Hendrik Witt

Medulloblastoma Comprises Four Distinct Molecular Variants

PURPOSE Recent genomic approaches have suggested the existence of multiple distinct subtypes of medulloblastoma. We studied a large cohort of medulloblastomas to determine how many subgroups of the disease exist, how they differ, and the extent of overlap between subgroups. METHODS We determined gene expression profiles and DNA copy number aberrations for 103 primary medulloblastomas. Bioinformatic tools were used for class discovery of medulloblastoma subgroups based on the most informative genes in the data set. Immunohistochemistry for subgroup-specific signature genes was used to determine subgroup affiliation for 294 nonoverlapping medulloblastomas on two independent tissue microarrays. RESULTS Multiple unsupervised analyses of transcriptional profiles identified the following four distinct, nonoverlapping molecular variants: WNT, SHH, group C, and group D. Supervised analysis of these four subgroups revealed significant subgroup-specific demographics, histology, metastatic status, and DNA copy number aberrations. Immunohistochemistry for DKK1 (WNT), SFRP1 (SHH), NPR3 (group C), and KCNA1 (group D) could reliably and uniquely classify formalin-fixed medulloblastomas in approximately 98% of patients. Group C patients (NPR3-positive tumors) exhibited a significantly diminished progression-free and overall survival irrespective of their metastatic status. CONCLUSION Our integrative genomics approach to a large cohort of medulloblastomas has identified four disparate subgroups with distinct demographics, clinical presentation, transcriptional profiles, genetic abnormalities, and clinical outcome. Medulloblastomas can be reliably assigned to subgroups through immunohistochemistry, thereby making medulloblastoma subclassification widely available. Future research on medulloblastoma and the development of clinical trials should take into consideration these four distinct types of medulloblastoma.

Delineation of two clinically and molecularly distinct subgroups of posterior fossa ependymoma.

Despite the histological similarity of ependymomas from throughout the neuroaxis, the disease likely comprises multiple independent entities, each with a distinct molecular pathogenesis. Transcriptional profiling of two large independent cohorts of ependymoma reveals the existence of two demographically, transcriptionally, genetically, and clinically distinct groups of posterior fossa (PF) ependymomas. Group A patients are younger, have laterally located tumors with a balanced genome, and are much more likely to exhibit recurrence, metastasis at recurrence, and death compared with Group B patients. Identification and optimization of immunohistochemical (IHC) markers for PF ependymoma subgroups allowed validation of our findings on a third independent cohort, using a human ependymoma tissue microarray, and provides a tool for prospective prognostication and stratification of PF ependymoma patients.

Combined molecular analysis of BRAF and IDH1 distinguishes pilocytic astrocytoma from diffuse astrocytoma

Separation of pilocytic astrocytoma from diffuse astrocytomas frequently poses problems mostly related to small sample size. Precise classification and grading are essential due to different therapeutic strategies prompted by diagnoses of pilocytic astrocytoma WHO grade I, diffuse astrocytomas WHO grade II or anaplastic astrocytoma WHO grade III. Recently, genomic aberrations with a high specificity for distinct glioma entities have been described. Pilocytic astrocytomas carry a duplication at chromosome band 7q34 containing a BRAF–KIAA1549 gene fusion in the majority of cases. IDH1 mutations are observed very frequently in adult astrocytomas and IDH2 mutations have been reported in some astrocytomas. We examined a series of 120 astrocytomas including 70 pilocytic astrocytomas WHO grade I and 50 diffuse astrocytomas WHO grade II for both, BRAF–KIAA1549 fusion with a newly developed FISH assay and mutations in IDH1 and IDH2 by direct sequencing. Pilocytic astrocytomas contained the BRAF fusion in 49 cases (70%) but neither IDH1 nor IDH2 mutations. Astrocytomas WHO grade II exhibited IDH1 mutations in 38 cases (76%) but neither IDH2 mutations nor BRAF fusions. Thus, combined molecular analysis of BRAF and IDH1 is a sensitive and highly specific approach to separate pilocytic astrocytoma from diffuse astrocytoma.

An Animal Model of MYC-Driven Medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor in children. Patients whose tumors exhibit overexpression or amplification of the MYC oncogene (c-MYC) usually have an extremely poor prognosis, but there are no animal models of this subtype of the disease. Here, we show that cerebellar stem cells expressing Myc and mutant Trp53 (p53) generate aggressive tumors following orthotopic transplantation. These tumors consist of large, pleiomorphic cells and resemble human MYC-driven MB at a molecular level. Notably, antagonists of PI3K/mTOR signaling, but not Hedgehog signaling, inhibit growth of tumor cells. These findings suggest that cerebellar stem cells can give rise to MYC-driven MB and identify a novel model that can be used to test therapies for this devastating disease.

