Jacob K. Kresovich

Exploring DNA methylation changes in promoter, intragenic, and intergenic regions as early and late events in breast cancer formation

BackgroundBreast cancer formation is associated with frequent changes in DNA methylation but the extent of very early alterations in DNA methylation and the biological significance of cancer-associated epigenetic changes need further elucidation.MethodsPyrosequencing was done on bisulfite-treated DNA from formalin-fixed, paraffin-embedded sections containing invasive tumor and paired samples of histologically normal tissue adjacent to the cancers as well as control reduction mammoplasty samples from unaffected women. The DNA regions studied were promoters (BRCA1, CD44, ESR1, GSTM2, GSTP1, MAGEA1, MSI1, NFE2L3, RASSF1A, RUNX3, SIX3 and TFF1), far-upstream regions (EN1, PAX3, PITX2, and SGK1), introns (APC, EGFR, LHX2, RFX1 and SOX9) and the LINE-1 and satellite 2 DNA repeats. These choices were based upon previous literature or publicly available DNA methylome profiles. The percent methylation was averaged across neighboring CpG sites.ResultsMost of the assayed gene regions displayed hypermethylation in cancer vs. adjacent tissue but the TFF1 and MAGEA1 regions were significantly hypomethylated (p ≤0.001). Importantly, six of the 16 regions examined in a large collection of patients (105 – 129) and in 15-18 reduction mammoplasty samples were already aberrantly methylated in adjacent, histologically normal tissue vs. non-cancerous mammoplasty samples (p ≤0.01). In addition, examination of transcriptome and DNA methylation databases indicated that methylation at three non-promoter regions (far-upstream EN1 and PITX2 and intronic LHX2) was associated with higher gene expression, unlike the inverse associations between cancer DNA hypermethylation and cancer-altered gene expression usually reported. These three non-promoter regions also exhibited normal tissue-specific hypermethylation positively associated with differentiation-related gene expression (in muscle progenitor cells vs. many other types of normal cells). The importance of considering the exact DNA region analyzed and the gene structure was further illustrated by bioinformatic analysis of an alternative promoter/intron gene region for APC.ConclusionsWe confirmed the frequent DNA methylation changes in invasive breast cancer at a variety of genome locations and found evidence for an extensive field effect in breast cancer. In addition, we illustrate the power of combining publicly available whole-genome databases with a candidate gene approach to study cancer epigenetics.

Associations of lead and cadmium with sex hormones in adult males

Heavy metal exposures are ubiquitous in the environment and their relation to sex hormones is not well understood. This paper investigates the associations between selected heavy metals (lead and cadmium) and sex hormones (testosterone, free testosterone, estradiol, free estradiol) as well as other major molecules in the steroid biosynthesis pathway (androstanedione glucuronide and sex-hormone binding globulin (SHBG)). Blood lead and cadmium were selected as biomarkers of exposure, and tested for associations in males using National Health and Nutritional Examination Survey (NHANES) data from 1999-2004. After adjustment for age, race, body mass index, smoking status, diabetes and alcohol intake, blood lead was positively associated with testosterone and SHBG while blood cadmium was positively associated with SHBG. After controlling for additional heavy metal exposure, the associations between lead and testosterone as well as cadmium and SHBG remained significant. Furthermore, the association between blood lead and testosterone was modified by smoking status (P for interaction=0.011), diabetes (P for interaction=0.021) and blood cadmium (P for interaction=0.029). The association between blood cadmium and SHBG levels was modified by blood lead (P for interaction=0.004). This study is the most comprehensive investigation to date regarding the association between heavy metals and sex hormones in males.

