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DESCRIPTION: Postmenopausal obesity is associated with increased circulating levels of androgens and estrogens and elevated breast cancer risk. Crown-like structures CLS; microscopic foci of dying adipocytes surrounded by macrophages are proposed to represent sites of increased aromatization of androgens to estrogens.

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Postmenopausal obesity is associated with increased circulating levels of androgens and estrogens and elevated breast cancer risk. Crown-like structures CLS; microscopic foci of dying adipocytes surrounded by macrophages are proposed to represent sites of increased aromatization of androgens to estrogens. Accordingly, we examined relationships between CLS and sex-steroid hormones in breast adipose tissue and serum from postmenopausal breast cancer patients. Tissues were immunohistochemically stained for CD68 30 min group boob sex determine the presence of CLS per unit area of adipose tissue.

Relationships were assessed between CD68 density and CLS and previously reported sex-steroid hormones quantified using radioimmunoassays in serum taken at the time of diagnosis and in fresh frozen adipose tissue taken at the time of surgery. Detection of CLS was not related to individual hormone levels or breast tumor pathology characteristics.

However, detection of CLS was associated with hormone ratios. Compared with women in the highest tertile of estrone: A similar pattern was observed with estradiol: CLS were more frequently identified in the breast fat of obese women and were associated with increased 30 min group boob sex of select estrogens: Additional studies on CLS, tissue and blood hormone levels, and breast cancer risk are needed to understand and confirm these findings.

The online version of this article doi: Among postmenopausal women, most endogenous estrogen is derived from aromatization of androgens in peripheral adipose tissues, and those in the highest quintile of circulating estrogen levels experience a twofold increased risk of developing breast cancer compared with those who have the lowest levels [ 56 ].

Limited data suggest that androgen levels are higher in the breast tissue than in circulation, and that concentrations are higher in benign versus malignant tissues, suggesting that local aromatization of androgens to estrogens within the breast could contribute to carcinogenesis [ 7 ]. Further, within breast adipose tissue, the ratio of testosterone to estradiol was statistically significantly lower than in serum, suggesting that hormone metabolism or other tissue factors might affect hormone concentrations locally in the breast, and could affect the 30 min group boob sex of immediately adjacent epithelium.

Specifically, these findings support the hypothesis that aromatization of androgens to estrogens in breast fat might increase estrogen levels, and potentially exert local influences on carcinogenesis [ 89 ]. We hypothesize that crown-like structures CLS may represent tissue factors that influence local hormone aromatization.

CLS are histological features in the breast adipose tissue that are recognized by the organized accumulation of CDpositive macrophages that surround dead or 30 min group boob sex adipocytes shown in Fig.

Studies using animal models and breast tissues of women suggest that their presence is associated with obesity and a pro-inflammatory, pro-carcinogenic process characterized by increased aromatase expression and activity and elevated signaling through ER-mediated pathways [ 10 — 12 ].

Data also suggest that the presence of CLS adversely affects outcomes among women with ER-positive breast cancers, and, in mice, CLS-related pro-inflammatory and carcinogenic processes may be reversible with weight-loss or chemopreventive agents [ 1314 ]. Accordingly, CLS in breast adipose tissue may represent one mechanism of increased local estrogen production, which would support the hypothesized role of local hormone levels in breast cancer development and progression.

In order to investigate whether the identification of CLS is related to concentrations and ratios of sex-steroid hormones in breast adipose tissue relative to systemic circulation, we analyzed the presence of CLS in benign breast tissue collected from surgical pathology specimens of postmenopausal breast cancer patients and compared results with previously measured hormone levels in breast fat and serum [ 9 ].

We hypothesized that 30 min group boob sex with the presence of CLS would be associated with elevated ratios of estrogens to androgens, consistent with elevated aromatase activity. Consenting women completed 30 min group boob sex detailed risk factor questionnaire, donated blood, and provided access to tissues for research.

These cases had not received neoadjuvant treatment and had formalin-fixed paraffin embedded FFPE tissues of benign breast tissue collected per protocol at the time of breast cancer surgery [ 9 ].

Benign breast tissue collected 30 min group boob sex varying 30 min group boob sex from the tumor, and thus physically separated from the tumor, was used for CLS assessment as outlined in Fig.

Tissue hormone levels were measured in fresh grossly homogeneous-appearing breast adipose tissue that was frozen in liquid nitrogen according to protocol until testing as further described below.

30 min group boob sex, frozen fat used for hormone measurements was consumed by the assay and CLS were enumerated in different pieces of tissue from the same breast.

