Santen, Richard J.

Abstract Context Prior reports of plasma estradiol concentrations and their reference ranges in postmenopausal women identified marked variation, in part due to assay method and body mass index (BMI). Objective To develop an international reference range for plasma estradiol in postmenopausal women, support accurate assessment of concentrations during systemic and local (eg, vaginal) estradiol therapy, and improve risk prediction for diseases, including osteoporosis, breast, and endometrial cancers. Methodology Estradiol concentrations were measured using primarily liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays in 5 international cohorts of community-dwelling, postmenopausal women of primarily European ancestry aged 38-100 years who were not using exogenous estrogens (n = 7206). Measurements were harmonized to the Centers for Disease Control and Prevention (CDC) reference measurement procedure (RMP) by re-assaying a subset of samples for each cohort and generating recalibration equations. Reference intervals were determined for all women, non-obese women, and specific BMI categories. Results Estradiol concentrations from separate LC-MS/MS assays correlated closely with the CDC RMP, and harmonization minimized inter-assay variability. Estradiol concentrations correlated with BMI but not with chronological age. The reference range (2.5th-97.5th percentile) was 1.1-18.2 pg/mL (4.0-66.8 pmol/L) with median of 4.9 pg/mL (18.0 pmol/L) for all postmenopausal women and 1.1-12.5 pg/mL (4.0-45.9 pmol/L) with median of 4.1 pg/mL (15.1 pmol/L) for non-obese women (BMI < 30). Reference ranges increased progressively with higher BMI categories. Conclusion Cross-calibration of estradiol measurements with the CDC RP enabled the development of harmonized plasma estradiol reference ranges for postmenopausal women who were not using exogenous estrogens, suitable for future studies to establish clinical utility. Increasing obesity in postmenopausal women was associated with increasing estradiol concentration and BMI category-specific reference ranges.

Abstract Atypical hyperplasia is a high-risk premalignant lesion of the breast, but its biology is poorly understood. Many believe that atypical ductal hyperplasia (ADH) is a direct precursor for low-grade ductal breast cancer, whereas atypical lobular hyperplasia (ALH) serves as a risk indicator. These assumptions underlie current clinical recommendations. We tested these assumptions by studying the characteristics of the breast cancers that develop in women with ADH or ALH. Using the Mayo Benign Breast Disease Cohort, we identified all women with ADH or ALH from 1967 to 2001 and followed them for later breast cancers, characterizing side of breast cancer versus side of atypia; time to breast cancer; type, histology, and grade of breast cancer, looking for patterns consistent with precursors versus risk indicators. A total of 698 women with atypical hyperplasia were followed a mean of 12.5 years; 143 developed breast cancer. For both ADH and ALH, there is a 2:1 ratio of ipsilateral to contralateral breast cancer. The ipsilateral predominance is marked in the first 5 years, consistent with a precursor phenotype for both ADH and ALH. For both, there is a predominance of invasive ductal cancers with 69% of moderate or high grade. Twenty-five percent are node positive. Both ADH and ALH portend risk for ductal carcinoma in situ and invasive breast cancers, predominantly ductal, with two thirds moderate or high grade. The ipsilateral breast is at especially high risk for breast cancer in the first 5 years after atypia, with risk remaining elevated in both breasts long term. ADH and ALH behave similarly in terms of later breast cancer endpoints. Cancer Prev Res; 7(2); 211–7. ©2014 AACR.

Abstract Abstract #4033 Background: Aromatase activity within the breast is a source of estrogen that may cause breast cancer. Mammographic density (MD) is a risk factor for breast cancer whose biologic basis is unknown. Our study compared aromatase expression in tissue from dense and non-dense areas of the breasts of healthy volunteers.
 Methods: Participants were 40+ yrs, had a screening mammogram with visible MD, no history of cancer and were not on endocrine therapy. Ultrasound-guided core biopsies were done within 6 months of mammography to obtain three paired cores from mammographically dense and non-dense regions of the breast. Immunostaining for aromatase expression employed the streptavidin-biotin amplification method using the recently developed 677 mouse monoclonal antibody. Immunoreactivity (IR) was scored in terms of proportion of cells staining positive for aromatase (PPC) (0=<1%, 1=1-25%, 2=26-50%, 3=51+% (and 4=75%+ for adipocytes)) and relative immunointensity (0=none, 1=weak, 2=moderate, 3=intense) for each cell type (stroma, normal ductal epithelium, adipocytes). A composite score weighting the PPC by their relative intensity (range 0-9; 0-12 for adipocytes) was also calculated. The sum of the composite score across the three cell types provided a global assessment of aromatase IR. Repeated measures analyses evaluated differences in aromatase IR for dense compared to non-dense tissue within and across cell types. Parameter estimates (β) indicated the average difference between dense and non-dense IR.
 Results: 18 (37%) of the 49 participants were premenopausal (median age 46 yrs). Summing across cell types, the global composite score showed increased aromatase IR on sections sampled from dense vs. non-dense regions (β=5.3,p<0.001). This global difference reflected increased aromatase IR in the stroma and normal ductal epithelium from dense sections (vs. non-dense). For 42 women with >1% stromal cells on each section, there was evidence for increased IR on dense sections as indicated by the greater PPCs (β=0.9), relative intensity (β=0.7) and composite score (β=2.7)(p's<0.001). Similarly, for 20 women with >1% normal ductal epithelium in both dense and non-dense sections, there was a greater composite score (β=1.4,p=0.004) for cells on dense sections although differences for PPC (β=0.3,p=0.11) and relative intensity (β=0.3,p=0.09) did not reach statistical significance. No differences were seen in IR for adipocytes from the two density regions (46 women). Findings were unchanged with adjustment for covariates, including proportions of each cell type per section.
 Conclusions: There is strong evidence for increased aromatase expression in the stromal and normal ductal epithelium of dense vs. non-dense tissue. These results support ongoing research into mammographic density as a biomarker of effect of aromatase inhibitors. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4033.

Abstract The majority of candidates for breast cancer prevention have not accepted tamoxifen because of the perception of an unfavorable risk/benefit ratio and the acceptance of raloxifene remains to be determined. One means of improving this ratio is to identify women at very high risk of breast cancer. Family history, age, atypia in a benign biopsy, and reproductive factors are the main parameters currently used to determine risk. The most powerful risk factor, mammographic density, is not presently employed routinely. Other potentially important factors are plasma estrogen and androgen levels, bone density, weight gain, age of menopause, and fracture history, which are also not currently used in a comprehensive risk prediction model because of lack of prospective validation. The Breast Cancer Prevention Collaborative Group (BCPCG) met to critically examine and prioritize risk factors that might be selected for further testing by multivariate analysis using existing clinical material. The BCPCG reached a consensus that quantitative breast density, state of the art plasma estrogen and androgen measurements, history of fracture and height loss, BMI, and waist–hip ratio had sufficient priority for further testing. As a practical approach, these parameters could be added to the existing Tyrer–Cuzick model which encompasses factors included in both the Claus and Gail models. The BCPCG analyzed potentially available clinical material from previous prospective studies and determined that a large case/control study to evaluate these new factors might be feasible at this time.