Retinal Oximetry and Vessel Diameter Measurements With a Commercially Available Scanning Laser Ophthalmoscope in Diabetic Retinopathy

Purpose To test the hypothesis that retinal vascular diameter and hemoglobin oxygen saturation alterations, according to stages of diabetic retinopathy (DR), are discernible with a commercially available scanning laser ophthalmoscope (SLO). Methods One hundred eighty-one subjects with no diabetes (No DM), diabetes with no DR (No DR), nonproliferative DR (NPDR), or proliferative DR (PDR, all had photocoagulation) underwent imaging with an SLO with dual lasers (532 nm and 633 nm). Customized image analysis software determined the diameters of retinal arteries and veins (DA and DV) and central retinal artery and vein equivalents (CRAE and CRVE). Oxygen saturations of hemoglobin in arteries and veins (SO2A and SO2V) were estimated from optical densities of vessels on images at the two wavelengths. Statistical models were generated by adjusting for effects of sex, race, age, eye, and fundus pigmentation. Results DA, CRAE, and CRVE were reduced in PDR compared to No DM (P ≤ 0.03). DV and CRVE were similar between No DM and No DR, but they were higher in NPDR than No DR (P ≤ 0.01). Effect of stage of disease on SO2A differed by race, being increased relative to No DM in NPDR and PDR in Hispanic participants only (P ≤ 0.02). Relative to No DM, SO2V was increased in NPDR and PDR (P ≤ 0.05). Conclusions Alterations in retinal vascular diameters and SO2 by diabetic retinopathy stage can be detected with a widely available SLO, and covariates such as race can influence the results.

Assessment of Conjunctival Microvascular Hemodynamics in Stages of Diabetic Microvasculopathy

Diabetes impairs the microcirculation and function of various vital tissues throughout the body. The conjunctival microcirculation can be non-invasively imaged and thus enables assessment of microvascular hemodynamics. In this study, alterations in conjunctival microvascular hemodynamics were quantitatively assessed at stages of increasing diabetic microvasculopathy based on diabetic retinopathy (DR). Subjects were categorized into non-diabetic control (C, N = 34), no clinically visible DR (NDR, N = 47), non-proliferative DR (NPDR, N = 45), and proliferative DR (PDR, N = 35). Conjunctival hemodynamic descriptors, namely vessel diameter (D), blood velocity (V), blood flow (Q), wall shear rate (WSR), and wall shear stress (WSS) were measured in arterioles and venules, and compared between DR and C subjects using generalized linear mixed models. In arterioles, V, WSR, and WSS were lower in NDR (P ≤ 0.01). V was lower in NDR than NPDR and PDR subjects (P ≤ 0.02). In venules, D was higher in NDR and NPDR (P ≤ 0.03), while V was lower in PDR (P = 0.04). Venular V and Q were higher in NPDR than PDR subjects (P ≤ 0.04). WSR and WSS were lower in all stages of DR (P ≤ 0.05), suggestive of the potential of WSS as a marker of diabetic microvasculopathy. Quantitative assessment of conjunctival hemodynamics can potentially be useful for evaluation of diabetic microvasculopathy.

Abstract 5569: Ki67 is an independent prognostic marker in breast cancer even after accounting for molecular subtype

Ki67 labeling index (LI) has been demonstrated to be a prognostic marker in invasive breast cancer; a high Ki67 LI is associated with poorer survival among breast cancer patients. The goal of these analyses was to examine the extent to which Ki67 was associated with specific breast cancer subtypes, and whether it could serve as an additional prognostic marker above and beyond breast cancer subtype, in an ethnically diverse sample of 287 patients (86 Non-Hispanic White, 84 Hispanic and 116 African American). IHC analysis was performed on tissue microarrays constructed from invasive breast cancer samples obtained from the Breast Cancer Care in Chicago study (patients diagnosed between 2005-2008). Tissue samples were tested for the expression of estrogen receptor (ER), progesterone receptor (PR), HER2, EGFR and CK5/6. From these results, breast cancers were classified as Luminal A (ER+/PR+/HER2-), Luminal B (ER+/PR+/HER2+), HER2 enriched (ER-/PR-/HER2+), and triple negative (TN) (ER-/PR-/HER2-). TN tumors were subclassified into basal like (BL) if they expressed either EGFR or CK5/6 or otherwise classified as unspecified (US) if negative for both EGFR and CK5/6. Ki67 LI was classified as low ( In adjusted models, the proportion of tumors with a high Ki67 LI was much lower for luminal A (28%) than for luminal B, HER2, TN-US, and TN-BL subtypes (91,66, 77 and 89%, respectively, p In this multi-ethnic sample of breast cancer patients, we found that Ki67 was a significant independent predictor of more aggressive, higher grade disease, even after accounting for molecular subtype. Ki67 might serve as a useful additional marker for the classification of breast cancer into molecular subtypes. Citation Format: Abeer M. Mahmoud, Virgilia Macias, Umaima Al-alem, Jacob K. Kresovich, Galina Khramtsova, Andre Kajdacsy-Balla, Elizabeth L. Wiley, Garth H. Rauscher. Ki67 is an independent prognostic marker in breast cancer even after accounting for molecular subtype. [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 5569. doi:10.1158/1538-7445.AM2014-5569

