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Supplementary Figure from Systemic Levels of Estrogens and PGE2 Synthesis in Relation to Postmenopausal Breast Cancer Risk

Background:Prostaglandin E2 (PGE2) induces aromatase expression in adipose tissue, leading to increased estrogen production that may promote the development and progression of breast cancer. However, few studies have simultaneously investigated systemic levels of PGE2 and estrogen in relation to postmenopausal breast cancer risk.Methods:Here, we determined urinary estrogen metabolites (EM) using mass spectrometry in a case–cohort study (295 incident breast cancer cases and 294 subcohort members), and using linear regression estimated the effect of urinary levels of a major PGE2 metabolite (PGE-M) on EMs. HRs for the risk of developing breast cancer in relation to PGE-M and EMs were compared between Cox regression models with and without mutual adjustment.Results:PGE-M was a significant predictor of estrone (E1), but not estradiol (E2) levels in multivariable analysis. Elevated E2 levels were associated with an increased risk of developing breast cancer [HRQ5vs.Q1, 1.54; 95% confidence interval (CI), 1.01–2.35], and this association remained unchanged after adjustment for PGE-M (HRQ5vs.Q1, 1.52; 95% CI, 0.99–2.33). Similarly, elevated levels of PGE-M were associated with increased risk of developing breast cancer (HRQ4vs.Q1, 2.01; 95% CI, 1.01–4.29), and this association was only nominally changed after consideration of E1 or E2 levels.Conclusions:Urinary levels of PGE-M and estrogens were independently associated with future risk of developing breast cancer among these postmenopausal women.Impact:Increased breast cancer risk associated with PGE-M might not be fully explained by the estrogens–breast cancer association alone but also by additional effects related to inflammation. Cancer Epidemiol Biomarkers Prev; 26(3); 383–8. ©2016 AACR.

Supplementary Table from Systemic Levels of Estrogens and PGE2 Synthesis in Relation to Postmenopausal Breast Cancer Risk

Supplementary Table 1. Sample Size of each participated study, by case-control status and genotype platform. Supplementary Table 2. Association between fourteen environmental factors and the risk of breast cancer. Supplementary Table 3. Interactions between genes and fourteen environmental factors.

Genome-wide association studies (GWAS) have identified more than 200 susceptibility loci for breast cancer, but these variants explain less than a fifth of the disease risk. Although gene–environment interactions have been proposed to account for some of the remaining heritability, few studies have empirically assessed this.We obtained genotype and risk factor data from 46,060 cases and 47,929 controls of European ancestry from population-based studies within the Breast Cancer Association Consortium (BCAC). We built gene expression prediction models for 4,864 genes with a significant (P < 0.01) heritable component using the transcriptome and genotype data from the Genotype-Tissue Expression (GTEx) project. We leveraged predicted gene expression information to investigate the interactions between gene-centric genetic variation and 14 established risk factors in association with breast cancer risk, using a mixed-effects score test.After adjusting for number of tests using Bonferroni correction, no interaction remained statistically significant. The strongest interaction observed was between the predicted expression of the C13orf45 gene and age at first full-term pregnancy (PGXE = 4.44 × 10−6).In this transcriptome-informed genome-wide gene–environment interaction study of breast cancer, we found no strong support for the role of gene expression in modifying the associations between established risk factors and breast cancer risk.Our study suggests a limited role of gene–environment interactions in breast cancer risk.

Funding and acknowledgements.

Quantile-Quantile plot (Q-Q plot) of the aMiSTi p-values for each set of the GxE interactions.

Supplementary Data from Association between Urinary Prostaglandin E2 Metabolite and Breast Cancer Risk: A Prospective, Case–Cohort Study of Postmenopausal Women

Supplementary Methods and Tables

Background: We previously observed that high serum 25-hydroxyvitamin D (25(OH)D; >38.0 ng/mL) was inversely associated with breast cancer. Here, we examined effect modification by SNPs in vitamin D–related genes.Methods: The Sister Study enrolled 50,884 U.S. women who had a sister with breast cancer, but who had never had breast cancer themselves. Using a case–cohort design, we compared 1,524 women who developed breast cancer within 5 years to 1,810 randomly selected participants. We estimated ratios of HRs (RHRs) for the 25(OH)D–breast cancer association per copy of the minor allele using Cox proportional hazards models. We considered 82 SNPs in 7 vitamin D–related genes (CYP24A1, CYP27B1, CYP2R1, GC, DHCR7/NADSYN1, RXRA, and VDR). We also tested gene-based interactions with 25(OH)D.Results: The SNP with the smallest interaction P value was rs4328262 in VDR (P = 0.0008); the 25(OH)D HR was 0.92 [95% confidence interval (CI), 0.68–1.24] among those homozygous for the common allele, and the minor allele was estimated to decrease the HR by 33% per copy (RHR = 0.67; 95% CI, 0.53–0.85). Five other VDR SNPs showed evidence of interaction at P < 0.05, as did one SNP in CYP2R1 and one in RXRA. As a group, the 82 SNPs showed evidence of multiplicative interaction with 25(OH)D (P = 0.04). In gene-based tests, only VDR showed strong evidence of interaction (P = 0.04).Conclusions: SNPs in vitamin D–related genes may modify the association between serum 25(OH)D and breast cancer.Impact: This work strengthens the evidence for protective effects of vitamin D. Cancer Epidemiol Biomarkers Prev; 26(12); 1761–71. ©2017 AACR.

