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Breast cancer is an important contributor to morbidity and mortality in society, but factors that affect the cause of the disease are poorly defined. Genomic instability drives tumorigenic processes in invasive carcinomas and premalignant breast lesions, and might promote the accumulation of genetic alterations in apparently normal tissues before histological abnormalities are detectable. Evidence suggests that genomic changes in breast parenchyma affect the behaviour of epithelial cells, and ultimately might affect tumour growth and progression. Inherent instability in genes that maintain genomic integrity, as well as exogenous chemicals and environmental pollutants, have been implicated in breast-cancer development. Although molecular mechanisms of tumorigenesis are unclear at present, carcinogenic agents could contribute to fields of genomic instability localised to specific areas of the breast. Understanding the functional importance of genomic instability in early carcinogenesis has important implications for improvement of diagnostic and treatment strategies. [ABSTRACT FROM AUTHOR]

Purpose: Insulin resistance and visceral adiposity are associated with increased risk of type 2 diabetes. In this report, we describe the methods of the IRAS Family Study, which was designed to identify the genetic and environmental risk factors for insulin resistance and visceral adiposity.Methods: Families from two ethnic groups (African American and Hispanic) have been recruited from three clinical sites. Blood samples for DNA as well as other standard measures were collected. A CT scan (visceral adiposity) and a frequently sampled glucose tolerance test (insulin resistance) were performed. Preliminary estimates of heritability for indirect measures related to insulin resistance and visceral adiposity were obtained using a variance components approach in the first 93 families (approximately 1000 individuals).Results: Estimates of heritability ranged from low (0.08) for fasting insulin and HOMA, to moderate (0.28) for fasting glucose, to high (0.54) for BMI. After adjustment for age, gender and ethnicity, all heritability estimates were significantly greater than zero (p < 0.05).Conclusions: These results are consistent with the expectation that intermediate measures of insulin resistance and visceral adiposity are heritable, and that the IRAS Family Study has statistical power to detect these intermediate phenotypes of type 2 diabetes and atherosclerosis. [ABSTRACT FROM AUTHOR]

Triple negative breast cancer (TNBC), representing 10-15% of breast tumors diagnosed each year, is a clinically defined subtype of breast cancer associated with poor prognosis. The higher incidence of TNBC in certain populations such as young women and/or women of African ancestry and a unique pathological phenotype shared between TNBC and BRCA1-deficient tumors suggest that TNBC may be inherited through germline mutations. In this article, we describe genes and genetic elements, beyond BRCA1 and BRCA2 , which have been associated with increased risk of TNBC. Multigene panel testing has identified high- and moderate-penetrance cancer predisposition genes associated with increased risk for TNBC. Development of large-scale genome-wide SNP assays coupled with genome-wide association studies (GWAS) has led to the discovery of low-penetrance TNBC-associated loci. Next-generation sequencing has identified variants in noncoding RNAs, viral integration sites, and genes in underexplored regions of the human genome that may contribute to the genetic underpinnings of TNBC. Advances in our understanding of the genetics of TNBC are driving improvements in risk assessment and patient management.

Agricultural pesticides are abundant environmental contaminants worldwide, prompting interest in studying their possible detrimental health effects. We examined organochlorine residues by quadrant (n = 245) in breast adipose tissues from 51 women with various stages of breast health to determine patterns of bioaccumulation within the breast and to assess relationships with patient clinical characteristics. Three organochlorine residues-2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE), hexachlorobenzene (HCB), and mirex-assayed by high resolution gas chromatography were abundant in breast tissue. p,p'-DDE (745 ± 1054 ng/g lipid) was the most prevalent residue, comprising 97.5% of the total chemical burden. Mean levels of p,p'-DDE and HCB were significantly correlated (P < .001) with patient age at mastectomy, and levels of p,p'-DDE were correlated (P < .05) with BMI. Pesticide concentrations did not differ significantly by breast quadrant and were not different in the quadrant(s) where the primary tumor was located compared to other cancer-free quadrants. In invasive cancer patients, organochlorine levels differed significantly based on clinical characteristics of the primary carcinoma, including stage, grade, ER status, and HER2 status, indicating that body burden of organochlorines may influence the development of specific subtypes of breast cancer. Potentially carcinogenic organochlorines were present at high levels within the human breast warranting further research to determine the impact of organochlorines in the etiology of breast cancer., (© 2018 Wiley Periodicals, Inc.)

Extensive genomic and transcriptomic heterogeneity in human cancer often negatively impacts treatment efficacy and survival, thus posing a significant ongoing challenge for modern treatment regimens. State-of-the-art DNA- and RNA-sequencing methods now provide high-resolution genomic and gene expression portraits of individual cells, facilitating the study of complex molecular heterogeneity in cancer. Important developments in single-cell sequencing (SCS) technologies over the past 5 years provide numerous advantages over traditional sequencing methods for understanding the complexity of carcinogenesis, but significant hurdles must be overcome before SCS can be clinically useful. In this review, we: (1) highlight current methodologies and recent technological advances for isolating single cells, single-cell whole-genome and whole-transcriptome amplification using minute amounts of nucleic acids, and SCS, (2) summarize research investigating molecular heterogeneity at the genomic and transcriptomic levels and how this heterogeneity affects clonal evolution and metastasis, and (3) discuss the promise for integrating SCS in the clinical care arena for improved patient care.

