Anja zur Mühlen

Nuclear Exclusion of TET1 Is Associated with Loss of 5-Hydroxymethylcytosine in IDH1 Wild-Type Gliomas

The recent identification of isocitrate dehydrogenase 1 (IDH1) gene mutations in gliomas stimulated various studies to explore the molecular consequences and the clinical implications of such alterations. The Cancer Genome Atlas Research Network showed evidence for a CpG island methylator phenotype in glioblastomas that was associated with IDH1 mutations. These alterations were associated with the production of the oncometabolite, 2-hydroxyglutarate, that inhibits oxygenases [ie, ten-eleven translocation (TET) enzymes involved in the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC)]. We investigated 60 gliomas for 5hmC presence, 5-methylcytosine content, TET1 expression, and IDH1 mutation to gain insight into their relationships on a histological level. Of gliomas, 61% revealed no immunoreactivity for 5hmC, and no correlation was observed between IDH1 mutations and loss of 5hmC. Interestingly, expression of TET1 showed remarkable differences regarding overall protein levels and subcellular localization. We found a highly significant (P = 0.0007) correlation between IDH1 mutations and nuclear accumulation of TET1, but not with loss of 5hmC. Of 5hmC-negative gliomas, 70% showed either exclusive or dominant cytoplasmic expression, or no detectable TET1 protein (P = 0.0122). Our data suggest that the loss of 5hmC is a frequent event in gliomas, independent of IDH1 mutation, and may be influenced by the nuclear exclusion of TET1 from the nuclei of glioma cells.

Molecular stratification of medulloblastoma: comparison of histological and genetic methods to detect Wnt activated tumours

Wnt activation in medulloblastomas is associated with good outcome. Upfront testing and risk‐adapted stratification of patients will be done in future clinical studies. In a cohort of 186 paediatric medulloblastomas our aim was to identify the optimal methods in standard clinical practice to detect this subgroup.

Biomarker-driven stratification of disease-risk in non-metastatic medulloblastoma: Results from the multi-center HIT-SIOP-PNET4 clinical trial

Purpose To improve stratification of risk-adapted treatment for non-metastatic (M0), standard-risk medulloblastoma patients by prospective evaluation of biomarkers of reported biological or prognostic significance, alongside clinico-pathological variables, within the multi-center HIT-SIOP-PNET4 trial. Methods Formalin-fixed paraffin-embedded tumor tissues were collected from 338 M0 patients (>4.0 years at diagnosis) for pathology review and assessment of the WNT subgroup (MBWNT) and genomic copy-number defects (chromosome 17, MYC/MYCN, 9q22 (PTCH1) and DNA ploidy). Clinical characteristics were reviewed centrally. Results The favorable prognosis of MBWNT was confirmed, however better outcomes were observed for non-MBWNT tumors in this clinical risk-defined cohort compared to previous disease-wide clinical trials. Chromosome 17p/q defects were heterogeneous when assessed at the cellular copy-number level, and predicted poor prognosis when they occurred against a diploid (ch17(im)/diploid(cen)), but not polyploid, genetic background. These factors, together with post-surgical tumor residuum (R+) and radiotherapy delay, were supported as independent prognostic markers in multivariate testing. Notably, MYC and MYCN amplification were not associated with adverse outcome. In cross-validated survival models derived for the clinical standard-risk (M0/R0) disease group, (ch17(im)/diploid(cen); 14% of patients) predicted high disease-risk, while the outcomes of patients without (ch17(im)/diploid(cen)) did not differ significantly from MBWNT, allowing re-classification of 86% as favorable-risk. Conclusion Biomarkers, established previously in disease-wide studies, behave differently in clinically-defined standard-risk disease. Distinct biomarkers are required to assess disease-risk in this group, and define improved risk-stratification models. Routine testing for specific patterns of chromosome 17 imbalance at the cellular level, and MBWNT, provides a strong basis for incorporation into future trials.

