Olga Bogatyrova

HPV-related methylation signature predicts survival in oropharyngeal squamous cell carcinomas

High-risk types of human papilloma virus (HPV) are increasingly associated with oropharyngeal squamous cell carcinoma (OPSCC). Strikingly, patients with HPV-positive OPSCC are highly curable with ionizing radiation and have better survival compared with HPV-negative patients, but the underlying molecular mechanisms remain poorly understood. We applied an array-based approach to monitor global changes in CpG island hypermethylation between HPV-negative and HPV-positive OPSCCs and identified a specific pattern of differentially methylated regions that critically depends on the presence of viral transcripts. HPV-related alterations were confirmed for the majority of candidate gene promoters by mass spectrometric, quantitative methylation analysis. There was a significant inverse correlation between promoter hypermethylation of ALDH1A2, OSR2, GATA4, GRIA4, and IRX4 and transcript levels. Interestingly, Kaplan-Meier analysis revealed that a combined promoter methylation pattern of low methylation levels in ALDH1A2 and OSR2 promoters and high methylation levels in GATA4, GRIA4, and IRX4 promoters was significantly correlated with improved survival in 3 independent patient cohorts. ALDH1A2 protein levels, determined by immunohistochemistry on tissue microarrays, confirmed the association with clinical outcome. In summary, our study highlights specific alterations in global gene promoter methylation in HPV-driven OPSCCs and identifies a signature that predicts the clinical outcome in OPSCCs.

Evolution of DNA methylation is linked to genetic aberrations in chronic lymphocytic leukemia

Although clonal selection by genetic driver aberrations in cancer is well documented, the ability of epigenetic alterations to promote tumor evolution is undefined. We used 450k arrays and next-generation sequencing to evaluate intratumor heterogeneity and evolution of DNA methylation and genetic aberrations in chronic lymphocytic leukemia (CLL). CLL cases exhibit vast interpatient differences in intratumor methylation heterogeneity, with genetically clonal cases maintaining low methylation heterogeneity and up to 10% of total CpGs in a monoallelically methylated state. Increasing methylation heterogeneity correlates with advanced genetic subclonal complexity. Selection of novel DNA methylation patterns is observed only in cases that undergo genetic evolution, and independent genetic evolution is uncommon and is restricted to low-risk alterations. These results reveal that although evolution of DNA methylation occurs in high-risk, clinically progressive cases, positive selection of novel methylation patterns entails coevolution of genetic alteration(s) in CLL.

DNA methylation dynamics during B cell maturation underlie a continuum of disease phenotypes in chronic lymphocytic leukemia

Charting differences between tumors and normal tissue is a mainstay of cancer research. However, clonal tumor expansion from complex normal tissue architectures potentially obscures cancer-specific events, including divergent epigenetic patterns. Using whole-genome bisulfite sequencing of normal B cell subsets, we observed broad epigenetic programming of selective transcription factor binding sites coincident with the degree of B cell maturation. By comparing normal B cells to malignant B cells from 268 patients with chronic lymphocytic leukemia (CLL), we showed that tumors derive largely from a continuum of maturation states reflected in normal developmental stages. Epigenetic maturation in CLL was associated with an indolent gene expression pattern and increasingly favorable clinical outcomes. We further uncovered that most previously reported tumor-specific methylation events are normally present in non-malignant B cells. Instead, we identified a potential pathogenic role for transcription factor dysregulation in CLL, where excess programming by EGR and NFAT with reduced EBF and AP-1 programming imbalances the normal B cell epigenetic program.

Global alterations of DNA methylation in cholangiocarcinoma target the Wnt signaling pathway

