Mendelian Randomization and mediation analysis of leukocyte telomere length and risk of lung and head and neck cancers.

BACKGROUND: Evidence from observational studies of telomere length (TL) has been conflicting regarding its direction of association with cancer risk. We investigated the causal relevance of TL for lung and head and neck cancers using Mendelian Randomization (MR) and mediation analyses. METHODS: We developed a novel genetic instrument for TL in chromosome 5p15.33, using variants identified through deep-sequencing, that were genotyped in 2051 cancer-free subjects. Next, we conducted an MR analysis of lung (16 396 cases, 13 013 controls) and head and neck cancer (4415 cases, 5013 controls) using eight genetic instruments for TL. Lastly, the 5p15.33 instrument and distinct 5p15.33 lung cancer risk loci were evaluated using two-sample mediation analysis, to quantify their direct and indirect, telomere-mediated, effects. RESULTS: The multi-allelic 5p15.33 instrument explained 1.49-2.00% of TL variation in our data (p = 2.6 × 10-9). The MR analysis estimated that a 1000 base-pair increase in TL increases risk of lung cancer [odds ratio (OR) = 1.41, 95% confidence interval (CI): 1.20-1.65] and lung adenocarcinoma (OR = 1.92, 95% CI: 1.51-2.22), but not squamous lung carcinoma (OR = 1.04, 95% CI: 0.83-1.29) or head and neck cancers (OR = 0.90, 95% CI: 0.70-1.05). Mediation analysis of the 5p15.33 instrument indicated an absence of direct effects on lung cancer risk (OR = 1.00, 95% CI: 0.95-1.04). Analysis of distinct 5p15.33 susceptibility variants estimated that TL mediates up to 40% of the observed associations with lung cancer risk. CONCLUSIONS: Our findings support a causal role for long telomeres in lung cancer aetiology, particularly for adenocarcinoma, and demonstrate that telomere maintenance partially mediates the lung cancer susceptibility conferred by 5p15.33 loci.

Association of BRCA2 K3326* With Small Cell Lung Cancer and Squamous Cell Cancer of the Skin.

Background: Most pathogenic mutations in the BRCA2 gene carry a high risk of hereditary breast and ovarian cancer (HBOC). However, a stop-gain mutation, K3326* (rs11571833), confers risk of lung cancer and cancers of the upper-aero-digestive tract but only a modest risk of breast or ovarian cancer. The Icelandic population provides an opportunity for comprehensive characterization of the cancer risk profiles of K3326* and HBOC mutations because a single mutation, BRCA2 999del5, is responsible for almost all BRCA2-related HBOC in the population. Methods: Genotype information on 43 641 cancer patients and 370 971 control subjects from Iceland, the Netherlands, and the United States was used to assess the cancer risk profiles of K3326* and BRCA2 999del5. BRCA2 expression was assessed using RNAseq data from blood (n = 2233), as well as 52 tissues reported in the GTEx database. Results: The cancer risks associated with K3326* are fundamentally different from those associated with 999del5. We report for the first time an association between K3326* and small cell lung cancer (odds ratio [OR] = 2.06, 95% confidence interval [CI] = 1.35 to 3.16) and squamous cell carcinoma of the skin (OR = 1.69, 95% CI = 1.26 to 2.26). Individuals homozygous for K3326* reach old age and have children. Unlike BRCA2 999del5, the K3326* allele does not affect the level of BRCA2 transcripts, and the allele is expressed to the same extent as the wild-type allele. Conclusions: K3326* associates primarily with cancers that have strong environmental genotoxic risk factors. Expression of the K3326* allele suggests that a variant protein may be made that retains the DNA repair capabilities important to hormone-responsive tissues but may be less efficient in responding to genotoxic stress.

Genome-wide association analyses identify new susceptibility loci for oral cavity and pharyngeal cancer.

