Many tumour types have been reported to have deletion of 9p21 (refs 1-6). A candidate target suppressor gene, p16 (p16INK4a/MTS-1/CDKN2), was recently identified within the commonly deleted region in tumour cell lines. An increasing and sometimes conflicting body of data has accumulated regarding the frequency of homozygous deletion and the importance of p16 in primary tumours. We tested 545 primary tumours by microsatellite analysis with existing and newly cloned markers around the p16 locus. We have now found that small homozygous deletions represent the predominant mechanism of inactivation at 9p21 in bladder tumours and are present in other tumour types, including breast and prostate cancer. Moreover, fine mapping of these deletions implicates a 170 kb minimal region that includes p16 and excludes p15.

Renal Cell Carcinoma (RCC) has the highest mortality rate of the genitourinary cancers and the incidence of RCC has risen steadily. If detected early, RCC is curable by surgery although a minority are at risk of recurrence. Increasing incidental detection and an ageing population has led to active surveillance as an option for patients with small renal masses. RCC is heterogeneous and comprises several histological cell types with different genetics, biology and behavior. The identification of the genes predisposing to inherited syndromes with RCC has provided much of our knowledge of the molecular basis of early sporadic RCC. Many of the oncogenes and tumor suppressor genes that are mutated leading to pathway dysregulation in RCC remain to be elucidated. Global studies of copy number, gene sequencing, gene expression, miRNA expression and gene methylation in primary RCC will lead towards this goal. The natural history of RCC indicated by candidate precursor lesions, multifocal or bilateral disease, growth rate of small renal masses under surveillance, and high risk populations provide insight into the behavior of this disease. The use of molecular markers for early detection and prognosis merits more attention with ongoing advances in omics technologies. This review focuses on early RCC, that is disease confined within the renal capsule.

less steel tube attached to a tungsten microwire recording electrode while TAN activity was recorded 0.6 mm beyond the outlet of the tube.

Loss of heterozygosity (LOH) studies have suggested that somatic mutations of a tumour suppressor gene or genes on chromosome 3p are a critical event in the pathogenesis of non-familial renal cell carcinoma (RCC). Germline mutations of the von Hippel-Lindau (VHL) disease gene predispose to early onset and multifocal clear cell renal cell carcinoma, and the mechanism of tumorigenesis in VHL disease is consistent with a one-hit mutation model. To investigate the role of somatic VHL gene mutations in non-familial RCC, we analysed 99 primary RCC for VHL gene mutations by SSCP and heteroduplex analysis. Somatic VHL gene mutations were identified in 30 of 65 (46%) sporadic RCC with chromosome 3p allele loss and one of 34 (3%) tumours with no LOH for chromosome 3p. The VHL gene mutations were heterogeneous (17 frameshift deletions, eight missense mutations, four frameshift insertions, one nonsense and one splice site mutation), but no mutations were detected in the first 120 codons of cloned coding sequence. Most RCCs with somatic VHL mutations (23 of 27 (85%) informative cases) had chromosome 3p25 allele loss in the region of the VHL gene so that both alleles of the VHL gene had been inactivated as expected from a two-hit model of tumorigenesis. Detailed histopathology was available for 59 of the tumours investigated: 18 of 43 (42%) RCC with a clear cell appearance had a somatic VHL gene mutation but none of 16 non-clear cell RCC (eight chromophilic, three chromophobe and five oncocytoma) (chi2 = 7.77, P < 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)

Because existing surgical and management methods can consistently cure only early-stage ovarian cancer, novel strategies for early detection are required. Silencing of tumor suppressor genes such as p16 INK4a , VHL, and hMLH1 have established promoter hypermethylation as a common mechanism for tumor suppressor inactivation in human cancer and as a promising target for molecular detection in bodily fluids. Using sensitive methylation-specific PCR, we screened matched tumor, preoperative serum or plasma, and peritoneal fluid (washes or ascites) DNA obtained from 50 patients with ovarian or primary peritoneal tumors for hypermethylation status of the normally unmethylated BRCA1 and RAS association domain family protein 1A tumor suppressor genes. Hypermethylation of one or both genes was found in 34 tumor DNA (68%). Additional examination of one or more of the adenomatous polyposis coli, p14 ARF , p16INK4a , or death associated protein-kinase tumor suppressor genes revealed hypermethylation in each of the remaining 16 tumor DNA, which extended diagnostic coverage to 100%. Hypermethylation was observed in all histologic cell types, grades, and stages of ovarian tumor examined. An identical pattern of gene hypermethylation was found in the matched serum DNA from 41 of 50 patients (82% sensitivity), including 13 of 17 cases of stage I disease. Hypermethylation was detected in 28 of 30 peritoneal fluid DNA from stage IC-IV patients, including 3 cases with negative or atypical cytology. In contrast, no hypermethylation was observed in nonneoplastic tissue, peritoneal fluid, or serum from 40 control women (100% specificity). We conclude that promoter hypermethylation is a common and relatively early event in ovarian tumorigenesis that can be detected in the serum DNA from patients with ovary-confined (stage IA or B) tumors and in cytologically negative peritoneal fluid. Analysis of tumor-specific hypermethylation in serum DNA may enhance early detection of ovarian cancer.

