Stewart L. MacLeod

Association of genetic variation in tamoxifen-metabolizing enzymes with overall survival and recurrence of disease in breast cancer patients

SummaryTamoxifen has been a mainstay of adjuvant therapy for breast cancer for many years. We sought to determine if genetic variability in the tamoxifen metabolic pathway influenced overall survival in breast cancer patients treated with tamoxifen. We examined functional polymorphisms in CYP2D6, the P450 catalyzing the formation of active tamoxifen metabolites, and UGT2B15, a Phase II enzyme facilitating the elimination of active metabolite in a retrospective study of breast cancer patients. We also examined whether the combination of variant alleles in SULT1A1 and UGT2B15 had more of an impact on overall survival in tamoxifen-treated patients than when the genes were examined separately.We conducted a retrospective study using archived paraffin blocks for DNA extraction and data from pathology reports and hospital tumor registry data for information on clinical characteristics, treatment, and outcomes (162 patients receiving tamoxifen and 175 who did not). Genotypes for CYP2D6 and UGT2B15 were obtained and Cox proportional hazards modeling was performed.After adjusting for age, race, stage of disease at diagnosis, and hormone receptor status, we found no significant association between CYP2D6 genotype and overall survival in either group of breast cancer patients. Tamoxifen-treated patients with UGT2B15 high activity genotypes had increased risk of recurrence and poorer survival. When UGT2B15 and SULT1A1 ‘at-risk’ alleles were combined, women with two variant alleles had significantly greater risk of recurrence and poorer survival than those with common alleles. These studies indicate that genetic variation in Phase II conjugating enzymes can influence the efficacy of tamoxifen therapy for breast cancer.

Analysis of total meat intake and exposure to individual heterocyclic amines in a case-control study of colorectal cancer : contribution of metabolic variation to risk

A case-control study of colorectal cancer, consisting of 157 cases and 380 controls matched by sex, ethnicity, decade of age and county of residence was performed to explore the associations between environmental exposure, metabolic polymorphisms and cancer risk. Participants were required to provide a blood sample, undergo caffeine phenotyping and complete an in-person interview that evaluated meat consumption, cooking methods and degree of doneness. A color atlas of foods cooked to different degrees of doneness was used to estimate food preparation techniques and food models were used to estimate serving portion sizes. Data was analyzed using a reference database of heterocyclic amine (HCA) exposure based on the food preferences chosen from the atlas. Data regarding individual food items cooked to different levels of doneness, as well as summary variables of foods and of food groups cooked to different degrees of doneness were also evaluated in a univariate analysis for association with colorectal cancer case status. Three measures of metabolic variation, hGSTA1 genotype, SULT1A1 genotype and the phenotype for CYP2A6 were also evaluated for possible association with colon cancer. While higher exposure to HCAs was strongly associated with colorectal cancer risk, increased consumption of five red meats cooked well done or very well done produced comparable odds ratios (OR) for colorectal cancer risk (OR=4.36, 95% CI 2.08-9.60) for the highest quartile of exposure. Similarly, individuals in the most rapid CYP2A6 phenotype quartile showed an odds ratio (OR = 4.18, 95% CI 2.03-8.90). The ORs for the low activity hGSTA1 and low activity SULT1A1 alleles were 2.0, 95% CI 1.0-3.7 and 0.6, 95% CI 0.3-1.1, respectively. Individual measures of specific HCAs provided little improvement in risk assessment over the measure of meat consumption, suggesting that exposure to other environmental or dietary carcinogens such as nitrosamines or undefined HCAs may contribute to colorectal cancer risk.

Relationship of phenol sulfotransferase activity (SULT1A1) genotype to sulfotransferase phenotype in platelet cytosol.