The identification of extensive genetic heterogeneity in human breast carcinomas poses a significant challenge for designing effective treatment regimens. Significant genomic evolution often occurs during breast cancer progression, creating variability within primary tumors as well as between the primary carcinoma and metastases. Current risk allocations and treatment recommendations for breast cancer patients are based largely on characteristics of the primary tumor; however, genetic differences between disseminated tumor cells and the primary carcinoma may negatively impact treatment efficacy and survival. In this review we (1) present current information about genomic variability within primary breast carcinomas, between primary tumors and regional/distant metastases, among circulating tumor cells (CTCs) and disseminated tumor cells (DTCs), and in cell-free nucleic acids in circulation, and (2) describe how this heterogeneity affects clinical care and outcomes such as recurrence and therapeutic resistance. Understanding the evolution and functional significance of the composite breast cancer genome within each patient is critical for developing effective therapies that can overcome obstacles presented by molecular heterogeneity., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Metastasis is a multistep process that is not well understood. Colonization of a secondary organ requires specific molecular alterations of the host microenvironment. To determine the temporal and spatial changes associated with metastatic dissemination to the axillary lymph nodes, gene expression profiles were compared between histologically normal lymph nodes from node-positive patients and tumor-free nodes from node-negative patients. Using a stringent false discovery rate correction (<0.05) for multiple hypothesis testing, we did not detect any differentially expressed genes between the lymph node groups. Thus, the presence of metastatic cells within the lymphatic system does not elicit widespread changes in gene expression through the axillary basin; rather, lymph nodes independently respond to disseminated tumor cells., Competing Interests: DECLARATION OF CONFLICTING INTERESTS: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objective: Metabolic dysfunction characterized by insulin resistance (IR) is an important risk factor for type-2 diabetes and coronary artery disease (CAD). The aim of this study was to determine if clinical lifestyle interventions differing in scope and intensity improve IR, defined by the lipoprotein IR (LPIR) score, in individuals differing in the severity of metabolic dysfunction., Methods: Subjects with diagnosed type-2 diabetes, CAD or significant risk factors participated in one of two clinical lifestyle modification interventions: (i) intensive non-randomized programme with a strict vegetarian diet ( n  = 90 participants, 90 matched controls) or (ii) moderate randomized trial following a Mediterranean-style diet ( n  = 89 subjects, 58 controls). On-treatment and intention-to-treat analyses assessed changes over 1 year in LPIR, lipoprotein profiles and metabolic risk factors in intervention participants and controls in both programmes., Results: In the on-treatment analysis, both interventions led to weight loss: [-8.9% (95% CI, -10.3 to -7.4), intensive programme; -2.8% (95% CI, -3.8 to -1.9), moderate programme; adjusted P  < 0.001] and a decrease in the LPIR score [-13.3% (95% CI, -18.2 to -8.3), intensive; -8.8% (95% CI, -12.9 to -4.7), moderate; adjusted P  < 0.01] compared with respective controls. Of the six lipoprotein parameters comprising LPIR, only large very-low-density lipoprotein particle concentrations decreased significantly in participants compared with controls in both programmes [-26.3% (95% CI, -43.0 to -9.6), intensive; -14.2% (95% CI, -27.4 to -1.0), moderate; P  < 0.05]. Intention-to-treat analysis confirmed and strengthened the primary results., Conclusion: A stringent lifestyle modification intervention with a vegetarian diet and a moderate lifestyle modification intervention following a Mediterranean diet were both effective for improving IR defined by the LPIR score.

Background: Obesity is a risk factor for breast cancer in postmenopausal women and is associated with decreased survival and less favorable clinical characteristics such as greater tumor burden, higher grade, and poor prognosis, regardless of menopausal status. Despite the negative impact of obesity on clinical outcome, molecular mechanisms through which excess adiposity influences breast cancer etiology are not well-defined., Methods: Affymetrix U133 2.0 gene expression data were generated for 405 primary breast tumors using RNA isolated from laser microdissected tissues. Patients were classified as normal-weight (BMI < 25), overweight (BMI 25-29.9) or obese (BMI ≥ 30). Statistical analysis was performed by ANOVA using Partek Genomics Suite version 6.6 using a false discovery rate <0.05 to define significance., Results: Obese patients were significantly more likely to be diagnosed ≥50 years or with African American ancestry compared to lean or overweight women. Pathological characteristics including tumor stage, size or grade, lymph node status, intrinsic subtype, and breast cancer mortality did not differ significantly between groups. No significant gene expression differences were detected by BMI in a non-stratified analysis which included all subtypes or within luminal B, HER2-enriched or basal-like subtypes. Within luminal A tumors, however, 44 probes representing 42 genes from pathways such as cell cycle, p53 and mTOR signaling, DNA repair, and transcriptional misregulation were differentially expressed., Conclusions: Identification of transcriptome differences in luminal A tumors from normal-weight compared to obese women suggests that obesity alters gene expression within ER+ tumor epithelial cells. Alterations of pathways involved in cell cycle control, tumorigenesis and metabolism may promote cellular proliferation and provide a molecular explanation for less favorable outcome of obese women with breast cancer. Targeted treatments, such as mTOR inhibitors, may allow for improved treatment and survival of obese women, especially African American women, who are more likely to be obese and suffer outcome disparities.

Molecular heterogeneity within primary breast carcinomas and among axillary lymph node (LN) metastases may impact diagnosis and confound treatment. In this study, we used short tandem repeated sequences to assess genomic heterogeneity and to determine hereditary relationships among primary tumor areas and regional metastases from 30 breast cancer patients. We found that primary carcinomas were genetically heterogeneous and sampling multiple areas was necessary to adequately assess genomic variability. LN metastases appeared to originate at different time periods during disease progression from different sites of the primary tumor and the extent of genomic divergence among regional metastases was associated with a less favorable patient outcome (P = 0.009). In conclusion, metastasis is a complex process influenced by primary tumor heterogeneity and variability in the timing of dissemination. Genomic variation in primary breast tumors and regional metastases may negatively impact clinical diagnostics and contribute to therapeutic resistance.

Next-generation sequencing (NGS) technologies allow for the generation of whole exome or whole genome sequencing data, which can be used to identify novel genetic alterations associated with defined phenotypes or to expedite discovery of functional variants for improved patient care. Because this robust technology has the ability to identify all mutations within a genome, incidental findings (IF)- genetic alterations associated with conditions or diseases unrelated to the patient's present condition for which current tests are being performed- may have important clinical ramifications. The current debate among genetic scientists and clinicians focuses on the following questions: 1) should any IF be disclosed to patients, and 2) which IF should be disclosed - actionable mutations, variants of unknown significance, or all IF? Policies for disclosure of IF are being developed for when and how to convey these findings and whether adults, minors, or individuals unable to provide consent have the right to refuse receipt of IF. In this review, we detail current NGS technology platforms, discuss pressing issues regarding disclosure of IF, and how IF are currently being handled in prenatal, pediatric, and adult patients.

