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

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• dc.contributor.author Trinidad, Eva María
• dc.contributor.author Juan-Ribelles, Antonio
• dc.contributor.author Pisano, Giulia
• dc.contributor.author Castel, Victoria
• dc.contributor.author Cañete, Adela
• dc.contributor.author Gut, Marta
• dc.contributor.author Heath, Simon
• dc.contributor.author Font de Mora, Jaime
• dc.date.accessioned 2023-07-18T06:18:45Z
• dc.date.available 2023-07-18T06:18:45Z
• dc.date.issued 2023
• dc.description.abstract 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.
• dc.description.sponsorship Supported by TRANSCAN-2 consortium LIQUIDHOPE by Fundación Científica de la Asociación Española Contra el Cáncer (TRNSC18001FON), by Spanish Ministry of Science and Innovation (PID2020-119323RB-I00 grant) and by “Sumemos muchas manos por los niños enfermos” non-profit organization.
• dc.format.mimetype application/pdf
• dc.identifier.citation Trinidad EM, Juan-Ribelles A, Pisano G, Castel V, Cañete A, Gut M, Heath S, Font de Mora J. Evaluation of circulating tumor DNA by electropherogram analysis and methylome profiling in high-risk neuroblastomas. Front Oncol. 2023 May 12;13:1037342. DOI: 10.3389/fonc.2023.1037342
• dc.identifier.doi http://dx.doi.org/10.3389/fonc.2023.1037342
• dc.identifier.issn 2234-943X
• dc.identifier.uri http://hdl.handle.net/10230/57596
• dc.language.iso eng
• dc.publisher Frontiers
• dc.relation.ispartof Front Oncol. 2023 May 12;13:1037342
• dc.relation.projectID info:eu-repo/grantAgreement/ES/2PE/PID2020-119323RB-I00
• dc.rights © 2023 Trinidad, Juan-Ribelles, Pisano, Castel, Cañete, Gut, Heath and Font de Mora. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.rights.uri http://creativecommons.org/licenses/by/4.0/
• dc.subject.keyword DNA mehtylation
• dc.subject.keyword ctDNA / normal cfDNA ratio
• dc.subject.keyword Enzymatic methyl-sequencing (EM-seq)
• dc.subject.keyword High-risk neuroblastoma
• dc.subject.keyword Liquid biopsy
• dc.title Evaluation of circulating tumor DNA by electropherogram analysis and methylome profiling in high-risk neuroblastomas
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion