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1.  Patient-Specific Screening Using High-Grade Glioma Explants to Determine Potential Radiosensitization by a TGF-β Small Molecule Inhibitor12 
Neoplasia (New York, N.Y.)  2016;18(12):795-805.
High-grade glioma (HGG), a deadly primary brain malignancy, manifests radioresistance mediated by cell-intrinsic and microenvironmental mechanisms. High levels of the cytokine transforming growth factor-β (TGF-β) in HGG promote radioresistance by enforcing an effective DNA damage response and supporting glioma stem cell self-renewal. Our analysis of HGG TCGA data and immunohistochemical staining of phosphorylated Smad2, which is the main transducer of canonical TGF-β signaling, indicated variable levels of TGF-β pathway activation across HGG tumors. These data suggest that evaluating the putative benefit of inhibiting TGF-β during radiotherapy requires personalized screening. Thus, we used explant cultures of seven HGG specimens as a rapid, patient-specific ex vivo platform to test the hypothesis that LY364947, a small molecule inhibitor of the TGF-β type I receptor, acts as a radiosensitizer in HGG. Immunofluorescence detection and image analysis of γ-H2AX foci, a marker of cellular recognition of radiation-induced DNA damage, and Sox2, a stem cell marker that increases post-radiation, indicated that LY364947 blocked these radiation responses in five of seven specimens. Collectively, our findings suggest that TGF-β signaling increases radioresistance in most, but not all, HGGs. We propose that short-term culture of HGG explants provides a flexible and rapid platform for screening context-dependent efficacy of radiosensitizing agents in patient-specific fashion. This time- and cost-effective approach could be used to personalize treatment plans in HGG patients.
PMCID: PMC5156509  PMID: 27978994
7.  Pilocytic astrocytoma and glioneuronal tumor with histone H3 K27M mutation 
PMCID: PMC4983033  PMID: 27519587
Glioblastoma; Histone H3 K27M; Pilocytic astrocytoma; Glioneuronal tumor
9.  Medulloblastoma subgroups remain stable across primary and metastatic compartments 
Acta neuropathologica  2015;129(3):449-457.
Medulloblastoma comprises four distinct molecular variants with distinct genetics, transcriptomes, and outcomes. Subgroup affiliation has been previously shown to remain stable at the time of recurrence, which likely reflects their distinct cells of origin. However, a therapeutically relevant question that remains unanswered is subgroup stability in the metastatic compartment. We assembled a cohort of 12-paired primary-metastatic tumors collected in the MAGIC consortium, and established their molecular subgroup affiliation by performing integrative gene expression and DNA methylation analysis. Frozen tissues were collected and profiled using Affymetrix gene expression arrays and Illumina methylation arrays. Class prediction and hierarchical clustering were performed using existing published datasets. Our molecular analysis, using consensus integrative genomic data, establishes the unequivocal maintenance of molecular subgroup affiliation in metastatic medulloblastoma. We further validated these findings by interrogating a non-overlapping cohort of 19-pairs of primary-metastatic tumors from the Burdenko Neurosurgical Institute using an orthogonal technique of immunohistochemical staining. This investigation represents the largest reported primary-metastatic paired cohort profiled to date and provides a unique opportunity to evaluate subgroup-specific molecular aberrations within the metastatic compartment. Our findings further support the hypothesis that medulloblastoma subgroups arise from distinct cells of origin, which are carried forward from ontogeny to oncology.
PMCID: PMC4333718  PMID: 25689980
Medulloblastoma; Metastasis; Molecular Subgroups; Integrative Genomics; Gene Expression; DNA Methylation
10.  Molecular Classification of Ependymal Tumors across All CNS Compartments, Histopathological Grades, and Age Groups 
Cancer cell  2015;27(5):728-743.
Ependymal tumors across age groups are currently classified and graded solely by histopathology. It is, however, commonly accepted that this classification scheme has limited clinical utility based on its lack of reproducibility in predicting patients’ outcome. We aimed at establishing a uniform molecular classification using DNA methylation profiling. Nine molecular subgroups were identified in a large cohort of 500 tumors, 3 in each anatomical compartment of the CNS, spine, posterior fossa, supratentorial. Two supratentorial subgroups are characterized by prototypic fusion genes involving RELA and YAP1, respectively. Regarding clinical associations, the molecular classification proposed herein outperforms the current histopathological classification and thus might serve as a basis for the next World Health Organization classification of CNS tumors.