We conducted a genome-wide association study of oral cavity and pharyngeal cancer in 6,034 cases and 6,585 controls from Europe, North America and South America. We detected eight significantly associated loci (P < 5 × 10-8), seven of which are new for these cancer sites. Oral and pharyngeal cancers combined were associated with loci at 6p21.32 (rs3828805, HLA-DQB1), 10q26.13 (rs201982221, LHPP) and 11p15.4 (rs1453414, OR52N2-TRIM5). Oral cancer was associated with two new regions, 2p23.3 (rs6547741, GPN1) and 9q34.12 (rs928674, LAMC3), and with known cancer-related loci-9p21.3 (rs8181047, CDKN2B-AS1) and 5p15.33 (rs10462706, CLPTM1L). Oropharyngeal cancer associations were limited to the human leukocyte antigen (HLA) region, and classical HLA allele imputation showed a protective association with the class II haplotype HLA-DRB1*1301-HLA-DQA1*0103-HLA-DQB1*0603 (odds ratio (OR) = 0.59, P = 2.7 × 10-9). Stratified analyses on a subgroup of oropharyngeal cases with information available on human papillomavirus (HPV) status indicated that this association was considerably stronger in HPV-positive (OR = 0.23, P = 1.6 × 10-6) than in HPV-negative (OR = 0.75, P = 0.16) cancers.

Genetic polymorphisms in UDP-glucuronosyltransferase 1A6 and 1A7 and the risk for benign Warthin's tumors of the parotid gland.

BACKGROUND: Warthin's tumors of the parotid gland are associated with smoking, whereas pleomorphic adenomas are not. Genetic polymorphisms in biotransformation enzymes, involved in detoxification of toxins and carcinogens in cigarette smoke, might modify the corresponding enzyme activity and influence detoxifying capacity. We hypothesize that these genetic polymorphisms may influence the individual risk for Warthin's tumor, but not for pleomorphic adenomas. METHODS: Blood from 146 patients with benign parotid gland tumors and 437 controls were investigated for polymorphisms in several biotransformation enzymes. Based on these polymorphisms, patients and controls were divided according to predicted enzyme activity (low, intermediate, and high). RESULTS: Prevalence of predicted intermediate and high activity UGT1A7 and UGT1A6 genotypes was significantly higher in the patients with Warthin's tumors, but not in patients with pleomorphic adenomas, compared with healthy controls. CONCLUSION: Predicted intermediate and high activity UGT1A7 and UGT1A6 genotypes are associated with an increased risk for Warthin's tumor. © 2015 Wiley Periodicals, Inc. Head Neck 38: E717-E723, 2016.

Identification of Patients With Variants in TPMT and Dose Reduction Reduces Hematologic Events During Thiopurine Treatment of Inflammatory Bowel Disease.

BACKGROUND & AIMS: More than 20% of patients with inflammatory bowel disease (IBD) discontinue thiopurine therapy because of severe adverse drug reactions (ADRs); leukopenia is one of the most serious ADRs. Variants in the gene encoding thiopurine S-methyltransferase (TPMT) alter its enzymatic activity, resulting in higher levels of thiopurine metabolites, which can cause leukopenia. We performed a prospective study to determine whether genotype analysis of TPMT before thiopurine treatment, and dose selection based on the results, affects the outcomes of patients with IBD. METHODS: In a study performed at 30 Dutch hospitals, patients were assigned randomly to groups that received standard treatment (control) or pretreatment screening (intervention) for 3 common variants of TPMT (TPMT*2, TPMT*3A, and TPMT*3C). Patients in the intervention group found to be heterozygous carriers of a variant received 50% of the standard dose of thiopurine (azathioprine or 6-mercaptopurine), and patients homozygous for a variant received 0%-10% of the standard dose. We compared, in an intention-to-treat analysis, outcomes of the intervention (n = 405) and control groups (n = 378) after 20 weeks of treatment. Primary outcomes were the occurrence of hematologic ADRs (leukocyte count < 3.0*10(9)/L or reduced platelet count < 100*10(9)/L) and disease activity (based on the Harvey-Bradshaw Index for Crohn's disease [n = 356] or the partial Mayo score for ulcerative colitis [n = 253]). RESULTS: Similar proportions of patients in the intervention and control groups developed a hematologic ADR (7.4% vs 7.9%; relative risk, 0.93; 95% confidence interval, 0.57-1.52) in the 20 weeks of follow-up evaluation; the groups also had similar mean levels of disease activity (P = .18 for Crohn's disease and P = .14 for ulcerative colitis). However, a significantly smaller proportion of carriers of the TPMT variants in the intervention group (2.6%) developed hematologic ADRs compared with patients in the control group (22.9%) (relative risk, 0.11; 95% confidence interval, 0.01-0.85). CONCLUSIONS: Screening for variants in TPMT did not reduce the proportions of patients with hematologic ADRs during thiopurine treatment for IBD. However, there was a 10-fold reduction in hematologic ADRs among variant carriers who were identified and received a dose reduction, compared with variant carriers who did not, without differences in treatment efficacy. ClinicalTrials.gov number: NCT00521950.

