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Drinking water arsenic exposure has been associated with increased bladder cancer susceptibility. Epidemiologic and experimental data suggest a co-carcinogenic effect of arsenic with exposure to DNA damaging agents, such as cigarette smoke. Recent evidence further supports the hypothesis that genetic variation in DNA repair genes can modify the arsenic-cancer relationship, possibly because arsenic impairs DNA repair capacity. We tested this hypothesis in a population-based study of bladder cancer with XRCC3, ERCC2 genotype/haplotype and arsenic exposure data on 549 controls and 342 cases. Individual exposure to arsenic was determined in toenail samples by neutron activation. Gene-environment interaction with arsenic exposure was observed in relation to bladder cancer risk for a variant allele of the double-strand break repair gene XRCC3 T241M (adjusted OR 2.8 (1.1-7.3)) comparing to homozygous wild type among those in the top arsenic exposure decile (interaction p-value 0.01). Haplotype analysis confirmed the association of the XRCC3 241. Thus, double-strand break repair genotype may enhance arsenic associated bladder cancer susceptibility in the U.S. population.

Purpose: The involvement of microRNAs in cancer and their potential as biomarkers of diagnosis and prognosis are becoming increasingly appreciated. We sought to identify microRNAs altered in head and neck squamous cell carcinoma (HNSCC) and to determine whether microRNA expression is predictive of disease., Experimental Design: RNA isolated from fresh-frozen primary tumors, fresh-frozen nondiseased head and neck epithelial tissues, and HNSCC cell lines was profiled for the expression of 662 microRNAs by microarray. The microRNAs that were both differentially expressed on the array and by quantitative reverse transcription-PCR were subsequently validated by quantitative reverse transcription-PCR using a total of 99 HNSCC samples and 14 normal epithelia., Results: A marked difference in microRNA expression pattern was observed between tumors and cell lines. Eighteen microRNAs were significantly altered in their expression between normal tissues and tumors. Four of these microRNAs were validated in the larger sample series, and each showed significant differential expression (P < 0.0001). Furthermore, an expression ratio of miR-221:miR-375 showed a high sensitivity (0.92) and specificity (0.93) for disease prediction., Conclusions: These data suggest that cultured tumor cell lines are inappropriate for microRNA biomarker identification and that the pattern of microRNA expression in primary head and neck tissues is reflective of disease status, with certain microRNAs exhibiting strong predictive potential. These results indicate that miR-221 and miR-375 should be evaluated further as diagnostic biomarkers because they may hold utility in defining broadly responsive prevention and treatment strategies for HNSCC.

Head and neck squamous cell carcinomas (HNSCCs) represent clinically and etiologically heterogeneous tumors affecting >40 000 patients per year in the USA. Previous research has identified individual epigenetic alterations and, in some cases, the relationship of these alterations with carcinogen exposure or patient outcomes, suggesting that specific exposures give rise to specific types of molecular alterations in HNSCCs. Here, we describe how different etiologic factors are reflected in the molecular character and clinical outcome of these tumors. In a case series of primary, incident HNSCC (n = 68), we examined the DNA methylation profile of 1413 autosomal CpG loci in 773 genes, in relation to exposures and etiologic factors. The overall pattern of epigenetic alteration could significantly distinguish tumor from normal head and neck epithelial tissues (P < 0.0001) more effectively than specific gene methylation events. Among tumors, there were significant associations between specific DNA methylation profile classes and tobacco smoking and alcohol exposures. Although there was a significant association between methylation profile and tumor stage (P < 0.01), we did not observe an association between these profiles and overall patient survival after adjustment for stage; although methylation of a number of specific loci falling in different cellular pathways was associated with overall patient survival. We found that the etiologic heterogeneity of HNSCC is reflected in specific patterns of molecular epigenetic alterations within the tumors and that the DNA methylation profiles may hold clinical promise worthy of further study.

Mechanisms of action of nonmutagenic carcinogens such as asbestos remain poorly characterized. As pleural mesothelioma is known to have limited numbers of genetic mutations, we aimed to characterize the relationships among gene-locus-specific methylation alterations, disease status, asbestos burden, and survival in this rapidly fatal asbestos-associated tumor. Methylation of 1505 CpG loci associated with 803 cancer-related genes were studied in 158 pleural mesotheliomas and 18 normal pleura. After false-discovery rate correction, 969 CpG loci were independently associated with disease status (Q < 0.05). Classifying samples based on CpG methylation profile with a mixture model approach, methylation classes discriminated tumor from normal pleura (permutation P < 0.0001). In a random forests classification, the overall misclassification error rate was 3.4%, with <1% (n = 1) of tumors misclassified as normal (P < 0.0001). Among tumors, methylation class membership was significantly associated with lung tissue asbestos body burden (P < 0.03), and significantly predicted survival (likelihood ratio P < 0.01). Consistent with prior work, asbestos burden was associated with an increased risk of death (hazard ratio, 1.4; 95% confidence interval, 1.1-1.8). Our results have shown that methylation profiles powerfully differentiate diseased pleura from nontumor pleura and that asbestos burden and methylation profiles are independent predictors of mesothelioma patient survival. We have added to the growing body of evidence that cellular epigenetic dysregulation is a critical mode of action for asbestos in the induction of pleural mesothelioma. Importantly, these findings hold great promise for using epigenetic profiling in the diagnosis and prognosis of human cancers.

Background: The loss of E-cadherin (ECAD) protein expression has been linked to aggressive head and neck squamous cell carcinoma (HNSCC). Promoter hypermethylation of the cadherin 1, type 1 (CDH1) gene (encoding ECAD) is 1 mechanism by which this protein can be inactivated, although this epigenetic alteration of the gene has not been linked conclusively to poorer patient outcome and, in fact, may be associated with better patient prognosis., Methods: The authors investigated the prevalence of CDH1 promoter hypermethylation in a population-based case series of 340 primary HNSCC tumors using methylation-specific polymerase chain reaction. They also studied the association between CDH1 hypermethylation and patient demographic characteristics using multivariate analysis and examined the impact of CDH1 hypermethylation on patient survival using both univariate and multivariate methods., Results: Hypermethylation of CDH1 was significantly more prevalent (P < .03) among individuals with a low smoking history independent of whether they were seropositive for human papillomavirus type 16 (HPV-16). Patients who had tumors with CDH1 hypermethylation had significantly better overall survival compared with patients who had tumors without hypermethylation (P < .02; log-rank test). This effect was independent of HPV-16 status and demonstrated a significant hazard ratio of 0.5 (95% confidence interval, 0.3-0.9) in a model that controlled for HPV-16 serology, age, sex, and tumor stage., Conclusions: The current results suggested that hypermethylation of CDH1 occurs more commonly in patients with HNSCC who are low smokers, suggesting that an additional factor may be driving this epigenetic alteration. Clinically, CDH1 hypermethylation may hold powerful prognostic potential in addition to that observed with HPV serology, and the authors concluded that it should be pursued in additional studies.

Background: Epigenetics is the study of heritable changes in gene function that cannot be explained by changes in DNA sequence. One of the most commonly studied epigenetic alterations is cytosine methylation, which is a well recognized mechanism of epigenetic gene silencing and often occurs at tumor suppressor gene loci in human cancer. Arrays are now being used to study DNA methylation at a large number of loci; for example, the Illumina GoldenGate platform assesses DNA methylation at 1505 loci associated with over 800 cancer-related genes. Model-based cluster analysis is often used to identify DNA methylation subgroups in data, but it is unclear how to cluster DNA methylation data from arrays in a scalable and reliable manner., Results: We propose a novel model-based recursive-partitioning algorithm to navigate clusters in a beta mixture model. We present simulations that show that the method is more reliable than competing nonparametric clustering approaches, and is at least as reliable as conventional mixture model methods. We also show that our proposed method is more computationally efficient than conventional mixture model approaches. We demonstrate our method on the normal tissue samples and show that the clusters are associated with tissue type as well as age., Conclusion: Our proposed recursively-partitioned mixture model is an effective and computationally efficient method for clustering DNA methylation data.

Background: As part of a population-based case-control study, we investigated the association of food groups and micronutrients estimated from a validated food frequency questionnaire (FFQ) with the risk of development of head and neck squamous cell carcinoma (HNSCC)., Methods: Incident cases were accrued through Boston area hospitals from 1999 to 2003, and neighborhood controls were selected and matched by location, age, and sex. There were 504 cases and 717 controls enrolled, who completed the FFQ., Results: We observed a positive association between the consumption of dairy products and HNSCC. The odds of HNSCC in the highest quintile of dairy intake was 1.64 (95% CI: 1.09-2.46), compared with subjects in the lowest quintile. There was a significant association between leanness with HNSCC. The odds of cancer among the leanest subjects was 5.8 (95%CI: 3.2-10.6) compared with a healthy BMI. Finally, intake of animal fat was positively associated with an elevation in cancer risk. The odds of HNSCC for high animal fat intake were 1.50 (0.99-2.27)., Conclusions: Our data suggest that consumption of fruits and vegetables is not universally protective for HNSCC and that other food groups and nutrients may influence the risk for developing this disease.

Malignant pleural mesothelioma (MPM) is a rapidly fatal tumor with increasing incidence worldwide responsible for many thousands of deaths annually. Although there is a clear link between exposure to asbestos and mesothelioma, and asbestos is known to be both clastogenic and cytotoxic to mesothelial cells, the mechanisms of causation of MPM remain largely unknown. However, there is a rapidly emerging literature that describes inactivation of a diverse array of tumor suppressor genes (TSGs) via promoter DNA CpG methylation in MPM, although the etiology of these alterations remains unclear. We studied the relationships among promoter methylation silencing, asbestos exposure, patient demographics and tumor histology using a directed approach; examining six cell cycle control pathway TSGs in an incident case series of 70 MPMs. Promoter hypermethylation of APC, CCND2, CDKN2A, CDKN2B, HPPBP1 and RASSF1 were assessed. We observed significantly higher lung asbestos body burden if any of these cell cycle genes were methylated (P < 0.02), and there was a significant trend of increasing asbestos body counts as the number of methylated cell cycle pathway genes increased from 0 to 1 to >1 (P < 0.005). This trend of increasing asbestos body count and increasing number of methylated cell cycle pathway genes remained significant (P < 0.05) after controlling for age, gender and tumor histology. These data suggest a novel tumorigenic mechanism of action of asbestos and may contribute to the understanding of precisely how asbestos exposure influences the etiology and clinical course of malignant mesothelioma.

Background: Malignant pleural mesothelioma (MPM) is a rapidly fatal asbestos-associated malignancy with a median survival time of <1 year following diagnosis. Treatment strategy is determined in part using known prognostic factors., Objective: The aim of this study was to examine the relationship between asbestos exposure and survival outcome in MPM in an effort to advance the understanding of the contribution of asbestos exposure to MPM prognosis., Methods: We studied incident cases of MPM patients enrolled through the International Mesothelioma Program at Brigham and Women's Hospital in Boston, Massachusetts, using survival follow-up, self-reported asbestos exposure (n=128), and a subset of cases (n=80) with quantitative asbestos fiber burden measures., Results: Consistent with the established literature, we found independent, significant associations between male sex and reduced survival (p<0.04), as well as between nonepithelioid tumor histology and reduced survival (p<0.02). Although self-reported exposure to asbestos was not predictive of survival among our cases, stratifying quantitative asbestos fiber burden [number of asbestos bodies per gram of lung (wet weight)] into groups of low (0-99 asbestos bodies), moderate (100-1,099), and high fiber burden (>1,099), suggested a survival duration association among these groups (p=0.06). After adjusting for covariates in a Cox model, we found that patients with a low asbestos burden had a 3-fold elevated risk of death compared to patients with a moderate fiber burden [95% confidence interval (CI), 0.95-9.5; p=0.06], and patients with a high asbestos burden had a 4.8-fold elevated risk of death (95% CI, 1.5-15.0; p<0.01) versus those with moderate exposure., Conclusion: Our data suggest that patient survival is associated with asbestos fiber burden in MPM and is perhaps modified by susceptibility.

