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Cancer and the Environment: Gene-Enviroment Interaction Abstracts of Talks GENE–ENVIRONMENT INTERACTIONS RELATED TO COLON CANCER2 David Alberts, M.D. and M. Elena Martinez, M.D. Rates of Colorectal Cancer and Adenomatous Polyps. Colorectal cancer remains the third leading cause of cancer deaths in each sex and second overall in the United States (Landis et al., 1999), despite the fact that it is largely a preventable disease. Approximately half of diagnosed individuals will die of this malignancy. Because adenomatous polyps are precursors to colorectal cancer, assessing the effect of environmental and genetic factors in adenoma occurrence and recurrence instead of cancer might help identify relatively asymptomatic individuals who are at increased risk of cancer and who would benefit most from an overall public health intervention. Additionally, the identification of risk factors for recurrence may help define follow-up screening protocols. Although we have obtained some clues regarding risk factors for newly diagnosed adenomas (Neugut et al., 1993), few data exist on predictors of adenoma recurrence among individuals with resected adenomas (Davidow et al., 1996; Tseng et al., 1997; Baron et al., 1998; Hyman et al., 1998; Whelan et al., 1999). Genetic Basis for Colorectal Neoplasia. The genetic basis for the development of colorectal cancer involves the accumulation of specific somatic mutations in proto-oncogenes and tumor suppressor genes with increasing age 2 Supported by a Cancer Research Foundation of America grant, Public Health Service grants CA41108 (Colon Cancer Prevention Program Project) and CA-23074 (Arizona Cancer Center Core Grant) from the National Cancer Institute, and a Career Development Award (KO1 CA-79069-10) grant to M.E. Martinez from the National Cancer Institute
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Cancer and the Environment: Gene-Enviroment Interaction (Kinzler and Vogelstein, 1996). However, only a small proportion of colorectal cancers are attributable to inheritance of these rare, highly penetrant mutated genes. Epigenetic changes, such as alterations in DNA methylation (e.g., CpG island methylator phenotype, or CIMP) and gene expression, also may play a critical role in the development of this malignancy (Baylin et al., 1998). It is evident too that variability in carcinogen-metabolizing genes influences the risk of colorectal neoplasia in humans (Gertig and Hunter, 1998; Hussain and Harris, 1998a; Pereira, 1998). It is clear that susceptibility to colorectal cancer is related to interindividual variability in biotransformation of endogenous and exogenous substances, as well as in DNA repair and cell cycle control. Common genetic variation may enhance susceptibility to environmental carcinogens by altering the rates of activation and detoxification of carcinogens. The interactions of environmental factors with metabolic polymorphisms may act via a model in which the exposure alone, but not the variant genotype alone, increases disease risk; however, exposure interacts with the variant genotype to further increase risk in the exposed individuals (Vineis, 1997). The same interaction can also modulate disease pathogenesis in that exposure and susceptibility factors may alter the effects of other risk factors, such as folate intake, methylenetetrahydrofolate reductase (MTHFR), CIMP, selenium, or celecoxib intervention, and cyclooxygenase (COX) upregulation, on adenoma recurrence. An example of such an interaction is the relationship between alcohol intake, folate, and MTHFR. Classification of subgroups of the population into those who may be more vulnerable to the effects of certain carcinogens may also have important implications beyond risk assessment. Through the identification of an increased risk in certain subgroups, disease risk factors may be better defined. However, to date, sample sizes for most studies attempting to uncover gene–environment interactions have been small, limiting the potential for detecting significant findings. CIMP as a Marker of Gene Methylation. As stated previously, a genetic basis for cancer has been established with the assumption that the age (and mutagen exposure) related accumulation of somatic mutations accounts for the increased incidence of cancer with age (Ames et al., 1993). The actual rate of mutation accumulation in aged tissues is more uncertain, with some investigators finding lower than expected mutation rates (Warner and Price, 1989; Bohr and Anson, 1995), possibly reflecting the presence of additional mechanisms for activation and/or inactivation of genes important in the carcinogenesis process. In the past few years, there has been renewed interest in epigenetic mechanisms in carcinogenesis (Jones, 1996; Baylin et al., 1998). Epigenetics refers to the study of changes in gene expression that can be mitotically inherited, without associated changes in the coding sequence of the affected genes. Aging and transformed cells show profound changes in gene expression, many of which cannot be accounted for genetically (Sager, 1997). Methylation of DNA within
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Cancer and the Environment: Gene-Enviroment Interaction promoter-associated CpG islands can be a powerful molecular mechanism for gene silencing (Razin and Riggs, 1980; Adams and Burdon, 1982). In mammals, 5-cytosine methylation of CpG dinucleotides is the only naturally occurring modification of DNA. DNA methylation patterns form early in development with the establishment of gene and tissue-specific patterns of methylation, which are relatively stable (Razin and Riggs, 1980). In humans, approximately 70% of CpG dinucleotides are methylated in adult cells (Adams and Burdon, 1982; Bird, 1992). The function of normal DNA methylation remains controversial, as suggested by the fact that highly expressed genes tend to be hypomethylated, and that silent genes tend to be hypermethylated (Cedar, 1988; Bird, 1992). Issa and coworkers (Toyota et al., 1999a, 1999b) have developed a definition for a methylated phenotype referred to as CIMP. This phenotype was developed after analysis of a number of colon cancers, colorectal adenomas, and normal colonic mucosa. The research group found that CIMP-positive colorectal cancers averaged 5.1 methylated loci (out of 7) versus 0.3 for CIMP-negative tumors. Additionally, some genes were methylated in an age-related manner, while others were more clearly associated with cancer. Based on this work, CIMP-positive adenomas were more likely to have Ki-ras mutations, while CIMP-negative adenomas were more likely to have mutations in p53. Furthermore, CIMP-positive adenomas were found to have lower levels of COX2 expression because of promoter methylation, and their dependence on expression of DNA methyltransferase to maintain tumor suppressor gene silencing via hypermethylation. Folate, MTHFR, and Gene Methylation. An increasing epidemiologic body of evidence from case-control (Ferraroni et al., 1994) and cohort studies (Giovannucci et al., 1995, 1998; Glynn et al., 1996) supports the important role of folate in reducing the risk of colorectal cancer. Another study (Ma et al., 1997), which did not have comprehensive dietary data, showed an inverse association between plasma folate and risk of colon cancer. Folate intake and blood levels have also been consistently associated with lower risk of colon adenomas (Giovannucci et al., 1993; Tseng et al., 1996). Recent results indicate that increased consumption of folic acid from supplements, after a period of 15 or more years, may decrease the risk of colon cancer by about 75% (Giovannucci et al., 1998). Giovannucci et al. (1993) have proposed that the increased risk associated with low folate levels is related to intracellular methylation defects. Additionally, these investigators proposed that alcohol consumption increases the risk of colorectal neoplasia by acting as a folate antagonist; this hypothesis is based on data demonstrating the modifying effect of folate and methionine on the alcohol and colorectal neoplasia relationship (Giovannucci et al., 1993, 1995; Ma et al., 1997). Given the epidemiologic evidence for the proposed protective effect of folate on colorectal neoplasia, some studies have explored the mechanisms involved in
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Cancer and the Environment: Gene-Enviroment Interaction this association. Because folate is the primary methyl donor in cellular metabolism (Hoffman, 1985), markers of folate status are important factors to address in the etiology of gene methylation. A critical role of folate is in synthesizing methionine from homocysteine (Hoffman, 1985). Methionine, in turn, is converted to S-adenosylmethionine (SAM), the primary methyl donor in the reaction transferring a methyl group to the enzyme 5’-cytosine-DNA methyltransferase. Transfer of a methyl group from SAM to methyltransferase produces S-adenosylhomocysteine (SAH), which is then hydrolyzed to homocysteine. Folate is also essential for nucleotide biosynthesis (Eto and Krumdieck, 1986). In folate deficiency, thymidylate shortages cause an imbalance in the thymidylate–deoxyuridylate pool and a resultant incorporation of uridylate into DNA. Excess uridylate incorporation into DNA results in unstable chromosomes, decreased DNA repair, and increased chromosome breaks (Barclay et al., 1982; Reidy, 1987; Everson et al., 1988; Dianov et al., 1991; James et al., 1992). Genetic Polymorphisms of MTHFR. A genetic factor that modifies the effects of folate status has recently been identified that includes the inherited variation in the activity of MTHFR, a critical enzyme involved in the production of the form of folate that supplies the methyl group for methionine synthesis (Kutzbach and Stokstad, 1971). Different endogenous forms of folate, 5-methyltetrahydrofolate, and 10-methylenetetrahydrofolate, are essential for DNA methylation and DNA synthesis, respectively. A common thermolabile polymorphism in the MTHFR gene (C677→T, alanine→valine) has been shown to be protective against colon cancer in some (Chen et al., 1996; Ma et al., 1997; Slattery et al., 1999; Ulrich et al., 1999) but not all (Chen et al, 1998) studies. Low MTHFR activity is thought to protect against colorectal cancer since less tetrahydrofolate is converted to 5-methyltetrahydrofolate, allowing more folate to be shunted toward DNA synthesis and repair. In these studies, an inverse association was shown for the presence of the val/val genotype and colorectal cancer among individuals with adequate folate intake, whereas this effect was not seen among those with low folate intake (Chen et al., 1996; Ma et al., 1997). Since MTHFR is required to convert 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, individuals with low MTHFR levels (val/val homozygotes) would be expected to have relatively high levels of 5,10-methylenetetrahydrofolate resulting from the low levels of the 5-methyltetrahydrofolate (Frosst et al., 1995). Therefore, in a low-folate environment where there are inadequate quantities of methyl groups for these pathways, individuals with the val/val genotype do not divert as much folate from the thymidylate pathway to the methylation pathway, resulting in lower SAM levels and high levels of homocysteine. Under conditions in which folic acid levels are insufficient to meet metabolic needs, val/val homozygotes would be less efficient at diverting folate metabolites into the 5-methyltetrahydrofolate product, resulting in a shortage of methyl groups. When levels of 5,10-methylenetetrahydrofolate (which is required
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Cancer and the Environment: Gene-Enviroment Interaction to convert deoxyuridylate to thymidylate) are low, misincorporation of uracil for thymidine may occur during DNA synthesis (Wickramasinghe and Fida, 1994), possibly increasing spontaneous mutation rates (Weinberg et al., 1981), sensitivity to DNA-damaging agents (Meuth, 1981), frequency of chromosomal aberrations (Sutherland, 1988; Fenech and Rinaldi, 1994), or errors in DNA replication (Hunting and Dresler, 1985; Fenech and Rinaldi, 1994; James et al., 1994). Folate Deficiency and CpG Island Gene Methylation. The proposed mechanism for the above-reviewed studies relates to dietary factors that influence methyl group availability, which can in turn affect DNA methylation. DNA methylation is an essential mechanism of gene regulation, and disturbances may cause differential gene expression (Cedar, 1988). In animal models, folate deficiency can cause imbalances in DNA methylation (Wainfan and Poirier, 1992; Kim et al., 1996). Furthermore, folate deficiency in rats has been shown to induce DNA strand breaks and altered methylation within the p53 tumor suppressor gene (Kim et al., 1997) and to result in deoxynucleotide pool disturbances (James et al., 1992). Given this literature, support for the potential effect of folate status and methyl group availability in the etiology of CIMP status exists. Additional supporting evidence for this association derives from our own study (Martinez et al., 2001), in which a low intake of folate was associated with a significantly higher risk of Ki-ras mutations in adenomatous polyps. Previous work by Toyota et al. (1999a) indicates that CIMP-positive adenomas are more likely to harbor Ki-ras mutations than CIMP-negative adenomas. Thus, the potential role of folate in the etiology of Ki-ras, mutations along with data supporting the high rate of these mutations in CIMP-positive adenomas, suggests that folate may be involved in the etiology of CIMP-positive adenomas. Dietary Folate Intake, MTHFR Status, and Colorectal Cancer. Published data on the interaction between folate and MTHFR in the etiology of colorectal neoplasia are inconsistent, suggesting that this interaction is more complex than originally proposed. In a recent report of more than 3,000 case and control participants (Slattery et al., 1999), a lower risk of colorectal cancer associated with higher intake of folate was shown among individuals with the val/val genotype as compared to those with the ala/ala genotype who had low folate intake (odds ratio (OR) = 0.6; 95% confidence interval = 0.4–1.0). Of particular interest, the effects of folate among the val/val genotypes appeared to be stronger for the proximal (OR = 0.5) compared to the distal (OR = 0.8) colon. Based on Issa’s work, CIMP-positive adenomas were more prevalent in the proximal colon, which may be related to factors affecting folate metabolism and methyl group availability. Plasma Homocysteine, MTHFR Status, and Polyp Recurrence. We prospectively examined whether plasma levels of homocysteine were associated