Allopurinol and 5-aminosalicylic acid influence thiopurine-induced hepatotoxicity in vitro.

INTRODUCTION: The use of thiopurines is frequently accompanied by hepatotoxicity. Studies on hepatocyte cultures showed a time- and dose-dependent increase of thiopurine toxicity. 5-Aminosalicylic acid (5-ASA) and allopurinol can influence thiopurine metabolism; however, it is unknown whether this affects in vitro cytotoxicity. METHODS: Human hepatoma cells (Huh7, HepG2 and HepaRG) were incubated with increasing concentrations of thiopurines, 5-ASA or allopurinol. Water-soluble tetrazolium salt-1 (WST-1) cytotoxicity assays were used to calculate cell survival curves and half maximal inhibitory concentrations (IC50). Combination experiments with thiopurines with a fixed dose of 200 µM 5-ASA or 100 µM allopurinol were conducted in HepaRG cells. Caspase-3/7 activation was evaluated, and single cell electrophoresis analysis was performed. RESULTS: A time- and dose-related cytotoxic effect was seen with azathioprine (AZA) in all hepatoma cells, whereas Huh7 and HepG2 cells did not show toxicity to 6-mercaptopurine (6-MP). HepaRG cells expressed the highest levels of drug metabolising enzymes, and therefore, combination experiments were conducted in HepaRG cells. Addition of a non-toxic dose of allopurinol resulted in a twofold to threefold increased cytotoxicity of all thiopurines, which seemed to be mediated by apoptosis/DNA damage. CONCLUSION: The addition of allopurinol to thiopurines leads to a two-threefold increased cytotoxicity in HepaRG cells.

Association between probiotics and enteral nutrition in an experimental acute pancreatitis model in rats.

BACKGROUND/OBJECTIVES: Recently, a randomized controlled trial showed that probiotic prophylaxis was associated with an increased mortality in enterally fed patients with predicted severe pancreatitis. In a rat model for acute pancreatitis, we investigated whether an association between probiotic prophylaxis and enteral nutrition contributed to the higher mortality rate. METHODS: Male Sprague-Dawley rats were allocated to four groups: 1) acute pancreatitis (n = 9), 2) acute pancreatitis and probiotic prophylaxis (n = 10), 3) acute pancreatitis and enteral nutrition (n = 10), and 4) acute pancreatitis, probiotic prophylaxis and enteral nutrition (n = 11). Acute pancreatitis was induced by intraductal glycodeoxycholate and intravenous cerulein infusion. Enteral nutrition, saline, probiotics and placebo were administered through a permanent jejunal feeding. Probiotics or placebo were administered starting 4 days before induction of pancreatitis and enteral nutrition 1 day before start until the end of the experiment, 6 days after induction of pancreatitis. Tissue samples and body fluids were collected for microbiological and histological examination. RESULTS: In all animals, serum amylase was increased six hours after induction of pancreatitis. After fulfilling the experiment, no differences between groups were found in histological severity of pancreatitis, degree of discomfort, weight loss, histological examination of small bowel and bacterial translocation (all p > 0.05). Overall mortality was 10% without differences between groups (p = 0.54). CONCLUSION: No negative association was found between prophylactic probiotics and enteral nutrition in acute pancreatitis. No new clues for a potential mechanism responsible for the higher mortality and bowel ischaemia in the PROPATRIA study were found.

GST Theta null genotype is associated with an increased risk for ulcerative colitis: a case-control study and meta-analysis of GST Mu and GST Theta polymorphisms in inflammatory bowel disease.

Glutathione S-transferases (GSTs) are important in the detoxification of many compounds, including reactive oxygen species. Polymorphisms in GSTs resulting in a decreased enzyme activity might enhance the risk for inflammatory bowel disease by eliciting a state of oxidative stress. Previous case-control studies showed divergent results and were frequently limited in sample size; therefore we conducted a meta-analysis including results from our case-control study. For the case-control study, we genotyped 552 patients with Crohn's disease (CD), 223 patients with ulcerative colitis (UC) and 972 healthy controls by PCR for functional deletions in GST Mu and GST Theta. Both were not analyzed in recent genome-wide association studies. For the meta-analysis, PubMed, EMBASE and Web of Science were searched. In this meta-analysis, we show an enhanced susceptibility for UC in individuals with the GSTT1null genotype (odds ratio (OR) 2.27, 95% confidence interval (CI) 1.31-3.92). In our case-control study, a reduced risk for CD was seen with the GSTT1null genotype (OR 0.58, 95% CI 0.43-0.77); however, pooled analysis showed an OR of 1.67, 95% CI 0.81-3.45. In this meta-analysis, we showed an increased risk for UC in individuals with the GSTT1null genotype.

