Abstract
BackgroundLung cancer is the major cause of cancer-related deaths worldwide. Differential diagnosis can be difficult, especially when only small samples are available. Epigenetic changes are frequently tissue-specific events in carcinogenesis and hence may serve as diagnostic biomarkers.Material and methods138 representative formalin-fixed, paraffin-embedded (FFPE) tissues (116 lung cancer cases and 22 benign controls) were used for targeted DNA methylation analysis via pyrosequencing of ten literature-derived methylation markers (APC, CDH1, CDKN2A, EFEMP1, FHIT, L1RE1, MGMT, PTEN, RARB, and RASSF1). Methylation levels were analyzed with the Classification and Regression Tree Algorithm (CART), Conditional Interference Trees (ctree) and ROC. Validation was performed with additional 27 lung cancer cases and 38 benign controls. TCGA data for 282 lung cancer cases was included in the analysis.ResultsCART and ctree analysis identified the combination of L1RE1 and RARB as well as L1RE1 and RASSF1 as independent methylation markers with high discriminative power between tumor and benign tissue (for each combination, 91% specificity and 100% sensitivity). L1RE1 methylation associated significantly with tumor type and grade (p<0.001) with highest methylation in the control group. The opposite was found for RARB (p<0.001). RASSF1 methylation increased with tumor type and grade (p<0.001) with strongest methylation in neuroendocrine tumors (NET).ConclusionHypomethylation of L1RE1 is frequent in tumors compared to benign controls and associates with higher grade, whereas increasing methylation of RARB is an independent marker for tumors and higher grade. RASSF1 hypermethylation was frequent in tumors and most prominent in NET making it an auxiliary marker for separation of NSCLC and NET. L1RE1 in combination with either RARB or RASSF1 could function as biomarkers for separating lung cancer and non-cancerous tissue and could be useful for samples of limited size such as biopsies.
Highlights
Lung cancer remains the number one cancer disease and main cause of cancer-related deaths worldwide
Classification and Regression Tree Algorithm (CART) and ctree analysis identified the combination of L1RE1 and RARB as well as L1RE1 and RASSF1 as independent methylation markers with high discriminative power between tumor and benign tissue
Hypomethylation of L1RE1 is frequent in tumors compared to benign controls and associates with higher grade, whereas increasing methylation of RARB is an independent marker for tumors and higher grade
Summary
Lung cancer remains the number one cancer disease and main cause of cancer-related deaths worldwide. A global hypomethylation of the cancer cell genome is prominent, but certain genes, tumor suppressor genes, present with de novo methylation leading to transcriptional inactivation. RASSF1 hypermethylation is another frequent event in lung cancer affecting approximately 60% of adenocarcinomas (ADC) and up to 100% of small cell lung carcinomas (SCLC) [7]. Hypomethylation of EFEMP1 was identified as independent prognostic marker of non-small cell lung carcinoma (NSCLC) [2]. Another potential methylation biomarker is L1RE1 ( known as LINE1), which is an autonomous transposable element in mammals and presents with hypomethylation in several cancers [8, 9]. Epigenetic changes are frequently tissue-specific events in carcinogenesis and may serve as diagnostic biomarkers
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