Meeting report from the joint IARC-NCI international cancer seminar series: a focus on colorectal cancer.

Despite significant progress in our understanding of the etiology, biology and genetics of colorectal cancer, as well as important clinical advances, it remains the third most frequently diagnosed cancer worldwide and is the second leading cause of cancer death. Based on demographic projections, the global burden of colorectal cancer would be expected to rise by 72% from 1.8 million new cases in 2018 to over 3 million in 2040 with substantial increases anticipated in low- and middle-income countries. In this meeting report, we summarize the content of a joint workshop led by the National Cancer Institute and the International Agency for Research on Cancer, which was held to summarize the important achievements that have been made in our understanding of colorectal cancer etiology, genetics, early detection and treatment and to identify key research questions that remain to be addressed.

International cancer seminars: a focus on esophageal squamous cell carcinoma.

The International Agency for Research on Cancer (IARC) and the US National Cancer Institute (NCI) have initiated a series of cancer-focused seminars [Scelo G, Hofmann JN, Banks RE et al. International cancer seminars: a focus on kidney cancer. Ann Oncol 2016; 27(8): 1382-1385]. In this, the second seminar, IARC and NCI convened a workshop in order to examine the state of the current science on esophageal squamous cell carcinoma etiology, genetics, early detection, treatment, and palliation, was reviewed to identify the most critical open research questions. The results of these discussions were summarized by formulating a series of 'difficult questions', which should inform and prioritize future research efforts.

International cancer seminars: a focus on kidney cancer.

Recent years have seen important advances in our understanding of the etiology, biology and genetics of kidney cancer. To summarize important achievements and identify prominent research questions that remain, a workshop was organized by IARC and the US NCI. A series of 'difficult questions' were formulated, which should be given future priority in the areas of population, genomic and clinical research.

Prediction of non-muscle invasive bladder cancer outcomes assessed by innovative multimarker prognostic models.

BACKGROUND: We adapted Bayesian statistical learning strategies to the prognosis field to investigate if genome-wide common SNP improve the prediction ability of clinico-pathological prognosticators and applied it to non-muscle invasive bladder cancer (NMIBC) patients. METHODS: Adapted Bayesian sequential threshold models in combination with LASSO were applied to consider the time-to-event and the censoring nature of data. We studied 822 NMIBC patients followed-up >10 years. The study outcomes were time-to-first-recurrence and time-to-progression. The predictive ability of the models including up to 171,304 SNP and/or 6 clinico-pathological prognosticators was evaluated using AUC-ROC and determination coefficient. RESULTS: Clinico-pathological prognosticators explained a larger proportion of the time-to-first-recurrence (3.1 %) and time-to-progression (5.4 %) phenotypic variances than SNPs (1 and 0.01 %, respectively). Adding SNPs to the clinico-pathological-parameters model slightly improved the prediction of time-to-first-recurrence (up to 4 %). The prediction of time-to-progression using both clinico-pathological prognosticators and SNP did not improve. Heritability (h (2)) of both outcomes was <1 % in NMIBC. CONCLUSIONS: We adapted a Bayesian statistical learning method to deal with a large number of parameters in prognostic studies. Common SNPs showed a limited role in predicting NMIBC outcomes yielding a very low heritability for both outcomes. We report for the first time a heritability estimate for a disease outcome. Our method can be extended to other disease models.

LINE-1 methylation in leukocyte DNA, interaction with phosphatidylethanolamine N-methyltransferase variants and bladder cancer risk.

BACKGROUND: Aberrant global DNA methylation is shown to increase cancer risk. LINE-1 has been proven a measure of global DNA methylation. The objectives of this study were to assess the association between LINE-1 methylation level and bladder cancer risk and to evaluate effect modification by environmental and genetic factors. METHODS: Bisulphite-treated leukocyte DNA from 952 cases and 892 hospital controls was used to measure LINE-1 methylation level at four CpG sites by pyrosequencing. Logistic regression model was fitted to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs). Interactions between LINE-1 methylation levels and environmental and genetic factors were assessed. RESULTS: The risk of bladder cancer followed a nonlinear association with LINE-1 methylation. Compared with subjects in the middle tertile, the adjusted OR for subjects in the lower and the higher tertiles were 1.26 (95% CI 0.99-1.60, P=0.06) and 1.33 (95% CI 1.05-1.69, P=0.02), respectively. This association significantly increased among individuals homozygous for the major allele of five single-nucleotide polymorphisms located in the phosphatidylethanolamine N-methyltransferase gene (corrected P-interaction<0.05). CONCLUSIONS: The findings from this large-scale study suggest that both low and high levels of global DNA methylation are associated with the risk of bladder cancer.