Graphical Abstract
PMCID: PMC4712639  PMID: 25965575
12.  BRAF Alterations in Primary Glial and Glioneuronal Neoplasms of the Central Nervous System With Identification of 2 Novel KIAA1549:BRAF Fusion Variants 
Recent studies highlight the importance of BRAF alterations resulting in mitogen activated protein kinase (MAK/ERK) pathway activation in low-grade CNS tumors. We studied 106 low-grade CNS neoplasms in a cohort of primarily pediatric patients to identify the prevalence and clinicopathologic significance of these alterations. Polymerase chain reaction testing identified KIAA1549:BRAF fusions in 51 (48%) tumors overall, including 42 (60%) pilocytic astrocytomas, 4 (17%) unclassifiable low-grade gliomas, 4 (36%) low-grade glioneuronal/neuroepithelial tumors, 0 (of 5) pleomorphic xanthoastrocytomas, 0 (of 4) diffuse astrocytomas (World Health Organization grade II), and 1 (of 3, 33%) pilomyxoid astrocytoma. KIAA1549:BRAF gene fusions confirmed by sequencing included the previously reported ones involving exons 1–16/9–18 (49%), 1–15/9–18 (35%), and 1–16/11–18 (8%) and 2 fusions with novel breakpoints: 1–15/11–18 (6%) and 1–17/10–18 (1%). DNA sequencing identified BRAFV600E mutations in 8% of tumors. BRAFG468A mutations were absent. KIAA1549:BRAF fusions were significantly more frequent in infratentorial (57%) and optic pathway (59%) tumors versus supratentorial (19%) tumors (p = 0.001). We did not identify significantly improved progression-free survival in tumors with fusions. In summary, KIAA1549:BRAF fusions predominate in pilocytic astrocytomas but are also present in some low-grade unclassifiable gliomas and glioneuronal tumors. The prognostic and therapeutic significance of this alteration is unclear and merits further study.
PMCID: PMC4629834  PMID: 22157620
Brain tumor; BRAF; Glioneuronal; MAPK; Pilocytic astrocytoma
Neuro-Oncology  2014;16(Suppl 5):v90.
BACKGROUND: Expression of chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1α (SDF-1α) is enhanced in glioblastoma, including at invasive edges, and associated with proliferation, invasion and angiogenesis (Zagzag, 2008; Ehtesham, 2013; Gagliardi, 2014). Using Protein Epitope Mimetics technology (Robinson, 2008), Polyphor Ltd. has developed selective, highly potent CXCR4 antagonists (De Marco, 2006), e.g., POL5551 (Ki: 0.5 nM). To address the problem of resistance to antiangiogenic therapy, we sought to determine whether POL5551 could inhibit the growth and/or invasion of gliomas in vivo. METHODS: Female adult C57BL/6 mice were implanted orthotopically with 1 x 105 CT-2A or GL261 glioma cells. On day 14, mice were randomized into 4 groups: 1) control, 2) POL5551 (5 mg/kg s.c.) twice daily, 3) anti-murine VEGF monoclonal antibody B20-4.1.1 (Genentech Inc.) (5 mg/kg i.p.) twice weekly (Bagri, 2010) and 4) combined POL5551 and B20-4.1.1. On day 28, brain tissues were processed, sections analyzed for tumor volume and invasion (H&E), vascular density (CD105) and apoptosis (caspase 3), and results compared (ANOVA). RESULTS: In CT-2A glioma-bearing mice, combined therapy significantly reduced tumor volume (17.3 ± 2.4 mm3, mean ± S.E.M.) compared with controls (57.2 ± 6.4, p < 0.01) and POL5551 (55.8 ± 11.2, p < 0.01), but not B20-4.1.1 (31.9 ± 8.7). The depth of tumor invasion was reduced by 47.5% (p < 0.07) with POL5551, but by only 10.3% with B20-4.1.1, and 24.4% with combined therapy. Comparable, but not statistically significant, results were obtained with GL261 gliomas. The concentration of POL5551 was higher in glioma tissues (87 ± 25.4 ng/g; 45.8 nM) than in brain adjacent to tumor (45 ± 4.8; 23.7) or normal brain (21 ± 1.5; 11.1) and sufficient to account for its biological effects. CONCLUSIONS: Although combined therapy did not reduce significantly glioma invasion, it did impair the growth of CT-2A and GL261 gliomas by 69.8% and 59.9%, respectively. Our results suggest the potential utility of POL5551 as adjunct therapy for glioblastoma.