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Cancer and the Environment: Gene-Enviroment Interaction with the risk of recurrence of adenomatous polyps (Martinez et al., 2001). Analyses were conducted among 1,014 men and women, 40 to 80 years of age, enrolled in a Phase III trial testing the effects of a wheat bran fiber intervention on adenoma recurrence. We also examined whether the association between plasma homocysteine and adenoma recurrence was modified by the MTHFR genotype among 961 participants with genotype data. Homocysteine in plasma was analyzed at baseline by high-performance liquid chromatography (HPLC). MTHFR genotyping was performed by high-throughput microarray technology. Compared to participants with lower plasma homocysteine levels, those with higher levels were older, were more likely to be male, had lower intakes of total (dietary plus supplemental) folate, had higher alcohol intakes, and had lower plasma folate levels. After adjustment for age, gender, number of colonoscopies, and a history of previous polyps, the odds ratio for adenoma recurrence for individuals with homocysteine levels >11.6 mmol/l was 1.45 (95% CI = 0.98–2.14; P-trend = 0.02) compared to those with levels <7.8. When we assessed the relationship between homocysteine levels and adenoma recurrence according to MTHFR status, individuals with the TT genotype and homocysteine levels above the median (>9.4) had a higher risk of recurrence (relative risk = 1.96) compared to those with the CC genotype and homocysteine levels below the median. The results of these analyses suggest a modest effect of plasma homocysteine levels on adenoma recurrence and a risk-enhancing effect of high homocysteine levels on adenoma recurrence among individuals with the TT MTHFR genotype. DIET AND RISK OF CHILDHOOD CANCER Greta Bunin, Ph.D. Epidemiologists often broadly define environment to encompass anything that is not genetics. Diet is an integral part of the environment. All solids including fish, foul, meat, grains, vegetables, and foods may contain trace contaminants (e.g., pesticides, heavy metals, polychlorinated biphenyls [PCBs]). The evidence is just beginning to emerge on the role of diet and cancer in children and, more specifically, the role of diet in the generation of cancer. Fewer than 20 studies have looked for a link between childhood cancer and diet. Most commonly, researchers have investigated the link between brain cancer and diet because of a hypothesis based on animal data. The hypothesis postulates that children with greater exposure to N-nitroso compounds (NOCs) and their precursors are more likely to develop a brain tumor compared to other children. In many species of animals, NOCs are highly potent carcinogens. Some NOCs induce nervous system tumors, and for a few NOCs, the risk of tumor development is multiplied when the exposure occurs in utero. Human exposure to NOCs is widespread, and they have been detected in many common products, including cigarette smoke, automobile interiors, and
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Cancer and the Environment: Gene-Enviroment Interaction cosmetics. Additionally, we are also exposed to precursors that combine to form NOCs in our stomachs and elsewhere in our bodies. In fact, most human exposure is thought to occur via synthesis in the body from precursors. Substances such as vitamins C and E inhibit the formation of NOCs and, thus, may be important for the prevention of brain tumors. Diet is a major source of NOCs, NOC precursors, and NOC inhibitors. Meats cured with nitrites, such as hot dogs and lunch meat, contain NOCs and NOC precursors, while fruits, vegetables, and vitamin supplements contain NOC inhibitors. Therefore, researchers hypothesize that a mother’s frequent eating of cured meats and infrequent eating of fruits and vegetables increase the risk of brain tumors in children. Of the eight studies investigating the NOC hypothesis, four found a significant doubling of risk of brain cancer when the mother frequently consumed cured meats during pregnancy. In a fifth study, a similar association was observed but it was not significant. Two studies had small numbers of children with brain tumors, which may explain why they failed to detect a difference. The last study looked at a less common type of brain tumor and observed no association with cured meat. This tumor type has a different sex and age distribution than the most common type and therefore might have a different etiology as well. Overall, the data are fairly consistent for an association between frequent consumption of cured meats during gestation and childhood brain tumors. Further research will be needed to determine if the brain tumors are due to the NOCs in cured meats or to a nutrient such as high fat or low folate in the diet. SIMILARITIES OF PROSTATE AND BREAST CANCER: EVOLUTION, DIET, AND ESTROGENS Donald S. Coffey, Ph.D. The risk of both prostate and breast cancer is similar and primarily determined by the environment in which one lives, and this risk can vary more than tenfold between countries. In contrast, no risk exists for human seminal vesicle cancer, thus demonstrating tissue specificity for cancer in the human. There is also species specificity because there is no risk for prostate cancer in any other of the thousand of aging mammal species except the dog. Evolution indicates that the prostate and breast appeared at the same time 65 million years ago with the development of mammals. All male mammals have a prostate; however, the presence of seminal vesicles is variable and is determined by the diet so that species primarily eating meat do not have seminal vesicles. The exception is the human, who has seminal vesicles and consumes meat, although this is a recent dietary change. Human lineage departed from other higher primates 8 million years ago. The closest existing primate to humans is the bonobo (pigmy chimpanzee), which does not eat meat but exists primarily on a high-fruit and fresh
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Cancer and the Environment: Gene-Enviroment Interaction vegetable diet. Homo sapiens evolved only about 150,000 years ago, and only in the last 10% of that time (10,000–15,000 years ago) did humans and dogs dramatically alter their diets. This is the time when humans domesticated the dog, bred animals, grew crops, and cooked, processed, and stored meats and vegetables. Current epidemiologic evidence and suggestions for preventing prostate and breast cancer in humans indicate that we should return to the original type of diets under which our ancestors evolved. The recent development of the Western-type diet is associated with breast and prostate cancer throughout the world. It is believed that the exposure to and metabolism of estrogen, and the dietary intake of phytoestrogens, combined with fat intake, obesity, and burned food processing, may all be related to hormonal carcinogenesis and oxidative DNA damage. An explanatory model is proposed. (For details see Coffey, 2001.) EFFECT OF HERBAL THERAPIES ON PROSTATE CANCER Robert S. DiPaola, M.D. Background. Herbal mixtures are popular alternatives to demonstrated therapies. PC-SPES, a commercially available combination of eight herbs, is used as a nonestrogenic treatment for cancer of the prostate. Since other herbal medicines have estrogenic effects in vitro, we tested the estrogenic activity of PC-SPES in yeast and mice and in men with prostate cancer. Methods. We measured the estrogenic activity of PC-SPES with transcriptional activation assays in yeast and biologic assay in mice. We assessed the clinical activity of PC-SPES in eight patients with hormone-sensitive prostate cancer by measuring serum prostate-specific antigen and testosterone concentrations during and after treatment. Results. PC-SPES had estrogenic activity similar to that of 1 nM estradiol, and in ovariectomized CD-1 mice, the herbal mixture increased uterine weights substantially. In six of six men with prostate cancer, PC-SPES decreased serum testosterone concentrations (P < 0.005), and in eight of eight patients, it decreased serum concentrations of PSA. All eight patients had breast tenderness and loss of libido, and one had venous thrombosis. HPLC, gas chromatography, and mass spectometry showed that PC-SPES contains estrogenic organic compounds that are distinct from diethylstilbestrol, estrone, and estradiol. Conclusions. PC-SPES has potent estrogenic activity. The use of this unregulated mixture of herbs may confound the results of standard or experimental therapies and may produce clinically significant adverse effects. Further studies to identify the estrogen(s) responsible for this activity are warranted.