Background: Common cancers develop through a multistep process often including inherited susceptibility. Collaboration among multiple institutions, and funding from multiple sources, has allowed the development of an inexpensive genotyping microarray, the OncoArray. The array includes a genome-wide backbone, comprising 230,000 SNPs tagging most common genetic variants, together with dense mapping of known susceptibility regions, rare variants from sequencing experiments, pharmacogenetic markers, and cancer-related traits.Methods: The OncoArray can be genotyped using a novel technology developed by Illumina to facilitate efficient genotyping. The consortium developed standard approaches for selecting SNPs for study, for quality control of markers, and for ancestry analysis. The array was genotyped at selected sites and with prespecified replicate samples to permit evaluation of genotyping accuracy among centers and by ethnic background.Results: The OncoArray consortium genotyped 447,705 samples. A total of 494,763 SNPs passed quality control steps with a sample success rate of 97% of the samples. Participating sites performed ancestry analysis using a common set of markers and a scoring algorithm based on principal components analysis.Conclusions: Results from these analyses will enable researchers to identify new susceptibility loci, perform fine-mapping of new or known loci associated with either single or multiple cancers, assess the degree of overlap in cancer causation and pleiotropic effects of loci that have been identified for disease-specific risk, and jointly model genetic, environmental, and lifestyle-related exposures.Impact: Ongoing analyses will shed light on etiology and risk assessment for many types of cancer. Cancer Epidemiol Biomarkers Prev; 26(1); 126–35. ©2016 AACR.

Supplementary Table 1. Between SNP correlations (r2) for VDR SNPs among sub-cohort sample from the Sister Study (n=1829); Supplementary Table 2. Joint effects of 25(OH)D and single nucleotide polymorphisms (SNPs) in vitamin D metabolism genes on the 5-year risk of post-menopausal breast cancer: Ratio of hazard ratios and 95% confidence intervals; Supplementary Table 3. Joint effects of 25(OH)D and single nucleotide polymorphisms (SNPs) in vitamin D metabolism genes on the 5-year risk of breast cancer according to first-degree family history: Minor allele frequencies (MAFs), Ratio of hazard ratios (RHR), 95% confidence intervals (95% CI), and interaction p-values (top 12 SNPs only); Supplementary Table 4. Top hits for joint effects of 25(OH)D and single nucleotide polymorphisms (SNPs) in other vitamin D-related genes on the 5-year risk of breast cancer; Supplementary Table 5. Gene-environment set association tests (GESAT) for 25(OH)D and other vitamin D-related genes on the 5-year risk of breast cancer

Tobacco smoking is the most important and well-established bladder cancer risk factor and a rich source of chemical carcinogens and reactive oxygen species that can induce damage to DNA in urothelial cells. Therefore, common variation in DNA repair genes might modify bladder cancer risk. In this study, we present results from meta-analyses and pooled analyses conducted as part of the International Consortium of Bladder Cancer. We included data on 10 single nucleotide polymorphisms corresponding to seven DNA repair genes from 13 studies. Pooled analyses and meta-analyses included 5,282 cases and 5,954 controls of non-Latino white origin. We found evidence for weak but consistent associations with ERCC2 D312N [rs1799793; per-allele odds ratio (OR), 1.10; 95% confidence interval (95% CI), 1.01–1.19; P = 0.021], NBN E185Q (rs1805794; per-allele OR, 1.09; 95% CI, 1.01–1.18; P = 0.028), and XPC A499V (rs2228000; per-allele OR, 1.10; 95% CI, 1.00–1.21; P = 0.044). The association with NBN E185Q was limited to ever smokers (interaction P = 0.002) and was strongest for the highest levels of smoking dose and smoking duration. Overall, our study provides the strongest evidence to date for a role of common variants in DNA repair genes in bladder carcinogenesis. [Cancer Res 2009;69(17):6857–64]

Supplementary Tables 1-8 from Polymorphisms in DNA Repair Genes, Smoking, and Bladder Cancer Risk: Findings from the International Consortium of Bladder Cancer

Supplementary Figure Legend from Polymorphisms in DNA Repair Genes, Smoking, and Bladder Cancer Risk: Findings from the International Consortium of Bladder Cancer

Supplementary Figure 1 from Polymorphisms in DNA Repair Genes, Smoking, and Bladder Cancer Risk: Findings from the International Consortium of Bladder Cancer

Clonal hematopoiesis of indeterminate potential (CHIP) has been associated with inflammation, which is a risk factor for cancer, including prostate cancer. We previously reported weak evidence of an association between CHIP and prostate cancer risk in men of European ancestry. However, little is known for African ancestry populations. We investigated the association of age-related CHIP with overall and aggressive prostate cancer risk in a large whole-exome sequencing study of 12,049 African ancestry men, including 7,176 prostate cancer cases (of which 3,283 had aggressive disease and 1,074 had metastatic disease) and 4,873 controls. Somatic variant calling was carried out using GATK Mutect2, and only variants with minor allele frequencies (MAF) 5% were included. Variants with a MAF ≥0.1% in gnomAD were excluded. CHIP variants were identified from a list of pre-specified mutations in 74 genes. Associations were tested using regression models adjusting for age, sub-study, and top 10 principal components, with statistical significance tested by the likelihood ratio test and applying a Bonferroni correction to account for multiple testing. In total, 998 variants in 57 CHIP genes were identified. Consistent with previous results, we observed a strong association between CHIP and age at blood draw. CHIP genes in aggregate were not statistically significantly associated with risks of total (OR=1.12, 95% CI=0.97-1.28), aggressive (OR=1.14, 95% CI=0.92-1.43) or metastatic (OR=1.17, 95% CI=0.91-1.49) prostate cancer. We observed that carriers of variants in DNMT3A, which is the gene that harbors the most CHIP driver mutations, had a nominally elevated risk of prostate cancer compared to non-carriers (OR=1.35, 95% CI=1.08-1.68, p=0.007). Additionally, carriers of variants in EZH2, which is implicated in cancer progression, showed a suggestive association with aggressive prostate cancer (OR=7.33, 95% CI=1.01-53.21, p=0.029). After adjusting for age at blood draw, CHIP genes in aggregate were not associated with age at prostate cancer diagnosis. However, we found that EZH2 variants carriers were diagnosed 12.9 years earlier on average than non-carriers (95% CI=6.1-19.7, adjusted p=0.01). A prostate cancer polygenic risk score (PRS) constructed using 269 risk variants was not associated with CHIP carrier status in aggregate (OR=0.99, 95% CI=0.92-1.06, p=0.70) or with any individual gene (all adjusted p>0.05). In summary, overall CHIP is not likely to be a risk factor of prostate cancer or aggressive disease in men of African ancestry. However, our results do confirm the association of CHIP in DNMT3A with prostate cancer risk as reported in previous studies in men of European ancestry. Future work will be needed to evaluate the biological causality of DNMT3A- and EZH2- related CHIP on prostate cancer. Citation Format: Anqi Wang, Yili Xu, Xin Sheng, Raymond Hughley, Ben Adusei, Mohamed Jalloh, Serigne Magueye Gueye, Andrew A Adjei, James Mensah, Pedro W. Fernandez, Akin Olupelumi Adebiyi, Oseremen Inokhoife Aisuodionoe-Shadrach, Lindsay Petersen, Maureen Joffe, Jeannette T. Bensen, James L. Mohler, Jack A. Taylor, Eboneé N. Butler, Sue A. Ingles, Benjamin A. Rybicki, Janet L. Stanford, Wei Zheng, Sonja I. Berndt, Chad D. Huff, Joseph Lachance, Luc Multigner, Caroline Andrews, Timothy R. Rebbeck, Laurent Brureau, Stephen J. Chanock, Adam de Smith, Fei Chen, Burcu F. Darst, David V. Conti, Christopher A. Haiman. Association between clonal hematopoiesis and risk of prostate cancer in a large sample of African ancestry men [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3508.

