Abstract 5364: DNA methylation profiles at precancerous stage cluster lung adenocarcinomas into subclusters associated with carcinogenetic pathway, clinicopathological aggressiveness and patient outcome.
Authors: Sato Takashi , Arai Eri , Kohno Takashi , Tsuta Koji , Watanabe Shun-ichi , Soejima Kenzo , Betsuyaku Tomoko , Kanai Yae 
Published: August 2014

Abstract
Background: We have reported DNA methylation alterations in non-cancerous lung tissue obtained from lung cancer patients. These previous data drew our attention to DNA methylation alterations at precancerous stage of lung adenocarcinoma (LADC). However, the impact of DNA methylation alterations at precancerous stage on the characteristics of established LADCs has been unclear. The purpose of this study is to clarify the significance of DNA methylation profiles during lung carcinogenesis. Methods: Using single-CpG resolution Infinium array, genome-wide DNA methylation analysis was performed in 36 samples of normal lung tissue (C), 139 samples of non-cancerous lung tissue (N) obtained from patients with LADCs, and the corresponding 139 samples of tumorous tissue (T) as a learning cohort. As a validation cohort, the 50 paired samples of N and the corresponding T were obtained from patients with primary LADCs.
Results: 3,621 probes showed significant differences in DNA methylation levels between the 36 C and 139 N samples in the learning cohort, suggesting that N was at precancerous stage with DNA methylation alterations. Unsupervised hierarchical clustering using DNA methylation levels of N on 26,447 autosomal probes subclustered 139 patients of the learning cohort into Cluster A (n=32), B (n=35) and C (n=72). Most of patients in Cluster A were heavy smokers and frequently showed severe pleural anthracosis which mainly reflects smoking history. LADCs in Cluster A were locally invasive tumors which develop in emphysematous lung tissue associated with inflammation. Most of patients in Cluster B were non-smokers and LADCs in Cluster B showed less aggressive features. Most of patients in Cluster C were light smokers and LADCs in Cluster C showed more frequent lymph node metastasis and higher Tumor-Node-Metastasis (TNM) stages. The cancer-free and overall survival rates of patients in Cluster C were significantly lower than those of Cluster B. We indetified 21, 26 and 35 probes as markers which characterize Clusters A, B and C, respectively. In the validation cohort, DNA methylation levels of Cluster A, B and C marker probes were significantly correlated with pleural anthracosis, never-smoking history, and lymph node metastases and higher TNM stages, respectively, i.e. correlation between DNA methylation profiles and each clinicopathological parameter were validated in the validation cohort.
Conclusion: DNA methylation profiles of Clusters A and C at precancerous stage may be established by the effects of smoking through or not thorough inflammation, respectively, whereas those of Cluster B may be associated with carcinogenesis in non-smokers. DNA methylation profiles at precancerous stage may underlie distinct pathways of lung carcinogenesis and determine tumor aggressiveness and patient outcome.
Citation Format: Takashi Sato, Eri Arai, Takashi Kohno, Koji Tsuta, Shun-ichi Watanabe, Kenzo Soejima, Tomoko Betsuyaku, Yae Kanai. DNA methylation profiles at precancerous stage cluster lung adenocarcinomas into subclusters associated with carcinogenetic pathway, clinicopathological aggressiveness and patient outcome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5364. doi:10.1158/1538-7445.AM2013-5364