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Cancer and the Environment: Gene-Enviroment Interaction COLORECTAL CANCER AND ENVIRONMENTAL RISK FACTORS Raymond N. DuBois, M.D., Ph.D. Risk factors for colorectal cancer include a positive family history, meat consumption, smoking, and alcohol consumption. A reduction in risk for the disease is associated with vegetable intake, use of nonsteroidal anti-inflammatory drugs (NSAIDs), hormone replacement therapy, and physical activity. There are several genetic and epigenetic alterations that are known to be involved in the development of colorectal cancer. These alterations are important in both inherited syndromes such as familial adenomatous polyposis (FAP) or hereditary nonpolyposis colorectal cancer (HNPCC) and in sporadic tumors. It will be important to understand the roles of environmental exposure and host susceptibility to develop better screening, prevention, and treatment strategies. Population-based studies indicate a 40–50% reduction in mortality from colorectal cancer in persons using NSAIDs on a regular basis (Smalley and DuBois, 1997). Colorectal cancer is a major cause of death from cancer in Western civilizations, claiming more than 55,000 lives in the United States each year. Environmental and dietary factors play an important role in the etiology of this disease as well as the known genetic components. Research efforts have been focused on understanding the molecular basis for the chemoprotective effects associated with use of aspirin and other NSAIDs. NSAIDs inhibit both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) activity (Williams and DuBois, 1996). Since COX-2 levels are increased in a number of solid tumors, this enzyme may serve as a molecular target for cancer prevention (Sheng et al., 1997). Recent clinical studies indicate that the presence of COX-2 in human lung and colon cancers is associated with a negative clinical prognosis (Achiwa et al., 1999; Sheehan et al., 1999). Therefore, COX-2 inhibitors are currently being evaluated for the prevention and/or treatment of cancer in humans (Steinbach et al., 1999). GENES AND THE ENVIRONMENT IN CANCER ETIOLOGY Joseph F. Fraumeni, Jr., M.D. The importance of environmental factors in human cancer has long been evident from the striking international variation reported in cancer incidence, resulting in estimates that perhaps 80% of all cancer in the United States is potentially preventable or avoidable. Further indications of environmental cancer come from the shifts in the cancer experience of migrant populations whose rates tend to approximate those of the host country, the geographic patterns of cancer within the United States, the changing incidence of certain cancers over time, ethnic and socioeconomic differentials, and the abundant epidemiologic evidence linking carcinogenic risks to a variety of lifestyle and other environ-
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Cancer and the Environment: Gene-Enviroment Interaction mental exposures. Not long ago, the role of inherited susceptibility in human cancer was considered to be quite small given the rarity of hereditary cancer syndromes, but recent progress in identifying and characterizing highly penetrant but relatively rare susceptibility genes in familial cancer has revolutionized our understanding of genetic mechanisms and their critical importance in cancer etiology. Of special significance to the public health burden of cancer, however, are the common polymorphic susceptibility or modifier genes that confer low relative and absolute risks, but high population-attributable risks in the presence of relevant environmental exposures. The two classes of genes represent parts of a continuum because even the highly penetrant genes responsible for hereditary cancer may involve environmental exposures for expression, as illustrated by the susceptibility to carcinogens in hereditary retinoblastoma and Li–Fraumeni syndrome. Especially exciting is the opportunity to parlay discoveries of polymorphic genes and their functions into a better understanding of environmental carcinogenesis. By incorporating careful exposure assessment and mechanistically plausible candidate susceptibility genes into epidemiological study designs, it should be possible to identify the more subtle risks due to specific dietary and nutritional factors, metabolic alterations, environmental pollutants, and other common exposures that have eluded traditional epidemiologic approaches. Although the molecular and statistical tools to examine complex gene–environment interactions are still in development, opportunities now exist for population and family-based studies using biomarkers that integrate the search for susceptibility genes and the exogenous and endogenous exposures that cause cancer. While the methodologic challenges of “molecular epidemiology” are formidable, this interdisciplinary approach to cancer etiology should provide unprecedented opportunities to enlarge our understanding of environmental and genetic risk factors and their biological pathways, and set the stage for new clinical and public health strategies aimed at preventing and controlling cancer. CANCER DISPARITIES IN APPALACHIA Gilbert H. Friedell, M.D. Despite recent good news about decreasing U.S. cancer mortality rates, not all population subgroups are sharing in this success story. Progress toward meeting the cancer-related Healthy People 2010 goals will be hampered by the nation’s inability to deal effectively with the greater cancer burden borne by certain vulnerable populations. These “special populations,” defined as population groups at higher-than-average risk of death, disease, and disability, include people with low incomes, African Americans, Hispanics, American Indians, and other ethnic minorities.
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Cancer and the Environment: Gene-Enviroment Interaction In addition, the National Cancer Institute (NCI) has stated that it considers rural residents to constitute “a special population.” Rural Americans tend to be older, poorer, less educated, and more likely to be uninsured than their urban counterparts. Rural communities have higher rates of chronic illness and disability, and report poorer overall health status than their urban neighbors. Residents in rural areas generally have less contact and fewer visits with physicians and, in general, lower levels of preventive care. In addition to factors related to rural health status and practices, there are systemic factors related to rural life in general (e.g., lack of public transportation and lower levels of other community services) that may also contribute to less than optimal cancer control. All of these factors are evident in the largely rural and predominantly white population of Appalachia, particularly in the Central Highlands, including the Appalachian counties of Ohio, West Virginia, Kentucky, Tennessee, and Virginia. Lung cancer is a leading cause of male cancer deaths in central Appalachia, with the highest rate in Appalachian Kentucky, the geographic area where the Behavioral Risk Factor Surveillance Survey data (BRFSS) indicate the highest rates of cigarette smoking in the state. Cervical cancer mortality rates are also higher in central Appalachia than in the U.S. population as a whole. Data from the Kentucky cancer registry showed that the incidence of invasive cervical cancer and lung cancer in eastern Kentucky is higher than the incidence of these cancers in the overall Kentucky population and in the population covered by the Surveillance, Epidemiology, and End Results (SEER) program. It is, however, quite similar to the incidence of lung cancer and cervical cancer in the predominantly urban, African American population of Kentucky. Poverty is a common characteristic in much of this region. Some of the counties in Appalachian Kentucky, for example, are among the poorest in the country. In the same geographic areas, the level of literacy—indicated by the highest grade of formal schooling completed—is also lower than in most of the country. Problems associated with poverty are similar to those confronting poor populations in other parts of the country, but the latter are often characterized by race or ethnicity rather than by socioeconomic status (SES). This use of race and ethnicity as surrogates for poverty has obscured the fact that the problems related to cancer in the poor white population are comparable in many ways to those seen in recognizable minority populations. Individuals living in poverty often do not receive quality health care, including cancer prevention, diagnosis, treatment, and appropriate follow-up care, because services are not available, accessible, and/or utilized. Behavioral risk factors, such as tobacco use, poor nutrition, obesity, and underutilization of cancer screening examinations are more evident in impoverished populations. The social environment in which poor people live also prevents the development of healthy behaviors. Freeman has pointed out that poverty “is a proxy for other elements of living, including lack of education, unemployment, substandard housing, poor nutrition, risk-promoting lifestyles and behaviors, and a dimin-
