Tissue Microarray-Based Digital Spatial Profiling of Benign Breast Lobules and Breast Cancers: Feasibility, Biological Coherence, and Cross-Platform Benchmarks.

Background: Biopsy diagnoses of benign breast disease (BBD) confer a 1.5- to 4-fold increased risk of developing breast cancer (BC) compared with women without BBD. Previously, we reported that decreased numbers of specific immune cell types in lobules of BBD biopsies predicted increased BC risk, suggesting the promise of future tissue biomarker studies to define BC risk markers among BBD patients. Thus, we applied protein-based GeoMx® Digital Spatial Profiling (DSP) to BBD biopsies preceding BC (cases) and to BBD biopsies from cancer-free patients (controls) to identify possible BC risk markers, which we then evaluated in subsequent BC tissues and in surrounding normal lobules of cases. Methods: Archived pathology slides of BBD biopsies were reviewed and used to guide preparation of TMAs containing 1.0-mm diameter FFPE cores of lobules from an age- and cohort-period-matched set of 91 cases and 88 controls from the Mayo Clinic BBD Cohort. For patients who later developed BC, we prepared TMAs of BC tissue and surrounding mapped normal lobules. We applied GeoMx® DSP (immune and canonical signaling proteins) to both sets of TMAs. Following QC and data normalization, associations of case status with log-transformed biomarker expression in lobules of BBD biopsies were carried out using linear mixed modeling approaches, accounting for multiple ROIs per individual. Biomarkers significantly associated with case status (p<0.05) were further examined in BCs and adjacent normal lobules, using similar approaches. Results: The mean age at BBD biopsy was 52 years, and at BC diagnosis of cases, 61.4 years (mean time from BBD to BC was 10.2 years). A family history of BC was more frequent among cases (70% versus 43%; chi-square p=0.002). Of 72 biomarkers tested, 46 (64.4%) were evaluable after QC and normalization and 5 were associated with BC risk after adjustment for family history of BC: BCL2 (p=0.005), STING (p=0.006), CD44 (p=0.02), S100 protein (p=0.03) and pan-AKT (p=0.05); each showed higher levels in lobules of BBD biopsies of controls than cases. Three unique patterns appeared when examining these biomarkers across tissue type within cases: for BCL2 (p=5 x 10-9) and STING (p=2 x 10-19), levels were high in both BC and lobules surrounding BC but low in preceding BBD; for GAPDH (p=4 x 10-53) and pan-AKT (p=2 x 10-33), levels were high in BC, low in preceding BBD, and moderate in lobules surrounding BC; and for CD44 (p=2 x 10-6) and S100B (p=2 x 10-49), levels were low in BC, high in lobules surrounding BC and moderate in preceding BBD. Conclusions: Using a novel TMA of lobules in combination with DSP, we preliminarily identified immune-based and PI3 kinase-related protein biomarkers in BBD biopsies associated with BC risk. In case-only analyses, these markers demonstrated complex differences between lobules in BBD biopsies, subsequent BCs and adjacent normal lobules. Citation Format: Robert Alan Vierkant, Jodi M. Carter, Stacey J. Winham, Chen Wang, Jennifer M. Kachergus, Ji Shi, Raymond M. Moore, Bryan M. McCauley, Laura M. Pacheco-Spann, E A. Thompson, Derek C. Radisky, Amy C. Degnim, Mark E. Sherman. Towards prediction of breast cancer risk in benign biopsies with high-plex GeoMx spatial protein profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2203.