BCAT1 promotes cell proliferation through amino acid catabolism in gliomas carrying wild-type IDH1

Here we show that glioblastoma express high levels of branched-chain amino acid transaminase 1 (BCAT1), the enzyme that initiates the catabolism of branched-chain amino acids (BCAAs). Expression of BCAT1 was exclusive to tumors carrying wild-type isocitrate dehydrogenase 1 (IDH1) and IDH2 genes and was highly correlated with methylation patterns in the BCAT1 promoter region. BCAT1 expression was dependent on the concentration of α-ketoglutarate substrate in glioma cell lines and could be suppressed by ectopic overexpression of mutant IDH1 in immortalized human astrocytes, providing a link between IDH1 function and BCAT1 expression. Suppression of BCAT1 in glioma cell lines blocked the excretion of glutamate and led to reduced proliferation and invasiveness in vitro, as well as significant decreases in tumor growth in a glioblastoma xenograft model. These findings suggest a central role for BCAT1 in glioma pathogenesis, making BCAT1 and BCAA metabolism attractive targets for the development of targeted therapeutic approaches to treat patients with glioblastoma.

Emerging Insights into the Ependymoma Epigenome

Ependymoma is the third most common pediatric brain tumor, yet because of the paucity of effective therapeutic interventions, 45% of patients remain incurable. Recent transcriptional and copy number profiling of the disease has identified few driver genes and in fact points to a balanced genomic profile. Candidate gene approaches looking at hypermethylated promoters and genome‐wide epigenetic arrays suggest that DNA methylation may be critical to ependymoma pathogenesis. This review attempts to highlight existing and emerging evidence implicating the ependymoma epigenome as a key player and that epigenetic modifiers may offer new targeted therapeutic avenues for patients.

Papillary Tumor of the Pineal Region: A Distinct Molecular Entity

Papillary tumor of the pineal region (PTPR) is a neuroepithelial brain tumor, which might pose diagnostic difficulties and recurs often. Little is known about underlying molecular alterations. We therefore investigated chromosomal copy number alterations, DNA methylation patterns and mRNA expression profiles in a series of 24 PTPRs. Losses of chromosome 10 were identified in all 13 PTPRs examined. Losses of chromosomes 3 and 22q (54%) as well as gains of chromosomes 8p (62%) and 12 (46%) were also common. DNA methylation profiling using Illumina 450k arrays reliably distinguished PTPR from ependymomas and pineal parenchymal tumors of intermediate differentiation. PTPR could be divided into two subgroups based on methylation pattern, PTPR group 2 showing higher global methylation and a tendency toward shorter progression‐free survival (P = 0.06). Genes overexpressed in PTPR as compared with ependymal tumors included SPDEF, known to be expressed in the rodent subcommissural organ. Notable SPDEF protein expression was encountered in 15/19 PTPRs as compared with only 2/36 ependymal tumors, 2/19 choroid plexus tumors and 0/23 samples of other central nervous system (CNS) tumor entities. In conclusion, PTPRs show typical chromosomal alterations as well as distinct DNA methylation and expression profiles, which might serve as useful diagnostic tools.

Abstract 3687: An integrative genomics approach identifies distinct molecular and epigenetic subgroups of pediatric glioblastoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pediatric glioblastoma (GBM) belongs to the comparably small group of childhood malignancies for which cure is still an exception. Histologically indistinguishable from their adult counterparts, they carry a similar dismal prognosis. Whereas genetic and epigenetic properties have been extensively studied in adult tumors, little is known about the molecular characteristics of pediatric GBM, although some reports indicate that it is likely a different entity in terms of tumor biology and molecular genetics. Thus, this study aimed to elucidate disease-defining molecular lesions by determining genomic, transcriptomic and epigenetic alteration profiles. Using an integrative genomics approach combining multiple screening strategies, we investigated primary tumor samples from 55 childhood GBM for copy-number aberrations (CNA), transcriptomic and epigenetic changes, complemented by sequencing analysis of TP53, IDH1/2 and further candidate genes. Methylome analysis revealed the existence of five separate clusters of childhood GBM with distinct molecular and clinico-pathological features. Methylation patterns correlated with novel recurrent, subgroup-specific driver mutations unique to the pediatric population, and with clearly distinguishable transcriptomic profiles. Integration of methylation and gene expression data suggested that different tumor subgroups are derived from at least two distinct precursor-cell populations, one of them without any signs of neural lineage commitment. Furthermore, distinct clusters were highly associated with the presence of balanced (13%) or aneuploid (33%) genomic profiles or with cases displaying highly-rearranged genomes (11%), or various high-level focal amplifications (43%) of known and novel oncogenes. Similar to adults, CNA frequently targeted GBM core signaling pathways such as RTK/PI3K, p53 and RB signaling. TP53 loss-of-function mutations were present in 46% of pediatric GBM. IDH1 mutations were detected in only six patients (11%), but these tumors displayed concerted hypermethylation at a large number of loci, resembling a CpG island methylator phenotype (CIMP). Relevant findings are being validated by immunohistochemistry or FISH analysis in an independent, large-scale cohort representing 130 uniformly-treated pediatric GBM. This study, one of the largest cohorts of pediatric GBM investigated for molecular alterations to date, describes frequent genetic and epigenetic features of this devastating disease and further emphasizes and differences between adult and pediatric GBM. The identification of distinct molecular subgroups and commonly altered pathways will help to characterize molecular biomarkers for improved prognostic assessment and risk-adapted treatment stratification, and may facilitate the development of suitable in vitro and in vivo models for defining novel therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3687. doi:1538-7445.AM2012-3687

