Targeted Deep DNA Methylation Analysis of Circulating Cell-free DNA in Plasma Using Massively Parallel Semiconductor Sequencing

Abstract

Aim: To set up a targeted methylation analysis using semiconductor sequencing and evaluate the potential for studying methylation in circulating cell-free DNA (cfDNA). Materials & Methods: Methylation of VIM, FBLN1, LTBP2, HINT2, h19 and IGF2 was analyzed in plasma cfDNA and white blood cell DNA obtained from eight hepatocellular carcinoma patients and eight controls using Ion Torrent™ PGM sequencer. Results:h19 and IGF2 showed consistent methylation levels and methylation was detected for VIM and FBLN1, whereas LTBP2 and HINT2 did not show methylation for target regions. VIM gene promoter methylation was higher in HCC cfDNA than in cfDNA of controls or white blood cell DNA. Conclusion: Semiconductor sequencing is a suitable method for analyzing methylation profiles in cfDNA. Furthermore, differences in cfDNA methylation can be detected between controls and hepatocellular carcinoma cases, even though due to the small sample set these results need further validation.

Keywords::

• cfDNA
• early cancer detection
• hepatocellular carcinoma
• methylation
• next-generation sequencing

Supplementary data

Acknowledgments

We thank R Sriraksa for providing the artificially methylated DNA and HS Lee for providing the primers for IGF2 and h19. Also D de Santos-Silva and P Guilloreau are thanked for their assistance in the sample collection.

Financial & competing interests disclosure

This work in the IARC Epigenetics Group is supported in part by grants from the l’Association pour la Recherche sur le Cancer (ARC), France and la Ligue Nationale Contre le Cancer, France (to Z Herceg). F Vaca-Paniagua and A Nogueira da Costa are recipients of a Postdoctoral Fellowship from the IARC, partially supported by the EC FP7 Marie Curie Actions – People – Co-funding of regional, national and international programs (COFUND). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Additional information

Funding

This work in the IARC Epigenetics Group is supported in part by grants from the l'Association pour la Recherche sur le Cancer (ARC), France and la Ligue Nationale Contre le Cancer, France (to Z Herceg). F Vaca-Paniagua and A Nogueira da Costa are recipients of a Postdoctoral Fellowship from the IARC, partially supported by the EC FP7 Marie Curie Actions – People – Co-funding of regional, national and international programs (COFUND). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.