Trinidad Eva María, Juan-Ribelles Antonio, Pisano Giulia, Castel Victoria, Cañete Adela, Gut Marta, Heath Simon, Font de Mora Jaime
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.
Front Oncol. 2023 May 12;13:1037342. doi: 10.3389/fonc.2023.1037342. eCollection 2023.
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.
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.
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.
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.
液体活检已成为肿瘤学中一种有前景的非侵入性诊断方法，因为对循环肿瘤DNA（ctDNA）的分析可反映疾病在诊断、进展和治疗反应时的精确状态。DNA甲基化谱分析也是许多癌症灵敏且特异检测的一种潜在解决方案。两种方法的结合，即对ctDNA进行DNA甲基化分析，为儿童癌症患者提供了一种极其有用且微创的工具，具有高度相关性。神经母细胞瘤是儿童最常见的颅外实体瘤，占癌症相关死亡的15%。这种高死亡率促使科学界寻找新的治疗靶点。DNA甲基化也为识别这些分子提供了新来源。然而，从癌症患儿获取的血液样本量有限，且ctDNA含量偶尔可能被非肿瘤游离DNA（cfDNA）稀释，这使得高通量测序研究的最佳材料量变得复杂。
在本文中，我们提出了一种改进的方法，用于对高危神经母细胞瘤患者血液来源的血浆进行ctDNA甲基化组研究。我们使用来自86例高危神经母细胞瘤患者的126个样本中10 ng血浆来源的ctDNA，评估了适合甲基化组研究的含ctDNA样本的电泳图谱，并评估了几种生物信息学方法来分析DNA甲基化测序数据。
我们证明，基于较低的PCR重复比例、较高的唯一映射读数百分比、平均覆盖率和基因组覆盖率，酶促甲基测序（EM-seq）优于基于亚硫酸氢盐转化的方法。对电泳图谱的分析揭示了核小体多聚体的存在，偶尔还有高分子量DNA。我们确定单核小体峰含量的10%就足以成功检测拷贝数变异和甲基化谱。单核小体峰的定量分析还表明，诊断时的样本比复发样本含有更多的ctDNA。
我们的结果完善了电泳图谱的使用，以优化后续高通量分析的样本选择，并支持采用液体活检，随后将未甲基化的半胱氨酸进行酶促转化，以评估神经母细胞瘤患者的甲基化组。