Sulfation catalysed by human cytosolic sulfotransferases is generally considered to be a detoxification mechanism. Recently, it has been demonstrated that sulfation of heterocyclic aromatic amines by human phenol sulfotransferase (SULT1A1) can result in a DNA binding species. Therefore, sulfation capacity has the potential to influence chemical carcinogenesis in humans. To date, one genetic polymorphism (Arg213His) has been identified that is associated with reduced platelet sulfotransferase activity. In this study, data on age, race, gender, SULT1A1 genotype and platelet SULT1A1 activity were available for 279 individuals. A simple colorimetric phenotyping assay, in conjunction with genotyping, was employed to demonstrate a significant correlation (r = 0.23, P < 0.01) of SULT1A1 genotype and platelet sulfotransferase activity towards 2-naphthol, a marker substrate for this enzyme. There was also a difference in mean sulfotransferase activity based on gender (1.28 nmol/min/mg, females; 0.94 nmol/min/mg, males, P = 0.001). DNA binding studies using recombinant SULT1A1*1 and SULT1A1*2 revealed that SULT1A1*1 catalysed N-hydroxy-aminobiphenyl (N-OH-ABP) DNA adduct formation with substantially greater efficiency (5.4 versus 0.4 pmol bound/mg DNA/20 min) than the SULT1A1*2 variant. A similar pattern was observed with 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5b]pyridine (N-OH-PhIP) (4.6 versus 1.8 pmol bound/mg DNA/20 min).

An Allele-Specific Polymerase Chain Reaction Method for the Determination of the D85Y Polymorphism in the Human UDPGlucuronosyltransferase 2B15 Gene in a Case-Control Study of Prostate Cancer

AbstractBackground: UDP-glucuronosyltransferase 2B15 (UGT2B15) catalyzes the inactivation of dihydrotestosterone (DHT) by forming the DHT-glucuronide and is expressed in normal and hyperplastic prostate tissue. Alterations in the activity of this enzyme could be a major contributing factor to the bioavailability of androgens in target tissue such as the prostate. Methods: A polymorphism (D85 to Y85) has been identified in the UGT2B15 gene1 that results in a 50% reduction in enzyme activity. Previously, detection of the polymorphic nucleotide has required direct sequencing. We have developed and validated an allele-specific polymerase chain reaction (PCR) assay to identify the polymorphic base pair in the UGT2B15 gene. This assay was used to examine the distribution of the UGT2B15 polymorphism in a small case-control group (64 cases and 64 controls) from a prostate cancer study. Results: The results of this analysis show that prostate cancer patients were significantly more likely to be homozygous for the lower activity D85 UGT2B15 allele than control individuals (41% versus 19%, respectively, odds ratio 5 3.0 (95% confidence intervals 1.3– 6.5)). Conclusions: These results suggest that individuals who are homozygous for the lower activity allele may be at increased risk for developing prostate cancer.

Pathophysiological effects of nicotine on the pancreas: an update.

Epidemiological evidence strongly suggests an association between cigarette smoking and pancreatic diseases. It is well recognized that nicotine, a major component in cigarette smoke, is an addictive agent and, therefore, reinforces smoking behavior. The current review update focuses on the genetics of nicotine dependence and its role on the development of pancreatic diseases. The role of smoking and nicotine in pancreatitis and pancreatic cancer development is also discussed. Exposure of laboratory animals to nicotine clearly supports the notion that nicotine can induce pancreatic injury. The mechanism by which nicotine induces such effects is perhaps mediated via signal transduction pathways in the pancreatic acinar cell, leading to enhanced levels of intracellular calcium release, resulting in cytotoxicity and eventual cell death. The induction of pancreatic injury by nicotine may also involve activation and expression of protooncogene, H-ras, which can increase cytosolic calcium via second messenger pathways. Development of pancreatic carcinoma in cigarette smokers as observed in human populations may be the result of activation and mutation of the H-ras gene. A possible pathogenetic mechanism of nicotine in the pancreas activating multiple signal transduction pathways is schematically summarized in Figure 1.