Purpose: Breast biopsies with benign findings (benign breast disease [BBD]) outnumber biopsies revealing in-situ or invasive cancer 4:1, providing an opportunity to identify histopathological features that predict breast cancer risk. Greater degrees of involution of terminal duct lobular units (TDLUs), the structures within the breast that produce milk and the primary source of breast cancer precursors, have been inversely associated with breast cancer risk among women with BBD. We recently developed measures to quantitate levels of TDLU involution, which demonstrate high inter/intra pathologist reproducibility. Here we assessed whether TDLU counts/100mm2 and median TDLU span (microns), two measures inversely related to degree of TDLU involution, are associated with subsequent breast cancer risk among women with BBD. Methods: From the Mayo BBD cohort (n = 9,376), we evaluated benign biopsies from 99 women who later developed breast cancer (cases) and 145 age-matched controls who did not develop breast cancer. Digitized images of biopsy sections were reviewed to enumerate TDLUs/mm2 and measure median TDLU span (microns) for up to ten normal TDLUs. Breast cancer risk factors were available from questionnaires or medical records. To assess associations with breast cancer risk, subjects were categorized into quartile levels of TDLU counts and median TDLU spans, based on data from controls. Odds ratios (ORs) and 95% confidence intervals (CI) were estimated using multivariable conditional logistic regression models, adjusted for histologic type of BBD, family history of breast cancer and menopausal hormone use. Results: Compared to controls, women who developed breast cancer had higher median number of TDLU counts/100mm2 (28 vs 20, p = 0.03) and larger TDLU spans (300 vs 267 microns, p = 0.14). In multivariable models higher TDLU counts (quartile4 vs. quartile1, OR = 2.44, 95%CI = 0.96-6.19, ptrend = 0.02) and larger TDLU span measures (quartile4 vs. quartile1, OR = 2.83, 95%CI = 1.13-7.06, ptrend = 0.03) were associated with subsequent diagnosis of breast cancer. Combinatorial metrics of TDLU counts with median TDLU span measures identified women at higher risk; specifically, women above the median for both TDLU span and TDLU counts had an OR = 3.75 (95%CI = 1.40-10.00, ptrend = 0.008), compared with women below the median for TDLU span and TDLU counts. Conclusion: These data show that lack of TDLU involution, as measured by increased persistence of TDLU counts, larger median TDLU spans and cross-classification using these measures was associated with increased breast cancer risk among women with BBD, extending prior work in this cohort based on other TDLU involution metrics. Future studies to identify determinants of TDLU involution, its association with breast cancer risk and its potential as an intermediate endpoint in prevention studies warrant consideration. Citation Format: Jonine D. Figueroa, Ruth Pfeiffer, Maya Palakal, Amy C. Degnim, Derek Radisky, Lynn C. Hartmann, Marlene Frost, Melody L. Stallings Mann, Louise A. Brinton, Daphne Papathomas, Daniel Visscher, Mark E. Sherman. Standardized measures of lobular involution and subsequent breast cancer risk among women with benign breast disease. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4682. doi:10.1158/1538-7445.AM2015-4682

Background Sex steroid hormone signaling is critical in the development and progression of breast cancers, though the role of androgens remains unspecified. Large epidemiologic studies have found a consistent association between circulating androgens and increased breast cancer risk, though it is unknown whether circulating androgens reflect the androgenic milieu in the breast. An interaction between androgen receptor (AR) and estrogen receptor (ER) signaling in the breast has been postulated, wherein AR signaling antagonizes ER signaling in estrogen-rich environments, and AR signaling induces proliferative effects in estrogen-deplete environments. Methods We evaluated the association between AR expression and subsequent breast cancer risk in a nested case-control study of women with benign breast disease (BBD) within the Nurses’ Health Studies. Cases were women with BBD that subsequently developed breast cancer (median 9 years later) while controls had BBD but did not develop breast cancer. Tissue microarrays were constructed containing normal terminal ductal lobular unit (TDLU) tissue from the BBD biopsy. AR expression was assessed by immunohistochemistry and the percent of positive-staining nuclei was digitally quantified for 61 breast cancer cases and 184 controls. Logistic regression models adjusting for the year of BBD biopsy, age at cancer diagnosis, years since BBD biopsy, and BBD lesion type were used to calculate odds ratios and 95% confidence intervals for the association between the tertile of AR expression and breast cancer risk. We further evaluated the impact of AR and ER co-expression, each dichotomized at the median, in a sub-analysis of 31 cases and 82 controls. Finally, we assessed AR expression as a predictor of subsequent ER tumor status using polytomous logistic regression. Results Overall, women in the highest tertile of AR expression experienced non-significant 1.32-fold increased odds of breast cancer (95% CI: 0.64-1.73, p-trend = 0.559) compared to the lowest tertile. A significant interaction was detected between AR and ER co-expression in normal breast TDLUs and subsequent breast cancer risk (p-interaction = 0.003). Among women with low ER expression, increased AR expression was associated with 2.52-fold increased odds (95% CI: 0.68-9.34) of developing breast cancer. In contrast, among women with high ER expression, high AR expression was associated with a 91% decrease in the odds (OR = 0.09, 95% CI: 0.01-0.62) of breast cancer. AR expression was not predictive of subsequent ER tumor status. Conclusions There was little evidence for an overall association between AR expression in normal breast tissue and breast cancer risk, though we observed a significant interaction between AR and ER expression. Our findings support the hypothesis that AR interacts with ER to promote cell proliferation in estrogen-deprived environments and inhibit growth in estrogen-rich environments. Citation Format: Kevin Kensler, Andrew Beck, Francisco Beca, Laura Collins, Stuart Schnitt, Aditi Hazra, Susan Hankinson, Myles Brown, Rulla Tamimi. Androgen receptor expression in normal breast TDLUs and subsequent breast cancer risk. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4295.