Genetic polymorphism 609C>T in NAD(P)H:quinone oxidoreductase 1 enhances the risk of proximal colon cancer.

Gastrointestinal (GI) cancer is responsible for the majority of deaths among all types of cancer. Lifestyle factors may not only be the main risk factor for GI cancer but reactive oxygen species (ROS) may also be involved. The single-nucleotide polymorphisms (SNPs) 609C>T (rs1800566) and 465C>T (rs1131341) in the NAD(P)H: quinone oxidoreductase 1 (NQO1) gene lead to a decline in NQO1 enzyme activity. NQO1 catalyzes the two-electron reduction of quinones to hydroquinones, thereby preventing the formation of ROS. Such polymorphisms in NQO1 may increase the risk of GI cancer. The aim of this study was to evaluate the influence of the SNPs rs1800566 and rs1131341 in the NQO1 gene on the risk of GI cancer in the Netherlands. Real-time polymerase chain reaction techniques were conducted to determine the NQO1 genotypes of 1457 patients with GI cancer and 1457 age- and gender-matched controls in a case-control study. Binary logistic regression analyses showed no statistically significant difference in genotype distributions between patients and controls: odds ratios (ORs) with 95% confidence interval (CI) for rs1800566 were 1.09 (0.93-1.28) and 1.17 (0.77-1.77) for the CT and TT genotypes, respectively. ORs for rs1131341 CT and TT genotypes were 1.21 (0.90-1.63) and 0.54 (0.05-5.94), respectively. For rs1800566, a significant association between the CT genotype and proximal colon cancer was detected (OR=1.60; 95% CI=1.09-2.35). The NQO1*2 T allele of SNP rs1800566 was found associated with an increased risk for proximal colorectal cancer, whereas SNP rs1131341 was rare in our Dutch population and was not associated with GI cancer.

Polymorphisms in the insulin-like growth factor axis are associated with gastrointestinal cancer.

INTRODUCTION: Numerous factors influence the development of gastrointestinal (GI) cancer. The insulin-like growth factor (IGF) axis plays a role in embryonic and postnatal growth and tissue repair. Elevated levels of IGFs, low levels of IGF binding proteins (IGFBPs) and over-expression of IGF receptor (IGFR-I) were associated with several stages of cancer. Here, the prevalence of the single nucleotide polymorphisms (SNPs) rs6214 in the IGF type I (IGF-I) gene and rs6898743 in the growth hormone receptor (GHR) gene in patients with GI cancer and controls was studied. MATERIALS & METHODS: In this Dutch case-control study, DNA isolated from blood of 1,457 GI cancer patients; 438 patients with head and neck cancer (HNC), 475 with esophageal cancer (EC) and 544 with colorectal cancer (CRC) and 1,457 matched controls, was used to determine the rs6214 and rs6898743 genotypes by polymerase chain reaction. The association between these SNPs and GI cancer, HNC, esophageal adenocarcinoma (EAC), esophageal squamous-cell carcinoma (ESCC) and proximal or distal CRC was studied. Odds ratios (ORs) with 95% confidence interval (95% CI) were calculated via unconditional logistic regression. RESULTS: Overall for GI cancer, the ORs for SNPs rs6214 and rs6898743 were approximately 1.0 (p-value>0.05), using the most common genotypes GG as reference. An OR of 1.54 (95% CI, 1.05-2.27) was found for EC for genotype AA of rs6214. The ORs for EAC were 1.45 (95% CI, 1.04-2.01) and 1.71 (95% CI, 1.10-2.68), for genotypes GA and AA, respectively. Genotype GC of rs6898743 showed an OR of 0.47 (95% CI, 0.26-0.86) for ESCC. CONCLUSION: The A allele of SNP rs6214 in the IGF-I gene was associated with EAC, and with HNC in women. The GC genotype of rs6898743 in the GHR gene was negatively associated with ESCC.

Over-expression of COX-2 mRNA in colorectal cancer.

