Purpose: To provide a comprehensive, thorough analysis of somatic mutation and promoter hypermethylation of the von Hippel-Lindau (VHL) gene in the cancer genome, unique to clear cell renal cancer (ccRCC). Identify relationships between the prevalence of VHL gene alterations and alteration subtypes with patient and tumor characteristics. Experimental Design: As part of a large kidney cancer case-control study conducted in Central Europe, we analyzed VHL mutations and promoter methylation in 205 well-characterized, histologically confirmed patient tumor biopsies using a combination of sensitive, high-throughput methods (endonuclease scanning and Sanger sequencing) and analysis of 11 CpG sites in the VHL promoter. Results: We identified mutations in 82.4% of cases, the highest VHL gene mutation prevalence reported to date. Analysis of 11 VHL promoter CpG sites revealed that 8.3% of tumors were hypermethylated and all were mutation negative. In total, 91% of ccRCCs exhibited alteration of the gene through genetic or epigenetic mechanisms. Analysis of patient and tumor characteristics revealed that certain mutation subtypes were significantly associated with Fuhrman nuclear grade, metastasis, node positivity, and self-reported family history of RCC. Conclusion: Detection of VHL gene alterations using these accurate, sensitive, and practical methods provides evidence that the vast majority of histologically confirmed ccRCC tumors possess genetic or epigenetic alteration of the VHL gene and support the hypothesis that VHL alteration is an early event in ccRCC carcinogenesis. These findings also indicate that VHL molecular subtypes can provide a sensitive marker of tumor heterogeneity among histologically similar ccRCC cases for etiologic, prognostic, and translational studies.Considerable progress has been made in understanding the genetic basis of kidney cancer (1, 2). The susceptibility genes associated with several forms of inherited renal cell cancer (RCC) have been identified by rigorous analysis of families using genetic linkage analysis and positional cloning (3 -7). The most common subtype of RCC is the conventional clear cell type (ccRCC), which accounts for f75% of cases. In both familial and sporadic ccRCC, allelic inactivation of the von

Renal tumor heterogeneity studies have utilized the von Hippel-Lindau VHL gene to classify disease into molecularly defined subtypes to examine associations with etiologic risk factors and prognosis. The aim of this study was to provide a comprehensive analysis of VHL inactivation in clear cell renal tumors (ccRCC) and to evaluate relationships between VHL inactivation subgroups with renal cancer risk factors and VHL germline single nucleotide polymorphisms (SNPs). VHL genetic and epigenetic inactivation was examined among 507 sporadic RCC/470 ccRCC cases using endonuclease scanning and using bisulfite treatment and Sanger sequencing across 11 CpG sites within the VHL promoter. Case-only multivariate analyses were conducted to identify associations between alteration subtypes and risk factors. VHL inactivation, either through sequence alterations or promoter methylation in tumor DNA, was observed among 86.6% of ccRCC cases. Germline VHL SNPs and a haplotype were associated with promoter hypermethylation in tumor tissue (OR = 6.10; 95% CI: 2.28–16.35, p = 3.76E-4, p-global = 8E-5). Risk of having genetic VHL inactivation was inversely associated with smoking due to a higher proportion of wild-type ccRCC tumors [former: OR = 0.70 (0.20–1.31) and current: OR = 0.56 (0.32–0.99); P-trend = 0.04]. Alteration prevalence did not differ by histopathologic characteristics or occupational exposure to trichloroethylene. ccRCC cases with particular VHL germline polymorphisms were more likely to have VHL inactivation through promoter hypermethylation than through sequence alterations in tumor DNA, suggesting that the presence of these SNPs may represent an example of facilitated epigenetic variation (an inherited propensity towards epigenetic variation) in renal tissue. A proportion of tumors from current smokers lacked VHL alterations and may represent a biologically distinct clinical entity from inactivated cases.

PurposeLeukocyte global DNA methylation levels are currently being considered as biomarkers of cancer susceptibility and have been associated with risk of several cancers. In this study, we aimed to examine the association between long interspersed nuclear elements (LINE-1) methylation levels, as a biomarker of global DNA methylation in blood cell DNA, and renal cell cancer risk.Experimental DesignLINE-1 methylation of bisulfite-converted genomic DNA isolated from leukocytes was quantified by pyrosequencing measured in triplicate, and averaged across 4 CpG sites. A total of 328 RCC cases and 654 controls frequency-matched(2∶1) on age(±5years), sex and study center, from a large case-control study conducted in Central and Eastern Europe were evaluated.ResultsLINE-1 methylation levels were significantly higher in RCC cases with a median of 81.97% (interquartile range[IQR]: 80.84–83.47) compared to 81.67% (IQR: 80.35–83.03) among controls (p = 0.003, Wilcoxon). Compared to the lowest LINE-1 methylation quartile(Q1), the adjusted ORs for increasing methylation quartiles were as follows: OR(Q2) = 1.84(1.20−2.81), OR(Q3) = 1.72(1.11−2.65) and OR(Q4) = 2.06(1.34−3.17), with a p-trend = 0.004. The association was stronger among current smokers (p-trend<0.001) than former or never smokers (p-interaction = 0.03). To eliminate the possibility of selection bias among controls, the relationship between LINE-1 methylation and smoking was evaluated and confirmed in a case-only analysis, as well.ConclusionsHigher levels of LINE-1 methylation appear to be positively associated with RCC risk, particularly among current smokers. Further investigations using both post- and pre-diagnostic genomic DNA is warranted to confirm findings and will be necessary to determine whether the observed differences occur prior to, or as a result of carcinogenesis.

