Every year in the EU, 45,300 women are diagnosed with ovarian cancer and 330,000 with breast cancer. Ovarian cancer has a mortality rate of around 64% and breast cancer around 27%*. The EU-funded research project EpiFemCare aims at improving cancer detection and subsequent patient care. The research project aims to develop blood tests based on epigenetic DNA modifications, i.e. the methylation of the base cytosine, for the identification of tumours. Konstanz-based GATC Biotech AG is developing a sequencing technology to analyse DNA methylation.
Breast cancer is the most common cancer in women in Europe and the number one cause of cancer-related deaths in women. Ovarian cancer is far less common than breast cancer, however a significantly larger proportion of patients die from this cancer. Ovarian cancer is usually diagnosed at a very advanced stage when the cancer has already spread to other areas of the body. “This results in poor prognosis, and more than 60% of ovarian cancer patients die within the first five years after diagnosis,” said Dr. Benjamin Wahl, a scientist in GATC Biotech AG’s development team.
In addition to GATC Biotech AG, the European EpiFemCare (Epigenetics for female personalised cancer care) consortium also involves the University College London (UK), the Ludwig Maximilian University in Munich (Germany), Charles University in Prague (Czech Republic), and the companies Genedata AG (Basel, Switzerland) and Euram Limited (Nottingham, UK). The project is led by Professor Dr. Martin Widschwendter from the Department of Women’s Cancer at the University College London. The EpiFemCare project aims to develop a blood test for the early detection of breast and ovarian cancer and improve subsequent patient treatment. The project will run for a period of 3.5 years and consists of three phases: first, the collaborators will identify cancer-specific markers using patient DNA samples; second, a serum-based test will be developed that will then be validated in the final phase using thousands of patient samples.
Epigenetic DNA modifications form the basis for the new test. “Such modifications belong to the natural spectrum of healthy cells and play a pivotal role in embryonic development and differentiation, to name but two examples,” Wahl says. In recent years, it has become increasingly clear that epigenetic modifications also play a key role in diseases such as cancer. Epigenetic modification is the covalent attachment of a methyl group to cytosine, which is one of the four main bases found in DNA.
The EpiFemCare project therefore seeks to identify characteristic methylation patterns of tumour DNA and use these as a marker for the diagnosis of breast and ovarian cancer. “Tumour tissue is an extremely modified tissue which is characterised by processes such as continuous growth and cell death resulting from apoptosis and necrosis,” Wahl explains. During these processes, the DNA of dying tumour cells enters the bloodstream. Tumour DNA is different from non-tumour DNA; it contains mutations that alter the DNA sequence as well as modifications such as the aforementioned epigenetic modifications (i.e. attachment of methyl tags). These epigenetic modifications differ between different types of cells, so that cancer cells have a different pattern from healthy cells.
GATC Biotech will manage sample processing and sequencing. Using several thousand patient serum samples provided by the clinical collaborators, GATC Biotech starts by isolating cell-free DNA. A particular difficulty is the fact that blood serum contains far less DNA than tissue biopsies. Only a small quantity of tumour DNA enters the bloodstream and is then present as cell-free DNA in blood serum. “While normal enrichment starts with DNA quantities in the microgramme range, our approach starts with only a few nanogrammes,” said Wahl highlighting the dimensions the team is working with. The protocol used to prepare and sequence DNA is rather complex and time-consuming, but simpler methods or commercial kits cannot be applied to such tiny quantities of DNA.
A technique known as reduced representation bisulphite sequencing (RRBS) is used to identify methylated bases in serum-derived DNA. In eukaryotic cells, DNA methylation is restricted to cytosine-phosphatidyl-guanine dinucleotide (CpGs) sequences which occur in so-called CpG islands, i.e. CpG-rich regions of the DNA. RRBS, which couples bisulphite conversion and next-generation sequencing, cleaves the DNA in the CpG islands in order to specifically enrich genomic regions with a high density of methylation sites. “After enrichment of the modified regions, the remaining 10 percent of the genome are sequenced using next-generation sequencing technologies with single-nucleotide resolution,” Wahl says. Bisulphite treatment preserves the existing methyl tags for sequencing. These tags would be lost during PCR (polymerase chain reaction) amplification, which is required for high-throughput sequencing.
The sequencing data are analysed by the Basel-based company Genedata, which is developing bioinformatic methods that enable tumour DNA to be differentiated from non-tumour DNA. “The problem is not finding the abnormalities or modifications, but identifying from the many modifications those that occur specifically in cancer patients,” Wahl says, referring to the observation that even cancer-specific modifications can vary considerably as breast cancer is not a homogeneous category of identical tumours.
The use of the blood test in regular preventive medical check-ups has the potential to reduce the number of deaths due to late diagnosis. The high resolution of the sequencing of the epigenetic modifications augments the high specificity and sensitivity of the test. “This would also considerably reduce the problem of false-positive diagnoses and subsequent unnecessary surgery, which can result from mammography, currently the main tool for identifying breast cancer,” said Wahl with a degree of confidence.
In addition to the early detection of breast and ovarian cancer, the new method also has the potential to be used for assessing therapeutic outcome, for example for finding out whether all tumour DNA, and hence the entire tumour, is completely gone after surgery or chemotherapy. The success of the EpiFemCare project would open up promising prospects for the diagnosis and therapy of cancer in general. “The test method is theoretically applicable to many types of cancer; the only thing required is the identification of markers that are specific to certain cancers,” said Wahl.
EpiFemCare 2012 - 2016
The European EpiFemCare (Epigenetics for female personalised cancer care) project is aimed at developing new methods for the screening, diagnosis and personalised therapy of breast and ovarian cancer patients. The consortium involves three clinical partners that provide the team with serum samples and, if necessary, tumour biopsies, for the identification of cancer markers: the University College London with Prof. Martin Widschwendter as project leader, the Ludwig Maximilian University in Munich and the Charles University in Prague. GATC Biotech AG is in charge of sample processing and sequencing and Basel-based Genedata is specifically focused on the development and application of bioinformatic techniques for the analysis of the data. Euram Limited is in charge of the administrative management of all non-scientific/non-technical aspects of the project.
The research that has led to the aforementioned results was funded under the European Seventh Framework Programme (RP7/2007-2013) according to grant agreement no. 305428.
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