BACKGROUND: Cyclooxygenase-2 (COX-2, PTGS2) is an enzyme involved in the synthesis of prostaglandins and thromboxanes, which are regulators of biologic processes such as inflammation, cell proliferation and angiogenesis. COX-2 over-expression was reported in many (pre) malignant tissues, but data strongly vary and seem to depend on the methodology used. METHODS: Normal colorectal mucosa and paired cancerous tissue from 60 patients with colorectal cancer was investigated for the levels of COX-2 mRNA by real-time quantitative Polymerase Chain Reaction (qPCR). COX-2 levels were expressed relative to either: tissue weight or levels of the housekeeping genes beta-2 microglobulin (B2M) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: COX-2 mRNA levels, normalized with respect to tissue weight or mRNA levels of the housekeeping genes B2M or GAPDH, were over-expressed in 80%, 70% and 40% of the colorectal tumor tissues, as compared to the paired adjacent normal colorectal mucosa samples, respectively. Highest mRNA COX-2 ratios tumor/normal were measured when expressed per mg tissue (mean ratio 21.6). When normalized with respect to the housekeeping genes B2M or GAPDH, mean tumor/normal ratios were 16.1 and 7.5, respectively. CONCLUSION: Expression of COX-2 mRNA levels per mg tissue is most simple in comparison to normalization with respect to the housekeeping genes B2M or GAPDH. Levels of COX-2 mRNA are found over-expressed in almost 80% of the colorectal tumors, compared to paired adjacent normal colorectal mucosa, suggesting a role of COX-2 as a potential biomarker for cancer risk, whereas inhibitors of COX-2 could be of value in chemoprevention of colon cancer.

The effect of iron loading and iron chelation on the innate immune response and subclinical organ injury during human endotoxemia: a randomized trial.

In this double-blind randomized placebo-controlled trial involving 30 healthy male volunteers we investigated the acute effects of iron loading (single dose of 1.25 mg/kg iron sucrose) and iron chelation therapy (single dose of 30 mg/kg deferasirox) on iron parameters, oxidative stress, the innate immune response, and subclinical organ injury during experimental human endotoxemia. The administration of iron sucrose induced a profound increase in plasma malondialdehyde 1 h after administration (433±37% of baseline; P<0.0001), but did not potentiate the endotoxemia-induced increase in malondialdehyde, as was seen 3 h after endotoxin administration in the placebo group (P=0.34) and the iron chelation group (P=0.008). Endotoxemia resulted in an initial increase in serum iron levels and transferrin saturation that was accompanied by an increase in labile plasma iron, especially when transferrin saturation reached levels above 90%. Thereafter, serum iron decreased to 51.6±9.7% of baseline at T=8 h in the placebo group versus 84±15% and 60.4±8.9% of baseline at 24 h in the groups treated with iron sucrose and deferasirox, respectively. No significant differences in the endotoxemia-induced cytokine response (TNF-α, IL-6, IL-10 and IL-1RA), subclinical vascular injury and kidney injury were observed between groups. However, vascular reactivity to noradrenalin was impaired in the 6 subjects in whom labile plasma iron was elevated during endotoxemia as opposed to those in whom no labile plasma iron was detected (P=0.029). In conclusion, a single dose of iron sucrose does not affect the innate immune response in a model of experimental human endotoxemia, but may impair vascular reactivity when labile plasma iron is formed. (Clinicaltrials.gov identifier:01349699).

Duodenal mucosal risk markers in patients with familial adenomatous polyposis: effects of celecoxib/ursodeoxycholic acid co-treatment and comparison with patient controls.

