Search

Supplementary Figures S1-S2 from HOP/OB1/NECC1 Promoter DNA Is Frequently Hypermethylated and Involved in Tumorigenic Ability in Esophageal Squamous Cell Carcinoma

Promoter DNA hypermethylation with gene silencing is a common feature of human cancer, and cancer-prone methylation is believed to be a landmark of tumor suppressor genes (TSG). Identification of novel methylated genes would not only aid in the development of tumor markers but also elucidate the biological behavior of human cancers. We identified several epigenetically silenced candidate TSGs by pharmacologic unmasking of esophageal squamous cell carcinoma (ESCC) cell lines by demethylating agents (5-aza-2′-deoxycitidine and trichostatin A) combined with ESCC expression profiles using expression microarray. HOP/OB1/NECC1 was identified as an epigenetically silenced candidate TSG and further examined for (a) expression status, (b) methylation status, and (c) functional involvement in cancer cell lines. (a) The HOP gene encodes two putative promoters (promoters A and B) associated with two open reading frames (HOPα and HOPβ, respectively), and HOPα and HOPβ were both down-regulated in ESCC independently. (b) Promoter B harbors dense CpG islands, in which we found dense methylation in a cancer-prone manner (55% in tumor tissues by TaqMan methylation-specific PCR), whereas promoter A does not harbor CpG islands. HOPβ silencing was associated with DNA methylation of promoter B in nine ESCC cell lines tested, and reactivated by optimal conditions of demethylating agents, whereas HOPα silencing was not reactivated by such treatments. Forced expression of HOP suppressed tumorigenesis in soft agar in four different squamous cell carcinoma cell lines. More convincingly, RNA interference knockdown of HOP in TE2 cells showed drastic restoration of the oncogenic phenotype. In conclusion, HOP is a putative TSG that harbors tumor inhibitory activity, and we for the first time showed that the final shutdown process of HOP expression is linked to promoter DNA hypermethylation under the double control of the discrete promoter regions in cancer. (Mol Cancer Res 2008;6(1):31–41)

Supplementary Table 1 from Pharmacologic Unmasking of Epigenetically Silenced Genes in Breast Cancer

Supplementary Table 2 from Pharmacologic Unmasking of Epigenetically Silenced Genes in Breast Cancer

Supplementary Table 3 from Pharmacologic Unmasking of Epigenetically Silenced Genes in Breast Cancer

Purpose: Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of various cancers including breast cancer. Many epigenetically inactivated genes involved in breast cancer development remain to be identified. Therefore, in this study we used a pharmacologic unmasking approach in breast cancer cell lines with 5-aza-2′-deoxycytidine (5-aza-dC) followed by microarray expression analysis to identify epigenetically inactivated genes in breast cancer.Experimental Design: Breast cancer cell lines were treated with 5-aza-dC followed by microarray analysis to identify epigenetically inactivated genes in breast cancer. We then used bisulfite DNA sequencing, conventional methylation-specific PCR, and quantitative fluorogenic real-time methylation-specific PCR to confirm cancer-specific methylation in novel genes.Results: Forty-nine genes were up-regulated in breast cancer cells lines after 5-aza-dC treatment, as determined by microarray analysis. Five genes (MAL, FKBP4, VGF, OGDHL, and KIF1A) showed cancer-specific methylation in breast tissues. Methylation of at least two was found at high frequency only in breast cancers (40 of 40) as compared with normal breast tissue (0 of 10; P < 0.0001, Fisher's exact test).Conclusions: This study identified new cancer-specific methylated genes to help elucidate the biology of breast cancer and as candidate diagnostic markers for the disease.

Supplementary Figures S1-S2 from A Promoter Methylation Pattern in the N-Methyl-d-Aspartate Receptor 2B Gene Predicts Poor Prognosis in Esophageal Squamous Cell Carcinoma

Purpose: To investigate whether the promoter methylation pattern in N-methyl-d-aspartate receptor 2B (NMDAR2B) is correlated with clinical features of human esophageal squamous cell carcinoma (ESCC), the methylation status of the gene was examined at three different sites (P1, P2, and P3) where two CpG islands reside within 1 kb upstream of the transcription start site.Experimental Design: Three independent modalities for methylation analysis (bisulfite sequencing, combined bisulfite restriction analysis, and TaqMan methylation-specific PCR) were done to analyze total 67 ESCC tissues that included 43 primary tumors with well-characterized clinicopathologic variables including patient outcome.Results: Using an optimized cutoff value based on quantitative methylation-specific PCR, we found that patients with higher NMDAR2B methylation ratio in the proximal region (P1) showed a worse 5-year disease-specific survival rate than those without NMDAR2B methylation (P < 0.006). A significant correlation was also seen between NMDAR2B promoter methylation and the presence of vascular permeation (P = 0.03).Conclusion: NMDAR2B promoter methylation could be a clinically applicable marker in ESCC.

Supplementary Table 6 from Genome-Wide Promoter Analysis Uncovers Portions of the Cancer Methylome

Supplementary Figure 1 from N-Methyl-d-Aspartate Receptor Type 2B Is Epigenetically Inactivated and Exhibits Tumor-Suppressive Activity in Human Esophageal Cancer

Supplementary Table 5 from Genome-Wide Promoter Analysis Uncovers Portions of the Cancer Methylome

Supplementary Figure 2 from N-Methyl-d-Aspartate Receptor Type 2B Is Epigenetically Inactivated and Exhibits Tumor-Suppressive Activity in Human Esophageal Cancer

Supplementary Table 4 from Genome-Wide Promoter Analysis Uncovers Portions of the Cancer Methylome

Primer sequence

Supplementary Table 8 from Genome-Wide Promoter Analysis Uncovers Portions of the Cancer Methylome

