A Genetic Locus within the FMN1/GREM1 Gene Region Interacts with Body Mass Index in Colorectal Cancer Risk

Colorectal cancer risk can be impacted by genetic, environmental, and lifestyle factors, including diet and obesity. Geneenvironment interactions (G x E) can provide biological insights into the effects of obesity on colorectal cancer risk. Here, we assessed potential genome-wide G x E interactions between body mass index (BMI) and common SNPs for colorectal cancer risk using data from 36,415 colorectal cancer cases and 48,451 controls from three international colorectal cancer consortia (CCFR, CORECT, and GECCO). The G x E tests included the conventional logistic regression using multiplicative terms (one degree of freedom, 1DF test), the two-step EDGE method, and the joint 3DF test, each of which is powerful for detecting G x E interactions under specific conditions. BMI was associated with higher colorectal cancer risk. The two-step approach revealed a statistically significant GxBMI interaction located within the Formin 1/Gremlin 1 (FMN1/GREM1) gene region (rs58349661). This SNP was also identified by the 3DF test, with a suggestive statistical significance in the 1DF test. Among participants with the CC genotype of rs58349661, overweight and obesity categories were associated with higher colorectal cancer risk, whereas null associations were observed across BMI categories in those with the TT genotype. Using data from three large international consortia, this study discovered a locus in the FMN1/GREM1 gene region that interacts with BMI on the association with colorectal cancer risk. Further studies should examine the potential mechanisms through which this locus modifies the etiologic link between obesity and colorectal cancer

Pre-Diagnostic Genome-Wide DNA Methylation in Blood and Risk of Bladder Cancer

Thesis (Ph.D.)--University of Washington, 2018Background: Differential DNA methylation as measured in blood is a promising marker of bladder cancer susceptibility. In fact, established bladder cancer risk factors such as smoking and various germline genetic variants may promote carcinogenesis in bladder tissue through processes that are detectable as differential DNA methylation in blood. Methods: Genome-wide methylation was measured in pre-diagnostic blood samples, using the Illumina Infinium HumanMethylation450 Bead Array, among 440 bladder cancer cases with the transitional cell carcinoma (TCC) subtype and 440 matched controls from the Women’s Health Initiative (WHI) cohort. After normalization and probe filtering, methylation measurements at 361,184 CpG sites remained for each study participant. We used conditional logistic regression models adjusted for potential confounders and for cell type composition to test for associations between the methylation level at each remaining CpG site and bladder cancer risk (Chapter 1). For each of three smoking-associated CpG sites, we conducted a regression-based mediation analysis to assess whether current smoking affects bladder cancer risk through differential methylation at the CpG site, while accounting for the smoking-CpG interaction using the four-way decomposition approach (Chapter 2). Since there are four single nucleotide polymorphisms (SNPs) that are known to be associated with bladder cancer and are also associated with proximal DNA methylation changes, we performed a regression-based, multiple-mediator mediation analysis for each SNP, which allowed us to determine whether the associated methylation changes at CpG sites are a path through which the SNP causes bladder cancer (Chapter 3). Results: Increased methylation at cg22748573, located in a CpG island within the 5'-UTR/first exon of the CITED4 gene, was associated with an 82% decreased risk of bladder cancer (OR = 0.18, q-value = 0.05) (Chapter 1). The result was robust to sensitivity analyses accounting for time between enrollment and diagnosis, race, tumor subtype, and secondhand smoke exposure. Most of the excess relative risk (ERR) associated with current smoking for a 30 pack-year smoking history as compared to never smoking was mediated through cg05575921 in an enhancer-like regulatory element within AHRR and cg19859270 in the first exon of GPR15 (Chapter 2). The largest components were the mediated interactions for both cg05575921 (ERR component = 2.29, p = 0.05; percent of ERR = 72%, p = 0.02) and cg19859270 (ERR component = 1.89, p-value = 0.05; percent of ERR = 72%, p-value = 0.04), where the mediated interaction capture the indirect effect of smoking through differential methylation as well as the smoking-CpG interaction. There was little evidence that the effect of smoking on bladder cancer risk is mediated through cg03636183. Though not statistically significant, our results suggest that large proportions of the modest effects of rs401681 (NIE = 1.05; NIE percent = 98.5%) and of rs2294008 (NIE = 1.10; NIE percent = 77.6%) on bladder cancer risk occur through their associated CpG sites (Chapter 3). Based on exploratory analyses, cg27028750, which is located in a long terminal repeat element, may drive the indirect effect for rs401681. The effect of rs2294008 may be primarily mediated by cg24023258 near LY6K and by cg17252645 in LY6D among non-smokers and primarily mediated by cg03405983 and cg17888033 in LYNX1 and by cg06565975 near SLURP1 among smokers. There was little evidence supporting mediation through changes in DNA methylation for the associations of rs8102137 and rs798766 with bladder cancer risk. Conclusions: While results need to be validated in additional prospective studies, differential methylation in the promoter region of CITED4, as measured in blood, is a promising marker of bladder cancer susceptibility. If confirmed, smoking may have effects on bladder cancer related to changes in AHRR and GPR15 expression. Our results also suggest that for some SNPs associated with bladder cancer, nearby methylation changes may be part of the underlying mechanisms of effect. Supplemental File: As supplementary information, “KM Jordahl Supplementary Table 1-1.xlsx” contains Supplementary Table 1.1 and provides the full results for all 361,184 CpG sites from the genome-wide study of DNA methylation and bladder cancer risk described in Chapter 1