BACKGROUND: Familial adenomatous polyposis (FAP) is a disease characterized by the development of hundreds to thousands of adenomatous polyps in the colorectum early in life. Virtually all patients with FAP will develop colorectal cancer before the age of 40 to 50 years, unless prophylactic colectomy is performed, which significantly improves their prognosis. The mortality pattern has changed and duodenal cancer now is one of the main cancer-related causes of death in these patients. Practically all patients with FAP develop premalignant duodenal adenomas, which may develop to duodenal cancer in approximately 3-7% of patients. Duodenal cancer in patients with FAP has a poor prognosis. The clinical challenge is to identify patients at high-risk for duodenal carcinoma. Chemoprevention would be desirable to avoid duodenectomy. The main goal of this study is to identify risk markers in normal duodenal mucosa of patients with FAP, that could help identify patients at increased risk for malignant transformation. METHODS: Messenger RNA (mRNA) levels of glutathione S-transferase A1 (GSTA1), glutathione S-transferase P1 (GSTP1), KIAA1199, E-cadherin, peroxisome proliferative activated receptor δ (PPARδ), caspase-3, cyclin D1, ß-catenin, and cyclooxygenase-2 (COX-2) were measured in duodenal mucosa, using the QuantiGene 2.0 Plex assay. Levels in normal appearing mucosa of patients with FAP (n = 37) were compared with levels in non-FAP patient controls (n = 16). In addition, levels before and after treatment with either celecoxib & ursodeoxycholic acid (UDCA, n = 14) or celecoxib & placebo (n = 13) were evaluated in patients with FAP. RESULTS: mRNA levels of glutathione S-transferase A1 (28.16% vs. 38.24%, p = 0.008) and caspase-3 (3.30% vs. 5.31%, p = 0.001) were significantly lower in patients with FAP vs. non-FAP patient controls, respectively. COX-2 mRNA levels in normal duodenal mucosa of patients with FAP were found to be unexpectedly low. None of the potential risk markers was influenced by celecoxib or celecoxib & UDCA. CONCLUSIONS: Protection against toxins and carcinogens (GSTA1) and apoptosis (caspase-3) is low in patients with FAP, which could contribute to increased susceptibility for malignant transformation of duodenal mucosa. TRIAL REGISTRATION: http://ClinicalTrials.gov number NCT00808743.

Disparities of conjugating protective enzyme activities in the colon of patients with adenomas and carcinomas.

AIM: To investigate the metabolic enzymatic capacity of the colon mucosa to detoxify noxious carcinogenic compounds. METHODS: We investigated the activity of 2 conjugating enzymes-the microsomal uridine glucuronosyltransferase (UGT) and the cytosomal glutathione S-transferase (GST) in the uninvolved mucosa of the colon transversum and sigmoideum in patients with adenomatous polyps and colorectal cancer. Biopsies were taken from the mucosa during colonoscopies which were done for clinical (diagnostic) reasons. After storage, the biopsy material was homogenized and after differential centrifugation the enzyme assays were performed with 4-nitrophenol (UGT) and 1-chloro 2,4-dinitrobenzene (GST) as substrates. RESULTS: About 48 patients were included of which 28 had adenomas and 20 had colorectal carcinomas confirmed by histopathology. Enzyme activities were expressed as nmol/mg per minute protein for the GST and as pmol/mg per minute protein for the UGT. Analysis of variance (F-test) indicated that both enzymes were more widely distributed in adenoma than in cancer patients. The means ± SD were smaller for cancer patients: GST for adenomas 268 ± 152 vs 241 ± 69 for carcinomas and UGT for adenomas 197 ± 200 vs 150 ± 86 for carcinomas. CONCLUSION: Compared to patients with adenomatous colon polyps those with colorectal carcinoma exhibited a lower capacity of detoxifying enzyme metabolism and their activities clustered over a smaller range.

Polymorphisms in alcohol-metabolizing enzymes and esophageal carcinoma susceptibility: a Dutch Caucasian case-control study.

Esophageal cancer (EC), mainly consisting of squamous cell carcinoma (ESCC) in the Eastern world and adenocarcinoma (EAC) in the Western world, is strongly associated with dietary factors such as alcohol use. We aimed to clarify the modifying role in EC etiology in Caucasians of functional genotypes in alcohol-metabolizing enzymes. In all, 351 Caucasian patients with EC and 430 matched controls were included and polymorphisms in CYP2E1, ADH and near ALDH2 genes were determined. In contrast to the results on ESCC in mainly Asian studies, we found that functional genotypes of alcohol-metabolizing enzymes were not significantly associated with EAC or ESCC in an European population.

Ursodeoxycholic acid counteracts celecoxib in reduction of duodenal polyps in patients with familial adenomatous polyposis: a multicentre, randomized controlled trial.