Purpose: The variant allele of CCND1 G870A encodes a splice variant of the cyclin D1 protein, which possesses an increased half-life. To confirm the phenotypic effect of the variant allele, we examined the immunohistochemical staining pattern of the protein in tumors from a case population of head and neck squamous cell carcinoma (HNSCC) and compared it with the genotype of these individuals. We also examined how this genotype was associated with the risk of HNSCC and if this genotype-phenotype association was related to patient outcome., Experimental Design: In a population-based case-control study of 698 cases and 777 controls, we both genotyped all participants for the CCND1 gene and did immunohistochemical staining of the cyclin D1 protein in the HNSCC tumors., Results: The variant AA genotype was significantly associated with positive immunohistochemical staining (P < 0.02), and this variant genotype was associated with a significantly elevated odds ratio of 1.5 (95% confidence interval, 1.1-2.0) for HNSCC overall, with risk greatest in oral and laryngeal sites. Positive immunohistochemical staining was inversely related to human papillomavirus 16 DNA present in the tumor (P < 0.03). The AA genotype and superpositive immunohistochemical staining for cyclin D1 also had independent and significant effects on patient survival., Conclusions: These results strongly suggest that this splice variant, when present in two copies, is a significant predictor of both the occurrence of HNSCC as well as patient survival after treatment. These data further indicate that this variant protein is an important determinant of individual response to therapy for this disease.

Genomic hypomethylation is a hallmark of essentially all cancers, but the degree of this hypomethylation differs among individual tumors. Little work has explored what leads to these differences and or asked whether they are clinically meaningful. In this study of head and neck squamous cell carcinoma, we assessed hypomethylation in tumors using a semiquantitative fragment analysis approach to determine the relative methylation status of the line retroviral element LRE1 (Line-1.2). Because this is an established marker of genomic methylation status, we examined the relationship between the relative methylation, patient demographics, and other risk factors for head and neck squamous cell carcinoma. We determined relative methylation status for 303 patients, 193 of which had complete data for all variables of interest. Using a generalized linear model, we found that patient body mass index was significantly positively associated with tumor LRE1 methylation level. Smoking duration, particularly in tumors lacking human papillomavirus (HPV) DNA, was significantly negatively associated with relative methylation level. Having previously assessed relative methylation in blood-derived DNA, we compared tumor with the blood DNA methylation level and observed these to be independent. Finally, the lower LRE1 methylation in patients whose tumors were HPV DNA negative was associated with poorer patient survival (hazard ratio, 1.6; 95% confidence interval, 1.0-2.6). These findings suggest that HPV-associated tumors differ molecularly from those arising after heavy tobacco use and that this epigenetic alteration may affect survival in HPV-negative patients already exhibiting a more aggressive disease.

Objective: There are limited data on the distribution of bladder cancers in the general population, classified by World Health Organization (WHO)/International Society of Urological Pathology (ISUP) criteria. This study evaluated the classification and stage of bladder cancers as part of a population-based epidemiological study of bladder cancer in the Northeastern United States., Material and Methods: All New Hampshire residents with bladder cancer newly diagnosed from 1998 to 2000 were identified through the state cancer registry. All slides were reviewed by a single pathologist. Tumors were classified by two sets of standard criteria., Results: The retrieval rate for cases was over 90%. Of 342 cases reviewed, 15 were excluded for technical reasons or because malignancy was not definitively diagnosed. According to WHO/ISUP criteria, 25.7% of tumors were papillary urothelial neoplasms of low malignant potential (PUNLMP), 34.3% low-grade papillary carcinomas, 22.6% high-grade papillary carcinomas, 10.1% non-papillary urothelial carcinomas and 5.5% carcinoma in situ. By WHO (1973) criteria, 52.5% of tumors were grade 1, 21.4% grade 2 and 26.1% grade 3. Two-thirds of all tumors were stage Ta, 20.8% stage T1 and 7.6% stage >or=T2. 100% of PUNLMPs were non-invasive, 6.3% of low-grade carcinomas were invasive and 64.9% of high-grade carcinomas were invasive., Conclusions: Compared to clinic or hospital referral-based series, this study documents a higher percentage of non-invasive tumors and a lower percentage of muscle-invasive tumors. There was also a higher percentage of PUNLMP tumors and fewer high-grade papillary carcinomas than in other series. These results may more accurately reflect prevalence data for bladder cancer grade and stage, although geographic variability may exist.

Both genetic and epigenetic alterations characterize human nonsmall cell lung cancer (NSCLC), but the biological processes that create or select these alterations remain incompletely investigated. Our hypothesis posits that a roughly reciprocal relationship between the propensity for promoter hypermethylation and a propensity for genetic deletion leads to distinct molecular phenotypes of lung cancer. To test this hypothesis, we examined promoter hypermethylation of 17 tumor suppressor genes, as a marker of epigenetic alteration propensity, and deletion events at the 3p21 region, as a marker of genetic alteration. To model the complex biology between these somatic alterations, we utilized an item response theory model. We demonstrated that tumors exhibiting LOH at greater than 30% of informative alleles in the 3p21 region have a significantly reduced propensity for hypermethylation. At the same time, tumors with activating KRAS mutations showed a significantly increased propensity for hypermethylation of the loci examined, a result similar to what has been observed in colon cancer. These data suggest that NSCLCs have distinct epigenetic or genetic alteration phenotypes acting upon tumor suppressor genes and that mutation of oncogenic growth promoting genes, such as KRAS, is associated with the epigenetic phenotype.