Abstract 2305: Residential airborne heavy metal concentrations and breast cancer characteristics

Background: Ambient air metal concentrations have recently been implicated in the etiology of breast cancer. Previous studies have shown airborne concentrations of arsenic and cadmium are associated with the development of estrogen receptor-negative tumors. This study aims to replicate these findings and examine the role of other toxic and essential heavy metals. Methods: Participants were women who were diagnosed with breast cancer in Chicago between 2005 and 2008. We examined estrogen and progesterone receptor-negative (ER-/PR-) and high-grade tumors as markers of aggressive breast cancer, and estimated 15-year markers of exposure of 11 heavy metals. Exposures were calculated using census tract-level airborne concentrations from the National-scale Air Toxics Assessment and participants’ residential histories. We adjusted all models for socioeconomic status and reproductive factors. Results: We found that prevalent ER-/PR- tumors were associated with increased residential exposure to airborne concentrations of antimony (Q4 vs Q1: OR= 1.81; 95% CI: 0.95, 3.44; P trend = 0.04), cobalt (Q4 vs Q1: OR= 2.37; 95% CI: 1.26, 4.45; P trend trend = 0.04), and selenium (Q4 vs Q1: OR= 1.85; 95% CI: 1.03, 3.29; P trend = 0.05), and also identified marginally significant trends for arsenic (P trend = 0.06), chromium (P trend = 0.08), lead (P trend = 0.08), and mercury (P trend = 0.07). We did not identify any overall associations with high-grade tumors, however when stratifying by menopausal status we found that antimony (Q4 vs Q1: OR= 6.97, 95% CI: 1.61-30.19) and arsenic (Q4 vs Q1: OR= 6.97, 95% CI: 1.61-30.19) were associated with prevalent high-grade tumors in premenopausal women only. Discussion: This study found further support for a role of airborne arsenic concentrations, and novel evidence implicating other airborne estrogen-pathway disrupting metal concentrations, in the development of aggressive breast cancer subtypes. Additionally, this is the first study to implicate heavy metal exposure in the etiology of high-grade tumors. These results suggest that long-term, low-dose exposures to certain heavy metals play a role in the etiology of aggressive breast cancer characteristics. Airborne exposures have the ability to affect large populations and findings from this and similar studies have large public health implications. Citation Format: Jacob K. Kresovich, Serap Erdal, Maria Argos, Hua Yun Chen, Peter H. Gann, Garth H. Rauscher. Residential airborne heavy metal concentrations and breast cancer characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2305. doi:10.1158/1538-7445.AM2017-2305

Abstract B49: The role of KCNK9 and TP53 on the racial disparity in biologically aggressive breast cancer subtype in The Cancer Genome Atlas (TCGA)