High-resolution genomic analysis does not qualify atypical plexus papilloma as a separate entity among choroid plexus tumors

Abstract Choroid plexus tumors are rare neoplasms that mainly affect children. They include papillomas, atypical papillomas, and carcinomas. Detailed genetic studies are rare, and information about their molecular pathogenesis is limited. Molecular inversion probe analysis is a hybridization-based method that represents a reliable tool for the analysis of highly fragmented formalin-fixed paraffin-embedded tissue–derived DNA. Here, analysis of 62 cases showed frequent hyperdiploidy in papillomas and atypical papillomas that appeared very similar in their cytogenetic profiles. In contrast, carcinomas showed mainly losses of chromosomes. Besides recurrent focal chromosomal gains common to all choroid plexus tumors, including chromosome 14q21–q22 (harboring OTX2), chromosome 7q22 (LAMB1), and chromosome 9q21.12 (TRPM3), Genomic Identification of Significant Targets in Cancer analysis uncovered focal alterations specific for papillomas and atypical papillomas (e.g. 7p21.3 [ARL4A]) and for carcinomas (16p13.3 [RBFOX1] and 6p21 [POLH, GTPBP2, RSPH9, and VEGFA]). Additional RNA expression profiling and gene set enrichment analysis revealed greater expression of cell cycle–related genes in atypical papillomas in comparison with that in papillomas. These findings suggest that atypical papillomas represent an immature variant of papillomas characterized by increased proliferative activity, whereas carcinomas seem to represent a genetically distinct tumor group.

Genome-wide DNA copy number analysis of desmoplastic infantile astrocytomas and desmoplastic infantile gangliogliomas.

Little is known about the molecular features of desmoplastic infantile ganglioglioma (DIG) and desmoplastic infantile astrocytoma (DIA). We performed a genome-wide DNA copy number analysis in combination with a multiplex ligation-dependent probe amplification-based analysis of copy number changes of candidate genes in 4 DIAs and 10 DIGs. Molecular inversion probe (MIP) assay showed that large chromosomal alterations were rare among DIG and DIA. Focal recurrent genomic losses were observed in chromosome regions such as 5q13.3, 21q22.11, and 10q21.3 in both DIA and DIG. Principal component analysis did not show any significant differences between the molecular profiles of DIG and DIA, and a hierarchical cluster analysis did not clearly separate the 2 tumor groups according to their molecular profiles. In 6 cases, gain of genomic material at 7q31 (corresponding to MET gene) was found in multiplex ligation-dependent probe amplification (MLPA) analysis. Furthermore, two cases showed gain at 4q12, and a single case showed BRAF mutation. In agreement with previous analyses, this study demonstrates the absence of consistent recurrent chromosomal alterations in DIA and DIG and overall rarity of the BRAF mutation in these tumors. Notably, these results suggest that DIA and DIG represent a histologic spectrum of the same tumor rather than 2 separate entities.

High-Resolution Genomic Analysis of Cribriform Neuroepithelial Tumors of the Central Nervous System

Abstract Cribriform neuroepithelial tumors (CRINET) are one of several recently characterized entities in the broad spectrum of solid tumors with SMARCB1-INI1 loss. This neoplasm seems to be exceedingly rare and displays unique neuropathologic and clinical features. To date, only a few cases of CRINET have been characterized from a molecular point of view. In this study, we investigated the molecular features of 3 cases of CRINET using multiplex ligation-dependent probe amplification and molecular inversion profiling approaches. Along with mutations and deletions of SMARCB1-INI1, molecular inversion profiling analysis revealed a stable genomic profile without significant large chromosomal changes. Focal alterations (gains) were observed in individual cases at chromosomes 4q12 (PDGFRA), 12q15 (MDM2), 7p15.1 (NPY), and 18q11.2 (CDH2). Genomic Identification of Significant Targets in Cancer analysis highlighted focal alterations, including gains at chromosomes 16q23.2 (MAF), 17q23 (AXIN2), and 8p12 (ADAM3A). No cases showed BRAFV600E or CTNNB1 mutations. These data indicate that CRINET present stable genetic features and lack alterations commonly identified in other pediatric brain tumors. Further studies are required to determine whether specific alterations and specific signaling pathways, in addition to SMARCB1-INI1, may be implicated in the biology of this rare tumor and whether there are additional molecular similarities between CRINET and atypical teratoid/rhabdoid tumors.