Purpose: The WHO/International Society of Urological Pathology classification of bladder cancer, introduced in 1998, differs from the traditional 1973 WHO classification. Few studies have reported survival data based on the WHO/International Society of Urological Pathology classification and none has demonstrated clear superiority compared to the 1973 WHO system. In a large, nonselected population of patients with bladder cancer we rated all incident tumors using each system and compared long-term patient survival., Materials and Methods: New Hampshire residents with bladder cancer diagnosed between 1994 and 2000 were identified through the State Cancer Registry. Slides were retrieved from more than 90% of cases and reviewed by a single pathologist. Tumors were classified according to WHO and WHO/International Society of Urological Pathology criteria. Overall patient survival was determined for the cohort of 504 patients after an average of 7 years using a national mortality database., Results: For both grading systems there was a gradient of progressively lower survival times from the lowest grade to the highest grade tumors. Hazard ratios and 95% confidence intervals for the WHO/International Society of Urological Pathology system were 1.9 (1.0-3.4) for low grade papillary urothelial carcinoma and 3.0 (1.5-6.0) for high grade papillary urothelial carcinoma, compared to papillary urothelial neoplasms of low malignant potential. For the WHO (1973) system compared to grade 1 tumors the hazard ratio for grade 2 tumors was 1.8 (1.1-3.1) and for grade 3 was 2.4 (1.2-4.7)., Conclusions: Advantages of the WHO/International Society of Urological Pathology bladder tumor classification include more detailed diagnostic criteria, the ability to define a lesion with minimal malignant potential and the ability to identify a larger group of patients needing closer surveillance. However, we found that the WHO/International Society of Urological Pathology tumor categories did not detect a clear overall survival advantage compared to the WHO (1973) classification system.

Hypermethylation of tumor suppressor genes is central to the pathogenesis of human malignancy, yet this alteration's etiology remains unclear, and its clinical impact has not yet been studied using an approach that can yield directly generalizable results. Therefore, we sought to examine both of these issues in bladder cancer, seeking to understand the characteristics of epidemiologically well-defined patient tumors with differing levels of methylation silencing. We analyzed epigenetic silencing of 16 genes in a population-based incident case series of 331 bladder transitional cell carcinomas. We utilized a novel item response theory (IRT) model, to examine, in an unbiased fashion, the relationship of patient characteristics, carcinogen exposure history and tumor characteristics with the underlying propensity for gene hypermethylation. Age, male gender and current cigarette smoking were significantly positively associated with the methylation latent trait. Promoter hypermethylation as a latent trait significantly predicted both non-invasive/high-grade and invasive stage disease and was also significantly associated with survival, with each unit increase in the latent trait resulting in a 30% increase in the risk of death. This work, studying all stages and grades of incident bladder cancer, provides definitive evidence that carcinogen exposures play a critical role in selecting these alterations in tumorous clones and that epigenetic silencing is a strong and significant predictor of tumor stage and overall patient survival. Finally, our novel approach provides insight into the etiology of promoter hypermethylation, suggesting that selected, carcinogen-exposed individuals have a greater propensity for hypermethylation that is associated with more aggressive, fatal disease.

Human papillomaviruses (HPV) are associated with nearly all cervical cancers, 20% to 30% of head and neck cancers (HNC), and other cancers. Because HNCs also arise in HPV-negative patients, this type of cancer provides unique opportunities to define similarities and differences of HPV-positive versus HPV-negative cancers arising in the same tissue. Here, we describe genome-wide expression profiling of 84 HNCs, cervical cancers, and site-matched normal epithelial samples in which we used laser capture microdissection to enrich samples for tumor-derived versus normal epithelial cells. This analysis revealed that HPV(+) HNCs and cervical cancers differed in their patterns of gene expression yet shared many changes compared with HPV(-) HNCs. Some of these shared changes were predicted, but many others were not. Notably, HPV(+) HNCs and cervical cancers were found to be up-regulated in their expression of a distinct and larger subset of cell cycle genes than that observed in HPV(-) HNC. Moreover, HPV(+) cancers overexpressed testis-specific genes that are normally expressed only in meiotic cells. Many, although not all, of the hallmark differences between HPV(+) HNC and HPV(-) HNC were a direct consequence of HPV and in particular the viral E6 and E7 oncogenes. This included a novel association of HPV oncogenes with testis-specific gene expression. These findings in primary human tumors provide novel biomarkers for early detection of HPV(+) and HPV(-) cancers, and emphasize the potential value of targeting E6 and E7 function, alone or combined with radiation and/or traditional chemotherapy, in the treatment of HPV(+) cancers.

Background: Head and neck squamous cell carcinoma (HNSCC) is commonly associated with tobacco and alcohol exposures, although dietary factors, particularly folate, and human papillomavirus, are also risk factors. Epigenetic alterations are increasingly implicated in the initiation and progression of cancer. Genome-wide (global) hypomethylation seems to occur in early neoplasia and is a feature of genomic DNA derived from solid tumor tissues, including HNSCC. This study aimed to determine whether global methylation in DNA derived from whole blood, a proxy tissue, is associated with HNSCC and to assess potential modification of this property by environmental or behavioral risk factors., Methods: Global DNA methylation levels were assessed using a modified version of the combined bisulfite restriction analysis of the LRE1 sequence in a population-based case-control study of HNSCC from the Boston area., Results: Hypomethylation lead to a significant 1.6-fold increased risk for disease (95% confidence interval, 1.1-2.4), in models controlled for other HNSCC risk factors. Smoking showed a significant differential effect (P < 0.03) on blood relative methylation between cases and controls. Furthermore, in cases, variant genotype in the MTHFR gene and low folate intake showed relationships with decreased global methylation, whereas in controls, antibody response to human papillomavirus 16 was associated with an increased global methylation level., Discussion: DNA hypomethylation in nontarget tissue was independently associated with HNSCC and had a complex relationship with the known risk factors associated with the genesis of HNSCC.

Recent work has begun to explore the instrumental role that small noncoding RNA species, particularly microRNAs (miRNA), have both in classifying human tumors and in directing embryonic development. These studies suggest that developmental programs in essentially all organisms studied are set, in part, by varied expressions of miRNAs and that neoplasia is characterized by altered expression of miRNAs. Reasoning that these observations are linked, we examined whether cellular exposures that induce both developmental anomalies and cancer alter miRNAs. Using microarrays of 385 known human miRNAs, we studied human lymphoblastoid cells grown under various conditions or treatments. Folate deficiency induced a pronounced global increase in miRNA expression. We observed no significant alteration in miRNA expression in cells treated with gamma-irradiation, whereas exposure to sodium arsenite led to global increases in miRNA expression. The miRNA hsa-miR-222 was identified from these arrays as significantly overexpressed under folate-deficient conditions, and this finding was confirmed in vivo in human peripheral blood from individuals with low folate intake. Alterations to cellular miRNA expression profiles represent a novel mode of action of folate deprivation and arsenic exposure, and specific alterations in miRNA expression may be a powerful biomarker for these and other toxins with serious effects on human health.