Background: There is a growing body of evidence supporting the contributions of germline rare variants to the susceptibility of prostate cancer (PCa), especially aggressive PCa. Our previous exome sequencing analysis highlighted 36 aggressive PCa candidate genes in populations of European ancestry. Here we investigated whether rare germline pathogenic, likely pathogenic, or deleterious (P/LD/D) variants in these genes were associated with overall and aggressive PCa risk in men of African ancestry. Methods: This exome sequencing analysis consists of 7,176 prostate cancer cases and 4,873 controls from the Research on Prostate Cancer in Men of African Ancestry (RESPOND) study. Among the PCa cases, 3,283 are aggressive cases (tumor stage T3/T4, regional lymph node involvement, metastatic disease, Gleason score >= 8.0, prostate-specific antigen [PSA] level >= 20 ng/mL or PCa as the underlying cause of death) including 1,074 metastatic cases, and 1,752 are non-aggressive cases (Gleason score ⇐ 7.0, PSA < 20 ng/mL, and tumor stage T1/T2). P/LP/D variants analyzed were rare (minor allele frequency < 1% in controls) and had either a Variant Effect Predictor impact score of “high” or a pathogenic or likely pathogenic ClinVar classification. The association between P/LP/D carrier status with risk of overall PCa, aggressive PCa, and metastatic PCa was evaluated in logistic regression models, adjusting for age and the top ten principal components. All statistical tests are two-sided. Results: Of the 36 PCa candidate genes, BRCA2 was the most frequently affected gene, with 1.7% of cases and 1.1% of controls harboring a germline P/LP/D variant, followed by MUTYH (1.5%/1.3%) ATM (0.93%/0.49%), MSH5 (0.70%/0.51%) and HOXB13 (0.70%/0.35%). Nominally significant associations with overall PCa were observed for ATM (OR=1.83, 95% CI=1.14-2.92, P=0.012), BRCA2 (OR=1.52, 95% CI=1.10-2.10, P=0.011), HOXB13 (OR=2.10, 95% CI=1.12-3.66, P=0.008), and PALB2 (OR=3.46, 95% CI=1.18-10.1, P=0.02). In case-case analyses (aggressive vs. non-aggressive cases), the association with aggressive PCa was nominally significant for ATM (OR=5.10, 95% CI=1.96-13.3, P=8.7 × 10−4) and BRCA2 (OR=2.00, 95% CI=1.19-3.38, P=0.009) and was suggestive for PALB2 (OR=2.99, 95% CI=0.83-10.7, P=0.09). Similar associations with metastatic PCa were also observed for these three genes. Conclusion: The associations of BRCA2, ATM, and PALB2 with overall PCa and aggressive PCa observed in men of African ancestry are consistent with findings from our previous study in men of European ancestry. These findings further support the importance of these genes in the consideration of screening and active surveillance for high-risk and advanced disease. Citation Format: Fei Chen, Burcu F. Darst, Xin Sheng, Anqi Wang, Yili Xu, Raymond Hughley, Ben Adusei, Mohamed Jalloh, Serigne Magueye Gueye, Andrew A. Adjei, James Mensah, Pedro W. Fernandez, Akindele O. Adebiyi, Oseremen Aisuodionoe-Shadrach, Lindsay Petersen, Maureen Joffe, Jo McBride, Jeannette T. Bensen, James L. Mohler, Jack A. Taylor, Eboneé N. Butler, Sue A. Ingles, Benjamin A. Rybicki, Janet L. Stanford, Wei Zheng, Sonja I. Berndt, Chad D. Huff, Joseph Lachance, Luc Multigner, Caroline Andrews, Timothy R. Rebbeck, Laurent Brureau, Stephen J. Chanock, David V. Conti, Christopher A. Haiman. Association of prostate cancer candidate genes with overall and aggressive prostate cancer in men of African ancestry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1182.