Heterogeneity within the PF-EPN-B ependymoma subgroup

Posterior fossa ependymoma comprise three distinct molecular variants, termed PF-EPN-A (PFA), PF-EPN-B (PFB), and PF-EPN-SE (subependymoma). Clinically, they are very disparate and PFB tumors are currently being considered for a trial of radiation avoidance. However, to move forward, unraveling the heterogeneity within PFB would be highly desirable. To discern the molecular heterogeneity within PFB, we performed an integrated analysis consisting of DNA methylation profiling, copy-number profiling, gene expression profiling, and clinical correlation across a cohort of 212 primary posterior fossa PFB tumors. Unsupervised spectral clustering and t-SNE analysis of genome-wide methylation data revealed five distinct subtypes of PFB tumors, termed PFB1-5, with distinct demographics, copy-number alterations, and gene expression profiles. All PFB subtypes were distinct from PFA and posterior fossa subependymomas. Of the five subtypes, PFB4 and PFB5 are more discrete, consisting of younger and older patients, respectively, with a strong female-gender enrichment in PFB5 (age: p = 0.011, gender: p = 0.04). Broad copy-number aberrations were common; however, many events such as chromosome 2 loss, 5 gain, and 17 loss were enriched in specific subtypes and 1q gain was enriched in PFB1. Late relapses were common across all five subtypes, but deaths were uncommon and present in only two subtypes (PFB1 and PFB3). Unlike the case in PFA ependymoma, 1q gain was not a robust marker of poor progression-free survival; however, chromosome 13q loss may represent a novel marker for risk stratification across the spectrum of PFB subtypes. Similar to PFA ependymoma, there exists a significant intertumoral heterogeneity within PFB, with distinct molecular subtypes identified. Even when accounting for this heterogeneity, extent of resection remains the strongest predictor of poor outcome. However, this biological heterogeneity must be accounted for in future preclinical modeling and personalized therapies.

Abstract 4347: Medulloblastoma comprises four distinct diseases

Prior attempts to subgroup medulloblastoma (MB) using genomics have identified 4-6 distinct molecular subtypes, including two subgroups driven by activated Wnt and Shh signaling. We performed an integrative analysis on a cohort of 103 primary MBs using a combination of Affymetrix expression and SNP genotyping arrays to determine how many subgroups of the disease exist, how they differ, and the extent of overlap between subgroups. Both unsupervised hierarchical clustering and principal component analysis of expression data reveals very high confidence for the existence of four medulloblastoma subgroups: WNT, SHH, Group C, and Group D. Additional bioinformatic analyses using non-negative matrix factorization (NMF) and subclass mapping (SubMap) further strengthens the support for the four subgroups. These subgroups exhibit distinct demographics: SHH tumors occur in infants and adults, Group C tumors are restricted to children, and WNT and Group D tumors are found across all age groups. While the sex ratio for the entire cohort is ∼1.5:1 (M:F), the sex ratio for WNT group tumors was ∼1:3. We identified a number of previously uncharacterized, subgroup-specific regions of chromosomal abnormality including 9p, 3q, 20q, and 21q gain in SHH tumors, 1q gain and 5q, 16q, and 10q loss in Group C tumors, and near universal isochromosome 17q and frequent loss of the X chromosome in Group D tumors. These regions likely harbor subgroup-specific oncogenes and tumor suppressor genes that could be targets for rationale therapy. Our demographic, transcriptional, and genetic data support the non-overlapping character of these four subgroups of MB. We identified ‘signature’ genes over-expressed in each subgroup for which there are high quality commercial antibodies available. Staining two separate MB tissue microarrays containing 294 non-overlapping tumors for DKK1 (WNT), SFRP1 (SHH), NPR3 (Group C), and KCNA1 (Group D) demonstrated that 288/294 (98%) tumors stained positive for only a single marker. Analysis of the demographics in these patients validated the results observed in our discovery series studied at the RNA level. Leptomeningeal dissemination was highly over-represented in Group C (47%) followed by Group D (30%) patients. A multivariate analysis that included age, extent of resection, histology, M stage, and subgroup revealed that only LCA histology and Group C affiliation were prognostic. As M0 Group C tumors have a very poor prognosis, we suggest that Group C patients include many of the children with ‘average-risk’ MB who relapse after current therapies. Our data highly support the existence of four independent subtypes of MB that differ in their demographics, transcription, genetic events, rate of metastases, and clinical outcome. Our novel ‘4 antibody’ technique is capable of determining MB subgroup through immunohistochemistry on formalin-fixed, paraffin-embedded material suggesting that it will be broadly generalizable across the globe. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4347.