Supplementary Table 7 from Genome-Wide Promoter Analysis Uncovers Portions of the Cancer Methylome

Supplementary Table 1 from Genome-Wide Promoter Analysis Uncovers Portions of the Cancer Methylome

Diffuse-type gastric cancer (DGC) is the most deadly form of gastric cancer and is frequently accompanied by peritoneal dissemination and metastasis. The specific molecular events involved in DGC pathogenesis remain elusive. Accumulating evidence of epigenetic inactivation in tumor suppressor genes led us to conduct a comprehensive screen to identify novel methylated genes in human cancers using pharmacologic unmasking and subsequent microarray analysis. We compared differential RNA expression profiles of DGC and intestinal-type gastric cancer (IGC) cell lines treated with 5-aza-2′-deoxycytidine using microarrays containing 22,284 genes. We identified 16 methylated genes, including many novel genes, in DGC cell lines and studied PGP9.5 with particular interest. In primary gastric cancers, PGP9.5 was found to be more frequently methylated in DGCs (78%) than in IGCs (36%; DGC versus IGC, P < 0.05). Furthermore, real-time methylation-specific PCR analysis of PGP9.5 showed relatively higher methylation levels in DGC than in IGC. Our data thus implicate a molecular event common in the DGC phenotype compared with IGC. (Cancer Res 2006; 66(7): 3921-7)

Supplementary Table 3 from Genome-Wide Promoter Analysis Uncovers Portions of the Cancer Methylome

DNA methylation has a role in mediating epigenetic silencing of CpG island genes in cancer and other diseases. Identification of all gene promoters methylated in cancer cells “the cancer methylome” would greatly advance our understanding of gene regulatory networks in tumorigenesis. We previously described a new method of identifying methylated tumor suppressor genes based on pharmacologic unmasking of the promoter region and detection of re-expression on microarray analysis. In this study, we modified and greatly improved the selection of candidates based on new promoter structure algorithm and microarray data generated from 20 cancer cell lines of 5 major cancer types. We identified a set of 200 candidate genes that cluster throughout the genome of which 25 were previously reported as harboring cancer-specific promoter methylation. The remaining 175 genes were tested for promoter methylation by bisulfite sequencing or methylation-specific PCR (MSP). Eighty-two of 175 (47%) genes were found to be methylated in cell lines, and 53 of these 82 genes (65%) were methylated in primary tumor tissues. From these 53 genes, cancer-specific methylation was identified in 28 genes (28 of 53; 53%). Furthermore, we tested 8 of the 28 newly identified cancer-specific methylated genes with quantitative MSP in a panel of 300 primary tumors representing 13 types of cancer. We found cancer-specific methylation of at least one gene with high frequency in all cancer types. Identification of a large number of genes with cancer-specific methylation provides new targets for diagnostic and therapeutic intervention, and opens fertile avenues for basic research in tumor biology. [Cancer Res 2008;68(8):2661–70]

Supplementary Table 2 from Genome-Wide Promoter Analysis Uncovers Portions of the Cancer Methylome

Background: Animal and epidemiologic studies suggest that exposure to the weed killer, glyphosate, and its primary metabolite aminomethylphosphonic acid (AMPA), is associated with increased risk for cancer, including breast cancer and non-Hodgkin's lymphoma. The International Agency for Research on Cancer (IARC) has categorized glyphosate as a “probable carcinogen,” yet glyphosate is the most highly used pesticide in the world. Biomonitoring studies assessing levels of human glyphosate and AMPA exposure in the United States have been limited. Thus, we examined urinary levels of these chemicals in a cohort of postmenopausal women residing in Southern California and evaluated associations with demographics, dietary intake, and other behavioral factors. Methods: 338 postmenopausal women provided two first-morning urine samples and at least one paired 24-hour dietary recall. Participants also reported on demographic and other factors. Urinary glyphosate and AMPA were measured using LC-MS/MS. Results: Glyphosate was detected in nearly 90% of urine samples and AMPA in 67.2%. Urinary glyphosate and AMPA levels were lower in women who reported high levels of organic eating (p=0.04 and p=0.01, respectively). Urinary glyphosate was lower in women who reported higher levels of physical activity (p=0.03). Multivariate analysis showed that grain consumption, particularly refined grains, was significantly associated with higher urinary glyphosate levels (adjusted p=0.004). Alcohol consumption (adjusted p=0.001) and frequency of eating fast-food (adjusted p=0.006) were associated with higher urinary AMPA levels, while fruit (adjusted p=0.03) and corn (adjusted p=0.008) consumption were associated with lower AMPA. Conclusions: In the largest study to date examining paired dietary recall data and measurements of urinary glyphosate and AMPA, the vast majority of women sampled had detectable levels. Consumption of certain foods, including grains and alcoholic beverages, were associated with higher urinary levels of glyphosate and AMPA. Citation Format: Rachel McFarland Lucia, Xiyue Liao, Wei-Lin Huang, Danielle Forman, Alexis Kim, Argyrios Ziogas, Trina M. Norden-Krichmar, Deborah Goodman, Andrea Alvarez, Irene Masunaka, Khyatiben Pathak, Marissa McGilvrey, Victoria David-Dirgo, Apurva Hegde, Patrick Pirrotte, Hannah Lui Park. Glyphosate and AMPA exposure: Associations with dietary intake and other factors. [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 4222.

