Evaluation of circulating tumor DNA by electropherogram analysis and methylome profiling in high-risk neuroblastomas.

Autor:	Trinidad EM; Laboratory of Cellular and Molecular Biology, Health Research Institute Hospital La Fe, Valencia, Spain.; Clinical and Translational Research in Cancer, Health Research Institute Hospital La Fe, Valencia, Spain., Juan-Ribelles A; Clinical and Translational Research in Cancer, Health Research Institute Hospital La Fe, Valencia, Spain.; Pediatric Oncology Unit, La Fe University Hospital, Valencia, Spain., Pisano G; Clinical and Translational Research in Cancer, Health Research Institute Hospital La Fe, Valencia, Spain.; Pediatric Oncology Unit, La Fe University Hospital, Valencia, Spain., Castel V; Clinical and Translational Research in Cancer, Health Research Institute Hospital La Fe, Valencia, Spain.; Pediatric Oncology Unit, La Fe University Hospital, Valencia, Spain., Cañete A; Clinical and Translational Research in Cancer, Health Research Institute Hospital La Fe, Valencia, Spain.; Pediatric Oncology Unit, La Fe University Hospital, Valencia, Spain.; School of Medicine, University of Valencia, Valencia, Spain., Gut M; National Center for Genomic Analysis - Centre for Genomic Regulation (CNAG-CRG), Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Universitat Pompeu Fabra (UPF), Barcelona, Spain., Heath S; National Center for Genomic Analysis - Centre for Genomic Regulation (CNAG-CRG), Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Universitat Pompeu Fabra (UPF), Barcelona, Spain., Font de Mora J; Laboratory of Cellular and Molecular Biology, Health Research Institute Hospital La Fe, Valencia, Spain.; Clinical and Translational Research in Cancer, Health Research Institute Hospital La Fe, Valencia, Spain.
Jazyk:	angličtina
Zdroj:	Frontiers in oncology [Front Oncol] 2023 May 12; Vol. 13, pp. 1037342. Date of Electronic Publication: 2023 May 12 (Print Publication: 2023).
DOI:	10.3389/fonc.2023.1037342
Abstrakt:	Background: Liquid biopsy has emerged as a promising, non-invasive diagnostic approach in oncology because the analysis of circulating tumor DNA (ctDNA) reflects the precise status of the disease at diagnosis, progression, and response to treatment. DNA methylation profiling is also a potential solution for sensitive and specific detection of many cancers. The combination of both approaches, DNA methylation analysis from ctDNA, provides an extremely useful and minimally invasive tool with high relevance in patients with childhood cancer. Neuroblastoma is an extracranial solid tumor most common in children and responsible for up to 15% of cancer-related deaths. This high death rate has prompted the scientific community to search for new therapeutic targets. DNA methylation also offers a new source for identifying these molecules. However, the limited blood sample size which can be obtained from children with cancer and the fact that ctDNA content may occasionally be diluted by non-tumor cell-free DNA (cfDNA) complicate optimal quantities of material for high-throughput sequencing studies. Methods: In this article, we present an improved method for ctDNA methylome studies of blood-derived plasma from high-risk neuroblastoma patients. We assessed the electropherogram profiles of ctDNA-containing samples suitable for methylome studies, using 10 ng of plasma-derived ctDNA from 126 samples of 86 high-risk neuroblastoma patients, and evaluated several bioinformatic approaches to analyze DNA methylation sequencing data. Results: We demonstrated that enzymatic methyl-sequencing (EM-seq) outperformed bisulfite conversion-based method, based on the lower proportion of PCR duplicates and the higher percentage of unique mapping reads, mean coverage, and genome coverage. The analysis of the electropherogram profiles revealed the presence of nucleosomal multimers, and occasionally high molecular weight DNA. We established that 10% content of the mono-nucleosomal peak is sufficient ctDNA for successful detection of copy number variations and methylation profiles. Quantification of mono-nucleosomal peak also showed that samples at diagnosis contained a higher amount of ctDNA than relapse samples. Conclusions: Our results refine the use of electropherogram profiles to optimize sample selection for subsequent high-throughput analysis and support the use of liquid biopsy followed by enzymatic conversion of unmethylated cysteines to assess the methylomes of neuroblastoma patients. Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2023 Trinidad, Juan-Ribelles, Pisano, Castel, Cañete, Gut, Heath and Font de Mora.)
Databáze:	MEDLINE
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