Covariate data included in this analysis were derived from the study interview as previously described [ 15 ]. To evaluate the presence of CLS, sections of designated benign tissues which were collected at varying distances from the tumor at the time of surgery and prepared as FFPE tissue blocks were sectioned and stained for CD68 1: The slides were then rinsed with water, rehydrated with alcohol, cleared in xylene, and mounted for CD68 assessment.

Using the lasso tool ImageScope, Aperioareas of adipose tissue visible on the slide were annotated. Areas affected by a previous biopsy were avoided. All annotated areas were summed to determine the total fat area mm 2 on each slide.

Within the fat area defined by the lasso tool 30 min group boob sex number of cells that showed CDpositive expression was counted. The number of CDpositive cells ImageScope, Aperio was normalized to total fat area mm 2 in the same section.

The presence and number of CLS were also 30 min group boob sex within the observed fat area on the whole tissue slide and defined as the complete encirclement of adipocytes 30 min group boob sex CDpositive macrophages Fig. Methods for measuring hormones in adipose tissue and serum with validation data are described in detail elsewhere [ 89 ]. Briefly, adipose tissue was enzymatically digested with collagenase and then centrifuged to yield oil for hormone measurements.

Partition chromatography using Celite columns was performed to elute sex-steroid hormones from serum and oil. Androstenedione, testosterone, estrone, and estradiol were quantified by radioimmunoasssy RIA [ 8 ]. Frequencies and percentages were used to describe selected characteristics of the study population. Sex-steroid hormone levels from both serum and tissue were transformed to the natural logarithm scale to normalize values.

Relationships between the number of CDpositive macrophages per unit fat area and 30 min group boob sex and clinical characteristics were analyzed using Mann Whitney U and Kruskal-Wallis analyses. Covariates that had a significant association with CLS through chi-square or Kruskal-Wallis tests were adjusted for in multivariable analysis. We examined whether tertiles of hormone levels were associated with the presence 30 min group boob sex CLS using logistic regression, with CLS as the outcome variable and tertiles of each hormone examined as explanatory variables.

Models were also adjusted for fat area, and the results of both unadjusted and adjusted models are presented. Potential effect modification of the association between tissue and blood hormone levels with the presence or absence of CLS was examined by including an interaction term in linear regression models. All analyses were conducted in SAS v9. The distribution of breast cancer risk factors and pathological characteristics of tumor samples are shown in Additional file 1 Table S1.

In univariate analyses, the number of CDpositive macrophages per unit area of fat was not significantly related to patient and clinical characteristics or to 30 min group boob sex levels in serum or adipose tissue Additional file 1: In analyses adjusted for age and fat tissue area, the frequency of CLS-positive cases was significantly higher among obese than normal weight women unadjusted OR 5. Analyses were adjusted for age as CLS were previously found to be associated with menopausal status [ 16 ].

Associations with other patient and tumor characteristics were not statistically significant. In adjusted analyses, estrone levels in adipose tissue were positively associated with the presence of CLS, but only in the middle as compared with the highest estrone tertile adjusted OR T2vsT3 4.

In contrast to the generally null relationships between individual hormone levels and CLS status, ratios of estrogens to precursor androgens were significantly associated with CLS status.

Compared with women whose estrone: In serum, similar relationships to those observed in fat were found in univariate but not in multivariate analyses for the estrone: In addition, in serum, as the ratio of estradiol: Hormone levels and ratios of hormone levels in adipose tissue and serum were positively correlated, but these relationships were not significantly modified by CLS status Fig. The aromatase enzyme is present in adipose tissue throughout the body where it converts androgens to estrogens, providing the main source of postmenopausal estrogens.

Similar to prior reports, we showed that CLS were more frequently found among obese compared with lean women, and therefore 30 min group boob sex have a greater effect on hormone levels among obese women, a group which is known to experience an increased risk of hormone receptor-positive breast cancer [ 101617 ] and adverse breast cancer prognosis [ 17 — 19 ].

Our analysis demonstrates that the presence of CLS in breast adipose tissue of postmenopausal patients with breast cancer is associated with increased ratios of estrogens to androgens in breast fat and serum, consistent with data showing that these microscopic inflammatory lesions are related to increased aromatase activity [ 10 — 12 ].

However, we were unable to directly measure hormones in the same piece of tissue in which CLS was assessed. Thus, we cannot exclude the possibility that there are important changes in hormone levels in tissues immediately surrounding CLS which could alter the microenvironment at the millimeter or submillimeter scale. This association was not statistically significant after adjusting for fat tissue area; therefore, our data do not support evaluating CDpositive cells as a surrogate of CLS.

Although CD68 counts are potentially evaluable by image analysis of stained sections, recognition of CLS was carried out using subjective visual assessment [ 20 ]. Among postmenopausal women, the ovaries and adrenals continue to synthesize androgens which can be converted to biologically active estrogens in peripheral tissues. The regulation of enzymes involved in this process is complex and not well understood. Within breast tissues, multiple mechanisms may affect hormone levels, including uptake from the circulation, inter-conversion of different hormones e.

The percentage of the breast comprised of adipose tissue increases with age, and increased adipose tissue has been associated with increased breast epithelial area [ 2223 ].

The presence of CLS was significantly associated with increased ratios of estrone to 30 min group boob sex in breast adipose tissue independent of age, BMI, and fat area on the slide. Our association between the presence of CLS and higher levels of estrogens 30 min group boob sex to androgens in serum and breast adipose suggests that uptake from the circulation and the local conversion of androgens to estrogens both influence the local hormonal milieu enveloping at-risk epithelium and cancer.

However, CLS status was generally not associated with absolute concentrations of individual hormone levels measured in tissue or blood, although these analyses were limited by small numbers of CLS-positive cases.

Accordingly, it remains uncertain whether the presence of CLS in breast fat implies information about risk for hormone receptor-positive breast cancer beyond that provided by measuring hormone levels in serum.

Furthermore, our findings of no association between CLS and ER status are in agreement with Iyengar and colleagues [ 17 ], and suggest that CLS may also be influencing additional inflammatory mechanisms associated with increased BMI. However, even if detection of CLS does not inform the magnitude of breast cancer risk, it may have implications for understanding mechanisms of hormonal carcinogenesis.

Our results showing no association between the presence of CLS and breast tumor clinical characteristics are largely consistent with prior studies. A recent analysis by Iyengar and colleagues that included a retrospective cohort of women who underwent mastectomy following a breast cancer diagnosis also found no relationship between the presence of CLS and tumor size, grade, and histology [ 17 ].

These findings 30 min group boob sex provide insight into why obese postmenopausal women suffer poorer breast cancer mortality compared with lean postmenopausal women [ 17 — 19 ]. Additional studies are needed to understand the biological importance of CLS in non-obese women, in particular with respect to hormone levels and risk of hormonally 30 min group boob sex cancers [ 17 ]. The focus of our 30 min group boob sex was to further understand the relationship between CLS and sex-steroid hormones.

However, CLS may also have additional roles in breast cancer development and progression other than those addressed in this analysis. For example, in the same study described above, Iyengar and colleagues observed significant associations between CLS and clinical features of metabolic syndrome in addition to obesity including hyperinsulinemia, hyperglycemia, and hypertriglyceridemia [ 17 ].

These findings were observed in two independent patient populations included in their study, suggesting that CLS may also influence breast cancer outcomes through alternative mechanisms. A key strength of our analysis was our ability to relate local hormone levels from the breast fat tissue to CLS presence, though a limitation of this approach was that CLS assessment was not carried out on the same frozen tissue piece from which hormone measurements were obtained.

This is an important limitation because our analysis assumes that hormone levels within the sampled adipose tissue are representative of levels within normal tissue near to the tumor, where CLS were characterized. It is possible that other histological features characteristic of the microenvironment are influencing either the hormonal levels or CLS presence.

Although technically challenging, future efforts comparing CD68 staining of frozen sections and hormonal measurements in the same tissue specimens could prove informative. In our analysis, we did not differentiate between M1 and M2 macrophages, which could have implications for cancer risk or prognosis. M1 macrophages mediate the release of many pro-inflammatory 30 min group boob sex including interleukin 1 beta and tumor necrosis factor-alpha.

M2 macrophages are largely involved in 30 min group boob sex anti-inflammatory [ 24 ]. 30 min group boob sex analysis also relied on visual assessment of CLS. However, more recent applications of novel imaging technologies, including Raman spectroscopy, have successfully detected CLS in ex vivo fresh frozen non-cancerous tissue from mouse models as well as women without cancer, and may enable future objective high-throughput assessments of CLS [ 25 ].

The use of such imaging methods will have important implications for the translation of CLS assessment into a clinical setting [ 25 ]. An additional limitation of our study was the limited sample size that had fresh frozen 30 min group boob sex available for inclusion.

Furthermore, this analysis of women with prevalent invasive carcinoma precluded the separation of etiologic influences from secondary effects related to the presence of 30 min group boob sex. However, the benign breast samples analyzed in this study, which were removed by visual 30 min group boob sex of surgical pathology specimens, were collected from areas that were physically separated from the tumors as outlined in Fig. Future studies examining 30 min group boob sex presence and function of CLS in cancer-free women, especially in the setting of obesity, are needed to determine if CLS are involved in breast cancer development.

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