BACKGROUND WISDOM (Women Informed to Screen Depending on Measures of risk) is a pragmatic trial comparing the safety, efficacy, cost, and patient acceptability of personalized versus annual breast cancer screening in women ages 40-74 years with no personal history of breast cancer. Cancer registry reporting is considered the gold standard for ascertaining cancer diagnosis data; however, the time lag between diagnosis and reporting (1-2 years or more) by the California Cancer Registry (CCR) presents challenges. The WISDOM study must quickly ascertain and report accurate cancer diagnoses to the Data Safety Monitoring Board; delays could lead to preventable harm, unnecessary costs, and unwarranted early trial termination. To ascertain cancer diagnoses in a timely manner, we used multiple data collection methods and compared self-reported with Electronic Health Records (EHR) information, and in some cases verified with chart review. Specifically, we conducted a procedural study with WISDOM participants who sought care at a University of California (UC) health system by extracting their data from the University of California Data Warehouse (UCDW) to 1) verify self-reported breast cancer diagnoses in WISDOM; 2) ensure that all on-study breast cancer diagnoses have been captured within the WISDOM study cohort; and 3) assess the accuracy and reliability of self-reported data. The UCDW provides harmonized EHR data from the six University of California (UC) health systems and constitutes ~120 billion data points from 7.8 million patient records. METHODS We provided the UCDW with a list of WISDOM participants who indicated they had sought care from a UC health system. Participants with recorded breast cancer diagnostic code (ICD-10CM C50.) were matched with the WISDOM self-reported cancer dataset to uncover participants who had no recorded breast cancer diagnosis in the study system. For those individuals with no recorded self-report diagnosis, coordinators conducted manual chart review to determine whether the participants had been diagnosed with breast cancer. RESULTS This cohort included 11,314 enrolled WISDOM participants who self-reported care at a UC and had at least one diagnostic or procedural breast care code. Among these participants, 160 had a ICD-10-CM C50 breast cancer diagnostic code of which 132 breast cancer cases were confirmed: 111 were already self-reported through WISDOM and 21 additional confirmed breast cancer cases were identified through the UCDW. As a standard process, only WISDOM self-reported cancer cases that were confirmed by chart review are entered into the study database. The percentage of confirmed UCDW-identified cases that were not also self-reported was greater for recent diagnoses (16% overall and 6% for period prior to June 2020). Among the 11,314 participants in our cohort, the CCR identified 61 pre-June 2020 cancer diagnoses. Of these 61 diagnoses, 92% (56) were identified by the UCDW procedural process. If the UCDW had been used as the single source for cancer diagnosis, 21 participants without cancer and 7 participants with unclear status (unlocated or inaccessible records) would have been classified as cancer cases. DISCUSSION/CONCLUSION Self-reported data provides quick ascertainment with relative accuracy compared to cancer registry. Cancer ascertainment can be further improved by combining self-reported data with EHR data from a health system data warehouse registry, particularly for self-reported questionnaire issues such as timing and lack of response. Accuracy of self-reported cancer diagnosis from annually distributed questionnaires improves over time. Identifying cancer diagnosis discordance between data sources can inform processes to improve self-reported study accuracy. Citation Format: Katherine Leggat-Barr, Rita H. Ryu, Allison Stover Fiscalini, Tomiyuri Lewis, Rohini S. Bulusu, Samrrah A. Raouf, Hannah Lui Park, Alyssa N. Rocha, Liliana Johansen, Laura Van ’t Veer, Laura J. Esserman, Michael A. Hogarth. The WISDOM Study: Early Ascertainment of Breast Cancer Diagnoses by Utilizing Self-Reported Questionnaires and Data Warehouse Electronic Health Records [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 P3-03-24.

Glyphosate is the most commonly used herbicide in the world and is purported to have a variety of health effects, including endocrine disruption and an elevated risk of several types of cancer. Blood DNA methylation has been shown to be associated with many other environmental exposures, but to our knowledge, no studies to date have examined the association between blood DNA methylation and glyphosate exposure.We conducted an epigenome-wide association study to identify DNA methylation loci associated with urinary glyphosate and its metabolite aminomethylphosphonic acid (AMPA) levels. Secondary goals were to determine the association of epigenetic age acceleration with glyphosate and AMPA and develop blood DNA methylation indices to predict urinary glyphosate and AMPA levels.For 392 postmenopausal women, white blood cell DNA methylation was measured using the Illumina Infinium MethylationEPIC BeadChip array. Glyphosate and AMPA were measured in two urine samples per participant using liquid chromatography-tandem mass spectrometry. Methylation differences at the probe and regional level associated with glyphosate and AMPA levels were assessed using a resampling-based approach. Probes and regions that had an false discovery rateWe identified 24 CpG sites whose methylation level was associated with urinary glyphosate concentration and two associated with AMPA. Four regions, within the promoters of theGlyphosate and AMPA exposure were associated with DNA methylation differences that could promote the development of cancer and other diseases. Further studies are warranted to replicate our results, determine the functional impact of glyphosate- and AMPA-associated differential DNA methylation, and further explore whether DNA methylation could serve as a biomarker of glyphosate exposure. https://doi.org/10.1289/EHP10174.

Background: Altered DNA methylation may be an intermediate phenotype between breast cancer risk factors and disease. Mammographic density is a strong risk factor for breast cancer. However, no studies to date have identified an epigenetic signature of mammographic density. We performed an epigenome-wide association study of mammographic density.Methods: White blood cell DNA methylation was measured for 385 postmenopausal women using the Illumina Infinium MethylationEPIC BeadChip array. Differential methylation was assessed using genome-wide, probe-level, and regional analyses. We implemented a resampling-based approach to improve the stability of our findings.Results: On average, women with elevated mammographic density exhibited DNA hypermethylation within CpG islands and gene promoters compared to women with lower mammographic density. We identified 250 CpG sites for which DNA methylation was significantly associated with mammographic density. The top sites were located within genes associated with cancer, including HDLBP, TGFB2, CCT4, and PAX8, and were more likely to be located in regulatory regions of the genome. We also identified differential DNA methylation in 37 regions, including within the promoters of PAX8 and PF4, a gene involved in the regulation of angiogenesis. Overall, our results paint a picture of epigenetic dysregulation associated with mammographic density.Conclusion: Mammographic density is associated with differential DNA methylation throughout the genome, including within genes associated with cancer. Our results suggest the potential involvement of several genes in the biological mechanisms behind differences in breast density between women. Further studies are warranted to explore these potential mechanisms and potential links to breast cancer risk.