Differential DNA methylation in blood as a mediator of the association between cigarette smoking and bladder cancer risk among postmenopausal women

Smoking accounts for approximately 52% of bladder cancer incidence among postmenopausal women, but the underlying mechanism is poorly understood. Our study investigates whether changes in DNA methylation, as measured in blood, mediate the impact of smoking on bladder cancer risk among postmenopausal women. We conducted analyses among 206 cases and 251 controls that were current or never smokers at baseline from a previous case-control study of bladder cancer and genome-wide DNA methylation nested within the Women’s Health Initiative. Separate mediation analyses were conducted for three CpG sites demonstrating robust associations with smoking in prior methylome-wide association studies: cg05575921 (AhRR), cg03636183 (F2RL3), and cg19859270 (GPR15). We estimated causal effects using the regression-based, four-way decomposition approach, which addresses the interaction between smoking and each CpG site. The overall proportion of the excess relative risk mediated by cg05575921 was 92% (p-value = 0.004) and by cg19859270 was 79% (p-value = 0.02). The largest component of the excess relative risk of bladder cancer due to 30 pack-years of smoking history in current smokers was the mediated interaction for both cg05575921 (72%, p = 0.02) and cg19859270 (72%, p-value = 0.04), where the mediated interaction is the effect of smoking on bladder cancer that both acts through differential methylation and depends on smoking history. There was little evidence that smoking was mediated through cg03636183. Our results suggest that differential methylation of cg05575921 and cg19859270 mediate the effects of smoking on bladder cancer, potentially revealing downstream effects of smoking relevant for carcinogenesis

Methylation Subtypes of Primary Prostate Cancer Predict Poor Prognosis.

BackgroundPatients with prostate cancer experience heterogeneous outcomes after radical prostatectomy. Genomic studies including The Cancer Genome Atlas (TCGA) have reported molecular signatures of prostate cancer, but few studies have assessed the prognostic effects of DNA methylation profiles.MethodsWe conducted the largest methylome subtyping analysis for primary prostate tumors to date, using methylome data from three patient populations: TCGA, a prostate cancer cohort study conducted at the Fred Hutchinson Cancer Research Center (FH; Seattle, WA), and the Canadian International Cancer Genome Consortium (ICGC) cohort. Four subtypes were detected in the TCGA dataset, then independently assigned to FH and ICGC cohort data. The identified methylation subtypes were assessed for association with cancer prognosis in the above three patient populations.ResultsUsing a set of hypermethylated CpG sites, four methylation subtypes were identified in TCGA. Compared with subtype 1, subtype 4 had an HR of 2.09 (P = 0.029) for biochemical recurrence (BCR) in TCGA patients. HRs of 2.76 (P = 0.002) for recurrence and 9.73 (P = 0.002) for metastatic-lethal (metastasis or prostate cancer-specific death) outcomes were observed in the FH cohort. A similar pattern of association was noted in the Canadian ICGC cohort, though HRs were not statistically significant.ConclusionsA hypermethylated subtype was associated with an increased hazard of recurrence and mortality in three studies with prostate tumor methylome data. Further molecular work is needed to understand the effect of methylation subtypes on cancer prognosis.ImpactThis study identified a DNA methylation subtype that was associated with worse prostate cancer prognosis after radical prostatectomy

Nightshift work, chronotype, and genome-wide DNA methylation in blood

Molecular mechanisms underlying the negative health effects of shift work are poorly understood, which remains a barrier to developing intervention strategies to protect the long-term health of shift workers. We evaluated genome-wide differences in DNA methylation (measured in blood) between 111 actively employed female nightshift and 86 actively employed female dayshift workers from the Seattle metropolitan area. We also explored the effect of chronotype (i.e., measure of preference for activity earlier or later in the day) on DNA methylation among 110 of the female nightshift workers and an additional group of 131 male nightshift workers. Methylation data were generated using the Illumina Infinium HumanMethylation450 BeadChip (450K) Array. After applying the latest methylation data processing methods, we compared methylation levels at 361,210 CpG loci between the groups using linear regression models adjusted for potential confounders and applied the false-discovery rate (FDR) ≤ 0.05 to account for multiple comparisons. No statistically significant associations at the genome-wide level were observed with shift work or chronotype, though based on raw P values and absolute effect sizes, there were suggestive associations in genes that have been previously linked with cancer (e.g., BACH2, JRK, RPS6KA2) and type-2 diabetes (e.g., KCNQ1). Given that our study was underpowered to detect moderate effects, examining these suggestive results in well-powered independent studies or in pooled data sets may improve our understanding of the pathways underlying the negative health effects of shift work and the influence of personal factors such as chronotype. Such an approach may help identify potential interventions that can be used to protect the long-term health of shift workers

DNA methylation and cis-regulation of gene expression by prostate cancer risk SNPs.

