Methylation Levels of LINE-1 As a Useful Marker for Venous Invasion in Both FFPE and Frozen Tumor Tissues of Gastric Cancer

  • Min, Jimin (Cancer Research Institute, Seoul National University College of Medicine) ;
  • Choi, Boram (Department of Life Science, Ewha Womans University) ;
  • Han, Tae-Su (Biotherapeutics Translational Research Center, Division of Biomedical Science, Korea Research Institute of Bioscience and Biotechnology) ;
  • Lee, Hyuk-Joon (Cancer Research Institute, Seoul National University College of Medicine) ;
  • Kong, Seong-Ho (Department of Surgery, Seoul National University College of Medicine) ;
  • Suh, Yun-Suhk (Department of Surgery, Seoul National University College of Medicine) ;
  • Kim, Tae-Han (Department of Surgery, Seoul National University College of Medicine) ;
  • Choe, Hwi-Nyeong (Department of Nursing, Seoul National University Hospital) ;
  • Kim, Woo Ho (Department of Pathology, Seoul National University College of Medicine) ;
  • Hur, Keun (Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University) ;
  • Yang, Han-Kwang (Cancer Research Institute, Seoul National University College of Medicine)
  • Received : 2017.01.31
  • Accepted : 2017.04.24
  • Published : 2017.05.31


Long interspersed nuclear element-1 (LINE-1) is a retrotransposon that contains a CpG island in its 5'-untranslated region. The CpG island of LINE-1 is often heavily methylated in normal somatic cells, which is associated with poor prognosis in various cancers. DNA methylation can differ between formalin-fixed paraffin-embedded (FFPE) and frozen tissues. Therefore, this study aimed to compare the LINE-1 methylation status between the two tissue-storage conditions in gastric cancer (GC) clinical samples and to evaluate whether LINE-1 can be used as an independent prognostic marker for each tissue-storage type. We analyzed four CpG sites of LINE-1 and examined the methylation levels at these sites in 25 FFPE and 41 frozen GC tissues by quantitative bisulfite pyrosequencing. The LINE-1 methylation status was significantly different between the FFPE and frozen GC tissues (p < 0.001). We further analyzed the clinicopathological features in the two groups separately. In the frozen GC tissues, LINE-1 was significantly hypomethylated in GC tissues compared to their corresponding normal gastric mucosa tissues (p < 0.001), and its methylation status was associated with gender, differentiation state, and lymphatic and venous invasion of GC. In the FFPE GC tissues, the methylation levels of LINE-1 differed according to tumor location and venous invasion of GC. In conclusion, LINE-1 can be used as a useful methylation marker for venous invasion in both FFPE and frozen tumor tissues of GC.


Supported by : Korean Health Industry Development Institute (KHIDI), National Research Foundation of Korea (NRF)


  1. Aparicio, A., North, B., Barske, L., Wang, X., Bollati, V., Weisenberger, D., Yoo, C., Tannir, N., Horne, E., Groshen, S., et al. (2009). LINE-1 methylation in plasma DNA as a biomarker of activity of DNA methylation inhibitors in patients with solid tumors. Epigenetics 4, 176-184.
  2. Baba, Y., Murata, A., Watanabe, M., and Baba, H. (2014). Clinical implications of the LINE-1 methylation levels in patients with gastrointestinal cancer. Surg. Today 44, 1807-1816.
  3. Bae, J.M., Shin, S.H., Kwon, H.J., Park, S.Y., Kook, M.C., Kim, Y.W., Cho, N.Y., Kim, N., Kim, T.Y., Kim, D., et al. (2012). ALU and LINE-1 hypomethylations in multistep gastric carcinogenesis and their prognostic implications. Int. J. Cancer 131, 1323-1331.
  4. Barchitta, M., Quattrocchi, A., Maugeri, A., Vinciguerra, M., and Agodi, A. (2014). LINE-1 hypomethylation in blood and tissue samples as an epigenetic marker for cancer risk: a systematic review and meta-analysis. PLoS One 9, e109478.