Mammographic density is an important risk factor for breast cancer and is known to differ in women by characteristics such as age and race/ethnicity. Women who experience longitudinal decreases in breast density have a reduced risk of breast cancer; however, besides age, which correlates with decreases in breast density, it is unclear what other factors contribute to changes in breast density over time. Understanding factors associated with density changes may enable a better understanding of breast cancer risk and potential strategies for prevention. This study investigated factors associated with changes in mammographic density in a cohort of 3,392 postmenopausal women (mean age=61.3±8.8 years) with no history of breast cancer who had at least two mammograms spaced at least three months apart at a University of California, Irvine Health breast imaging facility between 2011-2017. Self-reported information on demographics, breast and reproductive history, and lifestyle factors, including body mass index (BMI), alcohol intake, smoking, and physical activity, was collected by an electronic intake form, and Breast Imaging Reporting and Database System (BI-RADS) mammographic density scores were obtained from electronic medical records. 7.9% of women exhibited a longitudinal decrease in mammographic density, 6.7% exhibited an increase, and 85.4% exhibited no change. Longitudinal changes in mammographic density were statistically significantly correlated with age, race/ethnicity, and age at menopause by univariate analysis using the Fisher’s exact test. Multivariate conditional logistic regression using baseline mammographic density as strata was performed to identify factors associated with a longitudinal increase or decrease in mammographic density. Variables were selected if they were nominally significant (p < 0.10) in the initial univariate analyses of women who exhibited a longitudinal change in mammographic density (race/ethnicity, age at baseline, menopause age) or were otherwise selected a priori based on previous literature showing an association with mammographic density (alcohol, physical activity, BMI). Asian women were more likely to exhibit a longitudinal increase in mammographic density (OR=1.78, 95% CI=1.02-3.09, p=0.04) and less likely to exhibit a decrease (OR = 0.58, 95% CI = 0.40-0.83, p=0.003) compared to white women. On the other hand, overweight and obese women were less likely to exhibit an increase compared to normal weight women (OR = 0.52, 95% CI = 0.31-0.87, p=0.01; OR = 0.41, 95% CI = 0.23-0.75, p=0.004, respectively). Other lifestyle factors (alcohol intake, smoking, and physical activity) were not associated with longitudinal changes in mammographic density. The potential interactions of Asian race and BMI with longitudinal breast density changes in influencing breast cancer risk should be further studied. Citation Format: Roza L. Kirmizi, Christie J. Lee, Hannah Lui Park, Argyrios Ziogas, Andrea Alvarez, Rachel M. Lucia, Deborah Goodman, Kathryn M. Larsen, Richard Kelly, Hoda Anton-Culver, Stephen A. Feig. Factors associated with longitudinal changes in mammographic density in a multiethnic breast screening cohort of postmenopausal women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5892.

Objective: Alcohol intake is a known risk factor for breast cancer. National organizations recommend that women consume no more than one serving of alcohol per day, if at all; however, many women exceed this recommendation, and some are unwilling to decrease consumption. Our study sought to identify factors associated with women’s unwillingness to decrease their alcohol intake to decrease their breast cancer risk. Methods: 942 women in a screening mammography cohort were asked questions about their demographics, personal and family health history, lifestyle factors, and willingness/unwillingness to decrease alcohol intake to decrease their breast cancer risk. Univariate and multivariate analyzes of their responses were performed. Results: 13.2% of women in our cohort indicated they were unwilling to decrease their alcohol intake to reduce their breast cancer risk. After adjusting for potential confounders, women who were 60 years and older were more than twice as unwilling to decrease their alcohol intake compared to their younger counterparts ( P = .0002). Women who had an annual household income of more than $200,000 were 1.75 times more unwilling to decrease their alcohol intake compared to their less affluent counterparts ( P = .033). Unwillingness was not significantly associated with race/ethnicity, education, having a first-degree family member with cancer, health perception, breast cancer risk perception, or BMI. Conclusions: Levels of unwillingness to decrease alcohol intake differed by age and household income. An opportunity is present to potentially decrease breast cancer risk in the community by educating women, especially older and more affluent women, about alcohol as a risk factor for breast cancer and the importance of limiting one’s alcohol intake.

Background: Organophosphate pesticides (OPs) are one of the most commonly used classes of insecticides in the U.S., and metabolites of OPs have been detected in the urine of >75% of the U.S. population. While studies have shown that OP exposure is associated with risk of neurological diseases and some cancers, the relationship between OP exposure and breast cancer risk is not well understood. Methods: The aim of this rapid review was to systematically evaluate published literature on the relationship between OP exposure and breast cancer risk, including both epidemiologic and laboratory studies. Twenty-seven full-text articles were reviewed by searching on Pubmed, EMBASE, and Cochrane databases. Results: Some human studies showed that malathion, terbufos, and chlorpyrifos were positively associated with human breast cancer risk, and some laboratory studies demonstrated that malathion and chlorpyrifos have estrogenic potential and other cancer-promoting properties. However, the human studies were limited in number, mostly included agricultural settings in several geographical areas in the U.S., and did not address cumulative exposure. Conclusions: Given the mixed results found in both human and laboratory studies, more research is needed to further examine the relationship between OP exposure and breast cancer risk, especially in humans in non-agricultural settings.