A candidate gene approach to searching for low-penetrance breast and prostate cancer genes.

Most cases of breast and prostate cancer are not associated with mutations in known high-penetrance genes, indicating the involvement of multiple low-penetrance risk alleles. Studies that have attempted to identify these genes have met with limited success. The National Cancer Institute Breast and Prostate Cancer Cohort Consortium--a pooled analysis of multiple large cohort studies with a total of more than 5,000 cases of breast cancer and 8,000 cases of prostate cancer--was therefore initiated. The goal of this consortium is to characterize variations in approximately 50 genes that mediate two pathways that are associated with these cancers--the steroid-hormone metabolism pathway and the insulin-like growth factor signalling pathway--and to associate these variations with cancer risk.

Common genetic variants in the 9p21 region and their associations with multiple tumours.

BACKGROUND: The chromosome 9p21.3 region has been implicated in the pathogenesis of multiple cancers. METHODS: We systematically examined up to 203 tagging SNPs of 22 genes on 9p21.3 (19.9-32.8 Mb) in eight case-control studies: thyroid cancer, endometrial cancer (EC), renal cell carcinoma, colorectal cancer (CRC), colorectal adenoma (CA), oesophageal squamous cell carcinoma (ESCC), gastric cardia adenocarcinoma and osteosarcoma (OS). We used logistic regression to perform single SNP analyses for each study separately, adjusting for study-specific covariates. We combined SNP results across studies by fixed-effect meta-analyses and a newly developed subset-based statistical approach (ASSET). Gene-based P-values were obtained by the minP method using the Adaptive Rank Truncated Product program. We adjusted for multiple comparisons by Bonferroni correction. RESULTS: Rs3731239 in cyclin-dependent kinase inhibitors 2A (CDKN2A) was significantly associated with ESCC (P=7 × 10(-6)). The CDKN2A-ESCC association was further supported by gene-based analyses (Pgene=0.0001). In the meta-analyses by ASSET, four SNPs (rs3731239 in CDKN2A, rs615552 and rs573687 in CDKN2B and rs564398 in CDKN2BAS) showed significant associations with ESCC and EC (P<2.46 × 10(-4)). One SNP in MTAP (methylthioadenosine phosphorylase) (rs7023329) that was previously associated with melanoma and nevi in multiple genome-wide association studies was associated with CRC, CA and OS by ASSET (P=0.007). CONCLUSION: Our data indicate that genetic variants in CDKN2A, and possibly nearby genes, may be associated with ESCC and several other tumours, further highlighting the importance of 9p21.3 genetic variants in carcinogenesis.

GSTM1 null and NAT2 slow acetylation genotypes, smoking intensity and bladder cancer risk: results from the New England bladder cancer study and NAT2 meta-analysis.

Associations between bladder cancer risk and NAT2 and GSTM1 polymorphisms have emerged as some of the most consistent findings in the genetic epidemiology of common metabolic polymorphisms and cancer, but their interaction with tobacco use, intensity and duration remain unclear. In a New England population-based case-control study of urothelial carcinoma, we collected mouthwash samples from 1088 of 1171 cases (92.9%) and 1282 of 1418 controls (91.2%) for genotype analysis of GSTM1, GSTT1 and NAT2 polymorphisms. Odds ratios and 95% confidence intervals of bladder cancer among New England Bladder Cancer Study subjects with one or two inactive GSTM1 alleles (i.e. the 'null' genotype) were 1.26 (0.85-1.88) and 1.54 (1.05-2.25), respectively (P-trend = 0.008), compared with those with two active copies. GSTT1 inactive alleles were not associated with risk. NAT2 slow acetylation status was not associated with risk among never (1.04; 0.71-1.51), former (0.95; 0.75-1.20) or current smokers (1.33; 0.91-1.95); however, a relationship emerged when smoking intensity was evaluated. Among slow acetylators who ever smoked at least 40 cigarettes/day, risk was elevated among ever (1.82; 1.14-2.91, P-interaction = 0.07) and current heavy smokers (3.16; 1.22-8.19, P-interaction = 0.03) compared with rapid acetylators in each category; but was not observed at lower intensities. In contrast, the effect of GSTM1-null genotype was not greater among smokers, regardless of intensity. Meta-analysis of the NAT2 associations with bladder cancer showed a highly significant relationship. Findings from this large USA population-based study provided evidence that the NAT2 slow acetylation genotype interacts with tobacco smoking as a function of exposure intensity.

