Selected publications


LIQUORICE: Detection of epigenetic signatures in liquid biopsies based on whole-genome sequencing data

Fragmentation patterns of cell-free DNA reflect the chromatin structure of the cells from which these fragments are derived. Nucleosomes protect the DNA from fragmentation, resulting in decreased sequencing coverage in regions of open chromatin. LIQUORICE is a user-friendly software tool that takes aligned whole-genome sequencing data as input and calculates bias-corrected coverage signatures for predefined, application-specific sets of genomic regions. The tool thereby enables a blood-based analysis of cell death in the body, and it provides a minimally invasive assessment of tumor chromatin states and cell-of-origin. With user-defined sets of regions that exhibit tissue-specific or disease-specific open chromatin, LIQUORICE can be applied to a wide range of detection, classification and quantification tasks in the analysis of liquid biopsies.

LIQUORICE is freely and openly available as a Python package and command-line tool for UNIX-based systems from bioconda.

LIQUORICE: Detection of epigenetic signatures in liquid biopsies based on whole-genome sequencing data

Bioinformatics Advances, March 2022

by Peter Peneder, Christoph Bock, Eleni M Tomazou


Multimodal analysis of cell-free DNA whole
genome sequencing for pediatric cancers
with low mutational burden

Nature Communications, May 2021

Sequencing of cell-free DNA in the blood of cancer patients (liquid biopsy) provides attractive opportunities for early diagnosis, assessment of treatment response, and minimally invasive disease monitoring.

To unlock liquid biopsy analysis for pediatric tumors with few genetic aberrations, we introduce an integrated genetic/epigenetic analysis method and demonstrate its utility on 241 deep whole genome sequencing profiles of 95 patients with Ewing sarcoma and 31 patients with other pediatric sarcomas. Our method achieves sensitive detection and classification of circulating tumor DNA in peripheral blood independent of any genetic alterations. Moreover, we benchmark different metrics for cell-free DNA fragmentation analysis, and we introduce the LIQUORICE algorithm for detecting circulating tumor DNA based on cancer-specific chromatin signatures. Finally, we combine several fragmentation-based metrics into an integrated machine learning classifier for liquid biopsy analysis that is tailored to cancers with low mutation rates while exploiting widespread epigenetic deregulation. Clinical associations highlight the potential value of cfDNA fragmentation patterns as prognostic biomarkers in Ewing sarcoma.

In summary, our study provides a comprehensive analysis of circulating tumor DNA beyond recurrent somatic mutations, and it renders the
benefits of liquid biopsy more readily accessible for childhood cancers.

by Peter Peneder *, Adrian M Stütz *, [..], Marcus Tötzl, [..], Christoph Bock §, Eleni M Tomazou §

*: equal contribution

§: joint supervision

Ewing sarcoma

Ewing sarcoma is the second most frequent bone tumour of childhood and adolescence that can also arise in soft tissue. Ewing sarcoma is a highly aggressive cancer, with a survival of 70–80% for patients with standard-risk and localized disease and ~30% for those with metastatic disease. Treatment comprises local surgery , radiotherapy and polychemotherapy , which are associated with acute and chronic adverse effects that may compromise quality of life in survivors. Histologically , Ewing sarcomas are composed of small round cells expressing high levels of CD99. Genetically , they are characterized by balanced chromosomal translocations in which a member of the FET gene family is fused with an ETS transcription factor, with the most common fusion being EWSR1–FLI1 (85% of cases). Ewing sarcoma breakpoint region 1 protein (EWSR1)–Friend leukaemia integration 1 transcription factor (FLI1) is a tumour-specific chimeric transcription factor (EWSR1–FLI1) with neomorphic effects that massively rewires the transcriptome. Additionally , EWSR1–FLI1 reprogrammes the epigenome by inducing de novo enhancers at GGAA microsatellites and by altering the state of gene regulatory elements, creating a unique epigenetic signature. Additional mutations at diagnosis are rare and mainly involve STAG2, TP53 and CDKN2A deletions. Emerging studies on the molecular mechanisms of Ewing sarcoma hold promise for improvements in early detection, disease monitoring, lower treatment-related toxicity , overall survival and quality of life.

Nature Reviews Disease Primers,  July 2018

by Thomas Grünewald, Florencia Cidre-Aranaz, Didier Surdez, Eleni M Tomazou, [...]


MIRA: an R package for DNA methylation-based inference of regulatory activity.

Bioinformatics, August 2018

by John T Lawson, Eleni M Tomazou, Christoph Bock and Nathan C Sheffield

DNA methylation contains information about the regulatory state of the cell. MIRA aggregates genome-scale DNA methylation data into a DNA methylation profile for a given region set with shared biological annotation. Using this profile, MIRA infers and scores the collective regulatory activity for the region set. MIRA facilitates regulatory analysis in situations where classical regulatory assays would be difficult and allows public sources of region sets to be leveraged for novel insight into the regulatory state of DNA methylation datasets.


DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma

Developmental tumors in children and young adults carry few genetic alterations, yet they have diverse clinical presentation. Focusing on Ewing sarcoma, we sought to establish the prevalence and characteristics of epigenetic heterogeneity in genetically homogeneous cancers. We performed genome-scale DNA methylation sequencing for a large cohort of Ewing sarcoma tumors and analyzed epigenetic heterogeneity on three levels: between cancers, between tumors, and within tumors. We observed consistent DNA hypomethylation at enhancers regulated by the disease-defining EWS-FLI1 fusion protein, thus establishing epigenomic enhancer reprogramming as a ubiquitous and characteristic feature of Ewing sarcoma. DNA methylation differences between tumors identified a continuous disease spectrum underlying Ewing sarcoma, which reflected the strength of an EWS-FLI1 regulatory signature and a continuum between mesenchymal and stem cell signatures. There was substantial epigenetic heterogeneity within tumors, particularly in patients with metastatic disease. In summary, our study provides a comprehensive assessment of epigenetic heterogeneity in Ewing sarcoma and thereby highlights the importance of considering nongenetic aspects of tumor heterogeneity in the context of cancer biology and personalized medicine.

Nature Medicine, January 2017

by Nathan C Sheffield, [...] ,

Christoph Bock § and Eleni M Tomazou §

§: joint supervision


Epigenome Mapping Reveals Distinct Modes of Gene Regulation and Widespread Enhancer Reprogramming by the Oncogenic Fusion Protein EWS-FLI1

Transcription factor fusion proteins can transform cells by inducing global changes of the transcriptome, often creating a state of oncogene addiction. Here, we investigate the role of epigenetic mechanisms in this process, focusing on Ewing sarcoma cells that are dependent on theEWS-FLI1 fusion protein. We established reference epigenome maps comprising DNA methylation, seven histone marks, open chromatin states, and RNA levels, and we analyzed the epigenome dynamics upon downregulation of the driving oncogene. Reduced EWS-FLI1 expression led to widespread epigenetic changes in promoters, enhancers, and super-enhancers, and we identified histone H3K27 acetylation as the most strongly affected mark. Clustering of epigenetic promoter signatures defined classes of EWS-FLI1-regulated genes that responded differently to low-dose treatment with histone deacetylase inhibitors. Furthermore, we observed strong and opposing enrichment patterns for E2F and AP-1 among EWS-FLI1-correlated and anticorrelated genes. Our data describe extensive genome-wide rewiring of epigenetic cell states driven by an oncogenic fusion protein.

Cell Reports, February 2015

by Eleni M Tomazou, Nathan C Sheffield, [...] , Christoph Bock and Heinrich Kovar