Environmental factors have been linked to many diseases and health conditions, but reliable assessment of environmental exposures is challenging. Developing biomarkers of environmental exposures, rather than relying on self-report, will improve our ability to assess the association of such exposures with disease. Epigenetic markers, most notably DNA methylation, have been identified for some environmental exposures, but identification of markers for additional exposures is still needed. The rationale behind the Markers for Environmental Exposures (MEE) Study was to (1) identify biomarkers, especially epigenetic markers, of environmental exposures, such as pesticides, air/food/water contaminants, and industrial chemicals that are commonly encountered in the general population, and (2) support the study of potential relationships between environmental exposures and health and health-related factors. The MEE Study is a cross-sectional study with potential for record linkage and follow-up. The well-characterized cohort of 400 postmenopausal women has generated a repository of biospecimens, including blood, urine, and saliva samples. Paired data include an environmental exposures questionnaire, a breast health questionnaire, dietary recalls, and a food frequency questionnaire. This work describes the rationale, study design, and cohort characteristics of the MEE Study. In addition to our primary research goals, we hope that the data and biorepository generated by this study will serve as a resource for future studies and collaboration.

In epidemiology studies, identification of diabetes type (type 1 vs. type 2) among study participants with diabetes is important; however, conventional diabetes type identification approaches that include age at diabetes diagnosis as an initial criterion introduces biases. Using data from the National Health and Nutrition Examination Survey, we have developed a novel algorithm which does not include age at diagnosis to identify participants with self-reported diagnosed diabetes as having type 1 vs. type 2 diabetes. Methods A total of 5457 National Health and Nutrition Examination Survey participants between cycles 1999-2000 and 2015-2016 reported that a health professional had diagnosed them as having diabetes at a time other than during pregnancy and had complete information on diabetes-related questions. After developing an algorithm based on information regarding the treatment(s) they received, we classified these participants as having type 1 or type 2 diabetes. Results The treatment-based algorithm yielded a 6-94% split for type 1 and type 2 diabetes, which is consistent with reports from the Centers for Disease Control and other resources. Moreover, the demographics and clinical characteristics of the assigned type 1 and type 2 cases were consistent with contemporary epidemiologic findings. Conclusion Applying diabetes treatment information from the National Health and Nutrition Examination Survey, as formulated in our treatment-based algorithm, may better identify type 1 and type 2 diabetes cases and thus prevent the specific biases imposed by conventional approaches which include the age of diabetes diagnosis as an initial criterion for diabetes type classification.

Environmental and lifestyle factors are believed to account for &gt, 80% of breast cancers, however, it is not well understood how and when these factors affect risk and which exposed individuals will actually develop the disease. While alcohol consumption, obesity, and hormone therapy are some known risk factors for breast cancer, other exposures associated with breast cancer risk have not yet been identified or well characterized. In this paper, it is proposed that the identification of blood epigenetic markers for personal, in utero, and ancestral environmental exposures can help researchers better understand known and potential relationships between exposures and breast cancer risk and may enable personalized prevention strategies.

Younger women diagnosed with ductal carcinoma in situ (DCIS) are more likely to receive a more aggressive treatment compared with older women. Our analysis of Surveillance, Epidemiology, and End Results data (n = 3648) showed that young women who received mastectomy with or without contralateral prophylactic mastectomy did not have improved survival compared with breast-conserving surgery with radiation therapy. Thoughtful consideration should be given to treatment selection for DCIS in young women. BACKGROUND: Although it is known that the risk of a second breast cancer event among young women diagnosed with ductal carcinoma in situ (DCIS) is higher than in older women, the effect of current treatment options on long-term outcomes in this subgroup of women remains poorly defined. We aimed to evaluate national treatment trends and determine their effect on second breast cancer risk and overall survival among young women diagnosed with DCIS. MATERIALS AND METHODS: Surveillance, Epidemiology, and End Results data from 1998 to 2011 were used to analyze 3648 DCIS patients younger than age 40 years. RESULTS: Among all treatment options, breast-conserving surgery (BCS) with radiation therapy (BCS + RT) was the most prevalent (36.1%) followed by mastectomy (MTX) without contralateral prophylactic MTX (CPM; 25.8%), BCS alone (22.2%), and MTX with CPM (15.8%). Risk of a second ipsilateral event was > 5-fold and > 2-fold lower within 2 years and 5 years of initial DCIS diagnosis, respectively, in women who received BCS + RT compared with BCS alone; and overall survival was 3-fold higher in women who received BCS + RT. However, MTX with or without CPM did not show an increase in overall survival compared with BCS + RT. In addition, although the percentage of young women who receive MTX with CPM has increased in recent years, MTX with CPM did not show an increased benefit in survival compared with MTX without CPM. CONCLUSION: The results of our study suggest that more aggressive treatments do not offer survival benefits over BCS + RT; thus, clinical treatment options in young women with DCIS should be carefully considered.