Leukocyte DNA methylation (DNAm) at individual sites across the genome can be used to construct measures of biological age. Positive age acceleration—when biological age is older than chronological age—is associated with higher risk of age-associated diseases. Recent adjuvant therapy is reported to increase age acceleration, but the longer-term effects of a breast cancer diagnosis and treatment on age acceleration remain unknown. Here, we use blood samples collected at two timepoints to examine changes in age acceleration over time comparing women who did and did not develop breast cancer. Paired whole blood samples were drawn an average of 8 years apart (range: 5-11 years) in a sample of non-Hispanic White and Black (Hispanic and non-Hispanic) women. DNAm was profiled using Infinium MethylationEPIC BeadChips. Approximately half the women were diagnosed and treated for breast cancer between blood draws (cases; n= 190, baseline mean age= 57) whereas the other half remained breast cancer-free (controls; n= 227, baseline mean age= 56). Longitudinal changes in three age acceleration metrics were compared to determine whether an intervening breast cancer diagnosis and treatment was associated with trajectories in biological aging. On average, the cases were diagnosed with breast cancer 3.5 years after the initial blood draw and 4 years before the second blood draw. Among the cases, 36% were treated with chemotherapy, 65% with radiation therapy, and 70% with hormonal therapies; 45% of the cases received two types of therapy, and 13% received all three. Compared to women who remained cancer-free, women diagnosed and treated for breast cancer had increases in age acceleration over time as measured by PhenoAgeAccel (adjusted standardized mean difference (β)= 0.13, 95% CI: 0.00, 0.26), GrimAgeAccel (β= 0.13, 95% CI: 0.03, 0.24), and DunedinPACE (β= 0.35, 95% CI: 0.23, 0.48). The associations did not vary by timing of diagnosis between the blood draws or race; however, women diagnosed with estrogen receptor (ER) negative tumors appeared to experience faster increases in age acceleration than women diagnosed with ER positive tumors (GrimAgeAccel; ER negative β= 0.27, 95% CI: 0.07, 0.47; ER positive β= 0.10, 95% CI: -0.01, 0.21; P-interaction= 0.14). To investigate the impact of different types of breast cancer therapies, associations were examined using a case-only design. In models that simultaneously included chemotherapy, radiation therapy and hormone therapy, radiation therapy had the strongest associations with accelerated biological aging as measured by PhenoAgeAccel (β= 0.38, 95% CI: 0.18, 0.58), GrimAgeAccel (β= 0.27, 95% CI: 0.09, 0.45), and DunedinPACE (β= 0.25, 95% CI: 0.03, 0.47). We find that years after the initial diagnosis, breast cancer survivors have significantly accelerated biological aging; treatment modalities may differentially influence these rates. Citation Format: Jacob K. Kresovich, Katie M. O'Brien, Zongli Xu, Clarice R. Weinberg, Dale P. Sandler, Jack A. Taylor. Breast cancer diagnosis and treatment associated with acceleration of biological aging over time in a racially diverse cohort of women. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5757.

Purpose To estimate the risk of contralateral breast cancer (CBC) among women in the general population with germline pathogenic variants (PVs) in ATM, BRCA1, BRCA2, CHEK2, and PALB2. Methods Among 15,104 prospectively followed women within the CARRIERS study treated with ipsilateral surgery for invasive breast cancer, a subset of 14,237 women were identified from population-based studies. The risk of CBC was estimated for PV carriers in each gene compared to women without PVs in a multivariate proportional hazard regression analysis accounting for the competing risk of death and adjusting for patient and tumor characteristics. The primary analyses focused on the overall cohort and on women from the general population. Secondary analyses examined associations by race/ethnicity, age at primary breast cancer diagnosis, menopausal status, and tumor estrogen receptor status. Results Germline BRCA1, BRCA2, and CHEK2 PV carriers with breast cancer were at significantly elevated risk (Hazard ratio ≥ 1.9, p< 0.05) of CBC, whereas only the PALB2 PV carriers with ER-negative breast cancer had elevated risks. In contrast, ATM PV carriers did not have significantly increased CBC risks. African American PV carriers had similarly elevated risks of CBC as non-Hispanic White PV carriers. Among premenopausal women, the 15-year cumulative incidence of CBC was >20% for BRCA1, BRCA2 and CHEK2 PV carriers with breast cancer, and for PALB2 PV carriers with ER-negative breast cancer. The 15-year cumulative incidence of CBC among postmenopausal PV carriers was < 20% for PV carriers in any of the 5 genes. Conclusions Women diagnosed with breast cancer and known to carry germline PVs in BRCA1, BRCA2, CHEK2, or PALB2 are at substantially increased risk of CBC and may benefit from enhanced surveillance and risk-reduction strategies. Citation Format: Siddhartha Yadav, Nicholas J. Boddicker, Jie Na, Eric C. Polley, Chunling Hu, Steven N. Hart, Rohan D. Gnanaolivu, Nicole Larson, Carolyn Dunn, Susan Holtegaard, Huaizhi Huang, Lauren R. Teras, Alpa V. Patel, James V. Lacey Jr., Susan Neuhausen, Leslie Bernstein, Elena Martinez, Christopher Haiman, Fei Chen, Kathryn Ruddy, Janet Olson, Esther John, Allison W. Kurian, Dale P. Sandler, Katie M. O’Brien, Jack A. Taylor, Clarice R. Weinberg, Hoda Anton-Culver, Argyrios Ziogas, Gary R. Zirpoli, David E. Goldgar, Katherine L. Nathanson, Susan Domchek, Julie R. Palmer, Jeffrey Weitzel, Peter Kraft, Fergus J. Couch. Population-based Estimates of contralateral Breast Cancer Risk among Carriers of Germline Pathogenic Variants in ATM, BRCA1, BRCA2, CHEK2, and PALB2 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS4-04.