Objective: To examine relationships between weight loss through changes in lifestyle and peripheral blood gene expression profiles., Methods: A prospective nonrandomized trial was conducted over 1 year in participants undergoing intensive lifestyle modification to reverse or stabilize progression of coronary artery disease. Cardiovascular risk factors, inflammatory biomarkers, and gene expression as a function of weight loss were assessed in 89 lifestyle participants and 71 retrospectively matched controls undergoing usual care., Results: Substantial weight loss (-15.2 ± 3.8%) in lifestyle participants (n = 33) was associated with improvement in selected cardiovascular risk factors and significant changes in peripheral blood gene expression from pre- to post-intervention: 132 unique genes showed significant expression changes (false discovery rate corrected P-value <0.05 and fold-change ≥1.4). Altered molecular pathways were related to immune function and inflammatory responses involving endothelial activation. In contrast, participants losing minimal weight (-3.1 ± 2.5%, n = 32) showed only minor changes in cardiovascular risk factors and markers of inflammation and no changes in gene expression compared to non intervention controls after 1 year., Conclusions: Weight loss (≥10%) during lifestyle modification is associated with down-regulation of genetic pathways governing interactions between circulating immune cells and the vascular endothelium and may be required to successfully reduce CVD risk., (© 2015 The Obesity Society.)

Many environmental chemicals accumulate in human tissues and may contribute to cancer risk. Polychlorinated biphenyls (PCBs) are associated with adverse health effects, but relationships between PCB exposure and breast cancer are unclear. In this study, we sought to determine whether bioaccumulation of PCBs differs within regions of the human breast and whether PCB levels are associated with clinical and pathological characteristics in breast cancer patients. Tissue sections (n=245) were collected from breast quadrants from 51 women with a diagnosis ranging from disease-free to metastatic breast cancer. Ninety-seven PCB congeners were assayed by high resolution gas chromatography. ANOVA was used to examine PCB distribution within the breast and relationships with clinical/pathological variables. Pearson product-moment correlations assessed relationships between age at mastectomy and PCB levels. PCBs were abundant in breast tissues with a median concentration of 293.4ng/g lipid (range 15.4-1636.3ng/g). PCB levels in breast tissue were significantly different (p<0.001) among functional groupings of congeners defined by structure-activity properties: Group I (28.2ng/g), Group II (96.6ng/g), Group III (166.0ng/g). Total PCB concentration was highly correlated with age at mastectomy, but the distribution of PCBs did not differ by breast quadrant. PCB levels were not associated with patient status or tumor characteristics. In conclusion, PCB congeners with carcinogenic potential were present at high levels in the human breast, but were not associated with clinical or pathological characteristics in breast cancer patients., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Heart disease and related sequelae are a leading cause of death and healthcare expenditure throughout the world. Although many patients opt for surgical interventions, lifestyle modification programs focusing on nutrition and exercise have shown substantial health benefits and are becoming increasing popular. We conducted a year-long lifestyle modification program to mediate cardiovascular risk through traditional risk factors and to investigate how molecular changes, if present, may contribute to long-term risk reduction. Here we describe the lifestyle intervention, including clinical and molecular data collected, and provide details of the experimental methods and quality control parameters for the gene expression data generated from participants and non-intervention controls. Our findings suggest successful and sustained modulation of gene expression through healthy lifestyle changes may have beneficial effects on vascular health that cannot be discerned from traditional risk factor profiles. The data are deposited in the Gene Expression Omnibus, series GSE46097 and GSE66175.

Purpose: The cytochrome P450 (CYP) genes are oxygenases involved in estrogen biosynthesis and metabolism, generation of DNA damaging procarcinogens, and response to anti-estrogen therapies. Since lifetime estrogen exposure is an established risk factor for breast cancer, determining the role of CYP genes in breast cancer etiology may provide critical information for understanding tumorigenesis and response to treatment., Methods: This review summarizes literature available in PubMed published between 1993 and 2013 that focuses on studies evaluating the effects of DNA variants in CYP genes on estrogen synthesis, metabolism, and generation of procarcinogens in addition to response to anti-estrogen therapies., Results: Evaluation of DNA variants in estrogen metabolism genes was largely inconclusive. Meta-analyses of data from CYP19A1 support an association between the number of (TTTA) n repeats in intron 4 and breast cancer risk, but the biological mechanism for this relationship is unknown. Associations between single nucleotide polymorphism in CYP1B1 and DNA damage caused by procarcinogenic estrogen metabolites were ambiguous. Variants in CYP2D6 are associated with altered metabolism tamoxifen; however, current data do not support widespread clinical testing. The effect of variants in CYP19A1 in response to aromatase inhibitors is also questionable., Conclusion: Evaluation of DNA variants in CYP genes involved with estrogen metabolism or treatment response has been inconclusive, reflecting small samples sizes, tumor heterogeneity, and differences between populations. Better-powered studies that account for genetic backgrounds and tumor phenotypes are thus necessary.

Breast stroma plays an active role in tumorigenesis, undergoing both phenotypic and molecular changes that facilitate and promote tumor development and growth. The metastatic microenvironment also plays a role in successful colonization; however, genetic changes in these secondary microenvironments are not well described. To improve understanding of molecular changes associated with metastatic colonization, gene expression patterns from lymph node tissues from women with at least one positive, as well as one negative node, were compared. Lymph node tissue was microdissected and hybridized to U133A 2.0 gene expression arrays. Differential expression was detected using Partek(®) Genomics Suite™ 6.6 with FDR <0.05 and >2-fold change defining significance. Twenty-two genes were differentially expressed, 14 genes, including AZGP1, FOXA1 and PIP, were expressed at significantly higher levels in colonized lymph nodes and eight genes, such as CXCL2 and HPGDS, were expressed at significantly higher levels in non-metastatic lymph nodes. Thus, lymph node tissues harboring metastases have different gene expression patterns from those without metastases. Many differentially expressed genes are involved in cellular proliferation and survival, immune function and mesenchymal-epithelial transition, suggesting that repression of immune response and restoration of an epithelial phenotype in the host tissue are critical for successful establishment of lymph node metastases.