Evaluation of variation in the phosphoinositide-3-kinase catalytic subunit alpha oncogene and breast cancer risk.

BACKGROUND: Somatic mutations in phosphoinositide-3-kinase catalytic subunit alpha (PIK3CA) are frequent in breast tumours and have been associated with oestrogen receptor (ER) expression, human epidermal growth factor receptor-2 overexpression, lymph node metastasis and poor survival. The goal of this study was to evaluate the association between inherited variation in this oncogene and risk of breast cancer. METHODS: A single-nucleotide polymorphism from the PIK3CA locus that was associated with breast cancer in a study of Caucasian breast cancer cases and controls from the Mayo Clinic (MCBCS) was genotyped in 5436 cases and 5280 controls from the Cancer Genetic Markers of Susceptibility (CGEMS) study and in 30 949 cases and 29 788 controls from the Breast Cancer Association Consortium (BCAC). RESULTS: Rs1607237 was significantly associated with a decreased risk of breast cancer in MCBCS, CGEMS and all studies of white Europeans combined (odds ratio (OR)=0.97, 95% confidence interval (CI) 0.95-0.99, P=4.6 × 10(-3)), but did not reach significance in the BCAC replication study alone (OR=0.98, 95% CI 0.96-1.01, P=0.139). CONCLUSION: Common germline variation in PIK3CA does not have a strong influence on the risk of breast cancer.

Identification of a novel prostate cancer susceptibility variant in the KLK3 gene transcript.

Genome-wide association studies (GWAS) have identified more than 30 prostate cancer (PrCa) susceptibility loci. One of these (rs2735839) is located close to a plausible candidate susceptibility gene, KLK3, which encodes prostate-specific antigen (PSA). PSA is widely used as a biomarker for PrCa detection and disease monitoring. To refine the association between PrCa and variants in this region, we used genotyping data from a two-stage GWAS using samples from the UK and Australia, and the Cancer Genetic Markers of Susceptibility (CGEMS) study. Genotypes were imputed for 197 and 312 single nucleotide polymorphisms (SNPs) from HapMap2 and the 1000 Genome Project, respectively. The most significant association with PrCa was with a previously unidentified SNP, rs17632542 (combined P = 3.9 × 10(-22)). This association was confirmed by direct genotyping in three stages of the UK/Australian GWAS, involving 10,405 cases and 10,681 controls (combined P = 1.9 × 10(-34)). rs17632542 is also shown to be associated with PSA levels and it is a non-synonymous coding SNP (Ile179Thr) in KLK3. Using molecular dynamic simulation, we showed evidence that this variant has the potential to introduce alterations in the protein or affect RNA splicing. We propose that rs17632542 may directly influence PrCa risk.

Genetic variation in CLDN1 and susceptibility to hepatitis C virus infection.

Claudin-1 is a recently discovered co-receptor for hepatitis C virus (HCV) that is required for late-stage binding of the virus. Because variants in the gene that encodes claudin-1 (CLDN1) could play a role in HCV infection, we conducted a 'whole gene association study' among injection drug users (IDUs) to examine whether CLDN1 genetic variants were associated with the risk of HCV infection or with viral clearance. In a cross sectional study, we examined genotype results for 50 single nucleotide polymorphisms (SNPs) across the CLDN1 gene region, comparing genotypes among participants with chronic HCV (n = 658) to those in IDUs who had cleared HCV (n = 199) or remained HCV-uninfected (n = 68). Analyses were controlled for racial ancestry (African-American or European-American) by stratification and logistic regression modeling. We found that participants who remained uninfected more often carried CLDN1 promoter region SNPs -15312C [odds ratio (OR), 1.72; 95% confidence interval (CI) 1.00-2.94; P = 0.048], -7153A (OR, 2.13; 95% CI, 1.25-3.62; P = 0.006) and -5414C (OR, 1.78; 95% CI, 1.06-3.00; P = 0.03). HCV-uninfected participants less often carried CLDN1 IVS1-2983C (OR, 0.55; 95% CI, 0.31-0.97; P = 0.04), which lies in intron 1. CLDN1 -15312C, -7153A and -5414C formed a haplotype in both the African-American and European-American participants and a haplotype analysis supported the association of CLDN1 -7153A in the HCV-uninfected participants. The analyses of HCV clearance revealed no associations with any SNP. These results indicate that genetic variants in regulatory regions of CLDN1 may alter susceptibility to HCV infection.

Effect modification of endocrine disruptors and testicular germ cell tumour risk by hormone-metabolizing genes.