The CpG island methylator phenotype (CIMP), thoroughly described in colorectal cancer and to a lesser extent in other solid tumors, is important in understanding epigenetics in carcinogenesis and may be clinically useful for classification of neoplastic disease. Therefore, we investigated whether this putative phenotype exists in exposure-related solid tumors, where somatic gene alterations and enhanced clonal growth are selected for by carcinogens, and examined the ability of methylation profiles to classify malignant disease. We studied promoter hypermethylation of 16 tumor suppressor genes and 3 MINT loci (acknowledged classifiers of CIMP) in 344 bladder cancers, 346 head and neck squamous cell carcinomas (HNSCC), 146 non-small-cell lung cancer (NSCLC), and 71 malignant pleural mesotheliomas (MPM). We employed rigorous statistical methods to examine the distribution of promoter methylation and the usefulness of these profiles for disease classification. In bladder cancer, HNSCC, and NSCLC, there was a significant correlation (P < 0.0001) between methylation of the three MINT loci and methylation index, although the distribution of methylated loci varied significantly across these disease. Although there was a significant (P < 0.001) association between gene methylation profile and disease, rates of misclassification of each disease by their methylation profile ranged from 28% to 32%, depending on the classification scheme used. These data suggest that a form of CIMP exists in these solid tumors, although its etiology remains elusive. Whereas the gene profiles of hypermethylation among examined loci could not unequivocally distinguish disease type, the existence of CIMP and the relative preponderance of hypermethylation in these cancers suggest that methylation analysis may be clinically useful as a targeted screening tool.

Investigations of the ability of polymorphisms in the GSTM1, GSTT1, and GSTP1 genes to alter susceptibility to head and neck squamous cell carcinoma (HNSCC) have examined gene-environment interaction in their detoxification of tobacco-associated carcinogens. Little work has been done to ask if these variant genes also modify the interaction of tobacco and alcohol in the development of HNSCC. To test this hypothesis, we conducted a case-control study, enrolling 692 incident cases of HNSCC and 753 population controls. Information about lifetime tobacco and alcohol use was ascertained through questionnaires, and genotypes for GSTM1, GSTT1, and GSTP1 were determined from constitutional DNA. Genotype frequencies were compared among cases and controls, and the association between genotypes and tobacco use was evaluated on cancer risk through logistic regression. Deletion of GSTM1 was associated with an increased risk for HNSCC [odds ratio (OR), 1.3; 95% confidence interval (95% CI), 1.0-1.6]. GSTT1 deletion was associated with a slight decreased HNSCC risk (OR, 0.8; 95% CI, 0.6-1.0). Among those with GSTM1 present, the OR of cancer for heavy smoking was 2.6 (95% CI, 1.6-4.3) compared with 4.2 for those with the GSTM1 deleted (95% CI, 2.6-6.7). The combination of consuming 10 to 20 alcohol drinks weekly and smoking >45 pack-years was associated with a 13-fold elevated risk (OR, 12.6; 95% CI, 4.0-40.2) among the GSTM1 deleted subjects compared with an OR of 3.6 (95% CI, 1.5-8.7) among the GSTM1 present individuals. These data (showing that the GSTM1 deletion affects on the tobacco and alcohol synergy) suggest that the interaction of these carcinogens is, at least in part, driven by alcohol, enhancing the carcinogenic action of tobacco smoke.

The soluble frizzled receptor protein (SFRP) family encodes antagonists of the WNT pathway, and silencing of these genes, through promoter hypermethylation, leads to constitutive WNT signaling. In bladder cancers, hypermethylation of the SFRP genes occurs more often in current and former smokers and is a strong predictor of poor patient survival. Hence, we examined methylation of these genes in another tobacco-related epithelial cancer, head and neck squamous cell carcinoma (HNSCC), to determine if the pattern of tobacco exposure again predicts the epigenetic alteration of these genes. Using methylation-specific PCR, the prevalence of methylation of SFRP1, SFRP2, SFRP4 and SFRP5 was 35, 32, 35 and 29%, respectively among 350 HNSCC cases. Promoter methylation of SFRP1 occurred more often in both heavy (OR 3.5, 95% CI 0.9-13.7) and light drinkers (OR 3.7, 95% CI 1.0-14.3) compared to nondrinkers. SFRP4 promoter methylation, on the other hand, occurred at a higher prevalence in never smokers and former smokers than in current smokers, and also was independently associated with HPV16 viral DNA. A joint effects model of SFRP4 promoter methylation demonstrated that smoking status and HPV virus significantly interacted (p < 0.04) such that never smokers with HPV16 had an OR of SFRP4 methylation of 9.0 (95% CI 2.1-38.6). These results suggest that epigenetic alterations of the SFRP genes are highly prevalent in HNSCC, and that the clonal selection for these alterations is complex and may be related to the carcinogenic exposures that are known risk factors for this disease., (Copyright 2006 Wiley-Liss, Inc.)

Tobacco smoking, certain occupational exposures, and exposure to inorganic arsenic in drinking water have been associated with the occurrence of bladder cancer. However, in these tumors the exposure-associated pattern of somatic alterations in genes in the causal pathway for disease has been poorly characterized. Animal and in vitro studies have suggested that arsenic, tobacco carcinogens, and other exposures may act through epigenetic mechanisms. We, therefore, examined, in a population-based study of human bladder cancer (n = 351), the relationship between epigenetic silencing of the tumor-suppressor genes, p16(INK4A), RASSF1A, PRSS3, and the four SFRP genes and exposure to both tobacco and arsenic in bladder cancer. Promotor methylation silencing of each of these genes occurred in approximately 30-50% of bladder cancers. Epigenetic silencing of RASSF1A and PRSS3 and any of the SFRP genes were each significantly associated with advanced tumor stage (P < 0.001, P < 0.04, and P < 0.005, respectively). Arsenic exposure, measured as toenail arsenic, was associated with RASSF1A (P < 0.02) and PRSS3 (P < 0.1) but not p16(INK4A) or SFRP promotor methylation, in models adjusted for stage and other risk factors. Cigarette smoking was associated with a greater than twofold increased risk of promotor methylation of the p16(INK4A) gene, with greater risk seen in patients with exposures more recent to disease diagnosis, and smoking was also significantly associated with any SFRP gene methylation (P < 0.01). These results from human bladder tumors, add to the body of animal and in vitro evidence that suggests bladder carcinogens play a crucial role in the induction of important epigenetic alterations.

