Factors Associated with Increased Knowledge about Breast Density in South Australian Women Undergoing Breast Cancer Screening.

Background: There is growing awareness of breast density in women attending breast cancer screening; however, it is unclear whether this awareness is associated with increased knowledge. This study aims to evaluate breast density knowledge among Australian women attending breast cancer screening. Method: This cross-sectional study was conducted on women undergoing breast cancer screening at The Queen Elizabeth Hospital Breast/Endocrine outpatient department. Participants were provided with a questionnaire to assess knowledge, awareness, and desire to know their own breast density. Result: Of the 350 women who participated, 61% were familiar with 'breast density' and 57% had 'some knowledge'. Prior breast density notification (OR = 4.99, 95% CI = 2.76, 9.03; p = 0.004), awareness (OR = 4.05, 95% CI = 2.57, 6.39; p = 0.004), younger age (OR = 0.97, 95% CI = 0.96, 0.99; p = 0.02), and English as the language spoken at home (OR = 3.29, 95% CI = 1.23, 8.77; p = 0.02) were independent predictors of 'some knowledge' of breast density. A significant proportion of participants (82%) expressed desire to ascertain their individual breast density. Conclusions: While knowledge of breast density in this Australian cohort is generally quite low, we have identified factors associated with increased knowledge. Further research is required to determine optimal interventions to increase breast density knowledge.

CCL2-Mediated Stromal Interactions Drive Macrophage Polarization to Increase Breast Tumorigenesis.

CCL2 is an inflammatory cytokine that regulates macrophage activity and is implicated in increased mammographic density and early breast tumorigenesis. The role of CCL2 in mediating stromal interactions that contribute to breast tumorigenesis has yet to be fully elucidated. THP-1-derived macrophages and mammary fibroblasts were co-cultured for 72 h. Fibroblasts and macrophages were analysed for phenotype, expression of inflammatory and ECM-regulatory genes and collagen production. Mice overexpressing CCL2 in the mammary glands were analysed for global gene expression by RNAseq at 12 weeks of age. These mice were cross-bred with PyMT mammary tumour mice to examine the role of CCL2 in tumorigenesis. The co-culture of macrophages with fibroblasts resulted in macrophage polarization towards an M2 phenotype, and upregulated expression of CCL2 and other genes associated with inflammation and ECM remodelling. CCL2 increased the production of insoluble collagen by fibroblasts. A global gene expression analysis of CCL2 overexpressing mice revealed that CCL2 upregulates cancer-associated gene pathways and downregulates fatty acid metabolism gene pathways. In the PyMT mammary tumour model, CCL2 overexpressing mice exhibited increased macrophage infiltration and early tumorigenesis. Interactions between macrophages and fibroblasts regulated by CCL2 can promote an environment that may increase breast cancer risk, leading to enhanced early tumorigenesis.

Immune Regulation of Mammary Fibroblasts and the Impact of Mammographic Density.

Mammographic density is associated with a 4-6-fold increase in breast cancer risk independent of age and BMI. High mammographic density is characterized by breast tissue with high proportions of stroma comprised of fibroblasts, collagen, and immune cells. This study sought to investigate whether stromal fibroblasts from high mammographic density breast tissue contributes to increased extracellular matrix deposition and pro-tumorigenic signaling. Mammary fibroblasts were isolated from women with high and low mammographic density and exposed to immune factors myeloperoxidase (MPO), eosinophil peroxidase (EPO), transforming growth factor beta 1 (TGFB1) and tumour necrosis factor alpha (TNFA) for 72 h and profiled for expression of cancer-associated fibroblast and extracellular matrix regulation markers. No differences in gene expression profiles or collagen production were observed between fibroblasts with high or low mammographic density, and they did not have a differential response to immune mediators. MPO and EPO significantly increased the production of collagen 1. TGFB and TNFA induced variable changes in gene expression. Fibroblasts cultured in vitro from women with high mammographic density do not appear to be inherently different to those from women with low mammographic density. The function of fibroblasts in mammographic density-associated breast cancer risk is likely to be regulated by immune signals from surrounding cells in the microenvironment.

