First Indonesian Nasopharyngeal Cancer Whole Epigenome Sequencing Identify Tumour Suppressor CpG Methylation

Handoko,1– 3 Marlinda Adham,2,4 Lisnawati Rachmadi,2,5 Demak Lumban Tobing,6 Asmarinah,7 Fadilah,8 Wei Dai,9,10 Anne Wing Mui Lee,10 Soehartati A Gondhowiardjo2,3 1Doctoral Program in Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia; 2Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia; 3Department of Radiation Oncology, Cipto Mangunkusumo National General Hospital, Jakarta, Indonesia; 4Department of Otorhinolaryngology - Head and Neck Surgery, Cipto Mangunkusumo National General Hospital, Jakarta, Indonesia; 5Department of Anatomical Pathology, Cipto Mangunkusumo National General Hospital, Jakarta, Indonesia; 6Department of Clinical Pathology, Dharmais Cancer Hospital, Jakarta, Indonesia; 7Medical Biology Department, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia; 8Department of Medical Chemistry, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia-Bioinformatics Core Facilities, Indonesian Medical Education and Research Institute (IMERI), Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia; 9Department of Clinical Oncology, University of Hong Kong, Pokfulam, Hong Kong (SAR), People’s Republic of China; 10Clinical Oncology Center, University of Hong Kong – Shenzhen Hospital, Shenzhen, People’s Republic of ChinaCorrespondence: Soehartati A Gondhowiardjo; Handoko, Email [email protected]: Nasopharyngeal cancer (NPC) is a multifaceted disease characterized by genetic and epigenetic modifications. While Epstein–Barr virus (EBV) infection is a known risk factor, recent studies highlight the significant role of DNA methylation in NPC pathogenesis. Aberrant methylation, particularly at CpG sites, can silence tumour suppressor genes, promoting uncontrolled cell growth. This study aims to analyse the methylation patterns in Indonesian NPC patients through whole-epigenome sequencing.Methods: Seven clinical nasopharyngeal cancer samples were collected and confirmed histopathologically. DNA was extracted, sequenced using Oxford Nanopore technology, and aligned to the GRCh38 human reference genome. Methylation analysis was performed using modkit and statistical analysis with R software. Enriched pathways and processes were identified using ClusterProfiler in R, and gene overlap analysis was conducted.Results: The analysis identified both globally hypermethylated and hypomethylated NPC samples. Key tumour suppressor genes, such as PRKCB, PLCB3, ITGB3, EPHA2, PLCE1, PRKCD, CDKN2A, CDKN2B, RPS6KA2, ERBB4, LRRC4, AKT1, PPP2R5C, and STK11 were frequently hypermethylated and confirmed to have lower expression in an independent NPC transcriptome cohort, suggesting their role in NPC carcinogenesis. Enriched KEGG pathways included PI3K-Akt signalling, ECM–receptor interaction, and focal adhesion. The presence of EBV DNA was confirmed in all samples, implicating its role in influencing methylation patterns.Discussion: This study provides comprehensive insights into the epigenetic landscape of NPC, underscoring the role of CpG methylation in tumour suppressor gene silencing. These findings pave the way for targeted therapies and highlight the need for region-specific approaches in NPC management.Keywords: nasopharyngeal cancer, epigenome, methylation, Epstein–Barr virus, whole-genome sequencing