Introduction: Compared to non-Hispanic (nH) white, nH black women are more likely to present with biologically aggressive estrogen and progesterone receptor (ER/PR)-negative (ERN) and triple negative (TN) breast cancer subtypes which are associated with worse prognosis (black:white disparity). These subtypes also exhibit greater genetic instability, with TP53 being the single most recurrently mutated gene. KCNK9 is a maternally imprinted and functionally mono-allelic 2-pore domain potassium channel proto-oncogene located at chromosomal region 8q24.3, encoding TASK-3. KCNK9 overexpression may arise via loss of methylation (LOM) or copy number amplification (CNA). We examined KCNK9 and TP53 as potential mediators of the black:white racial disparity in aggressive breast cancer subtypes. Methods: Using publically available TCGA data on 1040 women with invasive breast cancer, we examined: associations of KCNK9 LOM (HM450 beta values; highest third loss vs. others), amplification (putative CNA from GISTIC; any vs. neutral), and mRNA overexpression (RNA-Seq from RSEM; z-score > +1 SD vs. others) with ERN and TN subtype, and overall survival (OS) and disease-free survival (DFS). General TP53 pathway function was examined using mRNA expression z-scores for six related genes ( TP53 MDM2 MDM4 CDKN2A CDKN2B TP53BP1 ). Prevalence risk ratios (RRs) with 95% confidence intervals (CIs) were estimated via logistic regression with model-based standardization (predictive margins) and hazard ratios (HRs) using Cox proportional hazards models, and all models were adjusted at baseline for age as a continuous variable and stage as categorical variable. In mediation analysis, we estimated the proportionate reduction in the racial disparity in aggressive subtypes after accounting for differences in KCNK9 and TP53 pathway biomarkers by comparison of rescaled coefficients using the method of Karlson, Holm and Breen in adjusted models. Death from any cause was considered an event for all-cause mortality (i.e. OS), and recurrence or progression of disease was considered an event for DFS. Results: KCNK9 LOM, amplification and overexpression were each significantly associated with ERN and TN subtype and black race (all p TP53 pathway markers and race, KCNK9 status significantly predicted outcome for both subtypes suggesting the importance of KCNK9 as a molecular driver of aggressive subtype (ERN: LOM RR, 3.69; 95% CI, 2.28-5.98; p TP53 pathway markers, no KCNK9 markers remained independent predictors of mortality or recurrence; however, TN subtype and CDKN2A overexpression remained significant predictors for both (TN: OS HR, 8.96; 95% CI, 2.65-30.28; p CDKN2A : OS HR, 0.29; 95% CI, 0.09-0.97; p=0.045. DFS HR, 0.21; 95% CI, 0.06-0.76; p=0.017). ERN and TN subtypes were more common among black patients (black:white disparity: ERN RR, 2.16; 95% CI, 1.6-2.8; p Conclusions: KCNK9 appears to be a molecular driver of aggressive breast tumor subtype, and in combination with aberrant TP53 pathway overexpression, largely accounts for observed black:white disparity in more biologically aggressive breast cancer subtypes. Citation Format: Keith A. Dookeran, Jacob K. Kresovich, Maria Argos, Garth H. Rauscher. The role of KCNK9 and TP53 on the racial disparity in biologically aggressive breast cancer subtype in The Cancer Genome Atlas (TCGA). [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B49.

Abstract B51: Exploring the role of reproductive factors and DNA methylation in ethnic disparities in breast cancer tumor aggressiveness