Tobacco smoking, certain occupational exposures, and exposure to inorganic arsenic in drinking water have been associated with the occurrence of bladder cancer. However, in these tumors the exposure-associated pattern of somatic alterations in genes in the causal pathway for disease has been poorly characterized. In particular, the mechanism by which arsenic induces bladder cancer and the effects of lower environmental levels of exposure remain uncertain. Animal and in-vitro studies have suggested that arsenic and other exposures may act through epigenetic mechanisms. We, therefore, examined, in a population-based study of human bladder cancer, the relationship between epigenetic silencing of three tumor suppressor genes, p16(INK4A), RASSF1A and PRSS3, and exposure to both tobacco and arsenic in bladder cancer. Promoter methylation of each of these genes occurred in approximately 30% of bladder cancers, and both RASSF1A and PRSS3 promoter methylation were associated with advanced tumor stage (P<0.001 and P<0.04, respectively). Arsenic exposure, measured as toenail arsenic, was associated with RASSF1A (P<0.02) and PRSS3 (P<0.1) but not p16INK4A promoter methylation, in models adjusted for stage and other factors. Cigarette smoking was associated with a >2-fold increased risk of promoter methylation of the p16INK4A gene only, with greater risk seen in patients with exposures more recent to disease diagnosis. These results, from human bladder tumors, add to the body of animal and in vitro evidence that suggests a role in epigenetic alterations for bladder carcinogens.

Background: Somatic loss of the 9q allele as well as alteration of the tumor suppressor p53 occurs commonly in bladder cancers. Although alteration of p53 has been strongly associated with invasive stage disease, the prognostic significance of 9q loss of heterozygosity (LOH) and the relations between these alterations are less well defined., Methods: The 9q LOH was examined at five microsatellites and p53 alterations (mutation and persistent immunohistochemical staining) in a population-based case series of 271 newly diagnosed bladder cancer patients. Loss of heterozygosity was scored quantitatively and p53 mutation completed using single-strand conformation polymorphism screening followed by sequencing., Results: Overall, allelic loss at 9q was detected in 74.5% (202/271) of cases and allele loss was associated with invasive disease (P < 0.05). Although based on small numbers, all nine in situ lesions contained 9q LOH. Age, gender, and smoking were not significantly associated with chromosome 9q allele loss. Both intense persistent p53 staining and LOH at 9q were independently associated with invasive disease (P < 10(-14) and P < 0.05, respectively)., Conclusions: These data, using a population-based sample, suggest a relation between 9q LOH and invasive stage bladder cancer, and thereby suggests that a tumor suppressor gene at this loci, in addition to p53, may be important in the development of this more aggressive form of the disease., ((c) 2005 American Cancer Society.)

Tobacco smoke is well recognized as the major etiological contributor to lung cancer, yet the relationship between tobacco smoke exposure and a specific pattern of molecular abnormalities at somatic loci is less well characterized. We analyzed 100 primary tumors from patients undergoing surgical resection of squamous cell carcinoma and adenocarcinoma of the lung for loss of heterozygosity (LOH) and homozygous deletions at two microsatellite markers in a recombinogenic region of 9p13. We describe the relationship of alterations at these markers with tumor characteristics (both clinical and molecular), patient demographics, survival, and measures of tobacco-smoke exposure. Homozygous deletions in this region occurred in 25% (21/85) and LOH in 33% (28/85) of informative tumors examined. These alterations occurred more often in tumors with intense TP53 protein staining by immunohistochemistry, suggesting that inactivation of the TP53 pathway may contribute to these LOH events. Duration of smoking was greatest in patients with the homozygous deletion, intermediate in patients with LOH, and shortest in patients whose tumor did not demonstrate loss in these markers. Unexpectedly, LOH at 9p13 was a significant predictor of improved survival in patients, while the homozygous deletion was associated with the poorest patient survival. Together, these results suggest that TP53 alteration and long-term tobacco smoke exposure may contribute to genetic alterations at 9p13, and that the mechanism and biologic consequences of allele loss reflect individual biologic differences that determine the extent of loss (LOH or homozygous deletion), such that those patients with the deletion of this region face a more aggressive and deadly disease.

TP53 is well-recognized as a mutational target in cancers and common variation in the TP53 gene has been investigated as potentially contributing to cancer susceptibility. The codon 72 polymorphism has been proposed to alter the phenotype of TP53 mutations, and TP53 mutations have been reported to occur preferentially on the arginine allele. Using a consecutive case series of non-small cell lung cancer we have investigated whether TP53 mutations occur preferentially on the arginine or proline allele, and whether the combination of mutation and allelism confers differences in the clinical phenotype. The overall prevalence of TP53 mutation was 26% (76/293). The majority of mutations occurred on the arginine allele (51/60, 85%), and there was corresponding strong selection for loss of the proline allele [87% of loss of heterozygosity (LOH) events were loss of proline]. However, there was no statistically significant difference in the prevalence of mutation by constitutional genotype and among heterozygotes with LOH, TP53 mutation prevalence did not differ by the codon 72 polymorphism (48% on arginine versus 40% on proline). Importantly, patient survival did significantly differ: those patients having a TP53 mutation on the proline allele had the worst survival outcomes (hazards ratio = 2.6, P < 0.03). Further, this phenotype was limited to those patients with advanced disease, where mutation on the proline allele was associated with a significantly worse outcome compared with those without mutation or with mutation on the arginine allele (P < 0.001). Our data suggest that there are selective pressures for loss of the TP53 proline allele in non-small cell lung cancer. Further, the combination of mutation with the codon 72 proline variant predicts poorer patient survival, particularly in a disease that has progressed outside the lung, a finding that warrants further investigation.