Obesity promotes 7,12-dimethylbenz(a)anthracene-induced mammary tumor development in female zucker rats

IntroductionHigh body mass index has been associated with increased risk for various cancers, including breast cancer. Here we describe studies using 7,12-dimethylbenz(a)anthracene (DMBA) to investigate the role of obesity in DMBA-induced mammary tumor susceptibility in the female Zucker rat (fa/fa), which is the most widely used rat model of genetic obesity.MethodFifty-day-old female obese (n = 25) and lean (n = 28) Zucker rats were orally gavaged with 65 mg/kg DMBA. Rats were weighed and palpated twice weekly for detection of mammary tumors. Rats were killed 139 days after DMBA treatment.ResultsThe first mammary tumor was detected in the obese group at 49 days after DMBA treatment, as compared with 86 days in the lean group (P < 0.001). The median tumor-free time was significantly lower in the obese group (P < 0.001). Using the days after DMBA treatment at which 25% of the rats had developed mammary tumors as the marker of tumor latency, the obese group had a significantly shorter latency period (66 days) than did the lean group (118 days). At the end of the study, obese rats had developed a significantly (P < 0.001) greater mammary tumor incidence (68% versus 32%) compared with the lean group. The tumor histology of the mammary tumors revealed that obesity was associated with a significant (P < 0.05) increase in the number of rats with at least one invasive ductal and lobular carcinoma compared with lean rats.ConclusionOur results indicate that obesity increases the susceptibility of female Zucker rats to DMBA-induced mammary tumors, further supporting the hypothesis that obesity and some of its mediators play a significant role in carcinogenesis.

Polymorphisms of CYP1A1 and GSTM1 influence the in vivo function of CYP1A2

Differences in human cancer susceptibility have been attributed to polymorphisms of carcinogen metabolizing enzymes. Our efforts have focused on the systems responsible for metabolism of aromatic and heterocyclic amines found in cigarette smoke and in cooked foods. Cytochrome P4501A2 (CYP1A2), which catalyzes aromatic and heterocyclic amine N-oxidation, has been implicated as a risk factor in both urinary bladder and colorectal cancer. In the present study we used the results of caffeine phenotyping experiments to measure the effects of cigarette smoke and compounds present in meat cooked at high temperature on CYP1A2 activity. Subjects in the smoking cessation study had mean CYP1A2 activity of 17.8 (expressed as the urinary molar ratio of [17X + 17U]/137X) while smoking: however, this activity decreased to 10.9 three weeks after cessation of smoking. Subjects in the cooked meat feeding study had mean CYP1A2 activity of 9.01 after 1 week of consuming meat cooked at low temperature, but this value increased to 12.7 after 1 week of consuming meat cooked at high temperature. Because no association has been identified between differences in CYP1A2 activity and variations in the CYP1A2 structural gene, we sought to determine whether the activities of other carcinogen metabolizing enzymes are involved in the regulation of CYP1A2 activity. CYP1A2 activity was higher in individuals who express the GSTM1 null allele compared to those expressing the GSTM1*A,B allele, 10.2 vs. 8.5 for unexposed conditions and 15.0 vs. 12.3 for exposed conditions. CYP1A1 genotyping demonstrated that individuals possessing the Ile/Ile CYP1A1 genotype had greater mean CYP1A2 activity than those who had the heterozygous Ile/Val allelic variant of the CYP1A1 gene. However, upon exposure to cigarette smoke or high-temperature cooked meat, individuals possessing the heterozygous form of the CYP1A1 gene had significantly increased CYP1A2 activity (18.1) compared to those with the more common Ile/Ile CYP1A1 genotype (13.3). These results indicate that CYP1A2, CYP1A1, and GSTM1 gene-gene interactions could be important confounders in the interpretation of molecular epidemiology studies.