Genome-wide association studies have identified more than 100 SNPs that increase the risk of prostate cancer (PrCa). We identify and compare expression quantitative trait loci (eQTLs) and CpG methylation quantitative trait loci (meQTLs) among 147 established PrCa risk SNPs in primary prostate tumors (n = 355 from a Seattle-based study and n = 495 from The Cancer Genome Atlas, TCGA) and tumor-adjacent, histologically benign samples (n = 471 from a Mayo Clinic study). The role of DNA methylation in eQTL regulation of gene expression was investigated by data triangulation using several causal inference approaches, including a proposed adaptation of the Causal Inference Test (CIT) for causal direction. Comparing eQTLs between tumors and benign samples, we show that 98 of the 147 risk SNPs were identified as eQTLs in the tumor-adjacent benign samples, and almost all 34 eQTL identified in tumor sets were also eQTLs in the benign samples. Three lines of results support the causal role of DNA methylation. First, nearly 100 of the 147 risk SNPs were identified as meQTLs in one tumor set, and almost all eQTLs in tumors were meQTLs. Second, the loss of eQTLs in tumors relative to benign samples was associated with altered DNA methylation. Third, among risk SNPs identified as both eQTLs and meQTLs, mediation analyses suggest that over two-thirds have evidence of a causal role for DNA methylation, mostly mediating genetic influence on gene expression. In summary, we provide a comprehensive catalog of eQTLs, meQTLs and putative cancer genes for known PrCa risk SNPs. We observe that a substantial portion of germline eQTL regulatory mechanisms are maintained in the tumor development, despite somatic alterations in tumor genome. Finally, our mediation analyses illuminate the likely intermediary role of CpG methylation in eQTL regulation of gene expression

Intracellular location of brca2 protein expression and prostate cancer progression in the swedish watchful waiting cohort

We found association between prognosis of prostate cancer patients and positive staining for the BRCA2 protein in diagnostic cancer tissue. Positive staining at the cell membrane was associated with improved survival whereas staining in the cytoplasm predicted adverse outcome.Prostate cancer patients with inherited BRCA2 mutations have a survival disadvantage. However, it is unknown whether progression is associated with BRCA2 protein expression in diagnostic prostate cancer tissue, among men without inherited mutations. We conducted a nested case-control study within the Swedish Watchful Waiting cohort. The case group included all 71 patients who died from prostate cancer within 5 years from diagnosis and controls were all patients (n = 165) who lived at least 7 years after diagnosis. Tissue microarrays were stained using antibodies for C- and N-terminal domains of the BRCA2 protein. Location (nuclear, cytoplasmic and membranous) and magnitude (intensity and percentage) of expression were assessed. Logistic regression models produced odds ratios (OR) and 95% confidence intervals (CI) adjusted for age, year of diagnosis and Gleason score. Positive BRCA2 staining at the cell membrane was associated with reduced risk of death within 5 years (N-terminal: OR = 0.47, 95% CI = 0.21-1.04, P = 0.06; C-terminal: OR = 0.41, 95% CI = 0.18-0.91, P = 0.03) and low Gleason scores (P = 0.006). Positive cytoplasmic C-terminal staining was associated with higher Gleason scores and increased lethality (OR = 3.61, 95% CI = 1.61-8.07, P = 0.002). BRCA2 protein expression at the cell membrane and lack of C-terminal expression in the cytoplasm were associated with a reduced risk of rapidly fatal prostate cancer. BRCA2 protein expression in prostate cancer tissue may have independent prognostic value. The potential biological significance of BRCA2 expression at the cell membrane warrants further investigation

Interpathologist concordance in the histological diagnosis of focal prostatic atrophy lesions, acute and chronic prostatitis, PIN, and prostate cancer

Epidemiological and biological evidence indicates a causal relationship between the presence of proliferative atrophic lesions and the development of prostatic intraepithelial neoplasia (PIN) and prostate cancer. The presence of inflammatory and atrophic lesions of the prostate is widely underestimated and they are not generally mentioned in pathology reports. We performed a histopathological concordance study among eight genitourinary specialists and seven generalist pathologists, using 116 histological slides of prostate lesions, including proliferative atrophic lesions, PIN, and cancer. The overall agreement between all possible pairs of reviewers was 80% for prostate cancer, 67% for PIN, and 49% for proliferative atrophic lesions. When using as gold standard the assessment of a single genitourinary pathologist, the mean agreement percentage increased to 97% for prostate cancer, 92% for PIN, and 72% for proliferative atrophic lesions