Background: Healthy lifestyle changes are thought to mediate cardiovascular disease risk through pathways affecting endothelial function and progression of atherosclerosis; however, the extent, persistence, and clinical significance of molecular change during lifestyle modification are not well known. We examined the effect of a rigorous cardiovascular disease risk reduction program on peripheral blood gene expression profiles in 63 participants and 63 matched controls to characterize molecular responses and identify regulatory pathways important to cardiovascular health., Methods and Results: Dramatic changes in dietary fat intake (-61%; P<0.001 versus controls) and physical fitness (+34%; P<0.001) led to significant improvements in cardiovascular disease risk factors. Analysis of variance with false discovery rate correction for multiple testing (P<0.05) identified 26 genes after 12 weeks and 143 genes after 52 weeks that were differentially expressed from baseline in participants. Controls showed little change in cardiovascular disease risk factors or gene expression. Quantitative reverse transcription polymerase chain reaction validated differential expression for selected transcripts. Lifestyle modification effectively reduced expression of proinflammatory genes associated with neutrophil activation and molecular pathways important to vascular function, including cytokine production, carbohydrate metabolism, and steroid hormones. Prescription medications did not significantly affect changes in gene expression., Conclusions: Successful and sustained modulation of gene expression through lifestyle changes may have beneficial effects on the vascular system not apparent from traditional risk factors. Healthy lifestyles may restore homeostasis to the leukocyte transcriptome by downregulating lactoferrin and other genes important in the pathogenesis of atherosclerosis. Clinical Trial Registration- URL: www.clinicaltrials.gov. Unique identifier: NCT01805492.

Background and Aims: Insulin and leptin are important markers of insulin resistance and vascular inflammation in metabolic and cardiovascular diseases. This study evaluated changes in circulating levels of insulin and leptin during a cardiovascular health program to improve our understanding of cardiometabolic risk reduction., Methods and Results: Participants (n=76) completed a prospective, nonrandomized program designed to stabilize or reverse progression of coronary artery disease through dietary changes, exercise, stress management, and group support. Controls (n=76) were matched to participants based on age, gender, and disease status. Traditional cardiovascular risk factors were assessed at baseline, 12 weeks, and 52 weeks by standard methods. Dietary data were collected by 72-h recall and evaluated by Food Processor® v8.4.0. Ultrasensitive insulin and leptin levels were measured by radioimmunoassay. Participants successfully reduced their total caloric intake from >2000 calories per day to ≈ 1700 calories per day (p<0.05 compared to controls), lowered daily fat intake by >60% (p<0.001 compared to controls), and increased carbohydrate intake by >30% (p<0.001). Repeated-measures ANOVA indicated significant beneficial changes (p<0.001 compared to controls) in plasma insulin (-19%) and leptin (-33%) during the lifestyle program, as well as improvement in traditional cardiovascular risk factors. Response was similar between men and women for most risk factors and was not markedly influenced by medication use., Conclusion: Lifestyle changes focusing on diet, physical activity, and stress reduction can successfully modify both cardiovascular and metabolic risk factors, with the potential to mediate cardiometabolic risk through beneficial anti-inflammatory and anti-oxidative effects on the vasculature., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Psychosocial stress profoundly impacts long-term cardiovascular health through adverse effects on sympathetic nervous system activity, endothelial dysfunction, and atherosclerotic development. Recreational Music Making (RMM) is a unique stress amelioration strategy encompassing group music-based activities that has great therapeutic potential for treating patients with stress-related cardiovascular disease., Material/methods: Participants (n=34) with a history of ischemic heart disease were subjected to an acute time-limited stressor, then randomized to RMM or quiet reading for one hour. Peripheral blood gene expression using GeneChip® Human Genome U133A 2.0 arrays was assessed at baseline, following stress, and after the relaxation session., Results: Full gene set enrichment analysis identified 16 molecular pathways differentially regulated (P<0.005) during stress that function in immune response, cell mobility, and transcription. During relaxation, two pathways showed a significant change in expression in the control group, while 12 pathways governing immune function and gene expression were modulated among RMM participants. Only 13% (2/16) of pathways showed differential expression during stress and relaxation., Conclusions: Human stress and relaxation responses may be controlled by different molecular pathways. Relaxation through active engagement in Recreational Music Making may be more effective than quiet reading at altering gene expression and thus more clinically useful for stress amelioration.

Advances in diagnostic screening and adjuvant therapy have dramatically increased the number of breast cancer survivors in the USA, who may face changes in physical and mental health, social support, quality of life and economics. Women living with breast cancer are increasingly interested in lifestyle modification to decrease the risk of recurrence and mortality while increasing physical and emotional wellbeing. Although organizations such as the American Cancer Society support a healthy diet, frequent physical activity and stress reduction for decreasing breast cancer risk, studies examining the effects of lifestyle on clinical outcomes including survival and prognosis have been inconclusive. With the number of breast cancer survivors predicted to increase to 3.4 million by 2015, it is important to develop effective treatment paradigms that overcome barriers to behavioral modification to improve clinical outcomes and survivorship in breast cancer patients.

Microarray-based gene expression profiling is revolutionizing biomedical research by allowing expression profiles of thousands of genes to be interrogated in a single experiment. In cancer research, the use of laser microdissection (LM) to isolate RNA from tissues provides the ability to accurately identify molecular profiles from different cell types that comprise the tumor and its surrounding microenvironment. Because RNA is an unstable molecule, the quality of RNA extracted from tissues can be affected by sample preparation and processing. Thus, special protocols have been developed to isolate research-quality RNA after LM. This chapter provides detailed descriptions of protocols used to generate micro-array data from high-quality frozen breast tissue specimens, as well as challenges associated with formalin-fixed paraffin-embedded specimens.

