Riichiroh Maruyama

Smoke exposure, histologic type and geography-related differences in the methylation profiles of non-small cell lung cancer.

Aberrant methylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of lung cancers. There are major smoke exposure, histology, geography and gender‐related changes in non‐small cell lung cancer (NSCLC). We investigated smoking‐related, histologic, geographic and gender differences in the methylation profiles of resected NSCLCs. We examined 514 cases of NSCLC and 84 corresponding nonmalignant lung tissues from 4 countries (USA, Australia, Japan and Taiwan) for the methylation status of 7 genes known to be frequently methylated in lung cancers [p16, RASSF1A (RAS association domain family 1), APC, RARβ, CDH13, MGMT and GSTP1]. Multivariate analyses were used for data analysis. Adenocarcinoma was the major histologic type in women and never smokers; analyses that involved smoke exposure and gender were limited to this histology. Our major findings are a) methylation status of any single gene was largely independent of methylation status of other genes; b) the rates of methylation of p16 and APC and the mean Methylation Index (MI), a reflection of the overall methylation status, were significantly higher in ever smokers than in never smokers; c) the mean MI of tumors arising in former smokers was significantly lower than the mean of current smokers; d) the methylation rates of APC, CDH13 and RARβ were significantly higher in adenocarcinomas than in squamous cell carcinomas; e) methylation rates of MGMT and GSTP1 were significantly higher in the USA and Australian cases than in those from Japan and Taiwan; and (f) no significant gender‐related differences in methylation patterns were noted. Our findings demonstrate important smoke exposure, histologic type and geography‐related differences in the methylation profiles of NSCLC tumors.

Aberrant promoter methylation and silencing of the RASSF1A gene in pediatric tumors and cell lines

Aberrant promoter methylation of tumor suppressor genes has not been fully investigated in pediatric tumors. Therefore, we examined the methylation status of nine genes (p16INK4A, MGMT, GSTP1, RASSF1A, APC, DAPK, RARβ, CDH1 and CDH13) in 175 primary pediatric tumors and 23 tumor cell lines using methylation-specific PCR. We studied the major forms of pediatric tumors – Wilms tumor, neuroblastoma, hepatoblastoma, medulloblastoma, rhabdomyosarcoma, osteosarcoma, Ewings sarcoma, retinoblastoma and acute leukemia. The most frequently methylated gene in both primary tumors and cell lines was RASSF1A (40, 86%, respectively). However, the rates of RASSF1A methylation in individual tumor types varied from 0 to 88%. RASSF1A methylation was tumor specific and was absent in adjacent non-malignant tissues. Methylation of the other genes was relatively rare in tumors and non-malignant tissues (less than 5%). Neuroblastoma patients with methylation of RASSF1A were significantly older than patients without methylation (P=0.008). There was no relationship between methylation status and other clinico-pathologic parameters. We treated six cell lines lacking RASSF1A mRNA with 5-aza-2′deoxycytidine to examine the relationship between methylation and transcriptional silencing. In five of six cell lines, restoration of RASSF1A mRNA was confirmed by RT–PCR. Our findings indicate that aberrant promoter methylation of RASSF1A may contribute to the pathogenesis of many different forms of pediatric tumors.

Dose effect of smoking on aberrant methylation in non-small cell lung cancers.

Shinichi TOYOOKA, Makoto SUZUKI, Toshihide TSUDA, Kiyomi O. TOYOOKA, Riichiroh MARUYAMA, Kazunori TSUKUDA, Yasuro FUKUYAMA, Toshihiko IIZASA, Takehiko FUJISAWA, Nobuyoshi SHIMIZU, John D. MINNA and Adi F. GAZDAR* Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Cancer and Thoracic Surgery, Okayama University Medical School, Okayama, Japan Department of Hygiene and Preventive Medicine, Okayama University Medical School, Okayama, Japan Department of Surgery 2, Kyushu University Faculty of Medicine, Fukuoka, Japan Department of Thoracic Surgery, Chiba University Medical School, Chiba, Japan Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA

Molecular detection of noninvasive and invasive bladder tumor tissues and exfoliated cells by aberrant promoter methylation of laminin-5 encoding genes.

Laminin-5 (LN5) anchors epithelial cells to the underlying basement membrane, and it is encoded by three distinct genes: LAMA3, LAMB3, and LAMC2. To metastasize and grow, cancer cells must invade and destroy the basement membrane. Our previous work has shown that epigenetic inactivation is a major mechanism of silencing LN5 genes in lung cancers. We extended our methylation studies to resected bladder tumors (n = 128) and exfoliated cell samples (bladder washes and voided urine; n = 71) and correlated the data with clinicopathologic findings. Nonmalignant urothelium had uniform expression of LN5 genes and lacked methylation. The methylation frequencies for LN5 genes in tumors were 21–45%, and there was excellent concordance between methylation in tumors and corresponding exfoliated cells. Methylation of LAMA3 and LAMB3 and the methylation index were correlated significantly with several parameters of poor prognosis (tumor grade, growth pattern, muscle invasion, tumor stage, and ploidy pattern), whereas methylation of LAMC2 and methylation index were associated with shortened patient survival. Of particular interest, methylation frequencies of LAMA3 helped to distinguish invasive (72%) from noninvasive (12%) tumors. These results suggest that methylation of LN5 genes has potential clinical applications in bladder cancers.

The relationship between aberrant methylation and survival in non-small-cell lung cancers

The present study examined the relationship between methylation of five genes (p16INK4a, RASSF1A, APC, RARβ and CDH13) and patient survival in 351 cases of surgically resected lung cancers. While there was no relationship between the other genes and survival, p16INK4a methylation was significantly related to unfavourable prognosis in lung adenocarcinomas.