Combined DNA methylation analysis in size based CTC fraction and matched plasma-cfDNA provides prognostic information in early stage NSCLC

Abstract Background: Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) analysis represents a liquid biopsy approach for real-time monitoring of tumor evolution. DNA methylation is considered to be an early event in the process of cancer development and progression. The aim of the present study was to evaluate whether detection of DNA methylation of selected tumour suppressor genes in size-based CTC fraction and matched ctDNA could provide prognostic information in early stage NSCLC. Methods: The methylation status of five selected gene promoters (APC, RASSFIA1, FOXA1, SLFN11, SHOX2) was examined by highly specific and sensitive real time methylation specific PCR assays in: a) a training group of 35 primary tumours and their corresponding adjacent non-cancerous tissues of early stage NSCLC patients, b) a validation group of 22 primary FFPEs tumor tissues and 42 peripheral blood samples of early stage NSCLC patients that gDNA was isolated from FFPEs, sized-based CTCs and plasma, and c) a control group of healthy blood donors (n=12). Results: All five gene promoters tested were highly methylated in the training group of primary tumours; methylation of SHOX2 promoter in the primary tissues was associated with unfavorable outcome. RASSFIA and APC were found methylated in plasma-cfDNA samples at 14.3% and 11.9% respectively, whereas in the corresponding size-based CTC-enriched fractions SLFN11 and APC promoters were methylated in 7.1%. The incidence of relapse was higher in patients with i) promoter methylation of APC and SLFN11 in plasma-cfDNA (P=0.037 and P=0.042 respectively) and ii) at least one gene promoter was methylated in the sized-based CTC fraction or plasma-cfDNA. Conclusions: Although in liquid biopsy components methylation of these gene promoters was significantly lower than in the primary tumors, the combination of DNA methylation analysis in sized-based CTC fraction and plasma-cfDNA revealed clinical relevance. Additional studies are required to validate our findings in a large cohort of early stage NSCLC patients. Citation Format: Athina N. Markou, Dora Londra, Victoria Tserpeli, John Kollias, Emilia Tsaroucha, Ioannis Pateras, K Potaris, Ioannis Vamvakaris, Athanasios Kotsakis, Vasilis Georgoulias, Evi Lianidou. Combined DNA methylation analysis in size based CTC fraction and matched plasma-cfDNA provides prognostic information in early stage NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3384.