Purpose: Non-Hispanic (nH) Black and Hispanic (or minority) breast cancer patients tend to be diagnosed with more aggressive forms of breast cancer compared to their nH White counterparts. Prior research as well as analyses from the current study has identified hormonal and reproductive factors associated with breast cancer aggressiveness subtypes. We explored the potential role of hormonal and reproductive factors, and the potential contribution of DNA methylation, in explaining the racial/ethnic disparity in tumor aggressiveness in a population based study of breast cancer disparities. Methods: The breast Cancer Care in Chicago (BCCC) study included 989 recently diagnosed nH White, nH Black and Hispanic patients with first primary breast cancer. Analyses include a subset of 286 patients with available tumor immunohistochemistry (IHC) data on estrogen and progesterone receptor (ER/PR), HER2, p53 and Ki67 status. A tumor aggressiveness score (TAS) with a high internal reliability coefficient (Chronbach9s alpha=0.76) was created from tumor grade and IHC data on ER, PR, HER2, p53 and Ki67. Values were standardized to have a mean of 0 and standard deviation of 1, then summed together and re-standardized to create the score. Pyrosequencing assays for DNA methylation were conducted on a set of DNA sequences identified based on prior literature, ENCODE data for DNA methylation, and transcription for normal vs. cancer cell lines from the UCSC genome browser. Of the 286 patients with tumor aggressiveness data, 214 had available methylation data for BRCA1, GSTM2, EGFR, RASSF1, Sat2 and TFF1 genes. Multivariable linear regression models were estimated with standardized aggressiveness score as the dependent variable and using nested models to conduct likelihood ratio tests for both forward (type 1) and backwards (type 3) analyses. A method of rescaled-coefficients was then used to estimate an average controlled direct effect representing the ethnic disparity in breast cancer tumor aggressiveness and the extent to which the disparity might be explained by patient reproductive factors and tumor DNA methylation. Because stage at diagnosis is downstream of, and strongly influenced by, tumor aggressiveness, it was excluded from our analyses. Results: Factors significantly associated with having a higher TAS (p Conclusions: Our findings suggest that DNA methylation of specific genes may influence breast cancer tumor aggressiveness and may help to explain the preponderance of aggressive subtypes diagnosed in ethnic minority women. DNA methylation may represent a promising avenue for biomarker development for early detection for biologically aggressive tumor types in vulnerable populations. These findings require replication and validation in other studies. Citation Format: Keith A. Dookeran, Abeer M. Mahmoud, Matthew Poulin, Liying Yan, Melanie Ehrlich, Jacob K. Kresovich, Virgilia Macias, Andre Kajdacsy-Balla, Elizabeth Wiley, Garth H. Rauscher. Exploring the role of reproductive factors and DNA methylation in ethnic disparities in breast cancer tumor aggressiveness. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr B51.

Abstract LB-274: Mode of breast cancer detection: Do markers of tumor aggressiveness predict which tumors are “missed” at screening

Screening mammography reduces breast cancer stage at diagnosis by detecting it early before symptoms develop. Nonetheless, a breast cancer can arise as a lump despite a recent prior negative screen (“missed detection”). In the Breast Cancer Care in Chicago study, Non-Hispanic (nH) Black and Hispanic women were more likely than their nH White counterparts to report a missed detection despite a recent prior screening mammogram. We examined whether breast cancer subtype and immunohistochemical (IHC) markers of tumor aggressiveness predicted missed detection and whether they could explain racial/ethnic disparities in missed detection, in a subsample of 198 patients with a recent prior screening mammogram (63 Non-Hispanic White, 78 Hispanic and 57 African American). Missed detection was defined as symptomatic awareness despite a negative screen in the prior 24 months. IHC analyses were conducted for estrogen receptor (ER), progesterone receptor (PR), HER2, EGFR and CK5/6, Ki67 and p53 expression. Breast cancers were classified as Luminal A (ER+/PR+/HER2-), Luminal B (ER+/PR+/HER2+), HER2 enriched (ER-/PR-/HER2+), and triple negative (TN) (ER-/PR-/HER2-). Missed detection was highest for more aggressive TN and HER2 enriched subtypes (61% and 60%) and lowest for luminal A and B tumors (37% and 38%). Greater Ki67, EGFR and p53 expression were each associated with missed detection (p In this multi-ethnic sample of breast cancer patients, markers of tumor aggressiveness were strongly associated with missed detection at screening but they did not explain racial/ethnic disparities in missed detection. Citation Format: Abeer Mostafa Mahmoud, Virgilia Macias, Jacob K. Kresovich, Andre Kajdacsy-Balla, Elizabeth L. Wiley, Garth H. Rauscher. Mode of breast cancer detection: Do markers of tumor aggressiveness predict which tumors are “missed” at screening. [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 LB-274. doi:10.1158/1538-7445.AM2014-LB-274