Value of p16 INK4a and RASSF1A Promoter Hypermethylation in Prognosis of Patients with Resectable Non–Small Cell Lung Cancer Jie Wang, 1,3 J. Jack Lee, 2 Luo Wang, 1 Diane D. Liu, 2 Charles Lu, 1 You-Hong Fan, 1 Waun Ki Hong, 1 and Li Mao 1 1 Department of Thoracic/Head and Neck Medical Oncology, and the 2 Department of Biostatistics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas; and 3 Department of Oncology, Beijing University School of Oncology, Beijing Cancer Hospital, Beijing Institution of Cancer Research, Beijing, China ABSTRACT The p16 INK4a and RASSF1A are tumor suppressor genes frequently inactivated by de novo promoter hyperm- ethylation in non-small cell lung cancer. We studied 119 patients with non-small cell lung cancer (70 stage I/II and 49 stage IIIA) who had undergone surgery with curative intent. The p16 INK4a and RASSF1A promoter methylation statuses were determined by methylation-specific PCR. Statistical analyses, all two-sided, were performed to determine the prognostic effect of hypermethylation on various clinical parameters. Hypermethylation of the p16 INK4a and RASSF1A promoters was found in 58 (49%) and 46 (39%) tumors, respectively, and 30 tumors (25%) exhibited hyper- methylation of both gene promoters. In patients with stage I/II tumors, only p16 INK4a promoter hypermethylation was associated with a poor 5-year overall survival rate (P  0.002). In patients with stage IIIA disease, however, RASSF1A promoter hypermethylation was a stronger pre- dictor of a poor 5-year overall survival rate (P < 0.0001) than p16 INK4a promoter hypermethylation. Among the 49 patients with stage IIIA tumors, 16 (89%) of the 18 patients whose tumors showed RASSF1A promoter hypermethyla- tion died within 3 years after surgery, as compared with only 12 (39%) of the 31 patients whose tumors had no RASSF1A promoter hypermethylation (P < 0.0001). Multivariate anal- ysis indicated that RASSF1A promoter hypermethylation was the stronger independent predictor for survival in pa- tients with locally advanced non-small cell lung cancer. Our results indicate that p16 INK4a promoter hypermethylation predicts a poor 5-year survival rates for patients with re- sectable non-small cell lung cancer, particularly for those with early stage tumors, whereas RASSF1A promoter hy- permethylation is a profound prognostic predictor for pa- tients with locally advanced non-small cell lung cancer, sug- gesting an important role of RASSF1A in non-small cell lung cancer progression. INTRODUCTION Non–small cell lung cancer constitutes 80% of all primary lung cancers, which are the leading cause of cancer-related death in both men and women in the United States (1). Despite advances in the treatment of the disease over the past two decades, the prognosis of patients with non-small cell lung cancer has improved only modestly, with the 5-year overall survival rate increasing from 11% in the 1970s to 15% in the late 1990s (2). Patients with early stage non-small cell lung cancer generally have a better survival than those with ad- vanced-stage tumors. For example, patients with stage I non- small cell lung cancer are expected to have an approximate 60% 5-year overall survival rate after surgical resection of their primary tumors, whereas those with stage IIIA disease have an estimated 25% 5-year overall survival rate after surgery fol- lowed by radiation with or without chemotherapy. Biological features of non-small cell lung cancer are de- termined by underlying molecular alterations of the tumors, including inactivation of the tumor suppressor genes (3–5). Besides mutations and deletions of genes, it is now clear that de novo promoter hypermethylation is a common mechanism to inactivate tumor suppressor genes (6 – 8). The p16 INK4a tumor suppressor gene located on 9p21 encodes a cyclin-dependent kinase inhibitor important for G 1 cell cycle arrest (9, 10). Promoter hypermethylation of the gene has been observed fre- quently early in lung carcinogenesis, including in individuals exposed to tobacco carcinogens but without evidence of cancer (11–13). The RASSF1A tumor suppressor gene is located at 3p21, a region frequently deleted in non-small cell lung cancer (14). Another common mechanism to inactivate RASSF1A is promoter hypermethylation of the gene (15–17). RASSF1A has been shown to bind to the Ras-GTP binding protein Nore1, consistent with its role as a negative effector of Ras oncoprotein (18). In contrast to p16 INK4a , which is inactivated early in lung carcinogenesis (13, 19), hypermethylation of the RASSF1A pro- moter occurs relatively late (20), suggesting RASSF1A might be important in non-small cell lung cancer progression. Because of the difference in timing between methylation of the p16 INK4a and RASSF1A promoters in lung carcinogenesis, we wanted to determine the clinical impact of these abnormal- Received 4/4/04; revised 5/28/04; accepted 6/15/04. Grant support: Department of Defense Grant DAMD17-01-1-0689-1 and National Cancer Institute Grants PO1 CA91844, UO1 CA86390, and P30 CA 16672. J. Wang is a recipient of the Oncology Research Faculty Development Award, National Cancer Institute (USA). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Li Mao, Department of Thoracic/Head and Neck Medical Oncology, Unit 432, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 745-6363; Fax: (713) 796-8655; E-mail: lmao@ mdanderson.org. ©2004 American Association for Cancer Research. 6119 Vol. 10, 6119 – 6125, September 15, 2004 Clinical Cancer Research Research. on May 25, 2020. © 2004 American Association for Cancer clincancerres.aacrjournals.org Downloaded from