PMCID: PMC4218146
Neuro-Oncology  2014;16(Suppl 5):v197.
Glioblastoma stem cells (GSCs) have the ability to self-renew, differentiate into tumor lineages, and give rise to tumors in immunodeficient animal models. More importantly, GSCs are resistant to current therapies, leading to tumor recurrence. Glioblastoma (GBM) is highly heterogeneous at the molecular and cellular level. This heterogeneity also exhibits itself within the stem cell compartment, further impairing the development of effective therapies. CD133+ GSCs cells have increased tumorigenicity. However, the facts that CD133- cells also give rise to tumors in animal models and that some tumors do not have a CD133+ cell population suggest that CD133 is not a universal GSC marker. The Notch signaling pathway, previously believed to be activated in CD133+ GSCs, is important for GSC self-renewal and therapy resistance. To study GSC heterogeneity, we used genetic reporters for Notch activation and made the surprising observation that Notch signaling is active in small populations of cells that do not express CD133 (CD133-/Notch + ; n = 3 primary GBM cultures). Our in vitro tumorsphere formation and in vivo tumor formation assays showed that both CD133 + /Notch- and CD133-/Notch+ cells have stem cell properties. Global transcriptome analysis revealed that CD133 + /Notch- and CD133-/Notch+ GSCs utilize differential transcriptional programs, which prime CD133 + /Notch- GSCs to rely on anaerobic glycolysis. Furthermore, CD133/-Notch+ cells are able to give rise to CD133 + /Notch- cells, although the converse does not occur. This finding suggests that the two cell types have distinct differentiation potentials. Finally, preliminary data suggest that these two GSC subtypes behave differentially in response to temozolomide and radiation treatment, which are the standard of care for GBM. We are currently testing the contribution of distinct GSCs to tumor heterogeneity in vivo, using lineage-tracing systems. Understanding the biology of GSCs and the functional importance of heterogeneity within GSC populations is critical for the design of novel therapies.
PMCID: PMC4218599
16.  Phase II study of sorafenib in children with recurrent or progressive low-grade astrocytomas 
Neuro-Oncology  2014;16(10):1408-1416.
Activation of the RAS-RAF-MEK-ERK signaling pathway is thought to be the key driver of pediatric low-grade astrocytoma (PLGA) growth. Sorafenib is a multikinase inhibitor targeting BRAF, VEGFR, PDGFR, and c-kit. This multicenter phase II study was conducted to determine the response rate to sorafenib in patients with recurrent or progressive PLGA.
Key eligibility criteria included age ≥2 years, progressive PLGA evaluable on MRI, and at least one prior chemotherapy treatment. Sorafenib was administered twice daily at 200 mg/m2/dose (maximum of 400 mg/dose) in continuous 28-day cycles. MRI, including 3-dimensional volumetric tumor analysis, was performed every 12 weeks. BRAF molecular testing was performed on tumor tissue when available.
Eleven patients, including 3 with neurofibromatosis type 1 (NF1), were evaluable for response; 5 tested positive for BRAF duplication. Nine patients (82%) came off trial due to radiological tumor progression after 2 or 3 cycles, including 3 patients with confirmed BRAF duplication. Median time to progression was 2.8 months (95% CI, 2.1–31.0 months). Enrollment was terminated early due to this rapid and unexpectedly high progression rate. Tumor tissue obtained from 4 patients after termination of the study showed viable pilocytic or pilomyxoid astrocytoma.
