A Gene Hypermethylation Profile of Human Cancer 1

We are in an era where the potential exists for deriving comprehensive profiles of DNA alterations characterizing each form of human cancer. Such profiles would provide invaluable insight into mechanisms underlying the evolution of each tumor type and will provide molecular markers, which could radically improve cancer detection. To date, no one type of DNA change has been defined which accomplishes this purpose. Herein, by using a candidate gene approach, we show that one category of DNA alteration, aberrant methylation of gene promoter regions, can enormously contribute to the above goals. We have now analyzed a series of promoter hypermethylation changes in 12 genes (p16 , p15, p14, p73,APC, BRCA1, hMLH1, GSTP1,MGMT, CDH1, TIMP3, and DAPK), each rigorously characterized for association with abnormal gene silencing in cancer, in DNA from over 600 primary tumor samples representing 15 major tumor types. The genes play known important roles in processes encompassing tumor suppression, cell cycle regulation, apoptosis, DNA repair, and metastastic potential. A unique profile of promoter hypermethylation exists for each human cancer in which some gene changes are shared and others are cancer-type specific. The hypermethylation of the genes occurs independently to the extent that a panel of three to four markers defines an abnormality in 70–90% of each cancer type. Our results provide an unusual view of the pervasiveness of DNA alterations, in this case an epigenetic change, in human cancer and a powerful set of markers to outline the disruption of critical pathways in tumorigenesis and for derivation of sensitive molecular detection strategies for virtually every human tumor type. Unfolding a Gene Hypermethylation Profile of Human Cancer Mutations in individual genes have outlined critical aspects of tumorigenesis, including disruption of the Rb/p16, APC/bcatenin/Tcf, and p53/p14/MDM2 pathways. Global genome screens, such as for repeat microsatellite sequence alterations and for gene expression changes by serial analysis of gene expression (1) or cDNA microarrays (2), have also provided important information about molecular events important for tumorigenesis. Despite these above studies, no detection of any one type of DNA alteration, either by candidate gene approach or by genomic screening techniques, has provided universal markers for all tumor types. In the present study, we demonstrate how one single type of DNA alteration, aberrant methylation of gene promoters, can point to pathways disrupted in every type of cancer and can provide markers for sensitive detection of virtually all tumor types. The growing list of genes inactivated by promoter region hypermethylation provides an opportunity to examine the patterns of inactivation of such genes among different tumors (3, 4). Recently, a global pattern of methylation events in tumors using restriction landmark genomic scanning was reported (5). We have instead used a candidate gene approach. We have studied multiple key cancer genes undergoing epigenetic inactivation in a large set of primary human tumors with the aim of obtaining a map of this alteration in malignant transformation. A total of 12 genes, including well-characterized tumor suppressor genes (p16 , p15, p14, p73,APC, and BRCA1), DNA repair genes (hMLH1, GSTP1, andMGMT), and genes related to metastasis and invasion ( CDH1, TIMP3, andDAPK) were included in the study. Each of these genes possesses a CpG island in their 59region which is unmethylated in corresponding normal tissues, as expected for a typical CpG island (6). We and others have shown, in previous studies for such genes in individual tumor types, that when these CpG islands are hypermethylated in cancer cells, expression of the corresponding gene is silenced and the silencing can be partially relieved by demethylation of the promoter region (3, 4). However, the scope of these changes has not been easy to observe by examining each study individually. The primary tumor samples examined in the present study constitute over 600 specimens that cover 15 major tumor types (colon, stomach, pancreas, liver, kidney, lung, head and neck, breast, ovary, endometrium, kidney, bladder, brain, and leukemia and lymphomas). The profile of promoter hypermethylation for each of the above genes in each tumor type is shown in Fig. 1. Important features of the data are as follows. First, one or more of the genes studied is hypermethylated in every tumor type. However, the profile of promoter hypermethylation for the genes differs for each cancer type, providing a tumor-type and gene-specific profile. Some genes, such as the cell cycle inhibitor p16, are hypermethylated across many tumor types including colorectal, lung, and breast carcinomas as previously described (7–9). This alteration reflects the widespread contribution of disruptions of thecyclinD-Rbcell cycle control pathway in human cancer. The extent of p16 INK4a epigenetic silencing reported in the literature expands over the neoplasms described in Fig. 1 and also include bladder (10) and cervical tumors (11) or melanomas (12) and