected for during remission induction with glucocorticoids and contributed to the emergence of a glucocorticoid-resistant cell population.
Cancer Research 12/2005; 65(21):9712-8. · 7.86 Impact Factor
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ABSTRACT: Medulloblastoma arises in the cerebellum and is the most common malignant brain tumour of childhood, however its molecular basis is not well understood. To assess the role of aberrant epigenetic events in medulloblastoma and identify critical genes in its development, we profiled the promoter methylation status of 11 candidate tumour-suppressor genes (TSGs; p14(ARF), p15(INK4b), p16(INK4a), CASP8, HIC1, EDNRB, TIMP3, TP73, TSLC1, RIZ1 and RASSF1A) in medulloblastoma cell lines, primary tumours and the normal cerebellum. Gene-specific TSG methylation was a significant feature of both medulloblastomas and the cerebellum. Extensive hypermethylation of RASSF1A was detected frequently in medulloblastomas but not in the normal cerebellum (41/44 primary tumours versus 0/5 normal cerebella). In contrast, complete methylation of HIC1 and CASP8 in a subset of primary tumours (17/44 and 14/39) occurred against a consistent background of partial methylation in the normal cerebellum. These data therefore indicate that extensive methylation of RASSF1A, HIC1 and CASP8 are tumour-specific events in medulloblastoma. Moreover, methylation of these genes in medulloblastoma cell lines was associated with their epigenetic transcriptional silencing and methylation-dependent re-expression following treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine. The remaining genes studied showed either low frequency methylation (p14(ARF), p16(INK4a), RIZ1; <7% of cases), no evidence of methylation (p15(INK4b), TIMP3, TP73, TSLC1), or comparable patterns of methylation in the normal cerebellum (EDNRB), suggesting that their hypermethylation does not play a major role in medulloblastoma. Our data demonstrate that tumour-specific hypermethylation affects only a subset of genes, and does not support the existence of a concordant methylation phenotype in this disease. We conclude that epigenetic TSG inactivation is a significant feature of medulloblastoma, and identify RASSF1A, HIC1 and CASP8 as potentially critical genes in its pathogenesis. Furthermore, methylation observed in the normal cerebellum emphasises the requirement for appropriate control tissues when assessing the tumour-specificity of TSG hypermethylation.
Carcinogenesis 06/2004; 25(5):661-8. · 5.70 Impact Factor