The serine protease family member PRSS3 (trypsinogen-IV) has been implicated as a putative tumor suppressor gene due to its loss of expression, which is correlated with promoter hypermethylation, in esophageal squamous cell carcinoma and gastric adenocarcinoma. As epigenetic alteration is common in non-small cell lung cancer (NSCLC), we sought to determine if promoter hypermethylation of PRSS3 occurred in this disease, and if it was associated with clinical features of NSCLC or tobacco-related exposures in these patients. Using methylation-specific PCR, we determined the promoter hypermethylation status of PRSS3 in a case series study of primary NSCLC, and found methylation of this gene to be common, occurring in 53% (86 of 166) of tumors examined. There was no association of this alteration with patient demographics, tumor features, or exposure histories of the patients. The lack of association is of interest, as it may suggest a lack of specific selection for inactivation of this gene. On the other hand, the high prevalence of this alteration makes PRSS3 methylation an attractive biomarker for use in diagnostic or screening applications in NSCLC., ((c) 2005 Wiley-Liss, Inc.)

In the United States each year, almost 13,000 deaths are attributable to bladder cancer, with the majority of these deaths related to higher stage, muscle-invasive solid tumors. Epigenetic silencing of the secreted frizzled receptor proteins (SFRP), antagonists of the WNT pathway, leads to constitutive WNT signaling, altering cell morphology and motility. Identifying alterations in this pathway in bladder cancer may prove useful for defining the invasive phenotype and provide targets for guiding therapy. Using a population-based study of bladder cancer (n = 355), we examined epigenetic alterations, specifically gene promoter hypermethylation, of four SFRP genes in addition to immunohistochemical staining of TP53, which has been previously shown to be a predictor of invasive disease. We observed a significant linear trend (P < 0.0004) in the magnitude of the risk of invasive disease with the number of SFRP genes methylated. Both TP53 alteration and SFRP gene methylation showed significant independent associations with invasive bladder cancer. Strikingly, in examining the joint effect of these alterations, we observed a >30-fold risk of invasive disease for patients with both altered SFRP gene methylation and intense TP53 staining (odds ratio, 32.1; P < 10(-13)). Overall patient survival was significantly poorer in patients with any SFRP genes methylated (P < 0.0003) and in proportional hazards modeling, patients with methylation of any SFRP gene had significantly poorer overall survival (hazard ratio, 1.78; P < 0.02) controlled for TP53 staining intensity and other survival-associated factors. Classifying tumors based on SFRP methylation status and TP53 protein staining intensity may be a clinically powerful predictor of invasive, deadly disease.

SEMA3B has been implicated as important for neuronal development and as a tumor suppressor in lung cancer. A single nucleotide alteration of this gene leads to the amino acid substitution T415I, and functionally, this variant protein has a reduced ability to act as a tumor suppressor. The prevalence of this variant in populations is unclear and its role in inherited lung cancer susceptibility has not been tested. Utilizing case-control studies of head and neck squamous cell carcinoma in a Caucasian population and of lung cancer in African-American and Latino-American populations, we determined both the prevalence of this polymorphic variant and its association with the case status of these patients. The variant Ile allele occurs at an allele frequency of 0.18 in African-American and 0.39 in Latino-American control subjects but not in Caucasian subjects. In analyses controlling for ethnicity and known lung cancer risk factors, a significant association was observed between case status and possession of the variant allele (OR 0.71, 95% CI 0.51-0.99). In stratified analysis, both Latino-Americans (OR 0.56, 95% CI 0.32-1.01) and African-Americans (OR 0.75, 95% CI 0.50-1.13) also showed a reduced risk of disease associated with the variant Ile allele. Possessing either the heterozygous or homozygous variant genotype confers a >40% reduced relative risk of lung cancer in Latino Americans controlling for other lung cancer risk factors. This study points to the need for further examination of this gene and its variant in lung cancer and other diseases.

The tumor suppressor PTEN encodes a lipid phosphatase that negatively regulates the phosphatidylinositol 3-kinase/AKT cell survival pathway. Mutations of this gene are common in brain, prostate, endometrial, and gastric cancers but occur rarely in non-small-cell lung cancer (NSCLC), although the PTEN protein is often lost in lung tumors. We have studied hypermethylation of the PTEN promoter, loss of heterozygosity (LOH) at microsatellites in chromosome 10q23 (surrounding and intragenic to the PTEN locus), and hypermethylation of PTEN's highly homologous pseudogene, PTENP1, and their association with PTEN protein loss in a surgical case series study of primary NSCLC. PTEN protein expression was reduced or lost in 74% (86/117) of tumors, with loss occurring more often in well to moderately differentiated tumors. In squamous cell carcinomas, PTEN loss occurred significantly more often in early-stage (stage I or II) disease. PTEN protein loss also occurred more frequently in tumors with low to no aberrant TP53 staining. Methylation of PTEN occurred in 26% (39/151) of tumors, and LOH at 10q23 was rare, occurring in only 19% (17/90) of informative tumors. Neither methylation nor LOH was a significant predictor of PTEN protein expression, although LOH occurred exclusively in early-stage disease. In NSCLC, loss of PTEN protein expression occurs frequently, although the mechanism responsible for loss is not clearly attributable to deletion or epigenetic silencing. PTEN loss may also be a favorable prognostic marker, although further studies are needed to confirm this finding.

The putative tumor suppressors RASSF1A and BLU are mapped adjacent to one another on chromosome 3p21.3, a region frequently deleted in lung cancer. These genes are often inactivated by promoter hypermethylation, but the association of this inactivation with clinical features of the disease or with carcinogen exposure has been poorly studied. Early age starting smoking has been hypothesized as an independent risk factor for lung cancer, and mechanistically, adolescence may constitute a critical period for tobacco carcinogen exposure. To study the relationship of tobacco smoke exposure with hypermethylation of RASSF1A and BLU, methylation-specific PCR was performed on a case series study of incident, surgically resected non-small cell lung cancer (NSCLC), and the prevalence of this alteration was examined in relation to clinical and exposure information collected on the patients. Hypermethylation of the RASSF1A promoter occurred in 47% (83/178) and of the BLU promoter in 43% (68/160) of NSCLC tumors examined. There was no significant association between methylation of these 2 genes, but methylation of either of these genes tended to occur more often in the adenocarcinoma (AC) histology compared to squamous cell carcinoma (SCC). Controlling for pack-years smoked, age, gender and histology, starting smoking under age 18 was significantly related to RASSF1A methylation [prevalence ratio (PR) = 1.6, 95% confidence interval [CI] = 1.1-2.3]. These results indicate that starting smoking under age 18 is an independent risk for RASSF1A hypermethylation, thus identifying a molecular alteration related to the epidemiologic effect of teenage smoking as a lung cancer risk., ((c) 2004 Wiley-Liss, Inc.)