It has been hypothesized that the increased prevalence of testicular germ cell tumours (TGCT) may be attributable to endocrine disrupting chemicals, such as persistent organic pollutants (POPs); these may be modulated by hormone-metabolizing enzymes. Using data from 568 cases and 698 controls enrolled in the US Servicemen's Testicular Tumor Environmental and Endocrine Determinants Study, we examined associations between TGCT and POPs, including p,p'-dichlorodiphenyldichloroethylene, chlordane-related compounds and polychlorinated biphenyls (PCBs), modified by polymorphisms in five hormone-metabolizing genes (CYP17A1, CYP1A1, HSD17B1, HSD17B4 and AR). Odds ratios (OR) and 95% confidence intervals (CI) were estimated using logistic regression models that stratified associations of POP exposure and TGCT risk by genotype. Two polymorphisms in CYP1A1, rs1456432 and rs7495708, modified the association between trans-nonachlor and total chlordanes and TGCT risk. Among men with a minor allele for rs1456432, those with the highest quartiles had an increased risk of TGCT (OR = 1.90, 95% CI, 1.01-3.56) compared with those with the lowest; there was no increased risk among men with the homozygous major allele genotype (p-interactions = 0.024). Similar results were seen for rs7495708. HSD17B4 rs384346 modified the associations between TGCT risk and PCB-118 and PCB-138 concentrations: the 45-55% reductions in TGCT risk for men with the highest quartiles compared with the lowest quartiles were only present in those who had a major homozygous allele genotype (p-interactions < 0.04). Thus, there are suggestions that certain CYP1A1 and HSD17B4 polymorphisms may modify the associations between POPs and TGCT risk. With false discovery rate values >0.2, however, caution is advisable when interpreting the findings of this study.

Association between genetic variants in VEGF, ERCC3 and occupational benzene haematotoxicity.

INTRODUCTION: Benzene is an established human haematotoxin, with substantial interindividual variation in benzene-induced toxicity. METHODS: To further examine if genetic variation contributes to benzene haematotoxicity, we analysed 1023 tagSNPs in 121 gene regions important for benzene metabolism, haematopoiesis, leukaemia and lymphoma among 250 workers exposed to benzene and 140 unexposed controls in a cross-sectional study carried out in China. Linear regression was used to analyse the relationship between genetic polymorphisms and total white blood cell (WBC) count and its subtypes, adjusting for potential confounders and occupational exposure to benzene and toluene among exposed workers. The minp test assessed the association on the gene region level. The false discovery rate method was used to control for multiple comparisons. RESULTS: VEGF (minp = 0.0030) and ERCC3 (minp = 0.0042) were the most significantly associated gene regions with altered WBC counts among benzene-exposed workers, after accounting for multiple comparisons. Highly significant changes were also found for WBC subtype counts, including granulocytes, CD4+ T cells and lymphocytes for VEGF and granulocytes and NK cells for ERCC3. Further, in workers exposed to <1 ppm, a SNP in VEGF was associated with changes in WBC and granulocyte counts, and SNPs in ERCC3 were associated with changes in WBC, NK cell and granulocyte counts. DISCUSSION: Our findings suggest that genetic variation in VEGF, which plays an important role in blood vessel growth, and ERCC3, which is a member of the DNA repair pathway and is responsible for repairing bulky DNA adducts formed by chemicals, may contribute to individual susceptibility to benzene-induced haematotoxicity at relatively low levels of benzene exposure.

Lack of association between genetic variation in G-protein-coupled receptor for asthma susceptibility and childhood asthma and atopy.

G-protein-coupled receptor for asthma susceptibility (GPRA or GPR154) was identified as an asthma and atopy candidate gene by positional cloning. Some subsequent studies suggest associations of GPRA single nucleotide polymorphisms (SNPs) and haplotypes with asthma or atopy susceptibility. However, the associated SNPs or haplotypes vary among studies. The role of GPRA genetic variation in asthma and atopy remains unsolved. Published data on GRPA variants and asthma come exclusively from Caucasian and Asian populations. We examined whether GPRA SNPs and haplotypes are associated with asthma and atopy in a Mexican population. We genotyped and analyzed 27 GPRA SNPs in 589 nuclear families consisting of asthmatic children aged 4-17 years of age and their parents in Mexico City. Atopy was determined by skin prick tests to 25 aeroallergens. The 27 SNPs examined provided excellent coverage of the GPRA gene. GPRA SNPs and haplotypes were not associated with childhood asthma and the degree of atopy to aeroallergens in a Mexican population. Our review of studies of GPRA variants in relation to asthma phenotypes shows considerable heterogeneity. Accordingly, our results suggest that GPRA variants are not an important contributor to childhood asthma and atopy susceptibility in a Mexican population.