Sorafenib produced unexpected and unprecedented acceleration of tumor growth in children with PLGA, irrespective of NF1 or tumor BRAF status. In vitro studies with sorafenib indicate that this effect is likely related to paradoxical ERK activation. Close monitoring for early tumor progression should be included in trials of novel agents that modulate signal transduction.
PMCID: PMC4165419  PMID: 24803676
low-grade glioma; pediatric low-grade astrocytoma; phase II trial; sorafenib
17.  Decreased Expression of Cystathionine β-Synthase Promotes Glioma Tumorigenesis 
Molecular cancer research : MCR  2014;12(10):1398-1406.
Cystathionine β-synthase (CBS) catalyzes metabolic reactions that convert homocysteine to cystathionine. To assess the role of CBS in human glioma, cells were stably transfected with lentiviral vectors encoding short hairpin RNA (shRNA) targeting CBS or a non-targeting control shRNA and subclones were injected into immunodeficient mice. Interestingly, decreased CBS expression did not affect proliferation in vitro but decreased the latency period prior to rapid tumor xenograft growth after subcutaneous injection and increased tumor incidence and volume following orthotopic implantation into the caudate-putamen. In soft agar colony formation assays, CBS knockdown subclones displayed increased anchorage-independent growth. Molecular analysis revealed that CBS knockdown subclones expressed higher basal levels of the transcriptional activator hypoxia-inducible factor 2α (HIF-2α/EPAS1). HIF-2α knockdown counteracted the effect of CBS knockdown on anchorage-independent growth. Bioinformatic analysis of mRNA expression data from human glioma specimens revealed a significant association between low expression of CBS mRNA and high expression of angiopoietin-like 4 (ANGPTL4) and vascular endothelial growth factor (VEGF) transcripts, which are HIF-2 target gene products that were also increased in CBS knockdown subclones. These results suggest that decreased CBS expression in glioma increases HIF-2α protein levels and HIF-2 target gene expression, which promotes glioma tumor formation.
CBS loss of function promotes glioma growth.
PMCID: PMC4274393  PMID: 24994751
anchorage-independent growth; CBS; HIF-2α; hypoxia-inducible factor
18.  Merlin/NF2-loss Driven Tumorigenesis Linked to CRL4DCAF1-Mediated Inhibition of the Hippo Pathway Components Lats1 and 2 in the Nucleus 
Cancer cell  2014;26(1):48-60.
It is currently unclear if Merlin/NF2 suppresses tumorigenesis by activating upstream components of the Hippo pathway at the plasma membrane or by inhibiting the E3 ubiquitin ligase CRL4DCAF1 in the nucleus. We found that de-repressed CRL4DCAF1 promotes YAP and TEAD-dependent transcription by ubiquitylating and thereby inhibiting Lats1 and 2 in the nucleus. Genetic epistasis experiments and analysis of tumor-derived missense mutations indicate that this signaling connection sustains the oncogenicity of Merlin-deficient tumor cells. Analysis of clinical samples confirms that this pathway operates in NF2 mutant tumors. We conclude that de-repressed CRL4DCAF1 promotes activation of YAP by inhibiting Lats1 and 2 in the nucleus.
PMCID: PMC4126592  PMID: 25026211
Neuro-Oncology  2014;16(Suppl 3):iii4.