Allelic loss of chromosome region 3p21.3 occurs early and frequently in non-small-cell lung cancer, and numerous tumor suppressor genes at this locus may be targets of inactivation. Using an incident case series study of non-small-cell lung cancer, we sought to determine the prevalence of loss of heterozygosity (LOH) in the 3p21.3 region and to examine the associations between this alteration and patient outcome, exposure to tobacco smoke, occupational asbestos exposure, and additional molecular alterations in these tumors. We examined LOH at 7 microsatellite markers in the chromosome 3p21.3 region, and LOH was present in at least one of the loci examined in 60% (156 of 258) of the tumors, with the prevalence of LOH at individual loci ranging from 15 to 56%. Occupational asbestos exposure and TP53 mutation were significantly associated with more extensive 3p21 LOH. In squamous cell carcinomas, measures of cumulative smoking dose were significantly lower in patients with LOH at 3p21, particularly in TP53 mutant tumors. Examining patient outcome, we found that in squamous cell carcinomas, having any LOH in this region was associated with a better overall survival (log-rank test, P < 0.04). Together, these results indicate that allelic loss at 3p21 can affect patient outcome, and that this loss may initially be related to carcinogen exposure, but that extension of this loss is related to TP53 mutation status and occupational asbestos exposure.

Inactivation of the FANC-BRCA pathway via promoter methylation of the FANCF gene renders cells sensitive to DNA crosslinking agents, and has been identified in ovarian cancer cell lines and sporadic primary tumor tissues. We investigated epigenetic alterations in the FANC-BRCA pathway in head and neck squamous cell carcinomas (HNSCC) and non-small-cell lung cancers (NSCLC) using methylation-specific PCR. Promoter methylation of FANCF occurred in 15% (13/89) of HNSCCs and 14% (22/158) of NSCLCs. Methylation of BRCA1 occurred only in 6/158 NSCLC, and was limited to adenocarcinomas and large-cell carcinomas of the lung. No methylation of BRCA2 was detected. FANCF methylation was associated with a shorter duration of tobacco use (P=0.03) and a younger age of starting smoking (P=0.06) in NSCLC, and with a greater number of years of alcohol drinking (P=0.02) in HNSCC. In adenocarcinomas of the lung, FANCF promoter methylation was a significant predictor of poor survival with a hazard ratio of 3.1 (95% CI 1.2-7.9). This study demonstrates that inactivation of the FANC-BRCA pathway is relatively common in solid tumors and may be related to tobacco and alcohol exposure and survival of these patients.

High performance thin-layer chromatography (HPTLC) was used to analyse the neutral lipids in the rediae, cercariae, and encysted metacercariae of Echinostoma caproni from Biomphalaria glabrata snails. Visual observations of the chromatograms showed that the most abundant lipid fraction in all stages was free sterol. Quantification of the free sterol revealed mean weights of 2.7 +/- 0.64 ng per redia, 0.53 +/- 0.023 ng per cercaria, and 0.081 +/- 0.0098 ng per encysted metacercaria. Oil Red O staining of the larval stages confirmed the presence of lipids within the rediae and cercariae but did not show lipids in the encysted metacercariae. The dimunition in neutral lipids from the cercarial to the encysted metacercarial stage does not support a previous observation that fat increases in successive phases of the digenean life cycle.

High-performance thin-layer chromatography was used to analyze the neutral lipids in the rediae, cercariae, and encysted metacercariae of the paramphistomid trematode Zygocotyle lunata. Visual observations of the chromatograms showed that the most abundant lipid fractions were free sterols and free fatty acids in all larval stages and triacylglycerols in the metacercariae and rediae. The weight of free sterols (x +/- SE) was 120+/-20 ng/cercaria, 56+/-3.8 ng/redia, and 5.9+/-1.5 ng/encysted metacercaria; the weight of triacylglycerols was 13+/-0.88 ng/encysted metacercaria, 6.3+/-0.063 ng/redia, and was not detectable in the cercaria; the weight of free fatty acids was 160+/-17 ng/ cercaria, 76+/-9.1 ng/redia, and 4.2+/-0.46 ng/encysted metacercaria. Oil red O staining of whole larvae showed the presence of neutral lipids in the rediae but not in the cercariae or encysted metacercariae. A dramatic reduction was seen in the quantity of free sterols and free fatty acids in the encysted metacercariae as compared with the cercariae, suggesting that these neutral lipids are used in some way during the transformation from cercaria to metacercaria.

High-performance thin-layer chromatography (HPTLC) analysis was done on lutein and beta-carotene in the digestive gland-gonad complex (DGG) and whole body of uninfected Cerithidia californica snails and those infected with the larval trematodes Mesostephanus appendiculatis or Euhaplorichis californiensis. HPTLC of the DGG extract on C-18 reversed-phase plates developed in petroleum ether-acetonitrile-methanol (1:2:2) mobile phase showed 2 identifiable pigment zones; the least polar zone had a retention factor (Rf) of 0.07, identical to a beta-carotene standard, and the more polar zone had an Rf of 0.41, identical to a lutein standard. Densitometric scanning of the pigment zones in sample versus standard chromatograms showed that the weight percent of lutein in the uninfected DGGs (3.4x10(-3)%) was significantly greater (P<0.05) than that of DGGs infected with either M. appendiculatis (0.35x10(-3)%) or E. californiensis (0.82x10(-3)%). Changes in beta-carotene in the infected DGGs were insignificant compared to the uninfected controls. However, the beta-carotene content of whole snails was significantly reduced (P<0.05) by infection with either trematode.