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Cancer and the Environment: Gene-Enviroment Interaction At least part of this variation in response to dietary components may relate to the consumer’s genetic profile. It is now becoming apparent that the prevalence of polymorphisms is variable among studied populations and these differences could influence the response to diet. For example, in a random sample of participants in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC Study), there was a low prevalence of polymorphisms in genes coding for activation (phase I) enzymes CYP1A1 (0.07) and CYP2E1 (0.02) and a high prevalence in genes coding for detoxification (phase II) enzymes GSTM1 (0.40) and NQO1 (0.20). Evidence exists that several genetic polymorphisms may modulate cancer risk through their influence on folate metabolism, including two polymorphisms of the MTHFR gene C677 C–T (alanine –valine) and 1298 A–C (glutamate–alanine) and a polymorphism of MTR, the gene that codes for methionine synthase C2756 A–G (aspartate–glycine); all of these polymorphisms reduce enzyme activity. Epidemiologic studies have reported that when folate intake was adequate, colorectal cancer risk was reduced (abou 50%) in individuals with the MTHFR 677TT genotype compared with the MTHFR 677CC genotype, and the risk of adult acute lymphocytic leukemia (ALL) was reduced by 77%. Variation in the response to folate metabolism is not unique since other studies suggest that variation in receptors for vitamin D may also be linked to cancer risk. Considerably more information is needed about how genetic polymorphisms influence the response to dietary components and ultimately cancer risk. Unquestionably cancer is intertwined with environmental factors including diet. Strategies to prevent cancer through modification of either diet or specific dietary patterns, although intriguing and likely a low-cost health care strategy, will probably not be uniformly effective for all individuals. A better understanding of gene–nutrient interactions will be needed to unravel who might benefit most from dietary intervention and who might be placed at risk. Future research in nutrition and cancer prevention must give top priority to studies that seek to understand the basic molecular and genetic mechanisms by which nutrients influence the various steps in carcinogenesis. While the challenges to researchers will be enormous, the potential rewards in terms of reducing cancer morbidity and mortality will be of an equally great magnitude. MOLECULAR PATHOGENESIS OF LUNG CANCER John D. Minna, M.D. We and others have hypothesized that clinically evident lung cancers have accumulated 10–20 different genetic abnormalities in dominant oncogenes and/ or tumor suppressor genes (TSGs) (Sekido et al., 1998; Fong et al., 1999). If true, this hypothesis has important ramifications for the clinic. For example, it should be possible to discover carcinogen-exposed respiratory epithelial cells
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Cancer and the Environment: Gene-Enviroment Interaction with only a subset of these changes and intervene with very early treatment and/ or chemoprevention. A related hypothesis is that these changes are recurrent and common between different tumors. If true, this has implications for directing the search for specific diagnostic and therapeutic targets and indicating the likelihood that all of the changes are required for the malignant phenotype. There have been many studies published on searching for genetic abnormalities in lung cancer (Sekido et al., 1998; Fong et al., 1999). However, with few exceptions, these studies have not been global in nature either in testing for genome-wide abnormalities or in testing for multiple abnormalities in the same individual lung cancer. To approach these two hypothesis in a global and quantitative fashion, we have performed a high resolution (10 cM) genome-wide search for loss of heterozygosity (LOH, allele loss) in 36 lung cancer cell lines using 399 polymorphic markers. Individual tumors averaged 17–22 chromosomal regions involved in frequent, recurrent allele loss (“hot spots”), and these regions were significantly different between small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) (Girard et al., 2000). On average, 35% of the markers showed allele loss in individual tumors, with an average size of subchromosomal regions of loss of 50–60 cM. We found 22 different regions with more than 60% LOH, 13 with a preference for SCLC, 7 for NSCLC, and 2 affecting both histologic types. This provides clear evidence on a genome-wide scale that SCLC and NSCLC differ significantly in the TSGs that are inactivated during their pathogenesis. However, in all other aspects (e.g. fractional allele loss, number of breakpoints, number of microsatellite alterations) SCLC and NSCLC were not significantly different. The chromosomal arms with the most frequent LOH were 1p, 3p, 4p,4q, 5q, 8p, 9p (p16), 9q, 10p, 10q, 13q (Rb), 15q, 17p (p53), 18q, 19p, Xp, and Xq. We next conducted detailed high-density allelotyping (~5-cm level) on chromosome arms 3p, 4p, 4q, and 8p (average of 25 markers per chromosome arm) in 66 microdissected primary archival lung cancers (22 SCLC, 21 squamous, 22 adenocarcinomas), as well as microdissected respiratory preneoplastic lesions from patients with lung cancers and from cigarette smokers (Shivapurkar et al., 1999; Wistuba et al., 1999b, 2000a). Allelic losses of 3p were found in 96% of lung cancers and in 78% of the preneoplastic/or preinvasive lesions. The 3p allele losses were often multiple and discontinuous, with areas of LOH interspersed with areas of retention of heterozygosity. There was progressive increase in the frequency and size of 3p allele loss regions with increasing severity of histopathological preneoplastic/preinvasive changes. Analysis of all of the data indicated multiple regions of localized 3p allele loss. A panel of six markers in the 600-kb 3p21.3 homozygous deletion region showed loss in 77% of lung cancers (100% SCLC, 100% squamous, 90% adenocarcinomas), 70% of normal or preneoplastic/preinvasive lesions associated with lung cancer, and 49% of 47 normal, mildly abnormal, or preneoplastic/preinvasive lesions found in smokers without lung cancer (Wistuba et al., 2000a). This was in contrast to 0% loss in 18
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Cancer and the Environment: Gene-Enviroment Interaction epithelial samples from seven never smokers. We have identified all of the genes in this completely sequenced region, and several of them appear to suppress the tumorigenic phenotype when introduced back into lung cancers with multiple other genetic lesions (Lerman and Minna, 2000). Of these, the best studied is the RASSF1A mRNA isoform of the RASSF1 locus (Dammann et al., 2000; Burbee et al., 2001). This gene is rarely mutated in lung cancer, but its expression is lost by promoter-acquired hypermethylation in ~90% of SCLCs and 30–40% of NSCLCs. Methylation in NSCLCs is associated with adverse survival and treatment of lung cancer cells with 5-azacytidine reactivates RASSF1A expression. This isoform contains a RAS binding domain and a putative diacylglycerol binding domain and suppresses the tumorigenic phenotype of lung cancer cells (Dammann et al., 2000; Burbee et al., 2001). This 600-kb region and the 3p14.2 (FHIT/FRA3B) and 3p12 (U2020/DUTT1) regions were common, independent sites of breakpoints (Wistuba et al, 2000a). We conclude that 3p allele loss is nearly universal in lung cancer pathogenesis; involves multiple, discrete, 3p LOH sites that often show a “discontinuous LOH” pattern in individual tumors; occurs in preneoplastic/preinvasive lesions of smokers with and without lung cancer; and frequently involves breakpoints in