Introduction: Columnar cell lesions (CCL) and atypical ductal hyperplasia (ADH) frequently coexist and share molecular changes with in situ and invasive components, suggesting that CCL and ADH may be precursors to breast cancer. These conclusions are largely based on studies examining CCL and/or ADH from patients diagnosed with more advanced disease. We assessed allelic imbalance (AI) in pure CCL or ADH specimens to characterize molecular changes in nonneoplastic breast lesions., Methods: DNA samples were obtained from laser-microdissected pure CCL (n = 42) or ADH (n = 31). AI was assessed at 26 chromosomal regions commonly altered in breast cancer. Data were analyzed using Fisher's exact and Student's t-tests using a cutoff of P < 0.05., Results: The average AI frequency was 6.2% in CCL and 6.1% in ADH; approximately 33% of nonneoplastic lesions had no detectable genetic changes. Levels of AI in CCL and ADH were significantly (P < 0.0001) lower than observed in either low- or high-grade ductal carcinoma in situ (DCIS) lesions. Genetic changes characteristic of in situ and invasive disease, especially on chromosomes 16q and 17p, were infrequent in pure nonneoplastic lesions., Conclusions: Pure CCL and ADH lesions demonstrate lower levels of genetic alterations than DCIS, invasive carcinomas or CCL/ADH lesions from cancerous breasts; alterations of chromosomes 16q and 17p were not detected. Pure CCL and ADH lesions are not genetically advanced, and molecular profiles do not support these lesions as obligatory precursors to more advanced disease. Molecular differences between pure and synchronous lesions support re-evaluation of current models of disease initiation, progression, and risk.

Breast cancer is a heterogeneous disease with a complex etiology that develops from different cellular lineages, progresses along multiple molecular pathways, and demonstrates wide variability in response to treatment. The "standard of care" approach to breast cancer treatment in which all patients receive similar interventions is rapidly being replaced by personalized medicine, based on molecular characteristics of individual patients. Both inherited and somatic genomic variation is providing useful information for customizing treatment regimens for breast cancer to maximize efficacy and minimize adverse side effects. In this article, we review (1) hereditary breast cancer and current use of inherited susceptibility genes in patient management; (2) the potential of newly-identified breast cancer-susceptibility variants for improving risk assessment; (3) advantages and disadvantages of direct-to-consumer testing; (4) molecular characterization of sporadic breast cancer through immunohistochemistry and gene expression profiling and opportunities for personalized prognostics; and (5) pharmacogenomic influences on the effectiveness of current breast cancer treatments. Molecular genomics has the potential to revolutionize clinical practice and improve the lives of women with breast cancer.

Pathological grade is routinely used to stratify breast cancer patients into favorable and less favorable outcome groups. Mechanisms by which genomic changes in breast tumors specifically contribute to the underlying components of tumor grade - tubule formation, nuclear pleomorphism, and mitoses - are unknown. This study examined 26 chromosomal regions known to be altered in breast cancer in 256 invasive breast carcinomas. Differences in overall levels and patterns of allelic imbalance (AI) at each chromosomal region were compared for tumors with favorable (=1) and unfavorable (=3) scores for tubule formation, nuclear pleomorphism and mitotic count. Levels of AI were significantly different between samples with high and low scores for tubule formation (P < 0.001), nuclear pleomorphism (P < 0.001) and mitotic count (P < 0.05). Significantly higher levels of AI were detected at regions 11q23 and 13q12 for tumors with reduced tubule formation, chromosomes 9p21, 11q23, 13q14, 17p13 and 17q12 for those with high levels of nuclear atypia, and chromosomes 1p36, 11q23, and 13q14 for those with high mitotic counts. Region 16q11-q22 showed significantly more AI events in samples with low nuclear atypia. Patterns of genetic changes associated with poorly-differentiated breast tumors were recapitulated by the individual components of the Nottingham Histologic Score. While frequent alteration of 11q23 is common for reduced tubule formation, high nuclear atypia and high mitotic counts, suggesting that this is an early genetic change in the development of poorly-differentiated breast tumors, alterations at the other seven loci associated with poorly-differentiated tumors may specifically influence cell structure, nuclear morphology and cellular proliferation.

Tumor-associated stroma plays an active role in breast pathogenesis, with alterations in cell signaling, proliferation and angiogenesis contributing to successful tumorigenesis. Although epigenetic modifications to DNA, as well as changes in RNA and protein expression have been identified in tumor-associated stroma, there is considerable debate regarding the presence of chromosomal alterations, detected as loss of heterozygosity/allelic imbalance events in histologically normal stromal cells adjacent to the breast carcinomas. Previous studies have detected loss of heterozygosity/allelic imbalance at varying distances from the tumor margin, and patterns of chromosomal change have been linked to tumor grade and lymph node status. By contrast, recent reports challenge the presence of genomic instability in stromal cells of the breast tumor microenvironment, leading to the speculation that the loss of heterozygosity/allelic imbalance studies, based almost exclusively on microsatellite-based profiling of formalin-fixed, paraffin-embedded tissues, do not accurately measure the true genomic content of the tumor microenvironment but rather are a reflection of technological artifact. In this perspective, we present evidence surrounding the debate and critically evaluate arguments, both pros and cons, over the presence or absence of genomic instability tumor-associated stroma.

Pathological grade is a useful prognostic factor for stratifying breast cancer patients into favorable (low-grade, well-differentiated tumors) and less favorable (high-grade, poorly-differentiated tumors) outcome groups. Under the current system of tumor grading, however, a large proportion of tumors are characterized as intermediate-grade, making determination of optimal treatments difficult. In an effort to increase objectivity in the pathological assessment of tumor grade, differences in chromosomal alterations and gene expression patterns have been characterized in low-grade, intermediate-grade, and high-grade disease. In this review, we outline molecular data supporting a linear model of progression from low-grade to high-grade carcinomas, as well as contradicting genetic data suggesting that low-grade and high-grade tumors develop independently. While debate regarding specific pathways of development continues, molecular data suggest that intermediate-grade tumors do not comprise an independent disease subtype, but represent clinical and molecular hybrids between low-grade and high-grade tumors. Finally, we discuss the clinical implications associated with different pathways of development, including a new clinical test to assign grade and guide treatment options.