The United States Preventive Services Task Force recommends that women who are at increased risk for breast cancer and at low risk for adverse medication effects should be offered risk-reducing medications, such as tamoxifen or raloxifene, by their clinicians. In addition, the National Comprehensive Cancer Network recommends risk counseling for women with a 5-year risk of ≥1.7% as calculated by the NCI-developed Breast Cancer Risk Assessment Tool (BCRAT, based on the Gail model) or other risk model. Thus, breast cancer risk assessment is important for the identification of women at "high risk" who should be offered risk counseling and potentially intervention. The Athena Breast Health Network, which has served >120,000 breast screening patients across California and the midwest, has integrated breast cancer risk assessment into its clinical breast screening programs. The goal of our study was to characterize breast cancer risk for >10,000 mammography patients in the University of California Irvine Athena Breast Health Network, overall and by race/ethnicity, using several different risk models, including the BCRAT, BCSC, and IBIS models. Our cohort was comprised of 47% non-Hispanic White, 13% non-Hispanic Asian, 38% Hispanic, and 2% women of other race/ethnicities. Using data collected from electronic medical records and self-completed questionnaires, we determined that, as expected, non-Hispanic White and Asian women had higher breast cancer risk scores than Hispanic women for all risk models (5-year risks = 1.51-1.68% and 1.22-1.40% vs. 0.95-1.05%, respectively). In addition, when women were categorized as "increased risk" according to a given risk model if their 5-year risk score was ≥1.7%, the percentages of women at "increased risk" were higher in White women (26.5–42.2%) than in Asian (15.8–28.6%) and Hispanic (6.2–10.7%) women. However, the correlations between risk models were low to moderate in our cohort, overall (Pearson's r = 0.47-0.62) and especially for Asian women (Pearson's r = 0.29-0.49). Our results indicate that using only one risk model in a clinical breast cancer risk assessment program to identify "high risk" women would miss a significant proportion of women who would have been considered "high risk" according to another risk model. Conversely, some women who are identified as "high risk" according to one model may not need risk counseling and intervention since they are not considered "high risk" according to two other models. As our cohort expands and incident breast cancers occur, we will be able to determine which risk model or combination of risk models will have the highest discriminatory accuracy for predicting breast cancer risk in women of different race/ethnicities, which will enable our risk assessment programs to have a more targeted approach to risk counseling and intervention. Citation Format: Park HL, Columbus A, Athena Breast Health Network Investigators and Advocate Partners, Kelly R, Alvarez A, Goodman D, Larsen K, Ziogas A, Anton-Culver H. Breast cancer risk assessment in a multiethnic patient population [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-09-07.

The incidences of both T1D and T2D are increasing in the U.S., and CVD is the leading cause of death among both cohorts. Despite differences in the underlying pathophysiology of T1D and T2D, CVD prevention approaches for T1D have been widely extrapolated from T2D studies. A few prior studies have compared CVD incidence and mortality rates between patients with T1D and T2D; however, a comparison of CVD risk factors between T1D and T2D within the same study cohort, as a measure of differences in progression to CVD events, has not been done. We thus compared CVD risk factors between patients with T1D and T2D, among U.S. adults (≥age 20) with self-reported diabetes from NHANES 1999-2016. First, we classified diabetes cases (n=5347) into T1D (n=230) and T2D (n=4677) using a treatment-based approach we developed, regardless of age at diagnosis (n=440 excluded as unassigned). Next, we compared the demographics, classical CVD risk factors and diabetes-related risk factors by diabetes type using bivariate analysis, and stepwise model selection. The final model comprised race, age, education, diabetes duration, triglycerides, CKD, BMI, and HbA1c [Table]. The similarity between the final model and the bivariate association results, suggests that differences in CVD risk factors between T1D and T2D are mainly due to the underlying pathophysiology and cannot merely be explained by demographic and diabetes-related differences between the two types. Disclosure M. Mosslemi: None. H.L. Park: None. C.E. McLaren: None. N.D. Wong: Advisory Panel; Self; Novartis Pharmaceuticals Corporation. Consultant; Self; Akcea Therapeutics, AstraZeneca. Research Support; Self; Amarin Corporation, Amgen Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Novo Nordisk Inc.

Background: Mammographic density is a strong risk factor for breast cancer. While numerous genetic variations have been shown to be associated with mammographic density, an epigenetic signature of mammographic density has not yet been identified. The objective of this study was to identify a DNA methylation signature associated with mammographic density. Methods: Blood samples were collected from 197 postmenopausal women 45-65 years old with no personal history of breast cancer. For each participant, mammographic density according to the BI-RADs density classification was obtained from the most recent mammogram report. White blood cell DNA methylation at more than 850,000 CpG sites was measured using the Illumina HumanMethylationEPIC BeadChip. Differential methylation was assessed using genome-wide, probe-level, and regional approaches after filtering and normalization using the recommended steps for DNA methylation microarray data. All analyses were adjusted for appropriate confounding variables. Results: On average, genome-wide DNA methylation was higher in women with elevated mammographic density. Additionally, DNA methylation at 343 individual sites and in 34 regions was significantly associated with mammographic density after false discovery rate correction. Most density-associated sites were hypermethylated. The top differentially methylated sites were located near cell cycle regulatory genes (ICMT, DESI2, TCF21/TARID, ROBO3), as well as those encoding cytoskeletal or plasma membrane proteins (PALLD, ANO7, ATP8B3, MTUS2). We observed hypermethylation near the promoters of genes including RUFY1 and SLFN12 and within the bodies of genes including LCN6, SNED1, and LRFN1. Functional analysis of the differentially methylated sites revealed enrichment of gene ontology categories including angiogenesis and regulation of cell adhesion, as well as apoptotic signaling in response to DNA damage. Conclusions: White blood cell DNA methylation differed according to mammographic density in a sample of postmenopausal women. Probe-based and regional analysis identified differentially methylated regions located near genes implicated in cancer. These differentially methylated regions may serve as a signature of mammographic density, and future studies of the genes implicated may provide a deeper understanding of the connection between mammographic density and breast cancer risk. Citation Format: Rachel McFarland Lucia, Wei-Lin Huang, Andrea Alvarez, Irene Masunaka, Argyrios Ziogas, Deborah Goodman, Andrew O. Odegaard, Trina M. Norden-Krichmar, Hannah Lui Park. Association of mammographic density with blood DNA methylation [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 3661.