BACKGROUND: Due to prophylactic colectomy, mortality in patients with familial adenomatous polyposis (FAP) has changed, with duodenal cancer currently being the main cause of death. Although celecoxib reduces duodenal polyp density in patients with FAP, its long-term use may increase the risk of cardiovascular events and alternatives need to be explored. Preclinical studies suggest that the combination of celecoxib with ursodeoxycholic acid (UDCA) is a potentially effective strategy. We performed a randomized, double-blind, placebo-controlled trial to investigate the effect of celecoxib and UDCA co-treatment on duodenal adenomatosis in patients with FAP. METHODS: Patients with FAP received celecoxib (400 mg twice daily) and UDCA (1000-2000 mg daily, ~20-30 mg/kg/day, n=19) or celecoxib and placebo (n=18) orally for 6 months. Primary outcome was drug efficacy, assessed by comparing duodenal polyp density at pre- and post-intervention by blinded review of endoscopic recordings. As secondary outcomes, cell proliferation, apoptosis, and COX-2 levels in normal duodenal mucosa were assessed by immunohistochemistry or real-time quantitative polymerase chain reaction. RESULTS: In intention-to-treat analysis, deceased polyp density was observed after celecoxib/placebo treatment (p=0.029), whereas increased polyp density was observed after celecoxib/UDCA treatment (p=0.014). The difference in change in duodenal polyp density was statistically significant between the groups (p=0.011). No changes in secondary outcomes were observed. Thirty patients (81%) reported one or more adverse events, 16 patients (84%, Common Toxicity Criteria for Adverse Events version 3.0 (CTCAE) grade 1-3) treated with celecoxib/UDCA and 14 patients (78%, CTCAE grade 1-2) treated with celecoxib/placebo. Nine patients (24%) discontinued intervention prematurely, 5 patients (26%) treated with celecoxib/UDCA and 4 patients (22%) treated with celecoxib/placebo. CONCLUSIONS: Celecoxib reduces duodenal polyp density in patients with FAP, and unexpectedly, high dose UDCA co-treatment counteracts this effect. The benefit of long term use of celecoxib for duodenal cancer prevention needs to be weighed against the (risk of) adverse events. TRIAL REGISTRATION: http://ClinicalTrials.gov, identifier NCT00808743.

Assessment of plasma aminothiol levels and the association with recurrent atherothrombotic events in patients hospitalized for an acute coronary syndrome: a prospective study.

BACKGROUND: The aminothiols homocysteine and, to a lesser extent, cysteine have been associated with adverse cardiovascular outcome, whereas glutathione, as an antioxidant, may protect against atherosclerosis and thrombosis. Potentially, the combined assessment of these aminothiols may provide a more accurate association with future cardiovascular outcome. We evaluated the association between recurrent atherothrombotic events and the concentration of total plasma cysteine, homocysteine, and glutathione and their combination. METHODS: Respective aminothiols were measured by high-performance liquid chromatography in blood plasma of consecutive first-day survivors admitted for an acute coronary syndrome between April 2002 and January 2004. The combined score was calculated using the combination of the individual aminothiols. The end point was the composite of cardiovascular death, myocardial infarction, and/or stroke. RESULTS: A cohort of 375 consecutive patients (median age 66 years, 66% male) were followed for a median duration of 2.7 years. The end point occurred in 82 patients (22%). In univariate analyses, all aminothiols were significantly associated with the composite end point. After correction for possible confounders, only cysteine and glutathione remained significantly associated. The strongest association with the end point was observed for the combined score (adjusted hazard ratio, 1.40 per standard deviation increase; p=0.005). CONCLUSIONS: Although homocysteine is generally considered the aminothiol of interest with respect to cardiovascular disease, in our prospective study, only cysteine and glutathione appeared independently associated with recurrent atherothrombotic events. Moreover, we showed that an imbalance in the combination of aminothiols could be of more importance than investigating the individual metabolites.

No role for glutathione S-transferase genotypes in Caucasian esophageal squamous cell or adenocarcinoma etiology: an European case-control study.