A new tumor suppressor gene PTEN/MMAC1 was recently isolated at chromosome 10q23 and found to be inactivated by point mutation or homozygous deletion in glioma, prostate and breast cancer. PTEN/MMAC1 was also identi®ed as the gene predisposing to Cowden disease, an autosomal dominant cancer predisposition syndrome associated with an increased risk of breast, skin and thyroid tumors and occasional cases of other cancers including bladder and renal cell carcinoma. We screened 345 urinary tract cancers by microsatellite analysis and found chromosome 10q to be deleted in 65 of 285 (23%) bladder and 15 of 60 (25%) renal cell cancers. We then screened the entire PTEN/MMAC1 coding region for mutation in 25 bladder and 15 renal cell primary tumors with deletion of chromosome 10q. Two somatic point mutations, a frameshift and a splicing variant, were found in the panel of bladder tumors while no mutation was observed in the renal cell carcinomas. To screen for homozygous deletion, we isolated two polymorphic microsatellite repeats from genomic BAC clones containing the PTEN/MMAC1 gene. Using these new informative markers, we identi®ed apparent retention at the gene locus indicative of homozygous deletion of PTEN/MMAC1 in four of 65 bladder and 0 of 15 renal cell tumors with LOH through chromosome 10q. Identi®cation of the second inactivation event in six bladder tumors with LOH of 10q implies that the PTEN/MMAC1 gene is occasionally involved in bladder tumorigenesis. However, the low frequency of biallelic inactivation suggests that either PTEN/MMAC1 is inactivated by other mechanisms or it is not the only target of chromosome 10q deletion in primary bladder and renal cell cancer.

Purpose: Breast cancer is the most common malignancy in American women and the second leading cause of death from cancer. The genetic and epigenetic alterations that initiate and drive cancer can be used as targets for detection of neoplasia in bodily fluids. Tumor cell-specific aberrant promoter hypermethylation can be detected in nipple aspirate and ductal lavage from breast cancer patients. In this study, we examine serum, a more readily accessible bodily fluid known to contain neoplastic DNA from individuals with cancer, for methylation-based detection of breast neoplasia.Experimental Design: We examined the promoter methylation status of three normally unmethylated biologically significant cancer genes, RAS association domain family protein 1A (RASSF1A), adenomatous polyposis coli (APC), and death-associated protein kinase (DAP-kinase), by sensitive methylation-specific PCR in 34 breast tumor and paired preoperative serum DNA. The 34 patients comprised 7 ductal carcinoma in situ (CIS), 3 lobular CIS, 5 stage I and 15 stage II to IV invasive ductal carcinomas, and 4 invasive lobular carcinomas. Normal and benign tissue and serum control DNA were also examined to determine the specificity of hypermethylation.Results: Hypermethylation of one or more genes was found in 32 of 34 (94%) breast tumor DNA. APC was hypermethylated in 15 of 34 (47%), RASSF1A in 22 of 34 (65%), and DAP-kinase in 17 of 34 (50%) tumors. Twentysix (76%) of the corresponding serum DNA were positive for promoter hypermethylation, including ductal CIS, lobular CIS, stage I disease, and lobular carcinoma patients. No hypermethylation of APC, RASSF1A, or DAP-kinase was observed in serum DNA from normal healthy women and patients with inflammatory breast disease or nonneoplastic breast tissue specimens. A gene unmethylated in the tumor DNA was always found to be unmethylated in the matched serum DNA (100% specificity).Conclusions: Tumor cell specific promoter hypermethylation of APC, RASSF1A, and DAP-kinase is present in ductal CIS, lobular CIS, and all grades and stages of invasive breast cancer. Hypermethylation can be detected by methylation-specific PCR analysis in serum DNA from patients with preinvasive and early-stage breast cancer amenable to cure. If confirmed in additional studies, hypermethylation-based screening of serum, a readily accessible bodily fluid, may enhance early detection of breast cancer.

Purpose: Promoter hypermethylation is an important mechanism of inactivation of tumor suppressor genes in cancer cells. Kidney tumors are heterogeneous in their histology, genetics, and clinical behavior. To gain insight into the role of epigenetic silencing of tumor suppressor and cancer genes in kidney tumorigenesis, we determined a hypermethylation profile of kidney cancer.Experimental Design: We examined the promoter methylation status of 10 biologically significant tumor suppressor and cancer genes in 100 kidney tumors (50 clear cell, 20 papillary, 6 chromophobe, 5 collecting duct, 5 renal cell unclassified, 7 oncocytoma, 6 transitional cell carcinomas of the renal pelvis, and 1 Wilms' tumor) by methylationspecific PCR. The hypermethylation profile was examined with regard to clinicopathological characteristics of the kidney cancer patients.Results: Hypermethylation of one or more genes was found in 93 (93%) of 100 tumors. A total of 33% of kidney tumors had one gene, 35% two genes, 14% three genes, and 11% four or more genes hypermethylated. The frequency of hypermethylation of the 10 genes in the 100 tumor DNAs was VHL 8% (all clear cell), p16INK4a 10%, p14 ARF 17%, APC 14%, MGMT 7%, GSTP1 12%, RAR␤2 12%, RASSF1A 45%, E-cadherin 11%, and Timp-3 58%. Hypermethylation was observed in all of the histological cell types and grades and stages examined. No hypermethylation was observed in specimens of normal kidney or ureteral tissue from 15 patients. Hypermethylation of VHL was specific to clear cell tumors. RASSF1A methylation was detected at a significantly higher frequency in papillary renal cell tumors and in high-grade tumors of all cell types. MGMT methylation was more frequent in nonsmokers. Simultaneous methylation of five or more genes was observed in 3 (3%) of 100 tumors and may indicate a methylator phenotype in kidney cancer. In addition, the CpG island in the promoter of the fumarate hydratase (FH) tumor suppressor gene was bisulfite sequenced and was found to be unmethylated in 15 papillary renal tumors.Conclusions: Promoter hypermethylation is common, can occur relatively early, may disrupt critical pathways, and, thus, likely plays an important role in kidney tumorigenesis. A hypermethylation profile may be useful in predicting a patient's clinical outcome and provide molecular markers for diagnostic and prognostic approaches to kidney cancer.