DNA Methylation Analysis in Plasma Cell-Free DNA and Paired CTCs of NSCLC Patients before and after Osimertinib Treatment.

Aliki Ntzifa,Evi Lianidou,Dora Londra,Athanasios Kotsakis,Theodoros Rampias,Vassilis Georgoulias

doi:10.3390/cancers13235974

Abstract

Simple SummaryLiquid biopsy is a highly useful tool for the management of NSCLC patients and could provide valuable information on early detection of resistance to osimertinib. Epigenetic biomarkers are very promising for the early diagnosis, prognosis, and prediction of drug response in many types of cancer. We performed a DNA methylation analysis in plasma cell-free DNA and paired CTCs of NSCLC patients before osimertinib treatment and at progression of disease (PD). Our results revealed a significant increase in DNA methylation at PD. Epigenetic alterations should be further evaluated as a possible resistance mechanism to osimertinib and their detection in liquid biopsy samples can be valuable for the follow-up of patients in real time.Osimertinib has been an effective second-line treatment in EGFR mutant NSCLC patients; however, resistance inevitably occurs. DNA methylation has been previously implicated in NSCLC progression and often in therapy resistance, however its distinct role in osimertinib resistance is not elucidated as yet. In the present study, we directly compared DNA methylation of nine selected genes (RASSF1A, RASSF10, APC, WIF-1, BRMS1, SLFN11, RARβ, SHISA3, and FOXA1) in plasma-cfDNA and paired CTCs of NSCLC patients who were longitudinally monitored during osimertinib treatment. Peripheral blood (PB) from 42 NSCLC patients was obtained at two time points: (a) baseline: before treatment with osimertinib and (b) at progression of disease (PD). DNA methylation of the selected genes was detected in plasma-cfDNA (n = 80) and in paired CTCs (n = 74). Direct comparison of DNA methylation of six genes between plasma-cfDNA and paired CTC samples (n = 70) revealed a low concordance, indicating that CTCs and cfDNA give complementary information. DNA methylation analysis of plasma-cfDNA and CTCs indicated that when at least one of these genes was methylated there was a statistically significant increase at PD compared to baseline (p = 0.031). For the first time, DNA methylation analysis in plasma-cfDNA and paired CTCs of NSCLC patients during osimertinib therapy indicated that DNA methylation of these genes could be a possible resistance mechanism.

Highlights

The therapeutic landscape of non-small cell lung cancer (NSCLC) patients that carry somatic mutations in the tyrosine kinase (TK) domain of epidermal growth factor receptor (EGFR) has been successfully validated during the last two decades and the prolonged survival of those patients who benefit from EGFR TKIs has already proven through numerous clinical studies [1–4]
We conducted for the first time a DNA methylation analysis of nine selected genes in plasma-cfDNA and paired circulating tumor cells (CTCs) of NSCLC patients before administration of osimertinib and at progression of disease
The Pearson correlation analysi7sofr1e8vealed th mRNA expression was negatively associated with DNA methylation in lu adenocarcinoma tissues for all genes indicating that DNA methylation of these genes m lead to their epigenetic silencing in lung adenocarcinoma (Figure 2). tissues for all genes indicating that DNA methylation of these genes may lead to their epigenetic silencing in lung adenocarcinoma (Figure 2)

Summary

Introduction

The therapeutic landscape of non-small cell lung cancer (NSCLC) patients that carry somatic mutations in the tyrosine kinase (TK) domain of epidermal growth factor receptor (EGFR) has been successfully validated during the last two decades and the prolonged survival of those patients who benefit from EGFR TKIs has already proven through numerous clinical studies [1–4]. DNA methylation as part of these modifications has been an important mechanism especially when it occurs in CpG rich regions of the 5 ends of many genes. This is a common epigenetic process that leads to the silencing of tumor suppressor genes and there is evidence that this can either act as a sole inactivating mechanism or in combination with the presence of mutations [12,13]. There is accumulating evidence that the dominance of tumor cancer cells undergo epigenetic modifications and their transition to a more drug tolerant cell subpopulation leads to the rapid acquisition of treatment resistance [16]

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