at least three very small, defined genomic regions. These findings are consistent with previously reported LOH studies in a variety of tumors showing allele loss occurring by mitotic recombination and induced by oxidative damage. Similarly, high frequencies of LOH (86% SCLC, 100% squamous, 81% adenocarcinomas) for the 8p21–23 regions were detected in primary lung cancers (Wistuba etal., 1999b). The LOH commenced early during the multistage development of lung cancer at the hyperplasia/metaplasia stage in cancer patients and in smokers without cancer. Of interest, 8p21–23 allelic losses always followed 3p and usually followed 9p allele loss. In contrast to 3p LOH, no 8p LOH was found in histologically normal epithelium; however, 15% of mildly abnormal, 50% of dysplastic, and 92% of carcinoma in situ lesions had 8p21–23 LOH. Allelic loss occurred in 65% of smokers without cancer and persisted for up to 48 years after smoking cessation. Frequent LOH of 4p and 4q markers was seen in SCLC and mesotheliomas (Region 1 4q33–34 >80%, Region 2 4q25–26 >60%, and Region 3 4p15.1–15.3 >50%) but was much less frequent (20–30%) in NSCLC where the most frequent pattern was loss of Region 3 alone (Shivapurkar et al., 1999). For 3p, the regions of loss in SCLC and squamous cancers were usually quite large, often involving multiple markers, whereas those in adenocarcinomas were much smaller and usually involved only one or two markers. SCLC had significantly higher frequency of 4p and 4q allele loss (two separate regions on each arm) than did NSCLC (Shivapurkar et al., 1999). The converse was seen with the 8p allelotype (Wistuba et al., 1999b). Tumor-acquired promoter methylation is a new, important mutational mechanism for inactivating TSGs. We found tumor-acquired aberrant promoter methylation in nine genes in 107 resected primary NSCLCs (RAR beta 40%, TIMP-3 26%, p16ink4a 25%, MGMT 21%, FHIT 37%, DAPK 19%, ECAD 18%,
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Cancer and the Environment: Gene-Enviroment Interaction p14ARF 8%, and GSTP1 7%) (Park et al., 1999; Virmani et al., 2000; Zochbauer-Muller, et al., 2001a, 2001b). At least one gene was methylated in 86% of NSCLCs, whereas normal lung from these same patients was methylated in only a few patients. In addition, we found 63/87 (72%) of SCLCs to exhibit RAR promoter hypermethylation. The methylation events occurred independently of one another. However, about 13% of the NSCLCs exhibited more frequent promoter hypermethylation and thus are candidates for having a “global CpG island methylator phenotype.” In nonmalignant bronchial epithelium 218 foci (195 of histologically normal or slightly abnormal epithelium and 23 of dysplastic epithelium) were studied from 19 surgically resected lobectomy specimens (Park et al., 1999). Thirteen (68%) of the nineteen specimens had at least one focus of bronchial epithelium with molecular changes. At least one molecular abnormality was detected in 32% of the 195 histologically normal or slightly abnormal foci and in 52% of the 23 dysplastic foci. Extrapolating from a two-dimensional analysis, we estimate that most clonal patches contain approximately 90,000 cells. Although, in a given individual, tumors appeared homogeneous with respect to molecular changes, the clonally altered patches of mildly abnormal epithelium were heterogeneous. Our findings indicate that multiple small clonal or subclonal patches containing molecular abnormalities are present in normal or slightly abnormal bronchial epithelium of patients with lung cancer. In detailed studies of bronchial epithelium and bronchial biopsies from current or former smokers without lung cancer, we also find thousands of clonal patches showing allele loss in histologically normal-appearing respiratory epithelium. In fact, these patches can be detected more than 30 years after cessation of cigarette smoking. This would suggest the potential for damaged stem cells to repopulate. We also investigated the relationship between the amount of smoking and the degree of methylation of the p16, RASSF1A, RAR, APC, and HCAD(CDH13) genes in more than 200 resected NSCLCs from Japan with known smoking history. We found that p16 and RASSF1A developed promoter hypermethylation related to the amount of cigarette smoking, while RAR, APC, and HCAD(CDH13) methylation occurred independently of the amount of cigarette smoking (Zöchbauer-Müller et al., in preparation). To investigate whether methylation of genes such as RAR, HCAD(CDH13), p16, RASSF1A, and FHIT occurs in smoking-damaged epithelium before lung cancer develops, we analyzed oropharyngeal brushes, sputum samples, bronchial brushes, and bronchoalveolar lavage (BAL) samples from more than 100 heavy smokers without evidence of cancer (Zochbauer-Muller et al., 2001a). Methylation of at least one gene was present in one or more specimens from nearly 50% of the smokers. However, the frequency of methylation of these genes found in the epithelial samples from heavy smokers was lower than the frequency found in primary lung cancers. These findings indicate that promoter-acquired methy-
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Cancer and the Environment: Gene-Enviroment Interaction lation should be tested as an intermediate marker of risk assessment and response to chemoprevention. Small cell lung cancer has many distinct morphologic and biochemical features (such as neuroendocrine phenotype) distinguishing it from the non-small cell lung cancer histologic types (Sekido et al., 1998, 2001; Fong et al., 1999). These distinctions are of diagnostic importance and commit patients with different histologic types to quite different initial treatment regimens. With the exception of bronchoalveolar lung cancer, all histologic types have smoking and tobacco carcinogens as the major underlying etiologic factor. Clearly, SCLC etiology is strongly tied to cigarette smoking. Therefore, we have sought to answer the following major questions: Are there differences in the number or type of acquired molecular abnormalities between SCLC and NSLC; what are the specific genes involved; and what is the nature of the molecular changes that are found in smoking-damaged bronchial epithelium accompanying SCLC and NSLC? Finally, are there gene expression profiles that distinguish these two major histologic types? There is a wealth of information concerning molecular abnormalities in SCLC (Fong et al., 1999; Sekido et al., 2001). Ras mutations represent an obvious difference, they are found in ~30% of NSCLCs (predominantly adenocarcinomas) but, to our knowledge, have never been found in SCLCs (with more than 100 tumors analyzed). In fact, introducing a mutant ras allele into SCLCs in vitro has led to an alteration of the cellular phenotype to one more like NSCLC. A related component in the same signal transduction pathway is Her2/neu, which is overexpressed in ~30% of NSCLCs but rarely overexpressed in SCLCs. We have recently found a related signal pathway activation difference for the ERK/ MAP kinase pathway. While ERK1 and ERK2 proteins are expressed in all histologic types of lung cancer, we find constitutive activation (detection of the “active” phosphorylated forms of ERK1 and ERK2 using specific antibodies) in 80% of NSCLCs but <5% of SCLCs. Autocrine growth factors involving neuroendocrine regulatory peptides (e.g., bombesin–gastrin-releasing peptide) were first described for SCLC. However, recently it has become clear that both SCLC and NSCLC can express these peptides and their specific receptors (Sekido et al., 1998, 2001; Fong et al., 1999). While there are some differences related to histology (e.g., expression of neuromedin B in NSCLCs), it appears that both histologic types use this mechanisms. Myc oncogenes are overexpressed frequently in both SCLC and NSCLC. C-myc is overexpressed in both SCLC and NSCLC, but the overexpression of myc family members L-myc and N-myc is usually only found in SCLC. The p53 gene is frequently mutated in both SCLC and NSCLC, but this occurs in >90% of SCLCs and ~50% of NSCLCs. The other components of the p53 pathway (such as MDM2 and p14ARF) need to be studied. In comparing the type of mutations occurring in p53, there appear to be no differences (e.g., in nucleotide-type change or location in the p53 open reading frame) between SCLC