Biological Mechanisms and Therapeutic Opportunities in Mammographic Density and Breast Cancer Risk.

Mammographic density is an important risk factor for breast cancer; women with extremely dense breasts have a four to six fold increased risk of breast cancer compared to women with mostly fatty breasts, when matched with age and body mass index. High mammographic density is characterised by high proportions of stroma, containing fibroblasts, collagen and immune cells that suggest a pro-tumour inflammatory microenvironment. However, the biological mechanisms that drive increased mammographic density and the associated increased risk of breast cancer are not yet understood. Inflammatory factors such as monocyte chemotactic protein 1, peroxidase enzymes, transforming growth factor beta, and tumour necrosis factor alpha have been implicated in breast development as well as breast cancer risk, and also influence functions of stromal fibroblasts. Here, the current knowledge and understanding of the underlying biological mechanisms that lead to high mammographic density and the associated increased risk of breast cancer are reviewed, with particular consideration to potential immune factors that may contribute to this process.

Ovarian cycle stage critically affects 21-gene recurrence scores in Mmtv-Pymt mouse mammary tumours.

BACKGROUND: The Oncotype DX 21-gene Recurrence Score is predictive of adjuvant chemotherapy benefit for women with early-stage, estrogen receptor (ER)-positive, HER2-negative breast cancer. In premenopausal women, fluctuations in estrogen and progesterone during the menstrual cycle impact gene expression in hormone-responsive cancers. However, the extent to which menstrual cycling affects the Oncotype DX 21-gene signature remains unclear. Here, we investigate the impact of ovarian cycle stage on the 21-gene signature using a naturally cycling mouse model of breast cancer. METHODS: ER-positive mammary tumours were dissected from naturally cycling Mmtv-Pymt mice at either the estrus or diestrus phase of the ovarian cycle. The Oncotype DX 21-gene signature was assessed through quantitative real time-PCR, and a 21-gene experimental recurrence score analogous to the Oncotype DX Recurrence Score was calculated. RESULTS: Tumours collected at diestrus exhibited significant differences in expression of 6 Oncotype DX signature genes (Ki67, Ccnb1, Esr1, Erbb2, Grb7, Bag1; p ≤ 0.05) and a significant increase in 21-gene recurrence score (21.8 ± 2.4; mean ± SEM) compared to tumours dissected at estrus (15.5 ± 1.9; p = 0.03). Clustering analysis revealed a subgroup of tumours collected at diestrus characterised by increased expression of proliferation- (p < 0.001) and invasion-group (p = 0.01) genes, and increased 21-gene recurrence score (p = 0.01). No correlation between ER, PR, HER2, and KI67 protein abundance measured by Western blot and abundance of mRNA for the corresponding gene was observed, suggesting that gene expression is more susceptible to hormone-induced fluctuation compared to protein expression. CONCLUSIONS: Ovarian cycle stage at the time of tissue collection critically affects the 21-gene signature in Mmtv-Pymt murine mammary tumours. Further studies are required to determine whether Oncotype DX Recurrence Scores in women are similarly affected by menstrual cycle stage.

Comparison of hormone-induced mRNA and protein biomarker expression changes in breast cancer cells.

PURPOSE: Protein biomarkers estrogen receptor (ER), progesterone receptor (PR), and marker of proliferation (Ki67) are routinely assessed by immunohistochemistry to guide treatment decisions for breast cancer. Now, quantification of mRNA encoding these proteins is being adopted in the clinic. However, mRNA and protein biomarkers may be differentially regulated by fluctuations in estrogen and progesterone that occur across the menstrual cycle in premenopausal breast cancer patients. This study aimed to compare how estrogen and progesterone affect mRNA and protein biomarker expression in hormone-responsive breast cancer cells. METHODS: Hormone-responsive ZR-75-1 and T-47D human breast cancer cell lines were xenografted into the mammary fat pad of BALB/c nude mice supplemented with estrogen. Progesterone or vehicle was administered prior to dissection of tumors. Protein expression of ER, PR and Ki67 was quantified by immunohistochemistry, and mRNA encoding these proteins, ESR1, PGR and KI67, respectively, was quantified by real-time PCR. mRNA expression was also quantified in breast cancer cell lines treated with estrogen and progesterone in vitro. RESULTS: In T-47D-xenografted tumors, estrogen and progesterone treatment reduced PGR and KI67 mRNA expression, and reduced PR and Ki67 protein positivity, compared to estrogen treatment alone. In ZR-75-1 xenografted tumors, no significant differences in protein or mRNA biomarker expression were observed. In vitro, estrogen and progesterone co-treatment significantly reduced ESR1 and PGR mRNA expression in both T-47D and ZR-75-1 cell lines. CONCLUSIONS: Estrogen and progesterone similarly affect mRNA and protein biomarker expression in hormone-responsive breast cancer xenografts. Further research is needed to investigate concordance between protein and mRNA biomarkers in premenopausal breast cancer.

Pubertal mammary gland development is a key determinant of adult mammographic density.

Mammographic density refers to the radiological appearance of fibroglandular and adipose tissue on a mammogram of the breast. Women with relatively high mammographic density for their age and body mass index are at significantly higher risk for breast cancer. The association between mammographic density and breast cancer risk is well-established, however the molecular and cellular events that lead to the development of high mammographic density are yet to be elucidated. Puberty is a critical time for breast development, where endocrine and paracrine signalling drive development of the mammary gland epithelium, stroma, and adipose tissue. As the relative abundance of these cell types determines the radiological appearance of the adult breast, puberty should be considered as a key developmental stage in the establishment of mammographic density. Epidemiological studies have pointed to the significance of pubertal adipose tissue deposition, as well as timing of menarche and thelarche, on adult mammographic density and breast cancer risk. Activation of hypothalamic-pituitary axes during puberty combined with genetic and epigenetic molecular determinants, together with stromal fibroblasts, extracellular matrix, and immune signalling factors in the mammary gland, act in concert to drive breast development and the relative abundance of different cell types in the adult breast. Here, we discuss the key cellular and molecular mechanisms through which pubertal mammary gland development may affect adult mammographic density and cancer risk.

Discordance in 21-gene recurrence scores between paired breast cancer samples is inversely associated with patient age.

BACKGROUND: The Oncotype DX 21-gene Recurrence Score is a genomic-based algorithm that guides adjuvant chemotherapy treatment decisions for women with early-stage, oestrogen receptor (ER)-positive breast cancer. However, there are age-related differences in chemotherapy benefit for women with intermediate Oncotype DX Recurrence Scores that are not well understood. Menstrual cycling in younger women is associated with hormonal fluctuations that might affect the expression of genomic predictive biomarkers and alter Recurrence Scores. Here, we use paired human breast cancer samples to demonstrate that the clinically employed Oncotype DX algorithm is critically affected by patient age. METHODS: RNA was extracted from 25 pairs of formalin-fixed paraffin-embedded, invasive ER-positive breast cancer samples that had been collected approximately 2 weeks apart. A 21-gene signature analogous to the Oncotype DX platform was assessed through quantitative real-time PCR, and experimental recurrence scores were calculated using the Oncotype DX algorithm. RESULTS: There was a significant inverse association between patient age and discordance in the recurrence score. For every 1-year decrease in age, discordance in recurrence scores between paired samples increased by 0.08 units (95% CI - 0.14, - 0.01; p = 0.017). Discordance in recurrence scores for women under the age of 50 was driven primarily by proliferation- and HER2-associated genes. CONCLUSION: The Oncotype DX 21-gene Recurrence Score algorithm is critically affected by patient age. These findings emphasise the need for the consideration of patient age, particularly for women younger than 50, in the development and application of genomic-based algorithms for breast cancer care.

Timing of breast cancer surgery during the menstrual cycle-is there an optimal time of the month?

An intriguing relationship between menstrual cycle phase at the time of breast cancer surgery and clinical outcomes was first proposed in the late 1980s. Despite a number of clinical studies conducted to address this, as well as meta-analyses and systematic reviews, there remains significant controversy surrounding the effect of menstrual cycle phase at time of surgery on the prognosis of premenopausal breast cancer. While some studies have suggested that surgery performed during the luteal phase results in the most favourable outcome, other studies report the follicular phase is more favourable, and others show no association. Given the conflicting results, there remains insufficient evidence to determine whether there is an optimal time of the month to perform surgery. This issue has dogged breast cancer surgery for decades; knowledge of an optimal time of the month to conduct surgery would be a simple approach to improving patient outcomes. This review explores the potential biological mechanisms through which the hormonal milieu might contribute to differences in prognosis, and why clinical findings are so variable. It is concluded that a significant problem with current clinical research is the lack of insight from mechanistic studies. While there are a number of plausible biological mechanisms that could lead to altered survival, supporting evidence is limited. There are also variable approaches to defining the menstrual cycle phase and hormone receptor status of the tumour and few studies controlled for prognostic factors such as tumour size and stage, or addressed the impact of adjuvant treatments. Elucidation of the specific confounding factors, as well as biological mechanistic pathways that could explain the potential relationship between timing of surgery and survival, will greatly assist in designing robust well-controlled prospective clinical studies to evaluate this paradigm.

Foxp3 heterozygosity does not overtly affect mammary gland development during puberty or the oestrous cycle in mice.

Female mice heterozygous for a genetic mutation in transcription factor forkhead box p3 (Foxp3) spontaneously develop mammary cancers; however, the underlying mechanism is not well understood. We hypothesised that increased cancer susceptibility is associated with an underlying perturbation in mammary gland development. The role of Foxp3 in mammary ductal morphogenesis was investigated in heterozygous Foxp3Sf/+ and wildtype Foxp3+/+ mice during puberty and at specific stages of the oestrous cycle. No differences in mammary ductal branching morphogenesis, terminal end bud formation or ductal elongation were observed in pubertal Foxp3Sf/+ mice compared with Foxp3+/+ mice. During adulthood, all mice underwent normal regular oestrous cycles. No differences in epithelial branching morphology were detected in mammary glands from mice at the oestrus, metoestrus, dioestrus and pro-oestrus stages of the cycle. Furthermore, abundance of Foxp3 mRNA and protein in the mammary gland and lymph nodes was not altered in Foxp3Sf/+ mice compared with Foxp3+/+ mice. These studies suggest that Foxp3 heterozygosity does not overtly affect mammary gland development during puberty or the oestrous cycle. Further studies are required to dissect the underlying mechanisms of increased mammary cancer susceptibility in Foxp3Sf/+ heterozygous mice and the function of this transcription factor in normal mammary gland development.

Breast Density Notification: An Australian Perspective.

Breast density, also known as mammographic density, refers to white and bright regions on a mammogram. Breast density can only be assessed by mammogram and is not related to how breasts look or feel. Therefore, women will only know their breast density if they are notified by the radiologist when they have a mammogram. Breast density affects a woman's breast cancer risk and the sensitivity of a screening mammogram to detect cancer. Currently, the position of BreastScreen Australia and the Royal Australian and New Zealand College of Radiologists is to not notify women if they have dense breasts. However, patient advocacy organisations are lobbying for policy change. Whether or not to notify women of their breast density is a complex issue and can be framed within the context of both public health ethics and clinical ethics. Central ethical themes associated with breast density notification are equitable care, patient autonomy in decision-making, trust in health professionals, duty of care by the physician, and uncertainties around evidence relating to measurement and clinical management pathways for women with dense breasts. Legal guidance on this issue must be gained from broad legal principles found in the law of negligence and the test of materiality. We conclude a rigid legal framework for breast density notification in Australia would not be appropriate. Instead, a policy framework should be developed through engagement with all stakeholders to understand and take account of multiple perspectives and the values at stake.

InforMD: a new initiative to raise public awareness about breast density.

On a mammogram, breast density (also known as mammographic density) is shown as white and bright regions and is associated with reduced sensitivity in cancer detection and increased breast cancer risk. However, many Australian women are unaware of the significance of breast density as it is not routinely reported or discussed. In order to address this lack of knowledge, Australian breast cancer researchers with expertise in mammographic density formed the InforMD alliance (INformation FORum on Mammographic Density) in 2016. The alliance is working to raise awareness of breast density with the goal of improving breast cancer diagnosis and health outcomes for women. The InforMD website (www.InforMD.org.au) was launched in October 2016, coinciding with a major nationwide public awareness campaign by the alliance during breast cancer awareness month. The website contains unbiased, accurate, updated information on breast density. The website also provides summaries of major research articles in layperson language, recent news items related to breast density, links to relevant information for health professionals, events, and feature articles. Members of the public and health professionals can also subscribe for news updates. The interactive online Forum section facilitates discussion between health professionals, scientists and members of the public. To increase online traffic to the website, Facebook (www.facebook.com/BeInforMD) and Twitter (https://twitter.com/BeInforMD_) pages were launched in December 2016. Since its launch, InforMD has generated considerable interest. The public awareness campaign reached over 7 million Australians through a combination of newspaper, TV, radio, and online news. The website has attracted 13,058 unique visitors and 30,353 page views (data as of 19/12/2017). Breast cancer researchers have a significant role to play in disseminating information to the public on breast density. A combination of mainstream and social media, together with a well-informed and updated website, has laid the groundwork for the InforMD alliance to reach a wide audience.

Hormonal Modulation of Breast Cancer Gene Expression: Implications for Intrinsic Subtyping in Premenopausal Women.

Clinics are increasingly adopting gene-expression profiling to diagnose breast cancer subtype, providing an intrinsic, molecular portrait of the tumor. For example, the PAM50-based Prosigna test quantifies expression of 50 key genes to classify breast cancer subtype, and this method of classification has been demonstrated to be superior over traditional immunohistochemical methods that detect proteins, to predict risk of disease recurrence. However, these tests were largely developed and validated using breast cancer samples from postmenopausal women. Thus, the accuracy of such tests has not been explored in the context of the hormonal fluctuations in estrogen and progesterone that occur during the menstrual cycle in premenopausal women. Concordance between traditional methods of subtyping and the new tests in premenopausal women is likely to depend on the stage of the menstrual cycle at which the tissue sample is taken and the relative effect of hormones on expression of genes versus proteins. The lack of knowledge around the effect of fluctuating estrogen and progesterone on gene expression in breast cancer patients raises serious concerns for intrinsic subtyping in premenopausal women, which comprise about 25% of breast cancer diagnoses. Further research on the impact of the menstrual cycle on intrinsic breast cancer profiling is required if premenopausal women are to benefit from the new technology of intrinsic subtyping.

Dissecting the Biology of Menstrual Cycle-Associated Breast Cancer Risk.

Fluctuations in circulating estrogen and progesterone across the menstrual cycle lead to increased breast cancer susceptibility in women; however, the biological basis for this increased risk is not well understood. Estrogen and progesterone have important roles in normal mammary gland development, where they direct dynamic interactions among the hormonally regulated mammary epithelial, stromal, and immune cell compartments. The continuous fluctuations of estrogen and progesterone over a woman's reproductive lifetime affect the turnover of mammary epithelium, stem cells, and the extracellular matrix, as well as regulate the phenotype and function of mammary stromal and immune cells, including macrophages and regulatory T cells. Collectively, these events may result in genome instability, increase the chance of random genetic mutations, dampen immune surveillance, and promote tolerance in the mammary gland, and thereby increase the risk of breast cancer initiation. This article reviews the current status of our understanding of the molecular and the cellular changes that occur in the mammary gland across the menstrual cycle and how continuous menstrual cycling may increase breast cancer susceptibility in women.

Hormonal regulation of the cytokine microenvironment in the mammary gland.

The mammary gland is a unique organ that undergoes hormone-driven developmental changes over the course of the ovarian cycle during adult life. Macrophages play a role in regulating cellular turnover in the mammary gland and may affect cancer susceptibility. However, the immune microenvironment that regulates macrophage function has not been described. Hormonal regulation of the cytokine microenvironment across the ovarian cycle was explored using microbead multiplex assay for 15 cytokines in mammary glands from C57Bl/6 mice at different stages of the oestrous cycle, and in ovariectomised mice administered oestradiol and progesterone. The cytokines that were found to fluctuate over the course of the oestrous cycle were colony-stimulating factor (CSF)1, CSF2, interferon gamma (IFNG) and tumour necrosis factor alpha (TNFA), all of which were significantly elevated at oestrus compared with other phases. The concentration of serum progesterone during the oestrus phase negatively correlated with the abundance of cytokines CSF3, IL12p40, IFNG and leukaemia inhibitory factor (LIF). In ovariectomised mice, exogenous oestradiol administration increased mammary gland CSF1, CSF2, IFNG and LIF, compared with ovariectomised control mice. Progesterone administration together with oestradiol resulted in reduced CSF1, CSF3 and IFNG compared with oestradiol administration alone. This study suggests that the cytokine microenvironment in the mammary gland at the oestrus phase of the ovarian cycle is relatively pro-inflammatory compared with other stages of the cycle, and that the oestradiol-induced cytokine microenvironment is significantly attenuated by progesterone. A continuously fluctuating cytokine microenvironment in the mammary gland presumably regulates the phenotypes of resident leukocytes and may affect mammary gland cancer susceptibility.

Hormonal regulation of the immune microenvironment in the mammary gland.

It is well established that the development and homeostasis of the mammary gland are highly dependent upon the actions of ovarian hormones progesterone and estrogen, as well as the availability of prolactin for the pregnant and lactating gland. More recently it has become apparent that immune system cells and cytokines play essential roles in both mammary gland development as well as breast cancer. Here, we review hormonal effects on mammary gland biology during puberty, menstrual cycling, pregnancy, lactation and involution, and dissect how hormonal control of the immune system may contribute to mammary development at each stage via cytokine secretion and recruitment of macrophages, eosinophils, mast cells and lymphocytes. Collectively, these alterations may create an immunotolerant or inflammatory immune environment at specific developmental stages or phases of the menstrual cycle. Of particular interest for further research is investigation of the combinatorial actions of progesterone and estrogen during the luteal phase of the menstrual cycle and key developmental points where the immune system may play an active role both in mammary development as well as in the creation of an immunotolerant environment, thereby affecting breast cancer risk.

Expression of toll-like receptors on B lymphocytes.

Toll-like receptors (TLRs) are a family of trans-membrane receptors that play an important role in the innate immune system. Most studies examining the cellular expression of TLRs on immune cells have focussed on neutrophils, monocytes and dendritic cells, but there is little evidence of TLRs being expressed on lymphocytes. Using 3-colour flow cytometry, expression of TLR-1, TLR-2, TLR-3, TLR-4, and TLR-9 on peripheral blood lymphocyte populations was determined. Further examination of TLRs on CD5- and CD5+ CD19+ B cell subsets was performed. The binding of TLR1 and TLR9 antibodies was detected on 15-90% of resting B cells, but not on resting T-cells. The higher expression of TLR1 and TLR9 on CD5+ B cells compared to CD5- B cells may reflect the role of B1 cells in more primitive, less specific antibody responses.