Gene Promoter Hypermethylation in Tumors and Lymph Nodes of Stage I Lung Cancer Patients Susan V. Harden, Yutaka Tokumaru, William H. Westra, Steven Goodman, Steven A. Ahrendt, Stephen C. Yang, and David Sidransky 1 Department of Otolaryngology-Head and Neck Surgery, Division of Head and Neck Cancer Research [S. V. H., Y. T., W. H. W., D. S.] and Departments of Pathology [W. H. W.], Oncology/Biostatistics [S. G.], and Thoracic Surgery [S. C. Y.], The Johns Hopkins University School of Medicine, Baltimore, Maryland 21206-2198, and Department of Surgery, University of Rochester Medical Center, Rochester, New York [S. A. A.] ABSTRACT Promoter hypermethylation is an important pathway for repression of gene transcription in cancer cells and a promising marker for cancer detection. We tested five gene promoters [CDKN2A (p16), O 6 -methylguanine-DNA-meth- yltransferase, glutathione S-transferase P1 (GSTP1), ade- nomatous polyposis coli (APC), and death-associated pro- tein kinase (DAPK)] by real-time methylation-specific PCR in primary tumors from 90 stage I lung cancer patients for aberrant DNA methylation. We then used the presence of tumor methylation as a marker to investigate the presence of occult metastasis in corresponding histologically negative lymph nodes. Of the primary tumors, 73 of 90 (81%) dis- played promoter hypermethylation in at least one of the genes studied: 17% (15 of 90) at p16 (CDKN2A); 16% (14 of 90) at O 6 -methylguanine-DNA-methyltransferase; 8% (7 of 90) at GSTP1; 72% (65 of 90) at APC; and 17% (15 of 90) at DAPK. Squamous histology was predictive of worse overall survival (P  0.074, log-rank test). APC methylation and GSTP1 methylation in the primary tumor were both corre- lated with nonsquamous histology (P  0.02 and P  0.01 likelihood ratio respectively). The presence of both APC methylation and DAPK methylation in the primary tumor predicted a worse outcome, with 7 of 13 (54%) deaths in this group compared with 21 of 77 (27%) deaths in cases without both genes methylated (P  0.229, log-rank test). The same methylation pattern was detected in DNA from at least one of the corresponding lymph nodes in 11 of 73 (15%) cases. Five of 11 (45%) patients with occult metastasis detected by methylation analysis have died compared with 17 of 62 (27%) patients with negative lymph nodes, although sur- vival analysis did not reach statistical significance (P  0.632, log-rank test). Promoter hypermethylation is common in lung cancer and represents a promising marker for the molecular staging of lung cancer patients. Although this study showed important trends, a larger prospective study is required to better understand the value of methylation anal- ysis in detecting occult metastasis. INTRODUCTION Lung cancer is the leading cause of cancer-related death in the United States (1). Surgical resection is the most effective form of treatment for NSCLC, 2 although at the time of diagno- sis, only one-third of patients will have early-stage disease amenable to curative surgery. In addition, a large percentage of patients with early-stage disease undergoing surgical resection ultimately die of recurrent disease, suggesting the presence of occult metastatic disease at the time of diagnosis (2). Several studies have used a variety of molecular techniques in a range of different tumors to detect occult metastatic spread (3– 6). We have previously looked for occult metastasis to lymph nodes in lung cancer using p53 and K-ras mutations as markers of tumor DNA (7). However, the presence of p53 mutation in the primary tumor was such a strong predictor of poor prognosis that detec- tion of occult spread did not appear to affect outcome. Silencing of tumor suppressor or other cancer-associated genes by hypermethylation of CpG islands within the promoter and/or 5'-regions of many genes is a common feature of human cancer and is often associated with a transcriptional block and loss of the relevant protein (8 –11). Moreover, a number of gene promoters have been found to be hypermethylated in NSCLC (12, 13). In addition to the functional implications of gene inactivation in tumor development, the presence of epigenetic methylation has been shown to be useful as a molecular target for tumor cell detection in serum, plasma, and bronchioaveolar lavage fluid from NSCLC patients (12, 14, 15). We sought to detect occult metastasis by using a panel of genes known to be frequently hypermethylated in lung tumors. To detect the pres- ence of neoplastic DNA with a sensitivity of 1 cell in 1000 normal cells, we used real-time QMSP. This PCR approach is more sensitive than conventional PCR and more specific due to the use of an internally binding, fluorogenic hybridization probe Received 9/9/02; revised 12/27/02; accepted 1/10/03. 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. 1 To whom requests for reprints should be addressed, at Department of Otolaryngology-Head and Neck Surgery, Division of Head and Neck Cancer Research, The Johns Hopkins University School of Medicine, 818 Ross Research Building, 720 Rutland Avenue, Baltimore, MD 21205-2196. Phone: (410) 502-5153; Fax: (410) 614-1411; E-mail: dsidrans@jhmi.edu. 2 The abbreviations used are: NSCLC, non-small cell lung cancer; MGMT, O 6 -methylguanine-DNA-methyltransferase; GSTP1, glutathi- one S-transferase P1; APC, adenomatous polyposis coli; DAPK, death- associated protein kinase; MSP, methylation-specific PCR; QMSP, quantitative MSP; FAM, 6 carboxyfluoroscein; TAMRA, 5(6) carboxy- tetramethylrhodamine. 1370 Vol. 9, 1370 –1375, April 2003 Clinical Cancer Research Cancer Research. on October 23, 2021. © 2003 American Association for clincancerres.aacrjournals.org Downloaded from