Neuroinflammation has been proposed as a target for neuroprotection. Therefore significant opportunities for preventing neurologic decline exist. ClinicoPathological Correlation (CPC) ImmunoTechnologies are critically needed for clinical utility for acute and chronic immunological and inflammatory disorders (including autoimmune neurological disorders such as Parkinson’s disease and Alzheimer’s disease) to determine if an individual has variants associated with a higher risk of disease (Gain of Function [GoF] variants); and elevated levels of analytes (circulating biomarkers). We hypothesize the neurovascular compartment plays a critical role in neurodegeneration. Using a multipronged approach, we identified specific immune cell subsets and inflammatory factors in the blood, which can potentially play a critical role in the early stages of neurodegeneration by disrupting homeostatic regulation, destabilizing the neurovascular compartment; and elevated levels of analytes (proteomic multiplexed quantification) in the cerebrospinal fluid (CSF) and plasma, which can potentially serve as robust, indicative biomarkers consistent with model of neuroinflammation contributing to neurovascular dysfunction, potentially leading to neurodegeneration. More specifically, we identified analytes (lymphotoxin/TNF-beta/LTA) in the plasma (low levels in CSF); TNF-alpha levels were low in plasma and CSF. Next-generation sequencing enabled the identification of risk variants, pathogen/virus profiles and LTA GoF variant strongly associated with increased protein production. We included distinct LTA inflammatory/autoimmune disease controls (rheumatoid arthritis, multiple sclerosis, uveitis or vasculitis).

Due to an increasing proportion of older individuals and the adoption of a westernized lifestyle, the incidence rate of breast cancer is expected to rapidly increase within the next 10 years in Korea. The National Cancer Screening Program (NCSP) of Korea recommends biennial breast cancer screening through mammography for women aged 40–69 years old and according to individual risk and preference for women above 70 years old. There is an ongoing debate on how to most effectively screen for breast cancer, with many proponents of personalized screening, or screening according to individual risk, for women under 70 years old as well. However, to accurately stratify women into risk categories, further study using more refined personalized characteristics, including potentially incorporating a polygenic risk score (PRS), may be needed. While most breast cancer risk prediction models were developed in Western countries, the Korean Breast Cancer Risk Assessment Tool (KoBCRAT) was developed in 2013, and several other risk models have been developed for Asian women specifically. This paper reviews these models compared to commonly used models developed using primarily Caucasian women, namely, the modified Gail, Breast Cancer Surveillance Consortium, Rosner and Colditz, and Tyrer-Cuzick models. In addition, this paper reviews studies in which PRS is included in risk prediction in Asian women. Finally, this paper discusses and explores strategies toward development and implementation of personalized screening for breast cancer in Korea.

PURPOSE Metabolic, hormonal, and hemostatic changes associated with pregnancy loss (stillbirth and miscarriage) may contribute to the development of cardiovascular disease (CVD) in adulthood. This study evaluated prospectively the association between a history of pregnancy loss and CVD in a cohort of postmenopausal women. METHODS Postmenopausal women (77,701) were evaluated from 1993–1998. Information on baseline reproductive history, sociodemographic, and CVD risk factors were collected. The associations between 1 or 2 or more miscarriages and 1 or more stillbirths with occurrence of CVD were evaluated using multiple logistic regression. RESULTS Among 77,701 women in the study sample, 23,538 (30.3%) reported a history of miscarriage; 1,670 (2.2%) reported a history of stillbirth; and 1,673 (2.2%) reported a history of both miscarriage and stillbirth. Multivariable-adjusted odds ratio (OR) for coronary heart disease (CHD) for 1 or more stillbirths was 1.27 (95% CI, 1.07–1.51) compared with no stillbirth; for women with a history of 1 miscarriage, the OR = 1.19 (95% CI, 1.08–1.32); and for 2 or more miscarriages the OR = 1.18 (95% CI, 1.04–1.34) compared with no miscarriage. For ischemic stroke, the multivariable odds ratio for stillbirths and miscarriages was not significant. CONCLUSIONS Pregnancy loss was associated with CHD but not ischemic stroke. Women with a history of 1 or more stillbirths or 1 or more miscarriages appear to be at increased risk of future CVD and should be considered candidates for closer surveillance and/or early intervention; research is needed into better understanding the pathophysiologic mechanisms behind the increased risk of CVD associated with pregnancy loss.

Introduction: Statins (HMG CoA reductase inhibitors) are a class of cholesterol lowering drugs that affect many intracellular pathways and have implications for chemopreventive activity against cancer. Epidemiological data on statins and breast cancer risk are conflicting. We analyzed updated data from the Women's Health Initiative (WHI) to assess the relationship between statins and breast cancer risk. Methods: This analysis included 154,587 post-menopausal women ages 50–79 years at baseline, in which 7,430 incident cases of invasive breast cancer were identified over an average of 10.8 (SD 3.3) years of follow-up. All cases of breast cancer were confirmed by review of medical records and pathology reports. Participants were asked to bring all current medications to their baseline visits and information on statin use was recorded. Statins were classified as lipophilic (lovastatin, simvastatin, fluvastatin) or hydrophilic (pravastatin and atorvastatin). Self and interviewer-administered questionnaires were used to collect information on other breast cancer risk factors. Cox proportional hazards regression was used to calculate hazard ratios (HRs) with 95% confidence intervals (CIs). Analyses investigated the association of any statin use as well as, type of statin (lipophilic vs. hydrophilic), statin potency, and duration of use with breast cancer. To evaluate the effect of change in statin use over time, statin use was examined as a time-dependent exposure using updated information on statin use gathered during follow-up visits. Separate analyses were conducted by hormone receptor and HER2neu status, other tumor characteristics and use of postmenopausal hormone therapy. All statistical tests were two-sided. Results: Statins were used at baseline by 11,584 (7.5%) women in the cohort of whom 7,840 used lipophilic statins. The annualized rate of breast cancer was 0.42% among statin users and 0.42% among nonusers. The multivariable adjusted HR of breast cancer for statin users compared with non-users was 0.93 (95% C.I. 0.83–1.05), however for women using lipophilic statins the HR was 0.86 (95% CI, 0.74–1.00). Statin use for < 1 year was associated with a reduction in risk (HR 0.79, 95% C.I. 0.63–0.99) however there was no trend for overall duration of use. In the stratified analysis by tumor size, there was a marginal reduction in risk for tumors between 10 and 30 mm but not in smaller or larger tumors. There were no effect modifications by tumor stage, hormone receptor or HER2neu status, hormone therapy use, family history of breast cancer or body mass index. In the multivariable adjusted time-dependent model, the HR for simvastatin was 0.80 (95% CI, 0.64–0.99). Conclusion: Simvastatin was associated with a reduced risk of invasive breast cancer, and as a class, lipophilic statins were associated with a marginal benefit. This provides further evidence for possible class differences in statins with regard to chemo-preventive effects in breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD03-09.