Background: We previously observed that high serum 25-hydroxyvitamin D (25(OH)D; >38.0 ng/mL) was inversely associated with breast cancer. Here, we examined effect modification by SNPs in vitamin D–related genes. Methods: The Sister Study enrolled 50,884 U.S. women who had a sister with breast cancer, but who had never had breast cancer themselves. Using a case–cohort design, we compared 1,524 women who developed breast cancer within 5 years to 1,810 randomly selected participants. We estimated ratios of HRs (RHRs) for the 25(OH)D–breast cancer association per copy of the minor allele using Cox proportional hazards models. We considered 82 SNPs in 7 vitamin D–related genes (CYP24A1, CYP27B1, CYP2R1, GC, DHCR7/NADSYN1, RXRA, and VDR). We also tested gene-based interactions with 25(OH)D. Results: The SNP with the smallest interaction P value was rs4328262 in VDR (P = 0.0008); the 25(OH)D HR was 0.92 [95% confidence interval (CI), 0.68–1.24] among those homozygous for the common allele, and the minor allele was estimated to decrease the HR by 33% per copy (RHR = 0.67; 95% CI, 0.53–0.85). Five other VDR SNPs showed evidence of interaction at P < 0.05, as did one SNP in CYP2R1 and one in RXRA. As a group, the 82 SNPs showed evidence of multiplicative interaction with 25(OH)D (P = 0.04). In gene-based tests, only VDR showed strong evidence of interaction (P = 0.04). Conclusions: SNPs in vitamin D–related genes may modify the association between serum 25(OH)D and breast cancer. Impact: This work strengthens the evidence for protective effects of vitamin D. Cancer Epidemiol Biomarkers Prev; 26(12); 1761–71. ©2017 AACR.

Background: Prostaglandin E2 (PGE2) induces aromatase expression in adipose tissue, leading to increased estrogen production that may promote the development and progression of breast cancer. However, few studies have simultaneously investigated systemic levels of PGE2 and estrogen in relation to postmenopausal breast cancer risk. Methods: Here, we determined urinary estrogen metabolites (EM) using mass spectrometry in a case–cohort study (295 incident breast cancer cases and 294 subcohort members), and using linear regression estimated the effect of urinary levels of a major PGE2 metabolite (PGE-M) on EMs. HRs for the risk of developing breast cancer in relation to PGE-M and EMs were compared between Cox regression models with and without mutual adjustment. Results: PGE-M was a significant predictor of estrone (E1), but not estradiol (E2) levels in multivariable analysis. Elevated E2 levels were associated with an increased risk of developing breast cancer [HRQ5vs.Q1, 1.54; 95% confidence interval (CI), 1.01–2.35], and this association remained unchanged after adjustment for PGE-M (HRQ5vs.Q1, 1.52; 95% CI, 0.99–2.33). Similarly, elevated levels of PGE-M were associated with increased risk of developing breast cancer (HRQ4vs.Q1, 2.01; 95% CI, 1.01–4.29), and this association was only nominally changed after consideration of E1 or E2 levels. Conclusions: Urinary levels of PGE-M and estrogens were independently associated with future risk of developing breast cancer among these postmenopausal women. Impact: Increased breast cancer risk associated with PGE-M might not be fully explained by the estrogens–breast cancer association alone but also by additional effects related to inflammation. Cancer Epidemiol Biomarkers Prev; 26(3); 383–8. ©2016 AACR.

Germline variation at 8q24 is the strongest risk factor for prostate cancer (PCa) across all racial and ethnic populations. While most 8q24 associations have been observed across populations, rs72725854 [T risk allele frequency ~6%] is only found in men of African ancestry and is the strongest known genome-wide association signal for PCa in this population. We investigated whether the T allele of rs72725854 is associated with PCa family history and age at diagnosis, characteristics known to have a strong genetic component. Analyses were performed using a sample of 9,052 cases and 8,595 controls from the African Ancestry Prostate Cancer (AAPC) GWAS Consortium and the ELLIPSE/PRACTICAL OncoArray Consortium. Participants were unselected for PCa family history. Among cases, 23.7% carried at least one copy of the T allele versus 11.6% of controls. The OR was 2.29 (95% CI=2.10–2.49) for TA heterozygotes and 5.04 (95% CI=3.36–7.55) for TT homozygotes. The percentage of cases carrying the T allele was significantly greater for men with a PCa family history (27.4% vs. 22.7% without a family history, p=0.002) and for men diagnosed 100 ng/ml or death from PCa), 25.4% for high-risk disease (stage T3/T4, Gleason 8-10, or PSA=20-100 ng/ml), 24.6% for intermediate-risk disease (Gleason=7, stage T1/T2, and PSA=10-20 ng/ml), and 21.4% for low-risk disease (Gleason Citation Format: Burcu F. Darst, Jeannette T. Bensen, Sue A. Ingles, Benjamin A. Rybicki, Barbara Nemesure, Esther M. John, Jay H. Fowke, Victoria L. Stevens, Sonja I. Berndt, Chad D. Huff, Jong Y. Park, Wei Zheng, Elaine A. Ostrander, Shiv Srivastava, John Carpten, Thomas A. Sellers, Maureen Sanderson, Dana C. Crawford, Olivier Cussenot, Jennifer Cullen, Rick A. Kittles, Jianfeng Xu, Zsofia Kote-Jarai, Luc Multigner, Marie-Elise Parent, Florence Menegaux, Geraldine Cancel-Tassin, Adam S. Kibel, Eric A. Klein, Phyllis J. Goodman, Jennifer J. Hu, Graham Casey, Anselm J. Hennis, Ian M. Thompson, Robin Leach, James L. Mohler, Elizabeth T. Fontham, Gary J. Smith, Jack A. Taylor, Rosalind A. Eeles, Laurent Brureau, Stephen J. Chanock, Stephen Watya, Janet L. Stanford, Diptasri Mandal, William B. Isaacs, Kathleen A. Cooney, William J. Blot, David V. Conti, Christopher A. Haiman. A germline variant at 8q24 contributes to familial clustering of prostate cancer in men of African ancestry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3517.