Molecular heterogeneity characterizes glioblastoma with lipoblast/adipocyte-like cytology

Glioblastoma, the most common primary brain tumor in adults, may rarely show among unusual histological patterns lipoblast/adipocyte-like features. The genetic features of such cases are not yet well characterized, and molecular data are available for only few cases. In order to further expand our knowledge about their molecular profile, we analyzed four cases of glioblastoma with adipocyte-like features. Multiplex ligation-dependent probe amplification (MLPA) revealed loss of PTEN and MDM2 amplification in two cases while another case was characterized by CDKN2A deletion. Conversely, we did not find any evidence of EGFR amplification, BRAFV600E or IDH1/2 mutations. Our results, along with data published in previous studies, showed that glioblastoma with lipoblast/adipocyte-like cytology present a heterogeneous genetic background and therefore seem to represent more a rare phenotypic variant than a specific tumor subtype.

Abstract LB-83: Molecular stratification of medulloblastoma: comparison of histological and genetical methods to detect Wnt activated tumors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Wnt activation in medulloblastomas is associated with good outcome. Therefore, upfront testing and risk-adapted stratification of the patients will be done in future clinical studies. In a cohort of 188 pediatric medulloblastomas our aim was to identify the optimal methods in standard clinical practice to detect this subgroup. Experimental procedures: Nuclear accumulation of s-Catenin in tumor cells was analyzed by immunohistochemistry (IHC) of sections from formalin-fixed paraffin-embedded (FFPE) tumor samples with 3 different automated immunostaining systems and subsequent neuropathological evaluation. Genomic DNA of FFPE tumor tissue from each sample was amplified by PCR for SSCP (single-strand conformation polymorphism) analysis and direct Sanger sequencing of CTNNB1 exon 3. Copy number aberrations of chromosome 6 were analyzed by MLPA (multiplex ligation-dependent probe amplification) using the same genomic DNA and were confirmed in a subset of cases by molecular invasion profiling. Results: The different automated immunostaining systems showed similar results even though s-Catenin staining intensity varied. 22 of 188 samples showed nuclear accumulation in >5% of tumor cells and further 21 samples showed ≤5% s-Catenin positive nuclei. None of the latter 21 cases had mutations in exon 3 of the CTNNB1 gene, but 18 of the 22 cases with >5% nuclear accumulation did, clearly identifying these 18 cases as Wnt-subgroup medulloblastomas. SSCP analysis only showed DNA bands with aberrant migration in 3 of the 18 mutated samples representing 2 different mutations. With MLPA and molecular invasion profiling the 4 cases with >5% nuclear accumulation but without CTNNB1 mutation also lacked monosomy 6, which is another known marker for Wnt subgroup medulloblastomas. 15 of the 18 mutated cases showed monosomy 6, but 3 had a balanced chromosome 6, indicating that monosomy 6 is present in most but not all Wnt cases. On the other hand, none of the CTNNB1 wildtype tumors showed monosomy 6. Conclusion: Standard neuropathological evaluation of medulloblastoma samples should include IHC of s-Catenin because tumors with high nuclear accumulation of s-Catenin most probably belong to the Wnt subgroup of medulloblastomas associated with a good outcome. Still, IHC alone may be insufficient because of low quality of paraffin tissue, technical problems or differences in scoring s-Catenin positive nuclei. Similar, chromosome 6 aberrations in MLPA/molecular invasion profiling were shown not to be present in all CTNNB1 -mutated cases. Therefore, we conclude that sequencing analysis of CTNNB1 exon 3 in combination with s-Catenin IHC (possibly as pre-screening method) is a feasible, fast and cost-efficient way for the determination of Wnt subgroup medulloblastomas. Citation Format: Tobias Goschzik, Anja zur Muhlen, Glen Kristiansen, Torsten Pietsch. Molecular stratification of medulloblastoma: comparison of histological and genetical methods to detect Wnt activated tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-83. doi:10.1158/1538-7445.AM2013-LB-83