Renal cell carcinoma, occupational pesticide exposure and modification by glutathione S-transferase polymorphisms.

This study investigated associations between occupational pesticide exposure and renal cell carcinoma (RCC) risk. To follow-up on a previous report by Buzio et al., we also considered whether this association could be modified by glutathione S-transferase M1 and T1 (GSTM1 and GSTT1) genotypes. About 1097 RCC cases and 1476 controls from Central and Eastern Europe were interviewed to collect data on lifetime occupational histories. Occupational information for jobs held for at least 12 months duration was coded for pesticide exposures and assessed for frequency and intensity of exposure. GSTM1 and GSTT1 gene deletions were analyzed using TaqMan assays. A significant increase in RCC risk was observed among subjects ever exposed to pesticides [odds ratio (OR): 1.60; 95% confidence interval (CI): 1.00-2.55]. After stratification by genotypes, increased risk was observed among exposed subjects with at least one GSTM1 active allele (OR: 4.00; 95% CI: 1.55-10.33) but not among exposed subjects with two GSTM1 inactive alleles compared with unexposed subjects with two inactive alleles (P-interaction: 0.04). Risk was highest among exposed subjects with both GSTM1 and GSTT1 active genotypes (OR: 6.47; 95% CI: 1.82-23.00; P-interaction: 0.02) compared with unexposed subjects with at least one GSTM1 or T1 inactive genotype. In the largest RCC case-control study with genotype information conducted to date, we observed that risk associated with pesticide exposure was exclusive to individuals with active GSTM1/T1 genotypes. These findings further support the hypothesis that glutathione S-transferase polymorphisms can modify RCC risk associated with occupational pesticide exposure.

Characterization of a candidate locus for malaria susceptibility in human populations: a (TA)n microsatellite in the promoter region of the CYBB gene, the gp91 phox subunit of the NADPH oxidase.

We have analysed the linkage disequilibrium pattern between the promoter TA microsatellite and Single Nucleotide Polymorphism (SNP) haplotypes for the CYBB gene. None of the CYBB SNPs serve as good surrogates for the microsatellite alleles, previously associated with mild malaria. Thus, the candidate (TA)(n) microsatellite should be directly tested in genetic epidemiology studies.

Vitamin D receptor polymorphisms and renal cancer risk in Central and Eastern Europe.

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

Single-nucleotide polymorphisms in selected cytokine genes and risk of adult glioma.

A role of immunological factors in glioma etiology is suggested by reports of an inverse relationship with history of allergy or autoimmune disease. To test whether single-nucleotide polymorphisms (SNPs) in cytokine genes were related to risk of adult glioma, we genotyped 11 SNPs in seven cytokine genes within a hospital-based study conducted by the National Cancer Institute and an independent, population-based study by the National Institute for Occupational Safety and Health (overall 756 cases and 1190 controls with blood samples). The IL4 (rs2243248, -1098T>G) and IL6 (rs1800795, -174G>C) polymorphisms were significantly associated with risk of glioma in the pooled analysis (P trend = 0.006 and 0.04, respectively), although these became attenuated after controlling for the false discovery rate (P trend = 0.07 and 0.22, respectively). Our results underscore the importance of pooled analyses in genetic association studies and suggest that SNPs in cytokine genes may influence susceptibility to glioma.

The phosphorylation targets of p47phox, a subunit of the respiratory burst oxidase. Functions of the individual target serines as evaluated by site-directed mutagenesis.

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to O2- at the expense of NADPH. Dormant in resting cells, the oxidase is activated by exposing the cells to appropriate stimuli. During activation, p47phox, a cytosolic oxidase subunit, becomes extensively phosphorylated on a number of serines located between S303 and S379. To determine whether this phosphorylation is necessary for oxidase activation, we examined phorbol-elicited oxidase activity in EBV-transformed B lymphoblasts deficient in p47phox after transfection with plasmids expressing various S-->A mutants of p47phox. The mutant containing S-->A mutations involving all serines between S303 and S379 [S(303-379)A] was not phosphorylated, did not translocate to plasma membrane during activation and was almost devoid of function. As to individual serines, S379 was of special interest because (a) p47 phox S379 was phosphorylated in phorbol-activated lymphoblasts expressing wild-type p47phox, and (b) p47phox S379A failed to translocate to the membrane, and was as functionless as p47phox S(303-379)A; other single S-->A mutations had little effect on oxidase activity. These findings suggest that the phosphorylation of S379 may be important for oxidase activation in whole cells.