The molecular mechanisms underlying the genesis of cholangiocarcinomas (CCs) are poorly understood. Epigenetic changes such as aberrant hypermethylation and subsequent atypical gene expression are characteristic features of most human cancers. In CC, data regarding global methylation changes are lacking so far. We performed a genome‐wide analysis for aberrant promoter methylation in human CCs. We profiled 10 intrahepatic and 8 extrahepatic CCs in comparison to non‐neoplastic biliary tissue specimens, using methyl‐CpG immunoprecipitation (MCIp) combined with whole‐genome CpG island arrays. DNA methylation was confirmed by quantitative mass spectrometric analysis and functional relevance of promoter hypermethylation was shown in demethylation experiments of two CC cell lines using 5‐aza‐2′deoxycytidine (DAC) treatment. Immunohistochemical staining of tissue microarrays (TMAs) from 223 biliary tract cancers (BTCs) was used to analyze candidate gene expression at the protein level. Differentially methylated, promoter‐associated regions were nonrandomly distributed and enriched for genes involved in cancer‐related pathways including Wnt, transforming growth factor beta (TGF‐β), and PI3K signaling pathways. In CC cell lines, silencing of genes involved in Wnt signaling, such as SOX17, WNT3A, DKK2, SFRP1, SFRP2, and SFRP4 was reversed after DAC administration. Candidate protein SFRP2 was substantially down‐regulated in neoplastic tissues of all BTC subtypes as compared to normal tissues. A significant inverse correlation of SFRP2 protein expression and pT status was found in BTC patients. Conclusion: We provide a comprehensive analysis to define the genome‐wide methylation landscape of human CC. Several candidate genes of cancer‐relevant signaling pathways were identified, and closer analysis of selected Wnt pathway genes confirmed the relevance of this pathway in CC. The presented global methylation data are the basis for future studies on epigenetic changes in cholangiocarcinogenesis. (Hepatology 2014;59:544–554)

Abstract 3364: DNA methylation loss at an enhancer site of the DNA repair geneTREX2is an epigenetic feature in multiple cancers

The onset of numerous cancers is strongly associated with exposure to genotoxic agents and is counteracted by cellular DNA repair mechanisms. However, the tumorigenic potential of genotoxic carcinogens varies widely among individuals. It is still uncertain which genetic and epigenetic traits shape cancer onset and progression in the general population. While genetic aberrations in DNA repair genes have been linked to cancer risk, less is known about the importance of epigenetics for the regulation of these genes. In order to identify DNA methylation alterations in laryngeal cancer we carried out targeted DNA methylation analysis at single CpG sites via mass spectrometry. We focused our analysis on five DNA repair-associated gene loci previously found to be altered in head and neck squamous cell carcinoma. We report loss of DNA methylation at the three prime repair exonuclease 2 (TREX2) gene locus in laryngeal cancer (n=161) and adjacent normal tissue (n=58) samples of patients from a German population-based case-control study. Following screening of tumor tissues from Chinese colorectal cancer patients as well as previously published data from the Cancer Genome Atlas (TCGA), we identified TREX2 methylation loss as a frequent trait in multiple cancers. We further characterized the regulatory activity of the affected TREX2 site using chromatin immunoprecipitation and luciferase reporter assays in cell models from different tumor types. Differential TREX2 methylation affects a CCAAT/enhancer binding protein alpha (CEBPA) binding site serving as a gene enhancer which drives the expression of TREX2 from a previously uncharacterized gene promoter. We also observed a strong association between TREX2 methylation and TREX2 protein expression determined via immunohistochemistry in laryngeal tumors. Finally, we found a significant association between overall survival and loss of TREX2 methylation in laryngeal cancer, with TREX2 methylation loss being a protective factor. Our findings highlight a profound regulatory role of epigenetic mechanisms for TREX2 in tumors, and underline the usefulness of TREX2 DNA methylation as a biomarker for patient stratification. Citation Format: Christoph Weigel, Jittiporn Chaisaingmongkol, Christine Kuhmann, Irene Santi, Volker Winkler, Olga Bogatyrova, Justo L. Bermejo, Tsun L. Chan, Felix Lasitschka, Manfred H. Bohrer, Alexander Marx, Frank Autschbach, Roland Heyni-von Hausen, Gerhard Dyckhoff, Klaus-Wolfgang Delank, Karl Hoermann, Burkard M. Lippert, Gerald Baier, Andreas Dietz, Christopher C. Oakes, Christoph Plass, Heiko Becher, Peter Schmezer, Heribert Ramroth, Odilia Popanda. DNA methylation loss at an enhancer site of the DNA repair gene TREX2 is an epigenetic feature in multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3364. doi:10.1158/1538-7445.AM2017-3364

Abstract B25: Progressive epigenetic programming during B cell maturation yields a continuum of clonal disease phenotypes with distinct etiologies in chronic lymphocytic leukemia.