Background: Low-density lipoprotein (LDL) cholesterol lowering is a primary goal in clinical management of patients with cardiovascular disease, but traditional cholesterol levels may not accurately reflect the true atherogenicity of plasma lipid profiles. The size and concentration of lipoprotein particles, which transport cholesterol and triglycerides, may provide additional information for accurately assessing cardiovascular risk. This study evaluated changes in plasma lipoprotein profiles determined by nuclear magnetic resonance (NMR) spectroscopy in patients participating in a prospective, nonrandomized lifestyle modification program designed to reverse or stabilize progression of coronary artery disease (CAD) to improve our understanding of lipoprotein management in cardiac patients., Results: The lifestyle intervention was effective in producing significant changes in lipoprotein subclasses that contribute to CAD risk. There was a clear beneficial effect on the total number of LDL particles (-8.3%, p < 0.05 compared to matched controls), small dense LDL particles (-9.5%, p < 0.05), and LDL particle size (+0.8%; p < 0.05). Likewise, participants showed significant improvement in traditional CAD risk factors such as body mass index (-9.9%, p < 0.01 compared to controls), total cholesterol (-5.5%, p < 0.05), physical fitness (+37.2%, p < 0.01), and future risk for CAD (-7.9%, p < 0.01). Men and women responded differently to the program for all clinically-relevant variables, with men deriving greater benefit in terms of lipoprotein atherogenicity. Plasma lipid and lipoprotein responses to the lifestyle change program were not confounded by lipid-lowering medications., Conclusion: In at risk patients motivated to participate, an intensive lifestyle change program can effectively alter traditional CAD risk factors and plasma lipoprotein subclasses and may reduce risk for cardiovascular events. Improvements in lipoprotein subclasses are more evident in men compared to women.

Background: Genomic alterations of the proto-oncogene c-erbB-2 (HER-2/neu) are associated with aggressive behavior and poor prognosis in patients with breast cancer. The variable clinical outcomes seen in patients with similar HER2 status, given similar treatments, suggests that the effects of amplification of HER2 can be influenced by other genetic changes. To assess the broader genomic implications of structural changes at the HER2 locus, we investigated relationships between genomic instability and HER2 status in patients with invasive breast cancer., Methods: HER2 status was determined using the PathVysion assay. DNA was extracted after laser microdissection from the 181 paraffin-embedded HER2 amplified (n=39) or HER2 negative (n=142) tumor specimens with sufficient tumor available to perform molecular analysis. Allelic imbalance (AI) was assessed using a panel of microsatellite markers representing 26 chromosomal regions commonly altered in breast cancer. Student t-tests and partial correlations were used to investigate relationships between genomic instability and HER2 status., Results: The frequency of AI was significantly higher (P<0.005) in HER2 amplified (27%) compared to HER2 negative tumors (19%). Samples with HER2 amplification showed significantly higher levels of AI (P<0.05) at chromosomes 11q23, 16q22-q24 and 18q21. Partial correlations including ER status and tumor grade supported associations between HER2 status and alterations at 11q13.1, 16q22-q24 and 18q21., Conclusion: The poor prognosis associated with HER2 amplification may be attributed to global genomic instability as cells with high frequencies of chromosomal alterations have been associated with increased cellular proliferation and aggressive behavior. In addition, high levels of DNA damage may render tumor cells refractory to treatment. In addition, specific alterations at chromosomes 11q13, 16q22-q24, and 18q21, all of which have been associated with aggressive tumor behavior, may serve as genetic modifiers to HER2 amplification. These data not only improve our understanding of HER in breast pathogenesis but may allow more accurate risk profiles and better treatment options to be developed.

Background: Ductal carcinoma in situ (DCIS) is a preinvasive lesion of the breast with an inherent but nonobligatory tendency for progression to invasive breast cancer. Although the transition from in situ to invasive disease is critical to the development of breast cancer, molecular and biological changes responsible for this transition are not well characterized., Methods: Chromosomal alterations at 26 regions were assayed in 66 DCIS lesions and 111 invasive ductal carcinomas. Levels and patterns of allelic imbalance (AI) were compared between grade 1 DCIS and well-differentiated breast carcinomas, and between grade 3 DCIS and poorly differentiated invasive breast carcinomas, using Fisher's exact and Student's t-tests., Results: Levels of AI were significantly lower (P < 0.01) in grade 1 DCIS (11.9%) compared to well-differentiated carcinomas (19.2%), but were not significantly different between grade 3 DCIS and poorly differentiated tumors. No significant differences were detected at any of the 26 chromosomal regions between low-grade DCIS and invasive tumors; however, AI events at chromosomes 1p36, 11q23, and 16q11-q22 could discriminate high-grade in situ from invasive disease., Conclusion: Lower levels of AI in low-grade in situ compared with invasive disease may reflect the protracted time to progression associated with low-grade DCIS. Increased levels of AI at chromosomes 1p36 and 11q23 in poorly differentiated carcinomas may harbor genes associated with invasiveness, while loss of chromosome 16q11-q22 may prevent the transition from in situ to invasive disease. Further characterization of these changes may provide molecular assays to identify DCIS lesions with invasive potential as well as targets for molecular therapeutics.

Background: Molecular studies suggest that acquisition of metastatic potential occurs early in the development of breast cancer; mechanisms by which cells disseminate from the primary carcinomas and successfully colonize foreign tissues are, however, largely unknown. Thus, we examined levels and patterns of chromosomal alterations in primary breast tumors from node-negative (n = 114) and node-positive (n = 115) patients to determine whether specific genomic changes are associated with tumor metastasis., Methods: Fifty-two genetic markers representing 26 chromosomal regions commonly altered in breast cancer were examined in laser microdissected tumor samples to assess levels and patterns of allelic imbalance (AI). Real time-PCR (RT-PCR) was performed to determine expression levels of candidate genes. Data was analyzed using exact unconditional and Student's t-tests with significance values of P < 0.05 and P < 0.002 used for the clinicopathological and genomic analyses, respectively., Results: Overall levels of AI in primary breast tumors from node-negative (20.8%) and node-positive (21.9%) patients did not differ significantly (P = 0.291). When data were examined by chromosomal region, only chromosome 8q24 showed significantly higher levels (P < 0.0005) of AI in node-positive primary tumors (23%) versus node-negative samples (6%). c-MYC showed significantly higher levels of gene expression in primary breast tumors from patients with lymph node metastasis., Conclusions: Higher frequencies of AI at chromosome 8q24 in patients with positive lymph nodes suggest that genetic changes in this region are important to the process of metastasis. Because overexpression of c-MYC has been associated with cellular dissemination as well as development of the premetastatic niche, alterations of the 8q24 region, including c-MYC, may be key determinants in the development of lymph node metastasis.

