Promoter Methylation of Selected Genes in Non-Small-Cell Lung Cancer Patients and Cell Lines.

Specific gene promoter DNA methylation is becoming a powerful epigenetic biomarker in cancer diagnostics. Five genes (CDH1,CDKN2Ap16,RASSF1A,TERT, andWT1) were selected based on their frequently published potential as epigenetic markers. Diagnostic promoter methylation assays were generated based on bisulfite-converted DNA pyrosequencing. The methylation patterns of 144 non-small-cell lung cancer (NSCLC) and 7 healthy control formalin-fixed paraffin-embedded (FFPE) samples were analyzed to evaluate the applicability of the putative diagnostic markers. Statistically significant changes in methylation levels are shown forTERTandWT1. Furthermore, 12 NSCLC and two benign lung cell lines were characterized for promoter methylation. The in vitro tests involved a comparison of promoter methylation in 2D and 3D cultures, as well as therapeutic tests investigating the impact ofCDH1/CDKN2Ap16/RASSF1A/TERT/WT1promoter methylation on sensitivity to tyrosine kinase inhibitor (TKI) and DNA methyl-transferase inhibitor (DNMTI) treatments. We conclude that the selected markers have potential and putative impacts as diagnostic or even predictive marker genes, although a closer examination of the resulting protein expression and pathway regulation is needed.