BACKGROUND: Identifying and monitoring high-risk patients can aid the prevention of esophageal cancer (EC). The interaction of environmental risk factor exposure and genetic susceptibility may contribute to the etiology of EC. Biotransformation enzymes such as Glutathione S-Transferases (GSTs ) detoxify mutagenic and genotoxic compounds and therefore control the rate of detoxification of carcinogens. Functional polymorphisms in the genes coding for GSTs alter their enzyme activity in vitro, and were reported to modify EC risk in Asians. We hypothesized that altered enzyme activity GST genotypes influence the susceptibility for esophageal adeno- (EAC) and squamous cell carcinoma (ESCC) in Caucasians. METHODS: We performed a case-control study including 440 Caucasian patients with EC and 592 healthy Caucasian controls matched for age and sex. Functional polymorphisms were selected and genotypes were determined in GST classes Alpha, Mu, Theta and Pi by means of polymerase chain reaction. Genotypes were classified into predicted high, intermediate and low enzyme activity categories based on in vitro activity data. The distribution of the activity genotypes were compared between patients with EAC or ESCC, and controls. Odds ratios (OR) with 95% confidence intervals (CI) were calculated by logistic regression analyses. Gene-gene interactions were tested and for comparison purposes, the predicted low and intermediate activity genotypes were combined. Genotypes with similar risks for EAC or ESCC were combined and analyzed for multiplicative effects. RESULTS: Our analyses includes 327 patients with EAC and 106 patients with ESCC. Low or intermediate activity enzyme genotypes for GSTM1, GSTA1, GSTP1 I105V and A114V as well as for GSTT1, did not significantly modify the risk for ESCC or EAC in our Dutch population. CONCLUSION: Functional genotypes in GST genes are not involved in EAC or ESCC susceptibility in Caucasians, in contrast to results on ESCC from Asia or Africa.

Barrett associated MHC and FOXF1 variants also increase esophageal carcinoma risk.

Barrett's esophagus, with gastroesophageal reflux disease and obesity as risk factors, predisposes to esophageal adenocarcinoma (EAC). Recently a British genome wide association study identified two Barrett's esophagus susceptibility loci mapping within the major histocompatibility complex (MHC; rs9257809) and closely to the Forkhead-F1 (FOXF1; rs9936833) coding gene. An interesting issue is whether polymorphisms associated with Barrett's esophagus, are also implicated in esophageal carcinoma (EC), and more specifically EAC genesis. Assessing the individual genetic susceptibility can help identify high risk patients more prone to benefit from surveillance programs. Our hypothesis: Barrett associated MHC and FOXF1 variants modify EC risk in Caucasians. In a Dutch case-control study, 431 patients with EC and 605 healthy controls were included. Polymorphisms at chromosomes 6p21 (MHC) and 16q24 (FOXF1) were determined by means of real-time polymerase chain reaction (RT-PCR). Logistic regression analysis was used to calculate odds ratios with 95% confidence intervals. The FOXF1 rs9936833 variant C allele was associated with an increased EAC susceptibility; OR, [95% CI]; 1.21, [0.99-1.47]. A sex-stratified analysis revealed a similar association in males; 1.24 [1.00-1.55]. The variant MHC rs9257809 G allele as well as the MHC heterozygous AG genotype significantly increased ESCC risk; 1.76 [1.16-2.66] and 1.74 [1.08-2.80], respectively. Sex-stratification showed that the variant G allele was especially present in female patients; 2.32 [1.04-5.20]. In conclusion, this study provides evidence that MHC rs9257809 and FOXF1 rs9936833 variants, associated with Barrett's esophagus, also increase ESCC and EAC susceptibility in Caucasians. FOX proteins are transcription factors involved in organogenesis of the GI tract, while MHC haplotypes are strongly associated with smoking behavior, a crucial risk factor for ESCC. Assessing the individual genetic susceptibility can help identify high risk patients more prone to benefit from (Barrett) surveillance programs.

GWAS-uncovered SNPs in PLCE1 and RFT2 genes are not implicated in Dutch esophageal adenocarcinoma and squamous cell carcinoma etiology.

Susceptibility to esophageal carcinoma (EC) is influenced by the interaction between genetic and environmental factors. To clarify the etiology of EC, several genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in PCLE1 and RFT2 genes as esophageal squamous cell carcinoma (ESCC) susceptibility loci in Asian populations. This study aimed to determine whether these SNPs also modify the risk of esophageal adenocarcinoma (EAC) and ESCC in western populations of Caucasian ethnicity. A European case-control study including 349 EC patients and 580 controls matched for age, sex, geographical location, and race was carried out. The SNPs rs2274223 in the PCLE1 gene at chromosome 10q23 and rs13042395 in the RFT2 gene at chromosome 20p13 were determined using PCR. Genotype distributions were compared between patients and controls, and odds ratios with 95% confidence intervals were calculated. The total EC group included 86 patients with ESCC and 258 patients with EAC. The distribution of PLCE1 and RFT2 genotypes did not differ between patients with EAC or ESSC, and the controls. In contrast to the modulation of the risk of ESCC in Asians, it is unlikely that the PLCE1 rs2274223 and RFT2 13042395 SNPs play a role in EAC or ESCC susceptibility in Dutch Caucasians.