Defining genetic variation associated with complex human diseases requires standards based on high-quality DNA from well-characterized patients. With the development of robust technologies for whole-genome amplification, sample repositories such as serum banks now represent a potentially valuable source of DNA for both genomic studies and clinical diagnostics. We assessed the performance of whole-genome amplified DNA (wgaDNA) derived from stored serum/plasma on high-density single nucleotide polymorphism arrays. Neither storage time nor usage history affected either DNA extraction or whole-genome amplification yields; however, samples that were thawed and refrozen showed significantly lower call rates (73.9 +/- 7.8%) than samples that were never thawed (92.0 +/- 3.3%) (P < 0.001). Genotype call rates did not differ significantly (P = 0.13) between wgaDNA from never-thawed serum/plasma (92.9 +/- 2.6%) and genomic DNA (97.5 +/- 0.3%) isolated from whole blood. Approximately 400,000 genotypes were consistent between wgaDNA and genomic DNA, but the overall discordance rate of 4.4 +/- 3.8% reflected an average of 11,110 +/- 9502 genotyping errors per sample. No distinct patterns of chromosomal clustering were observed for single nucleotide polymorphisms showing discordant genotypes or homozygote conversion. Because the effects of genotyping errors on whole-genome studies are not well defined, we recommend caution when applying wgaDNA from serum/plasma to high-density single nucleotide polymorphism arrays in addition to the use of stringent quality control requirements for the resulting genotype data.

Background: Metastatic breast cancer is an aggressive disease associated with recurrence and decreased survival. To improve outcomes and develop more effective treatment strategies for patients with breast cancer, it is important to understand the molecular mechanisms underlying metastasis., Methods: We used allelic imbalance (AI) to determine the molecular heritage of primary breast tumors and corresponding metastases to the axillary lymph nodes. Paraffin-embedded samples from primary breast tumors and matched metastases (n = 146) were collected from 26 patients with node-positive breast cancer involving multiple axillary nodes. Hierarchical clustering was used to assess overall differences in the patterns of AI, and phylogenetic analysis inferred the molecular heritage of axillary lymph node metastases., Results: Overall frequencies of AI were significantly higher (P < 0.01) in primary breast tumors (23%) than in lymph node metastases (15%), and there was a high degree of discordance in patterns of AI between primary breast carcinomas and the metastases. Metastatic tumors in the axillary nodes showed different patterns of chromosomal changes, suggesting that multiple molecular mechanisms may govern the process of metastasis in individual patients. Some metastases progressed with few genomic alterations, while others harbored many chromosomal alterations present in the primary tumor., Conclusions: The extent of genomic heterogeneity in axillary lymph node metastases differs markedly among individual patients. Genomic diversity may be associated with response to adjuvant therapy, recurrence, and survival, and thus may be important in improving clinical management of breast cancer patients.

Pathological grade is a useful prognostic factor for stratifying breast cancer patients into favorable (well-differentiated tumors) and less favorable (poorly-differentiated tumors) outcome groups. The current system of tumor grading, however, is subjective and a large proportion of tumors are characterized as intermediate-grade tumors, making determination of optimal treatments difficult. To determine whether molecular profiles can discriminate breast disease by grade, patterns and levels of allelic imbalance (AI) at 26 chromosomal regions frequently altered in breast disease were examined in 185 laser microdissected specimens representing well-differentiated (grade 1; n = 55), moderately-differentiated (grade 2; n = 71), and poorly-differentiated (grade 3; n = 59) stage I-IV breast tumors. Overall levels of AI were significantly higher in grade 3 compared to grade 1 tumors (P < 0.05). Grades 1 and 3 showed distinct genetic profiles--grade 1 tumors were associated with large deletions of chromosome 16q22, while alterations at 9p21, 11q23, 13q14, 17p13.1 and 17q12 were characteristics of grade 3 carcinomas. In general, levels and patterns of AI in grade 2 carcinomas were intermediate between grade 1 and grade 3 tumors. Patterns of AI accurately categorized approximately 70% of samples into high- or low-grade disease groups, suggesting that the majority of breast tumors have genetic profiles consistent with high- or low-grade, and that molecular signatures of breast tumors can be useful for more accurate characterization of invasive breast cancer.

In modern industrialized societies, people are exposed to thousands of naturally occurring and synthetic chemicals throughout their lifetime. Although certain occupational chemicals are known to be carcinogenic in humans, it has been difficult to definitively determine the adverse health effects of many environmental pollutants due to their tremendous chemical diversity and absence of a consistent structural motif. Many environmental chemicals are metabolized in the body to reactive intermediates that readily react with DNA to form modified bases known as adducts, while other compounds mimic the biological function of estrogen. Because environmental chemicals tend to accumulate in human tissues and have carcinogenic and/or estrogenic properties, there is heightened interest in determining whether environmental chemicals increase risk for endocrine-related cancers, including breast cancer. Breast cancer is the most common cancer in women worldwide, but established risk factors account for a relatively small proportion of cases and causative factors remain ambiguous and poorly defined. In this review, we outline the structural chemistry of environmental contaminants, describe mechanisms of carcinogenesis and molecular pathways through which these chemicals may exert detrimental health effects, review current knowledge of relationships between chemicals and breast cancer risk, and highlight future directions for research on environmental contributions to breast cancer. Improved understanding of the relationship between environmental chemicals and breast cancer will help to educate the general public about real and perceived dangers of these pollutants in our environment and has the potential to reduce individual risk by changing corporate practices and improving public health policies.

Background: Histological grading of ductal carcinoma-in-situ (DCIS) lesions separates DCIS into three subgroups (well-, moderately, or poorly differentiated). It is unclear, however, whether breast disease progresses along a histological continuum or whether each grade represents a separate disease. In this study, levels and patterns of allelic imbalance (AI) were examined in DCIS lesions to develop molecular models that can distinguish pathological classifications of DCIS., Methods: Laser microdissected DNA samples were collected from DCIS lesions characterized by a single pathologist including well- (n = 18), moderately (n = 35), and poorly differentiated (n = 47) lesions. A panel of 52 microsatellite markers representing 26 chromosomal regions commonly altered in breast cancer was used to assess patterns of AI., Results: The overall frequency of AI increased significantly (P < .001) with increasing grade (well differentiated, 12%; moderately differentiated, 17%; poorly differentiated, 26%). Levels of AI were not significantly different between well- and moderately differentiated grades of disease but were significantly higher (P < .0001) in poorly differentiated compared with well- or moderately differentiated disease. No statistically significant differences in patterns of AI were detected between well- and moderately differentiated disease; however, AI occurred significantly more frequently (P < .05) in high-grade lesions at chromosomes 6q25-q27, 8q24, 9p21, 13q14, and 17p13.1, and significantly more frequently in low-grade lesions at chromosome 16q22.3-q24.3., Conclusions: The inability to discriminate DCIS at the genetic level suggests that grades 1 and 2 DCIS may represent a single, non-high-grade form of DCIS, whereas poorly differentiated DCIS seems to be a genetically more advanced disease that may represent a discrete disease entity, characterized by a unique spectrum of genetic alterations.

Background: Although recent data suggest that cells with metastatic potential disseminate from the primary breast tumor early in tumor development, the mechanism by which disseminated breast cancer cells proliferate within foreign tissues is not well understood. Here, we examined levels and patterns of allelic imbalance (AI) in metastatic lymph node (LN) tumors to identify molecular signals that promote the survival and growth of disseminated breast tumor cells., Methods: DNA from 106 metastatic LN tumors from 25 patients was isolated after laser microdissection of pure tumor cell populations. AI was assessed at 26 chromosomal regions frequently altered in breast cancer. Tumor burden was calculated by dividing the area of the metastatic tumor in the node by the area of the entire LN., Results: Metastatic tumor burden ranged from focal to complete replacement of the LN with tumor. Grouping the nodes as < 25% tumor, 25-50% tumor, 50-75% tumor, and > or = 75% tumor replacement revealed the average frequency of AI ranged from 0.13 (+/-0.11) in the < 25% group to 0.17 (+/-0.13) in LNs with > or = 75% tumor burden. The range of AI in both the < 25% and > 75% replacement group was 0.00-0.48. Allelic losses at chromosomal regions 1p36.1-36.2, 5q21.1-21.3, 6q15, 10q23.31-23.33, and 17p13.1 were significantly higher in metastatic LNs with > 75% compared with < 25% tumor burden., Conclusions: In metastatic LNs, levels of AI were not associated with tumor burden, suggesting that accumulation of genetic changes is not coincidental with tumor growth; rather the accumulation of specific genetic changes is a prerequisite to the transformation of disseminated breast cells into metastatic LN tumors.

Objectives: Whole-blood RNA for microarray analysis is easily accessible but contains a large proportion of globin mRNA that interferes with the accurate assessment of other genes. This study investigated the biological significance of genes whose expression was unmasked by globin mRNA reduction in peripheral blood., Design and Methods: Samples were collected from healthy subjects using the PAXgene Blood RNA System, and globin mRNA was depleted using GLOBINclear. Genes exhibiting consistent changes in expression on Affymetrix HU133A 2.0 arrays were characterized in three main areas of gene ontology--molecular function, biological process, and cellular component., Results: Globin reduction permitted detection of 2652+/-395 additional genes per assay. Genes unmasked by globin reduction include low abundance transcripts that function primarily as molecular binding proteins and catalytic enzymes in biological processes including transcription, replication, and intracellular transport and signalling. Protein products of these genes are preferentially associated with membranes and the nucleus., Conclusions: Additional genes detectable only after globin reduction in whole-blood RNA function in a variety of biological processes that may be important to diverse fields of study.

Human urine samples are ideal for proteomic profiling and have tremendous potential as sources of biomarkers. Multi-dimensional protein identification technology (MudPIT) is an effective approach to analyzing human urine or other fluids dominated by diverse metabolites. MudPIT analysis was used to identify 87 proteins in just 15 ml of human urine. A high throughput, reproducible, and sensitive technology, MudPIT may soon be used for more proteomic analyses of metabolites.

Background: Breast cancer development has been characterized as a nonobligatory sequence of histological changes from normal epithelium through invasive malignancy. Although genetic alterations are thought to accumulate stochastically during tumorigenesis, little is known about the timing of critical mutations. This study examined allelic imbalance (AI) in tissue samples representing a continuum of breast cancer development to examine the evolution of genomic instability., Methods: Laser-microdissected DNA samples were collected from histologically normal breast specimens (n = 25), atypical ductal hyperplasia (ADH, n = 16), ductal carcinoma-in-situ (DCIS, n = 37), and stage I to III invasive carcinomas (n = 72). Fifty-two microsatellite markers representing 26 chromosomal regions commonly deleted in breast cancer were used to assess patterns of AI., Results: AI frequencies were <5% in histologically normal and ADH specimens, 20% in DCIS lesions, and approximately 25% in invasive tumors. Mann-Whitney tests showed (1) that levels of AI in ADH samples did not differ significantly from those in histologically normal tissues and (2) that AI frequencies in DCIS lesions were not significantly different from those in invasive carcinomas. ADH and DCIS samples, however, differed significantly (P < .0001)., Conclusions: DCIS lesions contain levels of genomic instability that are characteristic of advanced invasive tumors, and this suggests that the biology of a developing carcinoma may already be predetermined by the in situ stage. Observations that levels of AI in ADH lesions are similar to those in disease-free tissues provide a genomic rationale for why prevention strategies at the ADH level are successful and why cases with ADH involving surgical margins do not require further resection.