BACKGROUND: The current World Health Organisation (WHO) classification of central nervous system tumors comprises over 100 entities. Most of these are defined by purely histological criteria, with varying and sometimes overlapping spectra. Histological diagnosis is often challenging, however, especially in cases with limited or non-representative biopsy material. Thus, molecular technologies that can complement standard pathology testing have the potential to greatly enhance diagnostic precision and improve clinical decision-making. DNA methylation profiling, acting as a 'fingerprint' of cellular origin and molecular alterations, is one such promising technology. METHODS: We have assembled a reference dataset of more than 2,000 methylation profiles using the Illumina HumanMethylation450 (450k) array, currently representing over 50 brain tumor entities or subgroups. The array platform is suitable for both frozen and paraffin-embedded material, with minimal DNA input required. Each new diagnostic case receives an entity prediction with an associated probability score as a confidence measure. Genome-wide copy number profiles (e.g. for scoring 1p/19q loss or gene amplifications) and target gene methylation data (e.g. MGMT) generated from the array provide important additional information. RESULTS: In addition to the reference cohort, more than 500 diagnostic samples from Heidelberg University Hospital and external institutions have been processed. Approximately 5-10% of cases displayed a discrepancy between histological and molecular diagnoses. Careful re-examination of these often resulted in refinement of the original diagnosis, and improved patient care.Furthermore, samples collected for the reference cohort have led to significant improvements in our understanding of the biology of several tumor types, including the identification of further subgroups for several entities and associations with recurrent copy number changes and/or mutations. CONCLUSIONS: Our understanding of the molecular alterations underlying brain tumors has grown enormously in recent years, and it is crucial that this is translated into the clinic promptly. DNA methylation profiling is one tool with the potential to become an important part of the diagnostic armoury of neuropathologists. This relatively inexpensive and robust method is well suited to complement standard histopathologic techniques and improve diagnostic accuracy, thereby optimising patient management. We are currently expanding our pipeline to include additional diagnostic centres, allowing for further refinement and validation as well as broader international access. SECONDARY CATEGORY: Tumor Biology.
PMCID: PMC4144477
Neuro-Oncology  2014;16(Suppl 3):iii27.
BACKGROUND: NELL2 expression has been shown to identify patients with posterior fossa ependymomas (PFEp) that exhibit prolonged survivals. We analyzed expression NELL2; KI-67; Tenascin C; CD34; VEGF; and CA IX as well as vascular density against post-operative survival (Progression Free and Overall Survival; PFS, OS) in 31 patients under the age of 22 years. METHODS: PFEp from patients aged 22 years or less were studied. H&E sections were reviewed for grade and vascular status and immunohistochemistry (IHC) was used to determine NELL2; KI-67; Tenascin C; CD34; VEGF; and CA IX status. RESULTS: 3 WHO Grade I, 19 Grade II and 9 Grade III PFEp were studied. Median follow-up time was 9.0 years; median survival was 6.1 years. NELL2 status correlated with PFS (Log Rank P-Value: 0.0220) and exhibited a strong trend towards prolonged OS (Log rank p = 0.0555). Peri-necrotic CAIX localization correlated with PFS (Log rank = 0.0041) and OS (Log rank p = 0.0010) All patients with a CA IX ≤ 5% total area localization were alive at last follow-up. Perinecrotic CAIX staining was also associated with CD34 density (p = .0057) though not with VEGF expression score. MIB-1 labeling index (LI) correlated with OS (p = 0.0185) and PFS (p = 0.0049). MIB-1 LI and perinecrotic CA IX individually correlated with PFS. The effect of perinecrotic CA IX remained when grade was added to a Cox model predicting PFS. There was a significant difference in the distributions of perinecrotic CA IX as stratified by NELL2 expression (Wilcoxon p = 0.0225) with NELL2 negative tumors exhibiting much stronger CA IX expression. CONCLUSIONS: Immunodetection of NELL2 expression was confirmed as a predictive biomarker for PFS in these tumors. This effect is related to the absence of necrosis, low vascular proliferation, low MIB-1 proliferation index, and the absence of CA IX expression. SECONDARY CATEGORY: Pediatrics.
PMCID: PMC4144561
21.  ERG is a novel and reliable marker for endothelial cells in central nervous system tumors  
Clinical Neuropathology  2015;34(3):117-127.