  5. Cash, H.L., Tao, L., Yuan, J.M., Marsit, C.J., Houseman, E.A., Xiang, Y.B., Gao, Y.T., Nelson, H.H., and Kelsey, K.T. (2012). LINE-1 hypomethylation is associated with bladder cancer risk among nonsmoking Chinese. Int. J. Cancer 130, 1151-1159.
  6. Chalitchagorn, K., Shuangshoti, S., Hourpai, N., Kongruttanachok, N., Tangkijvanich, P., Thong-ngam, D., Voravud, N., Sriuranpong, V., and Mutirangura, A. (2004). Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis. Oncogene 23, 8841-8846.
  7. Consortium, B. (2016). Quantitative comparison of DNA methylation assays for biomarker development and clinical applications. Nat. Biotechnol. 34, 726-737.
  8. Do, H., and Dobrovic, A. (2015). Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization. Clin. Chem. 61, 64-71.
  9. Estecio, M.R., Gharibyan, V., Shen, L., Ibrahim, A.E., Doshi, K., He, R., Jelinek, J., Yang, A.S., Yan, P.S., Huang, T.H., et al. (2007). LINE-1 hypomethylation in cancer is highly variable and inversely correlated with microsatellite instability. PLoS One 2, e399.
  10. Ferlay, J., Soerjomataram, I., Dikshit, R., Eser, S., Mathers, C., Rebelo, M., Parkin, D.M., Forman, D., and Bray, F. (2015). Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer 136, E359-386.
  11. Hamilton, M.G., Roldan, G., Magliocco, A., McIntyre, J.B., Parney, I., and Easaw, J.C. (2011). Determination of the methylation status of MGMT in different regions within glioblastoma multiforme. J. Neurooncol. 102, 255-260.
  12. Hur, K., Cejas, P., Feliu, J., Moreno-Rubio, J., Burgos, E., Boland, C.R., and Goel, A. (2014). Hypomethylation of long interspersed nuclear element-1 (LINE-1) leads to activation of proto-oncogenes in human colorectal cancer metastasis. Gut 63, 635-646.
  13. Ikeda, K., Shiraishi, K., Eguchi, A., Shibata, H., Yoshimoto, K., Mori, T., Baba, Y., Baba, H., and Suzuki, M. (2013). Long interspersed nucleotide element 1 hypomethylation is associated with poor prognosis of lung adenocarcinoma. Ann. Thorac. Surg. 96, 1790-1794.
  14. Irahara, N., Nosho, K., Baba, Y., Shima, K., Lindeman, N.I., Hazra, A., Schernhammer, E.S., Hunter, D.J., Fuchs, C.S., and Ogino, S. (2010). Precision of pyrosequencing assay to measure LINE-1 methylation in colon cancer, normal colonic mucosa, and peripheral blood cells. J. Mol. Diagn. 12, 177-183.
  15. Jasmine, F., Rahaman, R., Roy, S., Raza, M., Paul, R., Rakibuz-Zaman, M., Paul-Brutus, R., Dodsworth, C., Kamal, M., Ahsan, H., et al. (2012). Interpretation of genome-wide infinium methylation data from ligated DNA in formalin-fixed, paraffin-embedded paired tumor and normal tissue. BMC Res. Notes 5, 117.
  16. Kile, M.L., Baccarelli, A., Tarantini, L., Hoffman, E., Wright, R.O., and Christiani, D.C. (2010). Correlation of global and gene-specific DNA methylation in maternal-infant pairs. PLoS One 5, e13730.
  17. Kim, E.J., Chung, W.C., Kim, D.B., Kim, Y.J., Lee, J.M., Jung, J.H., and Lee, Y.K. (2016). Long interspersed nuclear element (LINE)-1 methylation level as a molecular marker of early gastric cancer. Dig. Liver Dis. 48, 1093-1097.
  18. Lander, E.S., Linton, L.M., Birren, B., Nusbaum, C., Zody, M.C., Baldwin, J., Devon, K., Dewar, K., Doyle, M., FitzHugh, W., et al. (2001). Initial sequencing and analysis of the human genome. Nature 409, 860-921.