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Cancer and the Environment: Gene-Enviroment Interaction and NSCLC, providing evidence that the carcinogenic insult was similar. Another major difference is seen in the RB/p16 signaling pathway. This pathway is inactivated in the vast majority of all histologic types of lung cancer. However, the target mutated various dramatically between histologic types. In SCLC, Rb is inactivated in >90% of cases, with loss of protein expression usually occurring with truncating mutations. It is very rare for SCLCs to have mutations inactivating the expression of p16. In contrast, NSCLCs inactivate p16 expression in ~50% of cases, while loss of expression of Rb protein occurs in <20% (Sekido et al., 1998). There appear to be no differences in mutational frequencies for inactivation of FHIT occurring in 50–70% of all lung cancers (Fong et al., 1999; Sekido et al., 2001). Finally, we have looked at the bronchial epithelium accompanying SCLC and NSCLC for the occurrence of clonal alterations using precise laser capture microdissection with subsequent allelotyping for polymorphic markers (Wistuba et al., 2000b). In NSCLC, we frequently find clones of cells with molecular abnormalities in histologically affected epithelium (e.g., carcinoma in situ, dysplasia, hyperplasia) and occasionally in normal-appearing epithelium in the case of current or former smokers. In SCLC, these histologic preneoplastic changes were minimal. However, in studies of histologically normal respiratory epithelium, we found a severalfold increased rate of allele loss in SCLC compared to NSCLC patients. Thus, the smoking-damaged histologically normal epithelium associated with SCLC appeared “genetically scrambled” and had incurred significantly more damage than the epithelium accompanying NSCLCs. We conclude that SCLCs and NSCLCs do not differ significantly in the number of genetic alterations that occur, however SCLCs do differ significantly from NSCLCs in the specific genetic alterations that occur. In addition, smoking-damaged bronchial epithelium accompanying SCLCs appears to have undergone significantly more acquired genetic damage than that accompanying NSCLCs. Future studies need to identify the specific genes involved at these multiple sites and determine whether these provide new tools for early molecular detection, for monitoring of chemoprevention efforts, and as specific targets for developing new therapies. We conclude from our global and quantitative studies that clinically evident lung cancers have acquired 20 or more clonal genetic alterations; SCLC and NSCLC have acquired different genetic lesions; alterations in 3p TSGs appear especially early, followed by changes in 9p, 8p, and then multiple other sites; tumor-acquired promoter hypermethylation is a frequent mutational mechanism in lung cancer; changes consistent with oxidative damage leading to mitotic recombination are frequently seen; smoking-damaged histologically normal epithelium, as well as epithelium with preneoplastic/preinvasive changes, has thousands of clonal patches containing genetic alterations; and correcting even single genetic abnormalities can reverse the malignant phenotype. All of these observa-
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Cancer and the Environment: Gene-Enviroment Interaction tions are ready for translation into the clinic for new methods of diagnosis, risk assessment, prevention, and treatment. BREAST CANCER GENETICS: BRCA1 AND BRCA2 GENES Olufunmilayo Olopade, M.D. Breast cancer is a genetic disease, caused by spontaneous mutations in somatic cells or by germline inheritance of mutations in breast cancer susceptibility genes. Germline mutations in the BRCA1 or BRCA2 susceptibility genes result in breast cancers characterized by young age of onset, bilaterality, association with ovarian cancer and other tumor types, vertical transmission, and distinct tumor phenotypes. Because breast cancer develops in 37–85% of women that carry BRCA1 or BRCA2 mutations, genetic testing of individuals with a high risk of familial breast cancer is an important part of a cancer control effort. Somatic genomic rearrangements that cause breast cancer include amplification of the HER-2/neu gene, which is associated with a poor prognosis, relative resistance to chemotherapy and tamoxifen, and sensitivity to Herceptin. Detection of HER-2/neu amplification in tumors is therefore an important factor in prognosis and choice of therapies. These examples reveal the clinical value of addressing the genetic basis of cancer and illustrate the importance of understanding genetic mechanisms in developing methods of cancer prevention, early detection, and targeted therapies. TYPES AND TRENDS OF CHILDHOOD CANCER; CANCER IN CHILDHOOD CANCER SURVIVORS Leslie Robison, Ph.D. In the United States, cancer is the leading cause of death due to disease among individuals between the ages of 1 and 20. The annual incidence rate is 15/ 100,000, which translates into a cumulative risk of cancer equivalent to 1 in 300 by the age of 20 years. The types of malignancies in children and adolescents differ from adults and include (annual rate per million and proportion): leukemia (37, 24.8%); lymphoma (24, 16.1%); brain and central nervous system (CNS) (25, 16.8%); neuroblastomoa (7, 4.7%); retinoblastoma (3, 2.0%); kidney, predominantly Wilms’ tumor (6, 4.0%); liver, predominaently hepatoblastoma (2, 1.3%); bone, primarily osteoscarcoma; and Ewing’s sarcoma (9, 6.0%); soft tissue, predominaently rhabdomyosarcoma (11, 7.4%); germ cell (10, 6.7%); and others (15, 10.0%). There are distinct age-specific patterns of incidence; most notable are the peaks in incidence of acute lymphoblastic leukemia that occur between the age
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Cancer and the Environment: Gene-Enviroment Interaction of 3 and 6 years; the aggregation of neuroblastoma, retinoblastoma, and Wilms’ tumor in children below the age of 5; the increasing incidence with age of lymphoma; and the relatively constant incidence of brain and CNS malignancies. Overall, males have a higher rate of malignancies than females, which is attributable primarily to a higher incidence among males of lymphomas and acute lympohoblastic leukemia. In the 15–20 years of age group, females have a higher incidence of cancer than males. Further, the annual incidence (per million population) of childhood and adolescent cancers differs by race: Caucasian (161.7), black (124.6), Hispanic (145.6), Asian/Pacific Islander (136.8), and Native American (79.6). Observations during the past several decades have identified a modest, but consistent, increase in the incidence of childhood cancers. Secular trends have varied with specific categories, but the most consistent increases have been seen in acute leukemia and tumors of the central nervious system. The survival rate for childhood and adolescent cancer has increased dramatically during the past three decades. Currently, more than 70% of individuals diagnosed with cancer before age 15 will survive five or more years from diagnosis, with the majority being cured of their original malignancy. With these improvements in treatment and survival, it is estimated that approximately 1 in every 900 individuals in the United States between the age of 15 and 45 is now a survivor of childhood cancer. These survivors are, however, at increased risk for long-term complications of their initial cancer and subsequent therapy. Late sequelae of childhood cancer can include an increased risk of second and subsequent malignancies, as well as serious organ dysfunction and psychosocial effects. As more patients survive and the length of follow-up grows, patterns of second and subsequent malignancies are being identified in survivors, including increased rates of breast cancer, thyroid malignancies, CNS tumors, and leukemia. CANCER TREATMENT BASED ON IMMUNE STIMULATION Steven Rosenberg, M.D., Ph.D. Tumor infiltrating lymphocytes (TILs) obtained from patients with melanoma have been used to clone the genes encoding the antigens recognized by these TILs. TILs have been identified that can recognize unique cancer antigens on murine and human cancers including melanoma, breast cancer, colon cancer, and lymphoma. The major histocompatibility complex MHC restricted recognition of human cancer antigens was detected by assaying panels of human leukocyte antigen (HLA)-typed target cells and by transfection into target cells of genes encoding the appropriate HLA specificities. In clinical trials of TILs administration, 36% of patients with metastatic melanoma underwent objective cancer remission. TILs trafficked to and accumulated in cancer deposits.