ETS-related gene (ERG) is a transcription factor that has been linked to angiogenesis. Very little research has been done to assess ERG expression in central nervous system (CNS) tumors. We evaluated 57 CNS tumors, including glioblastomas (GBMs) and hemangioblastomas (HBs), as well as two arteriovenous malformations and four samples of normal brain tissue with immunohistochemistry using a specific ERG rabbit monoclonal antibody. In addition, immunostains for CD31, CD34, and α-smooth muscle actin (α-SMA) were performed on all samples. CD31 demonstrated variable and sometimes weak immunoreactivity for endothelial cells. Furthermore, in 1 case of a GBM, CD34 stained not only endothelial cells, but also tumor cells. In contrast, we observed that ERG was only expressed in the nuclei of endothelial cells, for example, in the hyperplastic vascular complexes that comprise the glomeruloid microvascular proliferation seen in GBMs. Conversely, α-SMA immunoreactivity was identified in the abluminal cells of these hyperplastic vessels. Quantitative evaluation with automated methodology and custom Matlab 2008b software was used to calculate percent staining of ERG in each case. We observed significantly higher quantitative expression of ERG in HBs than in other CNS tumors. Our results show that ERG is a novel, reliable, and specific marker for endothelial cells within CNS tumors that can be used to better study the process of neovascularization.
PMCID: PMC4542182  PMID: 25881913
ERG; immunostain; endothelial cell; CNS tumor; neovascularization
22.  Selective Lentiviral Gene Delivery to CD133-Expressing Human Glioblastoma Stem Cells 
PLoS ONE  2014;9(12):e116114.
Glioblastoma multiforme (GBM) is a deadly primary brain malignancy. Glioblastoma stem cells (GSC), which have the ability to self-renew and differentiate into tumor lineages, are believed to cause tumor recurrence due to their resistance to current therapies. A subset of GSCs is marked by cell surface expression of CD133, a glycosylated pentaspan transmembrane protein. The study of CD133-expressing GSCs has been limited by the relative paucity of genetic tools that specifically target them. Here, we present CD133-LV, a lentiviral vector presenting a single chain antibody against CD133 on its envelope, as a vehicle for the selective transduction of CD133-expressing GSCs. We show that CD133-LV selectively transduces CD133+ human GSCs in dose-dependent manner and that transduced cells maintain their stem-like properties. The transduction efficiency of CD133-LV is reduced by an antibody that recognizes the same epitope on CD133 as the viral envelope and by shRNA-mediated knockdown of CD133. Conversely, the rate of transduction by CD133-LV is augmented by overexpression of CD133 in primary human GBM cultures. CD133-LV selectively transduces CD133-expressing cells in intracranial human GBM xenografts in NOD.SCID mice, but spares normal mouse brain tissue, neurons derived from human embryonic stem cells and primary human astrocytes. Our findings indicate that CD133-LV represents a novel tool for the selective genetic manipulation of CD133-expressing GSCs, and can be used to answer important questions about how these cells contribute to tumor biology and therapy resistance.
PMCID: PMC4277468  PMID: 25541984
23.  Activation of mTORC1/mTORC2 signaling in pediatric low-grade glioma and pilocytic astrocytoma reveals mTOR as a therapeutic target 
Neuro-Oncology  2013;15(12):1604-1614.
Previous studies support a role for mitogen-activated protein kinase pathway signaling, and more recently Akt/mammalian target of rapamycin (mTOR), in pediatric low-grade glioma (PLGG), including pilocytic astrocytoma (PA). Here we further evaluate the role of the mTORC1/mTORC2 pathway in order to better direct pharmacologic blockade in these common childhood tumors.
We studied 177 PLGGs and PAs using immunohistochemistry and tested the effect of mTOR blockade on 2 PLGG cell lines (Res186 and Res259) in vitro.
Moderate (2+) to strong (3+) immunostaining was observed for pS6 in 107/177 (59%) PAs and other PLGGs, while p4EBP1 was observed in 35/115 (30%), pElF4G in 66/112 (59%), mTOR (total) in 53/113 (47%), RAPTOR (mTORC1 component) in 64/102 (63%), RICTOR (mTORC2 component) in 48/101 (48%), and pAkt (S473) in 63/103 (61%). Complete phosphatase and tensin homolog protein loss was identified in only 7/101 (7%) of cases. In PA of the optic pathways, compared with other anatomic sites, there was increased immunoreactivity for pS6, pElF4G, mTOR (total), RICTOR, and pAkt (P < .05). We also observed increased pS6 (P = .01), p4EBP1 (P = .029), and RICTOR (P = .05) in neurofibromatosis type 1 compared with sporadic tumors. Treatment of the PLGG cell lines Res186 (PA derived) and Res259 (diffuse astrocytoma derived) with the rapalog MK8669 (ridaforolimus) led to decreased mTOR pathway activation and growth.