Background Evidence has accumulated showing that recreational physical activity reduces breast cancer risk. However, it is unclear whether risk reduction pertains to specific receptor-defined subtypes. Moreover, few studies have examined whether changes in the amount of recreational physical activity during adulthood influence breast cancer risk. Methods A total of 108,907 women, ages 22 to 79 years with no history of breast cancer when joining the California Teachers Study in 1995–1996, completed a baseline questionnaire and were eligible for the study. Through 2012, 5882 women were diagnosed with invasive breast cancer. Breast cancer subtypes were defined by the expression status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Multivariable Cox proportional hazards models provided adjusted hazard ratios (HRs) and 95 % confidence intervals (CIs) for breast cancer overall and ER/PR/HER2-defined subtypes associated with long-term (from high school through age 54 or age at cohort entry, whichever was younger) and baseline (during 3 years prior to baseline) recreational physical activity. Among women who also completed a follow-up questionnaire at 10 years after baseline in 2005–2008 (54,686 women, 1406 with invasive breast cancer), risk associated with changes in the amount of recreational physical activity from baseline to the 10-year follow-up (during 3 years prior to the 10-year follow-up) was determined. Results Both long-term and baseline strenuous recreational physical activity were inversely associated with risk of invasive breast cancer (Ptrend ≤0.03). The observed associations were mainly confined to women with triple negative breast cancer (TNBC, ER–/PR–/HER2–, Ptrend ≤0.02) or luminal A-like subtype (ER+ or PR+ plus HER2–) who were former users of menopausal hormone therapy at baseline (Ptrend = 0.02, Phomogeneity of trends ≤0.03). Moreover, women who consistently engaged in the highest level (≥3.51 h/wk/y) of strenuous recreational physical activity between baseline and 10-year follow-up had the lowest risk of breast cancer (HR = 0.71, 95 % CI = 0.52–0.98) when compared to those who were consistently low (≤0.50 h/wk/y). Conclusions Strenuous recreational physical activity is associated with lower breast cancer risk, especially TNBC. The benefit may be maximized by consistently engaging in high-intensity recreational physical activity during adulthood. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0723-3) contains supplementary material, which is available to authorized users.

PGP9.5 is a controversial molecule from an oncologic point of view. We recently identified frequent methylation of PGP9.5 gene exclusively in primary head and neck squamous cell carcinoma (HNSCC), suggesting that it could be a tumor suppressor gene. On the other hand, PGP9.5 was reported to be overexpressed in a subset of human cancers presumably due to intrinsic oncogenic properties or as a result of transformation. To demonstrate that PGP9.5 possesses tumor suppressive activity, we examined forced expression by stable transfection of PGP9.5 in 4 HNSCC cell lines. Although all 4 cell lines demonstrated reduced log growth rates in culture after transfection, only 2 cell lines with wild type p53 (011, 022) demonstrated decreased growth in soft agar. In 2 cell lines with mutant p53 (013, 019), we observed no altered growth in soft agar and increased sensitivity to UV irradiation. We then tested for and found a high frequency of promoter methylation in a larger panel of primary tumors including HNSCC, esophageal SCC, gastric, lung, prostate and hepatocellular carcinoma. Our data support the notion that PGP9.5 is a tumor suppressor gene that is inactivated by promoter methylation or gene deletion in several types of human cancers.

DNA methylation has a role in mediating epigenetic silencing of CpG island genes in cancer and other diseases. Identification of all gene promoters methylated in cancer cells “the cancer methylome” would greatly advance our understanding of gene regulatory networks in tumorigenesis. We previously described a new method of identifying methylated tumor suppressor genes based on pharmacologic unmasking of the promoter region and detection of re-expression on microarray analysis. In this study, we modified and greatly improved the selection of candidates based on new promoter structure algorithm and microarray data generated from 20 cancer cell lines of 5 major cancer types. We identified a set of 200 candidate genes that cluster throughout the genome of which 25 were previously reported as harboring cancer-specific promoter methylation. The remaining 175 genes were tested for promoter methylation by bisulfite sequencing or methylation-specific PCR (MSP). Eighty-two of 175 (47%) genes were found to be methylated in cell lines, and 53 of these 82 genes (65%) were methylated in primary tumor tissues. From these 53 genes, cancer-specific methylation was identified in 28 genes (28 of 53; 53%). Furthermore, we tested 8 of the 28 newly identified cancer-specific methylated genes with quantitative MSP in a panel of 300 primary tumors representing 13 types of cancer. We found cancer-specific methylation of at least one gene with high frequency in all cancer types. Identification of a large number of genes with cancer-specific methylation provides new targets for diagnostic and therapeutic intervention, and opens fertile avenues for basic research in tumor biology. [Cancer Res 2008;68(8):2661–70]