Archived serum can now be used in epigenetic studies
- The Janus samples are truly a goldmine, and it turns out that it is possible to extract more knowledge from them than anyone could have imagined when this biobank was established. This is a very exciting opportunity to find new biomarkers for cancer and to study environmental epigenetics, says Marcin Wojewodzic, an expert in systems biology at the Cancer Registry of Norway.
In a collaborative national and international effort, scientists conducted a study to test the feasibility of extracting even more information from DNA from the Janus samples than was previously thought possible
Innovative use of 50 year old biobank
The Janus Bank is a population-based collection of serum from ~300,000 Norwegians collected between 1972 and 2004. Since collection, ~100,000 of the participants have been diagnosed with cancer. When the biobank was established, the idea was to use serum to research how cancer develops.
Nearly 50 years ago, when the collection started, there was limited knowledge of epigenetics and no technology to uncover its behaviour. Epigenetics is the study of how the behaviour of genes can be regulated (switched on or off) without altering the DNA sequence itself. This regulation results in different cell types, but is also altered by age, external factors (such as environmental impact, diet, and chemical exposures) or cancer.
In theory, serum contains no cells at all, but trace amounts of DNA can still be found as leftovers from white blood cells and as circulating DNA from other cell types.
- When isolating serum from blood, you remove almost all of the cells, but this process is not perfect. Therefore, serum contains residues of DNA, and the Janus samples are thus useful in more contexts than previously thought, says Trine B. Rounge, head of the Section for Integrated Genomics at the Cancer Registry.
Advanced technology gave good results despite very scarce DNA material
Cytosine, one of the four bases found in DNA, can have a chemical called a methyl group attached to it that affects the readability of the nearby genes. This is called DNA methylation and it is one of the major forms of epigenetics.
To identify DNA methylation patterns, i.e. changes that affect how genes are expressed, the researchers have used DNA isolated from archived serum samples.
The average DNA yield from the samples was very low, corresponding to about 10 percent of the recommended amount for a commonly used technology (Infinium MethylationEPIC). Using more modern technology (Whole Genome Bisulfite Sequencing using the NovaSeq 6000) however, which requires far smaller amounts of DNA, the researchers conducted a pilot study of 96 samples.
An optimized protocol, accurate laboratory work, and robust quality controls meant that, despite the very low amounts of DNA, 94 out of 96 of the serum samples obtained results that were satisfactory for Epigenome-Wide Association Studies (EWAS).
The resulting DNA methylation profiles could then be combined with other data to look for signs that may indicate cancer or cancer susceptibility.
- Our study shows that whole genome bisulfite sequencing of very low amounts of DNA from serum samples, stored for up to 45 years, is possible. These results show the potential for using large biobank cohorts of serum in future epigenetic studies, says Tom Grotmol, an expert in cancer research at the Cancer Registry of Norway.
- We optimized the procedure, and our first analyses, where we used samples from testicular cancer patients, show that we can measure the methylation level of DNA from most of the 28 million methylation sites found in the human genome. These sites represent an important gene regulation mechanism, but we also know that the pattern changes with cancer, age or environmental exposure, says Wojewodzic.
Great opportunities in cancer research
- When we started the project, we were unsure whether we would succeed in producing methylation profiles from the old Janus samples, but our modified protocol turned out to work. The results open up new avenues for what serum biobanks can generally be used for in research on cancer biomarkers, Rounge points out.
The scientists now hope that more information about DNA and methylation will be available to cancer researchers, due to the method they have developed and refined.
- We hope that, in the future, we will be able to digitalize the epigenetic information contained in the Janus biobank and make it available to other researchers to facilitate the building of AI models, find new biomarkers, and to help save lives, Wojewodzic concludes.
The study is funded by the Norwegian Cancer Society.