Maternal Genome-Wide DNA Methylation Patterns and Congenital Heart Defects

The majority of congenital heart defects (CHDs) are thought to result from the interaction between multiple genetic, epigenetic, environmental, and lifestyle factors. Epigenetic mechanisms are attractive targets in the study of complex diseases because they may be altered by environmental factors and dietary interventions. We conducted a population based, case-control study of genome-wide maternal DNA methylation to determine if alterations in gene-specific methylation were associated with CHDs. Using the Illumina Infinium Human Methylation27 BeadChip, we assessed maternal gene-specific methylation in over 27,000 CpG sites from DNA isolated from peripheral blood lymphocytes. Our study sample included 180 mothers with non-syndromic CHD-affected pregnancies (cases) and 187 mothers with unaffected pregnancies (controls). Using a multi-factorial statistical model, we observed differential methylation between cases and controls at multiple CpG sites, although no CpG site reached the most stringent level of genome-wide statistical significance. The majority of differentially methylated CpG sites were hypermethylated in cases and located within CpG islands. Gene Set Enrichment Analysis (GSEA) revealed that the genes of interest were enriched in multiple biological processes involved in fetal development. Associations with canonical pathways previously shown to be involved in fetal organogenesis were also observed. We present preliminary evidence that alterations in maternal DNA methylation may be associated with CHDs. Our results suggest that further studies involving maternal epigenetic patterns and CHDs are warranted. Multiple candidate processes and pathways for future study have been identified.

Maternal folate-related gene environment interactions and congenital heart defects.

OBJECTIVE: To investigate whether women with congenital heart defect (CHD)–affected pregnancies were more likely to have functional single-nucleotide polymorphisms in genes encoding enzymes in folate-dependent pathways. METHODS: A population-based case–control study of 572 women with CHD-affected pregnancies and 363 women in the control group was conducted. DNA samples were genotyped for single-nucleotide polymorphisms in three genes encoding for folate pathway enzymes. Maternal lifestyle factor information was obtained using standardized interviews. RESULTS: Women in the case group were 1.5 times more likely to be obese (body mass index of 30 or higher) compared with those in the control group. Obese women carrying the MTHFR TT genotype were 4.6 times more likely to have an affected pregnancy compared with normal-weight women carrying a CC genotype. Obese women carrying one or two copies of the A allele in the BHMT polymorphism were 1.8 times more likely to have a CHD-affected pregnancy than were normal-weight women carrying a BHMT GG genotype. Among women who smoked, those carrying a TCII CG or GG genotype were 1.8 times more likely to have an affected fetus than were women who smoked and carried a CC genotype. Among women who drank alcohol, those carrying a TCII CG or GG genotype were 1.7 times more likely to have an affected fetus than were women who drank and carried a CC genotype. CONCLUSION: Results indicate that functional polymorphisms in folate-related genes increase the risk of having a fetus with CHD when maternal lifestyle factors that alter folate metabolism are present. LEVEL OF EVIDENCE: II

Antigenic properties of peptide mimotopes of HIV-1-associated carbohydrate antigens

The glycan shield of the human immunodeficiency virus (HIV) envelope protein presents many potential epitopes for vaccine development. To augment immune responses to HIV, type 1 (HIV-1), envelope-associated carbohydrate antigens, we are defining peptide mimics of HIV-associated carbohydrate antigens that function as antigen mimotopes that upon immunization will induce antibodies cross-reactive with carbohydrate antigens. We have previously defined peptides with a putative sequence tract RYRY that mimic concanavalin A-binding glycans. To imitate the multivalent binding of carbohydrates, we compared the avidity of a linear (911) and cyclic peptide (D002) reactive with concanavalin A presented in a multiple antigen peptide (MAP) format. The affinity of the MAP-D002 peptide was higher than that of the peptide MAP-911, whereas the avidity of D002 peptide was lower than that of 911. Serum from mice immunized with MAP-911 had lower titer for oligomannose-9 than those elicited by MAP-D002 under the same conditions, but both immunogens elicited antibodies that can block the binding of GP120 to dendritic cells. Antibodies that bind to the studied MAPs were found in a preparation of normal human immunoglobulin for intravenous use. Those that were purified on 911 bound back to 911 and D002, whereas anti-D002 antibodies were specific only for D002. Human antibodies reactive with both mimotopes and with a mannosyl preparation were observed to bind to envelope protein. These results suggested the potential to fine-tune the antibody response to carbohydrate antigens by modifying structural features of peptide mimotope-based immunogens.