Knowledge of the cell-of-origin is essential for the full understanding of the causes of a malignant disease and for the rational design of targeted therapies. The B cell compartment is composed of a highly complex mixture of subtypes, each with distinct phenotypes and roles within the immune system. In chronic lymphocytic leukemia (CLL), heterogeneity in the biology and clinical course of the disease is thought to be linked to divergent cellular origins. We and others have previously shown that the epigenome of CLL, as measured by the global pattering of DNA methylation, is highly clonal and remarkably stable over time and thus represents a powerful approach to trace founder subtype populations. Here we combine epigenomic and transcriptomic analysis using next-generation sequencing approaches to compare CLL cells to highly purified and specific B cell subpopulations at various stages of maturation. We find that B cell maturation involves substantial unidirectional epigenetic programming that occurs as a continuum throughout the transition between naive to fully-mature memory B cell subpopulations. Combining 258 CLL cases using Illumina 450K analysis reveals that all CLLs arise from a discrete window within the spectrum of B cell maturation that is more similar to mature B cells, with the majority of cases clustering at two distinct points correlating with unmutated IGHV versus highly mutated ( Citation Format: Christopher C. Oakes, Marc Seifert, Assenov Yassen, Lei Gu, Martina Przekopowitz, Amy Ruppert, Andrius Serva, Sandra Koser, David Brocks, Daniel Lipka, Olga Bogatyrova, Daniel Mertens, Marc Zapatka, Peter Lichter, Hartmut Doehner, Ralf Kueppers, Thorsten Zenz, Stephan Stilgenbauer, John Byrd, Christoph Plass. Progressive epigenetic programming during B cell maturation yields a continuum of clonal disease phenotypes with distinct etiologies in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B25.

Abstract B42: An integrated view on genetic and epigenetic mechanisms revealed aberrant DNA methylation as an important source for miRNA deregulation in prostate cancer

Prostate cancer (PCa) the most common malignant tumor in males and the third leading cause of cancer-related deaths in Western developed countries. The clinical spectrum of PCa ranges from indolent tumors requiring no therapy to highly aggressive and often metastatic diseases. MicroRNAs (miRNAs) have emerged as important regulators in human tumorigenesis and tumor progression during the last decade. miRNA expression is strongly deregulated in many human cancers including PCa. We investigated the contribution of genetic and epigenetic events to the deregulation of miRNAs in PCa by utilizing the profiling data of 19 Early-Onset Pca (EOPC) from International Cancer Genome Consortium (ICGC) (http://www.icgc.org). For 31% (154/491) of the downregulated miRNAs and for 23% (133/578) of the upregulated miRNAs, DNA hyper- and hypomethylation respectively of a promoter region was observed in at least one of the 13 PCa. Deletions were detected for 26% (126/491) of the downregulated miRNAs in at least one sample. We observed a single translocation event leading to disruption of miR-1244-2 from its putative regulatory region that might contribute to expression decrease. We did not detect any SNV within miRNA precursors. Only one miRNA precursor (miR-4307) was amplified in one patient, but the corresponding mature miRNA was not expressed at all. We demonstrated that, among deletions, methylation of miRNA promoters is one of the major mechanisms leading to miRNA silencing or activation, respectively in PCa. 37 miRNA were downregulated by hypermethylation of regulatory regions and 10 miRNAs were upregulated by hypomethylation in both the ICGC EOPC cohort and the validation dataset on 50 prostate cancer and 48 normal prostate tissues. 82% (9/11) of the miRNAs/miRNA clusters analyzed regulate genes that are involved in PI3K/AKT/PTEN signaling, which represents one of the PCa key pathways being deregulated in more than one third of primary PCa. Note: This abstract was not presented at the conference. Citation Format: Olga Bogatyrova, Daniela Wuttig, Lei Gu, Yassen Assenov, Ruprecht Kuner, Constance Baer, Lars Feuerbach, Clarissa Gerhauser, Dieter Weichenhan, Thorsten Schlomm, Ronald Simon, Guido Sauter, Holger Sultmann. An integrated view on genetic and epigenetic mechanisms revealed aberrant DNA methylation as an important source for miRNA deregulation in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B42.