Previous studies investigated the role of vitamin D intake and cancer risk. The kidney is a major organ for vitamin D metabolism, activity, and calcium homeostasis; therefore, it was hypothesized that dietary vitamin D intake and polymorphisms in the vitamin D receptor (VDR) gene may modify renal cell carcinoma (RCC) risk. Three common VDR gene polymorphisms (BsmI, FokI, TaqI) were evaluated among 925 RCC cases and 1192 controls enrolled in a hospital-based case-control study conducted in Central and Eastern Europe. Overall associations with RCC risk were not observed; however, subgroup analyses revealed associations after stratification by median age of diagnosis and family history of cancer. Among subjects over 60 yr, reduced risks were observed among carriers of the f alleles in the FokI single-nucleotide polymorphism (SNP) (odds ratio [OR] = 0.61 for Ff and OR = 0.74 for ff genotypes) compared to subjects with the FF genotype (P trend = 0.04; P interaction = 0.004). Subjects with the BB BsmI genotype and a positive family history of cancer had lower risk compared to subjects with the bb allele (OR = 0.60; 95% CI: 0.33-1.1; P trend = 0.05). Genotype associations with these subgroups were not modified when dietary sources of vitamin D or calcium were considered. Additional studies of genetic variation in the VDR gene are warranted.

In a case -control study of kidney cancer in four central European countries, with 1097 incident cases and 1476 controls, we found an increased risk for self-reported hypertension and for obesity. Additional unknown risk factors are likely to be responsible for the high rates of kidney cancer in this region. British Journal of Cancer (2008Cancer ( ) 99, 1912Cancer ( -1915 (Murai and Oya, 2004). Smoking is an established risk factor, although the increase in risk is moderate (IARC, 2004). Other possible risk factors are hypertension and obesity, with potentially differing risks for men and women (Shapiro et al, 1999;Bergstrom et al, 2001). To examine the risk factors for kidney cancer in central Europe, we conducted a large multicenter case -control study in Czech Republic, Poland, Russia, and Romania. We present here data on the role of smoking, hypertension, and body mass index (BMI) in this high-risk population. MATERIALS AND METHODSThis hospital-based case -control study was conducted in seven centres (Moscow (Russia), Bucharest (Romania), Lodz (Poland), and Prague, Olomouc, Ceske Budejovice, and Brno (Czech Republic)). A total of 1097 newly diagnosed, histologically confirmed, renal parenchymal cancers (ICD-O-2 code C64) between 20 and 79 years of age were recruited during August 1999 and January 2003. Trained medical staff reviewed medical records to extract relevant diagnostic information, including date and method of diagnosis, histologic type, tumour location, stage, and grade. Eligible controls (n ¼ 1476) were admitted to the same hospitals as the cases for conditions unrelated to smoking or genitourinary disorders (except for benign prostatic hyperplasia) between August 1998 and March 2003. No single disease made up more than 20% of the control group. Both cases and controls had to be residents of the study areas for at least 1 year. The response rate for cases ranged from 90 to 98.6% and for the controls, 90.3-96.1%.Trained interviewers used standardised questionnaires at all centres to elicit information on demographic background, smoking, alcohol drinking, dietary practices, height, weight, medical history, family history of cancer, residential history, and occupational history.Smoking status (never smoker, former smoker, current smoker) was defined as status 2 years before interview. Packyears were used as a measure of cumulative tobacco smoking and were calculated by the number of cigarettes smoked per day multiplied by years of smoking and divided by 20. History of hypertension was selfreported and a positive history was restricted to patients who reported being treated for hypertension. The weight in kilograms was ascertained for 2 years before the interview. Body mass index was calculated by dividing the weight by the square of the height in metres.Kidney cancer risks were estimated by odds ratios (ORs) and 95% confidence intervals (CIs) using logistic regression analysis, with adjustment for age (5-years interval), smoking (current, former, and never smokers), BMI in five categories (o25, 25...