Germline mutations in several cancer predisposition genes included in hereditary multigene testing panels have been associated with increased breast cancer risk. However, estimates of breast cancer risks for each gene have in large part been derived from studies of high-risk populations enriched for family history of breast and ovarian cancer, young age of breast cancer diagnosis, or founder mutations. The breast cancer risks associated with mutations in many of these genes in the general population remain to be clearly defined. As clinical hereditary cancer genetic testing is already provided to many women at increased risk and may soon be offered to all breast cancer patients, population-based risk estimates associated with predisposition gene mutations will be needed for appropriate clinical management of mutation carriers. The “CAnceR RIsk Estimates Related to Susceptibility” (CARRIERS) consortium of 17 cohort-based case-control, population-based case-control, and family studies focused on estimation of population-based breast cancer risks and penetrance analysis for mutations in known and candidate breast cancer predisposition genes. Germline DNA from 39486 breast cancer cases and 35868 matched controls was subjected to dual bar-coded QIAseq multiplex amplification of 1733 target regions in 37 predisposition genes. Products from sets of 768 samples were pooled and sequenced in each lane of a HiSeq 4000 system. Mutations were called by GATK Haplotype Caller and Verdict. High quality sequence data was obtained for 99.3% of target regions. Here we report on results from 34741 population-based breast cancer cases and 32728 matched unaffected controls. The mean age of breast cancer diagnosis for cases was 61.2 years and mean age for controls was 61.1 years. Overall, 20.9% of cases and 14.3% of controls had a family history of breast cancer. Furthermore, 74.3% of cases and 75.8% of controls were non-Hispanic white, whereas 13.3% of cases and 15.3% of controls were African American. Among cases with tumor information available, 81.7% were ER-positive and 7.7% were triple negative breast cancers (TNBC). We assessed mutation frequencies in 24 genes including 12 established breast cancer predisposition genes (ATM, BARD1, BRCA1, BRCA2, CDH1, CHEK2, NF1, PALB2, PTEN, RAD51C, RAD51D, andTP53). The overall mutation frequency was 6.2% in cases and 2.7% in controls. Case-control association analysis after adjusting for study, age, family history of breast cancer, and race/ethnicity showed that BRCA1(OR=7.45; 95%CI:5.24-10.95) and BRCA2(OR=5.31; 95%CI:4.15-6.88) were associated with high risks of breast cancer in the general population. However, PALB2mutations were only associated with moderate risks (OR=3.63; 95%CI:2.57-5.26). ATMmutations conferred attenuated risks (OR=1.83; 95%CI:1.47-2.28) relative to studies of high-risk families or young onset breast cancers. NBNmutations were not associated with increased breast cancer risk. Mutations in BARD1, RAD51C, RAD51Dand XRCC2were specifically significantly associated with moderate risks of ER-negative breast cancer and TNBC, whereas mutations in ATM, CDH1and CHEK2were specifically significantly associated with ER-positive disease. On the basis of these findings age-related absolute breast cancer risks for commonly mutated genes were estimated. Overall, results from the CARRIERS study establish that mutations in predisposition genes are associated with lower risks of breast cancer in the general population than in enriched populations including high-risk families and young onset, bilateral, or multiple primary cases. We anticipate that the results from this study will inform cancer screening and other risk management strategies for women in the general population with mutations in predisposition genes. Citation Format: Fergus J Couch, Chunling Hu, Steven N Hart, Rohan Gnanaolivu, Kun Y Lee, Jie Na, Chi Gao, Nicholas J Boddicker, Bruce Eckloff, Raed Samara, Josh Klebba, Christine B Ambrosone, Hoda Anton-Culver, Paul Auer, Leslie Bernstein, Mia M Gaudet, Christopher Haiman, Esther M John, Susan Neuhausen, Janet E Olson, Tuya Pal, Julie R Palmer, Dale P Sandler, Jack A Taylor, Amy Trentham-Dietz, Celine M Vachon, Clarice Weinberg, Song Yao, Jeffrey N Weitzel, David E Goldgar, Susan M Domchek, Katherine L Nathanson, Peter Kraft, Eric C Polley. Population-based breast cancer risk estimates for predisposition gene mutations: Results from the CARRIERS study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD3-01.

Prostaglandin E2 (PGE2) induces aromatase expression in adipose tissue leading to increased estrogen production that may promote the development and progression of breast cancer. However, few studies have simultaneously investigated systemic levels of PGE2 and estrogen in relation to postmenopausal breast cancer risk. In a case-cohort study of postmenopausal women (295 cases and 294 subcohort) we previously reported that high levels of PGE-M, a major metabolite of PGE2, were associated with an increased risk of breast cancer among postmenopausal women who did not regularly use nonsteroidal anti-inflammatory drugs (NSAIDs). Here we determined urinary estrogen metabolites (EMs) using mass spectrometry in the same case-cohort set and using linear regression estimated the effect of PGE-M on EMs. Hazard ratios (HRs) for the risk of developing breast cancer in relation to PGE-M and EMs were evaluated in Cox regression models with and without mutual adjustment. PGE-M was a significant predictor of estrone (E1), but not estradiol (E2) levels in multivariable analysis. Elevated E2 levels were associated with an increased risk of developing breast cancer (HRQ5vs.Q1 =1.54, 95% CI: 1.01-2.35), and this association remained unchanged after adjustment for PGE-M (HRQ5vs.Q1 =1.52, 95% CI: 0.99-2.33). Similarly, elevated levels of PGE-M were associated with increased risk of developing breast cancer (HRQ4vs.Q1 =2.01, 95% CI: 1.01-4.29), and this association was only nominally changed after consideration of E1 or E2 levels. Urinary levels of PGE-M and parent estrogens were independently associated with future risk of developing breast cancer among these postmenopausal women. Increased breast cancer risk associated with PGE-M might be attributable both to PGE2-mediated increases in estrogens, and to additional effects related to inflammation. Note: This abstract was not presented at the conference. Citation Format: Sangmi Kim, Jeff Campbell, Wonsuk Yoo, Jack A. Taylor, Dale P. Sandler. Urinary levels of PGE-M and estrogens are independently associated with postmenopausal breast cancer risk. [abstract]. In: Proceedings of the AACR Special Conference: Improving Cancer Risk Prediction for Prevention and Early Detection; Nov 16-19, 2016; Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(5 Suppl):Abstract nr A12.