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Cancer and the Environment: Gene-Enviroment Interaction GENETIC SUSCEPTIBILITY TO LUNG CANCER4 Margaret R. Spitz, M.D. Less than 20% of long-term smokers develop lung cancer by age 75. Genetically determined factors that abrogate the effects of environmental carcinogens may explain differences in susceptibility. The challenge in quantitative risk assessment is to account for this interindividual variation in susceptibility to carcinogens. Evidence of familial aggregation of lung cancer provides indirect support for the role of genetic predisposition to lung cancer. These patterns of inheritance studies suggest that a small proportion of lung cancer is due to “lung cancer genes” that are probably of low frequency, but high penetrance. However, the study of low-penetrance, high-frequency genes is likely to be more useful in elucidating the causal pathways for the vast majority of lung cancers. Lung cancer risk is dependent on the dose of tobacco carcinogens, which in turn is modulated by genetic polymorphisms in the enzymes responsible for carcinogen activation (e.g., myeloperoxidase) and detoxification (e.g., glutathione s-transferases), as well as by the efficiency of the host cells in monitoring and repairing tobacco carcinogen DNA damage. Individuals with susceptible genotypes (or adverse phenotypes) tend to develop lung cancer at earlier ages and with lower levels of tobacco exposure. On the other hand, the genetic component in risk tends to be lower at high dose levels, when environmental influences overpower genetic predisposition. We have applied phenotypic assays to measure DNA repair capacity (DRC) by means of the in vitro host cell reactivation assay using plasmids damaged with benzo[a]pyrene. DRC was significantly lower in cases than controls, lower in women compared to men, and lower in younger compared to older cases. There was a statistically significant trend for increasing risk with decreasing DRC and an odds ratio (OR) of 2.54 (P < 0.05) for lung cancer in the least efficient repair stratum. The mutagen sensitivity assay, in which in vitro mutagen-induced breaks are quantitated as a measure of carcinogen sensitivity, has also been identified as a significant risk factor for lung cancer. Mutagens used are bleomycin and benzo[a]pyrene. Higher risk estimates are evident for current compared to former smokers and lighter smokers (less than one pack per day) compared to heavier smokers. There was a dose–response relationship with adjusted ORs for increasing quartiles of induced chromatid breaks for both bleomycin sensitivity and benzo[a]pyrene sensitivity (trend P < 0.0001). On stratified analyses, the risk for both adverse phenotypes (suboptimal DRC and mutagen sensitivity) was fivefold. 4 Supported by National Cancer Institute grants CA55769 (to M.R.S.) and CA 68437 (to W.K.H.).
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Cancer and the Environment: Gene-Enviroment Interaction Genotype–phenotype and diet–gene interactions are also being studied intensively. For example, while the GSTM1 null genotype was not an apparent independent risk factor for lung cancer, in the presence of low isothiocyanate intake, the OR for the GSTM1 null genotype was 2.33. There was no increased risk in any stratum for former smokers. It is most likely that multiple susceptibility factors must be accounted for to represent the true dimensions of gene–environment interactions. In the near future, microarray technology will facilitate the performance of large-scale, lowcost genotyping. The ethical, educational, social, and informatics considerations that will result are challenging. However, the ability to identify smokers with the highest risks of developing cancer has substantial preventive implications for intensive screening and smoking cessation interventions and for enrollment into chemoprevention trials. ENVIRONMENT AND BREAST CANCER Mary S. Wolff, Ph.D. Wide variations are seen in the incidence of breast cancer, both internationally and nationally among ethnic groups. In the United States, Hispanic women have lower rates of breast cancer than black and white women, and lower rates than Japanese Americans but higher than other women of Asian ancestry. Genetics, diet, and reproductive factors do not explain all of the differences. Indeed, women with inherited genetic predisposition may never suffer from breast cancer, just as not all smokers get lung cancer. Environmental exposures have been implicated, but few specific agents are strongly related to breast cancer. However, individuals respond differently to exposures in terms of their innate ability to metabolize chemicals. Therefore, diet, lifestyle, and adverse exposures must collaborate with susceptibility factors to incur risk for breast cancer. Breast cancer etiology is complex because tumorigenesis can arise from a combination of many different mechanisms over a very long time. Because breast cancer risk is strongly associated with reproductive hormones, any role for environmental exposures must act in concert with endogenous hormones. Environment, genes, and hormones must work together at specific end points—extending from perinatal mammary cell development, to onset of puberty, through birth, lactation, and menopause—following the course of tumor progression and, after diagnosis, prognosis for recurrence. In this age of generally low exposures, relative risks for main effects are low, and many environmental exposures and genetic variants alone are not strong risk factors for breast cancer. Combinations of exposures may obscure exposure–disease relationships. Crucial exposures as well as critical reproductive end points may have happened years before a tumor is found. The second generation of studies will address whether complex genetic factors, hormonal milieu, or
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Cancer and the Environment: Gene-Enviroment Interaction dietary intakes alter environmental risk factors. Such effects may be responsible for differences in breast cancer among racial and ethnic groups who may have risks related to genetic polymorphisms and excessive exposures. PRIORITIES AND SPECIAL POPULATIONS: TIES THAT BIND Armin Weinberg, Ph.D. It becomes clear as we examine charts and data on health such as the those in Healthy People 2000 that socioeconomic status (SES) plays a role in cancer and many other health care issues. Individuals and groups with a higher SES (1) can obtain better housing, (2) can live in better neighborhoods, (3) have opportunities to engage in healthy behaviors, (4) have better access to health care, and (5) can more readily participate in clinical trials. Thus, in order to understand clinical data as it relates to gene–environment interactions, we need to include analysis of the communities and SES. Studies today use descriptive phrases such as “special populations,” “priority populations,” and “vulnerable populations.” These terms are used extensively in research and discussions, but there is a great diversity of opinion as to their definition. As we continue to use these phrases in our communities, research will have to refine the definitions to better describe—not label—these groups. Additionally, they will have to provide a certain degree of flexibility to accommodate the subgroups that emerge. More than 2,500 individuals immigrate to the United States each day—a trend that complicates gene–environment research. Many of these individuals are from Latin America and Mexico, as well as other countries throughout the world. As we start to include these foreign-born residents in our studies, we must pay attention to the fact that these individuals are mostly young and have had different exposures in their country of origin at a time when they were most vulnerable. Additionally, migratory patterns have shifted in the United States as individuals from different countries or regions of a country immigrate distinctly to geographic regions in the United States. The NCI’s national special population networks have been formed to consider factors related to these and other issues. The steering committee for one network Radas En Acum, which has research sites in California, Illinois, New York, Florida, and Texas, was formed to help establish both a research agenda and research priorities. In addition to the genetics and the gene–environment issues, language and health literacy will require special attention. As we try to close the gap in enrollment in clinical trials, these other issues will become more important. We will have to be sensitive to cultural differences in these communities and ensure that material is available in many languages. Further, as we talk to communities about gene–environment interactions, we must communicate what this means, what we want, and what we hope to learn.
Representative terms from entire chapter: