Minimally invasive classification of paediatric solid tumours using reduced representation bisulphite sequencing of cell-free DNA: a proof-of-principle study

ABSTRACT

In the clinical management of paediatric solid tumours, histological examination of tumour tissue obtained by a biopsy remains the gold standard to establish a conclusive pathological diagnosis. The DNA methylation pattern of a tumour is known to correlate with the histopathological diagnosis across cancer types and is showing promise in the diagnostic workup of tumour samples. This methylation pattern can be detected in the cell-free DNA. Here, we provide proof-of-concept of histopathologic classification of paediatric tumours using cell-free reduced representation bisulphite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. We determined the correct tumour type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free DNA. We demonstrate that the majority of misclassifications were associated with sample quality and not with the extent of disease. Our approach has the potential to help tackle some of the remaining diagnostic challenges in paediatric oncology in a cost-effective and minimally invasive manner.

KEYWORDS:

• DNA methylation
• cell-free DNA
• liquid biopsy
• pediatric oncology
• biomarker
• diagnosis
• infinium
• reduced representation bisulfite sequencing

Acknowledgments

A.D.K and R.V.P were funded by a predoctoral fellowship from the Research Foundation Flanders (FWO). B.D.W. is a senior clinical investigator for the FWO. This project was partially funded by Kom op tegen Kanker, a CRIG (Cancer Research Institute Ghent) Young Investigator Proof-of-Concept grant, vzw Kinderkankerfonds - a non-profit childhood cancer foundation under Belgian law (grant to T.L), KickCancer, Liquidhope (a TranScan-2 project by Koningin Wilhelmina Fund, Dutch Cancer Society KWF TRANSCAN 8352/TRS-2018- 00000715), and by the VIB Grand Challenges Program (N.C. lab). We thank Lennart Raman for assistance with WisecondorX and Peter Stockwell for assistance with mkrrgenome. We thank the Center for Medical Genetics Ghent lab technicians for assistance with plasma preparation, cfDNA extraction, shallow WGS library preparation, and sequencing (Thalia Van Laethem, Eveline Vanden Eynde, Kimberly Verniers, Peter Degrave, Dimitri Broucke, Machteld Baetens, Aline Eggermont, Els De Smet). We thank Landric Vautmans for assistance with the FEMTO Pulse. In France, this study was supported by the Annenberg Foundation, the Association Hubert Gouin Enfance et Cancer, the Association Enfants et Santé, and the SiRIC programme (Site de Recherche Integrée sur le Cancer). This study makes use of data generated by dr. Junko Takita, Department of Pediatrics, The University of Tokyo, and The Chinese University of Hong Kong (CUHK) Circulating Acids Research Group, as reported by Chan KC in Proc Natl Acad Sci USA (19 November 2013;110(47):18761-8), the TARGET database, Gene Expression Omnibus (GEO) identifiers GSE89041, GSE97529, GSE90496, GSE107946, GSE90496, and ArrayExpress identifier E-MTAB-4187.

Disclosure statement

A.D.K and N.C. are listed as inventors in patent application PCT/EP2017/056850 related to the cf-RRBS method.

Code and data availability

Scripts and supporting files are available at https://github.com/rmvpaeme/cfRRBS_manuscript.

Raw data are available at EGA identifier EGAD00001005928. Processed data are available at ArrayExpress identifier E-MTAB-8770.

The cf-RRBS protocol is available at https://www.protocols.io/view/cf-rrbs-protocol-pc6dize.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the Annenberg Foundation; Fonds Wetenschappelijk Onderzoek (FWO); Kom op tegen Kanker (KOTK); Association Enfants et Santé; Association Hubert Gouin Enfance et Cancer; VIB Grand Challenges; Site de Recherche Integrée sur le Cancer Program; Cancer Research Institute Ghent (CRIG); vzw Kinderkankerfonds, Dutch Cancer Society (KWF); KickCancer.