The association between use of hormone therapy (HT) and breast cancer risk has been consistently replicated in epidemiologic studies, but the biological effects of HT on breast tissue are poorly understood. We hypothesized that postmenopausal exposure to HT leads to changes in DNA methylation in breast tissue. We used fresh frozen samples from the Normal Breast Study (NBS), a hospital based study conducted between 2009 and 2013 at the University of North Carolina in Chapel Hill. Women undergoing breast surgery for reduction mammoplasty, prophylactic resection, or tumor resection donated samples of histologically normal-appearing breast tissue. Out of 474 enrolled participants 106 reported ever using hormone therapy, 37 of which had used it within one year of surgery (classified as current users) and 17 of which had used HT within 1-5 years prior to surgery (classified as recent users). Current and recent users were compared with 36 age frequency-matched controls that had never used HT. Frozen tissue DNA samples from these 90 women were evaluated using the Illumina HumanMethylation450 BeadChip. Genome wide DNA methylation data was analyzed using the reference-free cell mixture method to test for associations between HT-use and the methylation level at each CpG site. In the model we adjusted for SVA derived surrogate variables, to control for unobserved confounders, and multiple comparisons using the false discovery rate (FDR). Although methylation differences between current and never users were small in magnitude (β Citation Format: Sophia Harlid, Zongli Xu, Erin L. Kirk, Lauren E. Wilson, Melissa A. Troester, Jack A. Taylor. Hormone therapy use and DNA methylation in breast tissue. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-376.

Epigenetic modifications influence gene expression and have been implicated in the development of breast cancer. Few studies have evaluated breast cancer risk factors in relation to DNA methylation. We examined known reproductive and hormonal risk factors for breast cancer and epigenome-wide methylation patterns. Participants included 612 women enrolled in the Sister Study prospective cohort who did not have breast cancer. DNA methylation profiling was performed using an Illumina array at the NIH Center for Inherited Disease (CIDR) on DNA extracted from whole blood. Methylation data was obtained at single CpG site resolution for 27,578 CpG sites covering >14,000 genes across 23 chromosomes. Statistical analyses were performed using normalized methylation residuals from a linear model adjusting for age and experimental variables. Controlling for a false discovery rate of 5% (q < 0.05), 1,452 methylation sites (1,220 in CpG islands) were differentially methylated in postmenopausal women compared to premenopausal women. Average methylation was increased at 1,040 sites and decreased at 412 sites. Gene ontology (GO) analysis suggested enrichment of several biological pathways including lobular involution. Among parous women, only 2 sites (1 CpG island) were differentially methylated among women with older versus younger ages at first birth. A single CpG site demonstrated lower average methylation values among long-term users of postmenopausal hormones compared to short-term users. No further statistically significant differences in methylation patterns (q < 0.05) were observed according to age at menarche, parity, breastfeeding history, or postmenopausal hormone use. These data support the menopausal transition as an influential period for epigenetic modifications; few associations between DNA methylation and other classical reproductive and hormonal breast cancer risk factors were observed. The following are the 18 highest scoring abstracts of those submitted for presentation at the 37th Annual ASPO meeting held March 10–12, 2013, in Memphis, TN.

Smoking increases the risk of many diseases -including cancer, and is also linked to blood DNA methylation changes that may be important in disease etiology. The objective of the current study was to identify and confirm potentially novel CpG sites associated with cigarette smoking. To do this we used two epigenome-wide datasets from the Sister Study. One included 908 women with methylation measurements at 27,578 CpG sites using the Illumina HumanMethylation27 BeadChip; the other included 200 women with methylation measurements for 473,844 CpG sites using the Illumina HumanMethylation450 BeadChip. Significant CpGs from the second dataset that were not included in the 27k assay were validated by pyrosequencing in a subset of 476 samples from the first dataset. We were able to successfully confirm smoking associations for nine previously established CpGs and identify two potentially novel CpGs: cg26764244 in GNG12 (p= 9.0×10-10) and cg22335340 in PTPN6 (p=2.9×10-05).In addition to this we provide the first independent confirmation of the association between smoking status and cg02657160 in CPOX (p=7.28x10-7). All 12 CpGs were under-methylated in current smokers and showed increasing percent methylation in former and never smokers. The CPOX CpG proved to be of particular interest as the corresponding enzyme is involved in heme biosynthesis and smoking is known to increase heme production. Our study extends the evidence base for smoking-related changes in DNA methylation. Citation Format: Sara Sophia Harlid, Zongli Xu, Vijayalakshmi Panduri, Dale P. Sandler, Jack A. Taylor. Epigenome-wide study of sister study samples replicates and extends cpg sites associated with cigarette smoking. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 284. doi:10.1158/1538-7445.AM2014-284

SOD2 encodes manganese superoxide dismutase, a mitochondrial enzyme that protects the cell against damage from superoxide free radicals. A common polymorphism, a T → C transition (Val16Ala), was shown to alter the manganese superoxide dismutase mitochondrial targeting sequence ([1][1]). Animal

In utero diethylstilbestrol (DES) exposure has long term consequences including increased risk of vaginal and breast cancer. Epigenetic factors are suspected to contribute to some of these increased risks and animal studies show DES-mediated effects on DNA methylation in both uterine tissue and blood. However, there are currently no studies examining the effect of fetal DES exposure on DNA methylation in humans. Therefore, we investigated differences in DNA methylation between women exposed to DES in utero compared to unexposed women. All our study subjects were participants in the Sister Study -a prospective cohort focused on environmental and familial risk factors for breast cancer and other diseases. Within this study 1,156 women (2.3%) said that they were definitely or probably exposed to DES in utero. In a special sub study (including both women who reported positive exposure and women who reported no exposure), we confirmed exposure history with the woman's mother and from those with matching mother daughter reports we randomly selected 100 exposed and 100 unexposed women. We analyzed DNA methylation patterns in blood using the Illumina 450k array to assess methylation at 473,844 CpG sites. Primary analysis did not reveal any differentially methylated CpGs (dmCpGs) below the false discovery rate (FDR, q Citation Format: S Sophia Harlid, Zongli Xu, Vijayalakshmi Panduri, Aimee D'Aloisio, Lisa DeRoo, Dale Sandler, Jack A. Taylor. Fetal exposure to diethylstilbestrol and DNA methylation in adult women. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3643. doi:10.1158/1538-7445.AM2013-3643

