Webinar: Development of a Novel Targeted NGS Panel for Analysis of DNA Methylation Biomarkers in Lung Cancer Research

Webinar summary

Epigenetic modifications such as DNA methylation have long shown promise as potential biomarkers to diagnose and guide treatment of many human diseases, including multiple types of cancers. However, DNA methylation biomarkers have not yet been widely adopted in clinical settings due to some technical limitations of previous analysis methods.

In this webinar, Dr. Lucia Anna Muscarella, Head of Lung Cancer Research Unit and Executive Biologist at Casa Sollievo della Sofferenza Foundation, presents the development and analytical evaluation of a novel targeted next-generation sequencing (NGS) panel for DNA methylation analysis in lung cancer research.

This study demonstrates the feasibility of targeted NGS-based DNA methylation analysis using the OPERA METI custom panel for epigenetic biomarker research. The panel achieved robust analytical performance across multiple specimen types while enabling high-resolution quantification of methylation across promoter and intragenic CpG regions.

The findings further illustrate the capability of targeted bisulfite sequencing to characterize heterogeneous DNA methylation patterns and support development of integrated epigenetic models for future cancer research applications. 

Study overview

The presentation begins with an overview of epigenetic regulation, emphasizing DNA methylation as one of the earliest and most extensively studied epigenetic modifications. DNA methylation within CpG islands is described as an important regulator of gene expression and a contributor to multiple biological processes, including tissue development, genomic imprinting, aging, and disease pathogenesis.

Within oncology, aberrant DNA methylation patterns are presented as characteristic features of tumor biology, contributing to cellular transformation, invasion, metastasis, altered metabolism, immune evasion, and other cancer-associated processes. These observations have led to investigation of DNA methylation profiles as potential cancer biomarkers.

To support targeted epigenetic profiling, the research team developed the OPERA METI panel in collaboration with Thermo Fisher Scientific. The custom panel was designed to quantify DNA methylation at single-base resolution across cancer-associated CpG sites relevant to lung cancer and other solid tumor research. 

Key findings

DNA methylation represents a key epigenetic biomarker

• DNA methylation occurs predominantly at CpG dinucleotides and influences gene transcription through epigenetic regulation
• Aberrant methylation events are associated with multiple biological processes involved in tumor development and progression
• DNA methylation profiles have emerged as promising biomarker candidates for cancer research

Targeted NGS enables high-resolution epigenetic analysis

• Targeted bisulfite NGS provides single-base resolution measurement of DNA methylation across multiple genomic regions simultaneously
• The approach enables sensitive quantification of methylation density using relatively small amounts of biological material
• Targeted sequencing supports large-scale evaluation of candidate biomarker regions with increasing numbers of clinically relevant targets

OPERA METI Panel was designed for comprehensive targeted methylation profiling

• The custom panel targets 155 genomic regions comprising 1,107 CpG sites across 80 cancer-related genes
• Selected targets include genes associated with immunotherapy, oxidative stress, prognosis, lung cancer, and other solid tumor research
• Both promoter-associated and intragenic CpG islands were incorporated, with amplicons designed for both Watson and Crick DNA strands

Verification demonstrated robust library generation across sample types

• Analytical evaluation included FFPE tissue, OCT-embedded tissue, and cultured cell lines representing varying levels of DNA fragmentation.
• Bisulfite conversion was incorporated to distinguish methylated from unmethylated cytosines.
• Successful library generation was achieved across sample types, with improved performance observed using a minimum input of 10 ng bisulfite-converted genomic DNA.

Sequencing performance supported reliable methylation quantification

• Quality control metrics supported successful sequencing with a minimum target coverage of approximately 500 mapped reads per CpG site
• Up to six samples could be analyzed within a single sequencing run while maintaining target coverage requirements
• Sequencing performance demonstrated concordance between expected and observed global methylation measurements using commercially available methylated and unmethylated controls

Bioinformatic analysis enabled strand-specific methylation assessment

• The methylation analysis workflow quantified methylated and unmethylated reads for each targeted CpG site
• Mean bisulfite conversion efficiency exceeded 99% across control samples
• Analytical outputs included strand-specific methylation measurements for Watson and Crick strands, together with sample-level methylation summaries

Differential methylation patterns were observed across tumor and normal samples

• Analysis of paired FFPE samples demonstrated differential methylation across multiple gene regions between tumor and matched non-neoplastic tissue
• The panel also identified asymmetric methylation patterns between Watson and Crick strands, highlighting heterogeneity in CpG methylation distribution
• Strand-specific methylation assessment may provide additional insight into gene regulation and transcriptional biology in cancer research