These findings suggest that the mTOR pathway is active in PLGG but varies by clinicopathologic subtype. Additionally, our data suggest that mTORC2 is differentially active in optic pathway and neurofibromatosis type 1–associated gliomas. MTOR represents a potential therapeutic target in PLGG that merits further investigation.
PMCID: PMC3829599  PMID: 24203892
mTOR; neurofibromatosis; optic nerve; pediatric glioma; pilocytic astrocytoma
24.  Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations 
Nature genetics  2014;46(5):451-456.
Diffuse Intrinsic Pontine Glioma (DIPG) is a fatal brain cancer that arises in the brainstem of children with no effective treatment and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumors and choosing therapies based on assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic make-up of this brain cancer with nearly 80% harboring a K27M-H3.3 or K27M-H3.1 mutation. However, DIPGs are still thought of as one disease with limited understanding of the genetic drivers of these tumors. To understand what drives DIPGs we integrated whole-genome-sequencing with methylation, expression and copy-number profiling, discovering that DIPGs are three molecularly distinct subgroups (H3-K27M, Silent, MYCN) and uncovering a novel recurrent activating mutation in the activin receptor ACVR1, in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer.
PMCID: PMC3997489  PMID: 24705254 CAMSID: cams4215
25.  Recurrence patterns across medulloblastoma subgroups: an integrated clinical and molecular analysis 
The lancet oncology  2013;14(12):1200-1207.
Recurrent medulloblastoma is a daunting therapeutic challenge as it is almost universally fatal. Recent studies confirmed that medulloblastoma comprises four distinct subgroups. We sought to delineate subgroup specific differences in medulloblastoma recurrence patterns.
We retrospectively identified a discovery cohort of all recurrent medulloblastomas at the Hospital for Sick Children between 1994-2012, and performed molecular subgrouping on FFPE tissues using a nanoString-based assay. The anatomical site of recurrence (local tumour bed or leptomeningeal metastasis), time to recurrence and survival post-recurrence were determined in a subgroup specific fashion. Subgroup specific recurrence patterns were confirmed in two independent, non-overlapping FFPE validation cohorts. Where possible molecular subgrouping was performed on tissue obtained from both the initial surgery and at recurrence.
A screening cohort of 30 recurrent medulloblastomas was assembled; nine with local recurrences, and 21 metastatic. When re-analysed in a subgroup specific manner, local recurrences were more frequent in SHH tumours (8/9, 88%) and metastatic recurrences were more common in Group 3 and 4 (17/20 [85%] with one WNT, p=0.0014, local vs metastatic recurrence, SHH vs Group 3 vs Group 4). The subgroup specific location of recurrence was confirmed in a multicenter validation cohort (p=0·0013 for local vs metastatic recurrence SHH vs Group 3 vs Group 4, n=77), and a second independent validation cohort comprising 96 recurrences (p<0·0001 for local vs metastatic recurrence SHH vs Group 3 vs Group 4, n=96). Treatment with craniospinal irradiation at diagnosis was not significantly associated with the anatomical pattern of recurrence. Survival post recurrence was significantly longer in Group 4 patients (p=0·013) as confirmed in a multicenter validation cohort (p=0·0075). Strikingly, subgroup affiliation remained stable at recurrence in all 34 cases with available matched primary and recurrent pairs.
Medulloblastoma does not switch subgroup at the time of recurrence further highlighting the stability of the four principle medulloblastoma subgroups. Significant differences in the location and timing of recurrence across medulloblastoma subgroups were observed which have potential treatment ramifications. Specifically, intensified local (posterior fossa) therapy should be tested in the initial treatment of SHH patients. Refinement of therapy for Groups 3 and 4 should focus on the metastatic compartment, as it is the near universal cause of patient deaths.
PMCID: PMC3953419  PMID: 24140199

Results 1-25 (53)