MicroRNAs (miRNAs) are small, non-coding, single-stranded RNAs between 18-22 nucleotides that regulate gene expression by affecting mRNA translation and degradation. It has been proposed that miRNAs could be master regulators of gene expression by regulating entire gene networks. Expression of miRNAs is altered in tumor compared to normal tissue and there is growing evidence that miRNA profiles in serum may differ in cancer cases as compared to healthy controls. However, these studies use samples collected after diagnosis so treatment and/or disease effects cannot be excluded. We are using serum samples from the Sister Study, a prospective cohort study of 50,000 women who had never had breast cancer at the time of enrollment, but were at increased risk because they had a sister previously diagnosed with breast cancer. We are exploring whether serum miRNA profiles can be used as prediagnostic markers of breast cancer. In an initial pilot analysis comparing 5 women diagnosed with breast cancer 1-2 months after their blood draw to 5 controls, there were 25 miRNAs significantly different at the 0.05 level (paired T test). Of those 25 miRNAs, 7 have been previously associated with breast tumor tissue in published studies showing differential expression in tumor tissue or in breast cancer cell lines. We have now completed a larger follow up study examining 205 women diagnosed with breast cancer within 18 months of their blood draw compared to 205 controls frequency matched to cases by age, 2 month interval of blood draw and race. We are using Affymetrix miRNA microarrays to profile miRNA expression in serum samples, and have optimized this platform for use with less than .5mLs of serum. After normalization and background correction, we found 439 miRNAs (of 1105 human miRNAs on the chip) expressed above background in at least 50 samples. When the average expression of controls was compared to cases using logistic regression, we found 16 miRNAs significantly different at the .05 level. For cases, the mean time between blood draw and cancer diagnosis was 10 months. We hypothesize that, if miRNA is a prediagnostic marker, cases with a longer time between blood draw and diagnosis would have a more control-like miRNA profile. To examine this, we are conducting a more detailed analysis among cases with a short time between blood draw and diagnosis. The next steps of our analysis will be to examine how far in advance of cancer diagnosis can we detect case-control differences in miRNA expression profiles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5049. doi:1538-7445.AM2012-5049

Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is a divergent member of the transforming growth factor beta superfamily. NAG-1 has been implicated in many cellular processes, including inflammation, early bone formation, apoptosis, and tumorigenesis, in several types of cancers. Evidence from recent clinical studies suggests that a C to G single nucleotide polymorphism at position 6 (histidine to aspartic acid substitution, or H6D (G) of the NAG-1 protein is associated with lower incidence, but higher rate of mortality, of prostate cancer. The objective of the current study was to determine the activity of the NAG-1 H6D variant in prostate cancer using two different approaches. First, DU145 cells stably transfected with the H6D NAG-1 gene, wild-type NAG-1 gene, and empty pLXIN vector were injected into nude mice subcutaneously and tumor growth monitored for eight weeks. The H6D variant was less tumorigenic than the wild-type NAG-1, and significantly inhibited tumor growth, 67% by volume and 59% by tumor weight compared to control mice. The growth inhibition by the H6D variant of NAG-1 was associated with significant reduction of serum levels of adiponectin, leptin, and IGF-1, and expression of Cyclin D1 in tumor samples. Second, we examined whether the sequence variant in the NAG-1 gene was associated with prostate cancer tumor aggressiveness in blood samples from the North Carolina-Louisiana Prostate Cancer Project (PCaP). A total of 449 Caucasians Americans and 413 African Americans were examined for allele frequencies using Taqman SNP genotyping assay. Although statistically insignificant, African Americans carrying the H6D (G) allele had an increased risk of more aggressive prostate cancer than those carrying two copies of the C allele (Odds ratio, 1.63; 95% CI, 0.903-2.95; P = 0.25). This result may help to explain the higher mortality rate of prostate cancer in African Americans. In conclusion, our data suggest that the H6D polymorphism of NAG-1 may have a dual effect during the early and late stages of prostate cancer carcinogenesis. More studies are needed to further examine the association of H6D variant of NAG-1 with prostate cancer incidence and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5541. doi:10.1158/1538-7445.AM2011-5541

Background: Telomeres are repetitive DNA structure at the end of chromosomes that are required for genomic integrity and stability. Numerous epidemiologic studies have examined telomere length in peripheral blood in relation to cancer and other aging-related diseases. In many of these studies, a measurement of telomere length from a single blood draw is quantified using a real-time PCR technique, and assumed to reflect a replicative history of hematopoietic stem cells in individuals. We examined the reproducibility of telomere length measurement and the relative magnitudes of sources of variation using sequential samples collected over a 9-month period. Methods: Relative telomere length in peripheral blood was estimated using a single tube monochrome multiplex quantitative PCR assay in blood DNA samples from 27 non-pregnant adult women (aged 35 to 74 years) collected in 7 visits over a 9-month period. Each specimen was assayed in triplicate on each of two plates. Reproducibility of the technical triplicates within a plate was assessed using the coefficient of variation (%CV). A linear mixed model was used to estimate the components of variance for telomere length measurements attributed to variation among women and variation between time points within women. Results: Within-plate technical replicate variability ranged from 8.5 to 15% (mean %CV=12.9). Plates had a significant systematic influence on telomere length measurements, explaining 16% of the variance; however, telomere length measurements between different plates were highly correlated (Pearson's correlation coefficient r=0.93, p Conclusion: Our data demonstrate good reproducibility of telomere length measurement using blood from a single draw. However, the existence of technical variability, particularly plate effects, reinforces the need for technical replicates and balancing of case and control samples across plates. Citation Information: Cancer Prev Res 2010;3(12 Suppl):B44.

Discussion Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-185.