  19. Lee, J.R., Chung, W.C., Kim, J.D., Lee, K.M., Paik, C.N., Jung, S.H., Jung, J.H., Lee, Y.K., and Han, S.W. (2011). Differential LINE-1 hypomethylation of gastric low-grade dysplasia from high grade dysplasia and intramucosal cancer. Gut Liver 5, 149-153.
  20. Li, P., Ling, Y.H., Zhu, C.M., Hu, W.M., Zhang, X.K., Luo, R.Z., He, J.H., Yun, J.P., Li, Y.F., and Cai, M.Y. (2015). Vascular invasion as an independent predictor of poor prognosis in nonmetastatic gastric cancer after curative resection. Int. J. Clin. Exp. Pathol. 8, 3910-3918.
  21. Maehara, Y., Kabashima, A., Koga, T., Tokunaga, E., Takeuchi, H., Kakeji, Y., and Sugimachi, K. (2000). Vascular invasion and potential for tumor angiogenesis and metastasis in gastric carcinoma. Surgery 128, 408-416.
  22. Ogino, S., Kawasaki, T., Nosho, K., Ohnishi, M., Suemoto, Y., Kirkner, G.J., and Fuchs, C.S. (2008a). LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer. Int. J. Cancer 122, 2767-2773.
  23. Ogino, S., Nosho, K., Kirkner, G.J., Kawasaki, T., Chan, A.T., Schernhammer, E.S., Giovannucci, E.L., and Fuchs, C.S. (2008b). A cohort study of tumoral LINE-1 hypomethylation and prognosis in colon cancer. J. Natl. Cancer Inst. 100, 1734-1738.
  24. Rahman, R., Asombang, A.W., and Ibdah, J.A. (2014). Characteristics of gastric cancer in Asia. World J. Gastroenterol. 20, 4483-4490.
  25. Rodic, N., and Burns, K.H. (2013). Long interspersed element-1 (LINE-1): passenger or driver in human neoplasms? PLoS Genet. 9, e1003402.
  26. Shigaki, H., Baba, Y., Watanabe, M., Murata, A., Iwagami, S., Miyake, K., Ishimoto, T., Iwatsuki, M., and Baba, H. (2013). LINE-1 hypomethylation in gastric cancer, detected by bisulfite pyrosequencing, is associated with poor prognosis. Gastric Cancer 16, 480-487.
  27. Song, Y.S., Kim, Y., Cho, N.Y., Yang, H.K., Kim, W.H., and Kang, G.H. (2016). Methylation status of long interspersed element-1 in advanced gastric cancer and its prognostic implication. Gastric Cancer 19, 98-106.
  28. Suh, Y.S., Yu, J., Kim, B.C., Choi, B., Han, T.S., Ahn, H.S., Kong, S.H., Lee, H.J., Kim, W.H., and Yang, H.K. (2015). Overexpression of plasminogen activator inhibitor-1 in advanced gastric cancer with aggressive lymph node metastasis. Cancer Res. Treat. 47, 718-726.
  29. Sunami, E., de Maat, M., Vu, A., Turner, R.R., and Hoon, D.S. (2011). LINE-1 hypomethylation during primary colon cancer progression. PLoS One 6, e18884.
  30. Tangkijvanich, P., Hourpai, N., Rattanatanyong, P., Wisedopas, N., Mahachai, V., and Mutirangura, A. (2007). Serum LINE-1 hypomethylation as a potential prognostic marker for hepatocellular carcinoma. Clin. Chim. Acta 379, 127-133.
  31. van Hoesel, A.Q., van de Velde, C.J., Kuppen, P.J., Liefers, G.J., Putter, H., Sato, Y., Elashoff, D.A., Turner, R.R., Shamonki, J.M., de Kruijf, E.M., et al. (2012). Hypomethylation of LINE-1 in primary tumor has poor prognosis in young breast cancer patients: a retrospective cohort study. Breast Cancer Res. Treat. 134, 1103-1114.
  32. Wang, G., Gao, J., Huang, H., Tian, Y., Xue, L., Wang, W., You, W., Lian, H., Duan, X., Wu, B., et al. (2013). Expression of a LINE-1 endonuclease variant in gastric cancer: its association with clinicopathological parameters. BMC Cancer 13, 265.