Promoter methylation status of ASC/TMS1/PYCARD is associated with decreased overall survival and TNM status in patients with early stage non-small cell lung cancer (NSCLC)

Maja Šutić, Antje Motzek, Gordana Bubanović, Matthias Linke, Ivan Sabol, Oliver Vugrek, Petar Ozretić, Luka Brčić, Sven Seiwerth, Željko Debeljak, Antonija Jakovčević, Zoran Janevski, Dinko Stančić-Rokotov, Andrea Vukić-Dugac, Marko Jakopović, Miroslav Samaržija, Ulrich Zechner, Jelena Knežević


Background: Lung cancer is the leading cause of cancer-related death worldwide, with 5-year overall survival less than 15%. Therefore, it is essential to find biomarkers for early detection and prognosis. Aberrant DNA methylation is a common feature of human cancers and its utility is already recognized in cancer management. The aim of this study was to explore the diagnostic and prognostic value of the promoter methylation status of the ASC/TMS1/PYCARD and MyD88 genes, key adaptor molecules in the activation of the innate immune response and apoptosis pathways.
Methods: A total of 50 non-small cell lung cancer (NSCLC) patients were enrolled in the study. Meth- ylation of bisulphite converted DNA was quantified by pyrosequencing in fresh frozen malignant tissues and adjacent non-malignant tissues. Associations between methylation and lung function, tumor grade and overall survival were evaluated using receiver-operating characteristics (ROC) analysis and statistical tests of hypothesis.
Results: Methylation level of tested genes is generally low but significantly decreased in tumor tissues (ASC/ TMS1/PYCARD, P<0.0001; MyD88, P<0.0002), which correlates with increased protein expression. Three CpG sites were identified as promising diagnostic marker candidates; CpG11 (-63 position) in ASC/TMS1/ PYCARD and CpG1 (-253 position) and 2 (-265 position) in MyD88. The association study showed that the methylation status of the ASC/TMS1 CpG4 site (-34 position) in malignant and non-malignant tissues is associated with the overall survival (P=0.019) and the methylation status of CpG8 site (-92 position) is associ- ated with TNM-stage (P=0.011).
Conclusions: The methylation status of the ASC/TMS1/PYCARD and MyD88 promoters are promising prognostic biomarker candidates. However, presented results should be considered as a preliminary and should be confirmed on the larger number of the samples.