Liquid biopsy uncovers distinct patterns of DNA methylation and copy number changes in NSCLC patients with different EGFR-TKI resistant mutations

Targeted therapy with tyrosine kinase inhibitors (TKI) provides survival benefits to a majority of patients with non-small cell lung cancer (NSCLC). However, resistance to TKI almost always develops after treatment. Although genetic and epigenetic alterations have each been shown to drive resistance to TKI in cell line models, clinical evidence for their contribution in the acquisition of resistance remains limited. Here, we employed liquid biopsy for simultaneous analysis of genetic and epigenetic changes in 122 Vietnamese NSCLC patients undergoing TKI therapy and displaying acquired resistance. Genome-wide epigenetics, particularly DNA methylation changes, have been implicated in the acquisition of drug resistance by cancer cells. However, the relationship between DNA methylation and mutation alterations in mediating resistance to TKI drugs remains largely unknown. To profile the methylation changes at the genome-wide level of different resistance mutation groups, we employed the workflow previously reported by Jiang et al.. The assay was based on the combination of bisulfite conversion and massively parallel sequencing to analyze methylation density (MD) in bins of 1 Mb across 22 chromosomes. The analysis was performed for all cases with <i>EGFR</i> amplification (3 cases) and MET amplification (4 cases). For the remaining cases, only 3 out of 5 cases with co-occurring T790M and <i>EGFR</i> amplification and 3 out of 5 cases with <i>HER2</i> amplification had sufficient cell free DNA for the analysis. Among 23 patients carrying T790M mutation, we randomly selected 5 cases with sufficient plasma. The total 18 chosen cases were grouped into either EGFR dependent resistance mutations (on-target) or EGFR independent mutations (off-target). Samples from 3 healthy subjects were included as a reference set.