Lesser degrees of terminal duct-lobular unit (TDLU) involution predict higher breast cancer risk; however, standardized measures to quantitate levels of TDLU involution have only recently been developed. We assessed whether three standardized measures of TDLU involution, with high intra/inter pathologist reproducibility in normal breast tissue, predict subsequent breast cancer risk among women in the Mayo benign breast disease (BBD) cohort. We performed a masked evaluation of biopsies from 99 women with BBD who subsequently developed breast cancer (cases) after a median of 16.9years and 145 age-matched controls. We assessed three metrics inversely related to TDLU involution: TDLU count/mm(2), median TDLU span (microns, which approximates acini content), and median category of acini counts/TDLU (0-10; 11-20; 21-30; 31-50; >50). Associations with subsequent breast cancer risk for quartiles (or categories of acini counts) of each of these measures were assessed with multivariable conditional logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CI). In multivariable models, women in the highest quartile compared to the lowest quartiles of TDLU counts and TDLU span measures were significantly associated with subsequent breast cancer diagnoses; TDLU counts quartile4 versus quartile1, OR=2.44, 95%CI 0.96-6.19, p-trend=0.02; and TDLU spans, quartile4 versus quartile1, OR=2.83, 95%CI=1.13-7.06, p-trend=0.03. Significant associations with categorical measures of acini counts/TDLU were also observed: compared to women with median category of <10 acini/TDLU, women with >25 acini counts/TDLU were at significantly higher risk, OR=3.40, 95%CI 1.03-11.17, p-trend=0.032. Women with TDLU spans and TDLU count measures above the median were at further increased risk, OR=3.75 (95%CI 1.40-10.00, p-trend=0.008), compared with women below the median for both of these metrics. Similar results were observed for combinatorial metrics of TDLU acini counts/TDLU, and TDLU count. Standardized quantitative measures of TDLU counts and acini counts approximated by TDLU span measures or visually assessed in categories are independently associated with breast cancer risk. Visual assessment of TDLU numbers and acini content, which are highly reproducible between pathologists, could help identify women at high risk for subsequent breast cancer among the million women diagnosed annually with BBD in the US.

Background: Biological activity, including potential carcinogenic effects, of estrogen and progesterone in breast tissue is primarily mediated by their receptors in the tissue. Ki67 is a marker of cell cycle activation. We examined the associations of estrogen receptor (ER), progesterone receptor (PR), and Ki67 expression in normal breast tissue from benign biopsies with subsequent breast cancer risk. Methods: We conducted an analysis among 385 women (90 cases, 295 controls) with benign breast disease (BBD) in a nested case-control study within the Nurses’ Health Study (NHS) and the NHSII. Tissue microarrays (TMA) were constructed using cores obtained from benign biopsies containing normal terminal duct lobular units (TDLU). Immunohistochemical staining for ER, PR, and Ki67 was performed on sections cut from the TMAs. Staining results were interpreted by computational image analysis which scored the percentage of positively stained cells for each marker. Unconditional logistic regression models, adjusting for matching factors and benign lesion subtype, were used to estimate odds ratios (OR) for developing subsequent breast cancer by tertiles of marker expression. Results: ER and Ki67 expression (highest vs. lowest tertiles) in normal breast TDLUs was not significantly associated with subsequent breast cancer risk (≥14.7 vs. &amp;lt;7.3% ER-positive cells: OR = 0.55, 95% CI = 0.21-1.44, p-trend = 0.85; ≥6.2 vs. &amp;lt; 2.4% Ki67-positive cells: OR = 1.75, 95% CI = 0.87-3.50, p-trend = 0.15). PR expression was suggestively positively associated with breast cancer risk (≥9 vs. &amp;lt;4%: OR = 2.08, 95% CI = 1.00-4.31, p-trend = 0.06); the positive association was significant among women who were premenopausal at BBD biopsy (OR = 3.55, 95% CI = 1.28-9.87, p-trend = 0.03). Conclusion: PR expression in normal breast tissue was significantly positively associated with subsequent breast cancer risk in premenopausal women. Although we did not observe significant results with ER and Ki67, we cannot exclude associations given the limited power in this study. These findings may contribute to understanding of breast cancer biology and may suggest new targets for breast cancer risk assessment and prevention. However, further studies are required to confirm these results. Citation Format: Hannah Oh, A Heather Eliassen, Molin Wang, Stephanie A. Smith-Warner, Andrew H. Beck, Stuart J. Schnitt, Laura C. Collins, James L. Connolly, Laleh Montaser-Kouhsari, Rulla M. Tamimi. Expression of estrogen receptor, progesterone receptor, and ki67 in normal breast tissue and subsequent risk of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-179. doi:10.1158/1538-7445.AM2015-LB-179

The growth hormone and insulin-like growth factor (IGF) axis plays an essential role in the growth and development of the mammary gland. More recently it has become evident that IGF1 and IGF1 receptor (IGF1R) may play a role in the early transformation of mammary cells. Using a nested case-control design, we examined the association between IGF1R expression in normal breast tissue from benign biopsies and subsequent risk of breast cancer within the Nurses' Health Study. We constructed tissue microarrays (TMAs) containing normal terminal ductal lobular units (TDLUs) from benign breast biopsies. Immunostains for IGF1R were performed on sections cut from the TMAs. A total of 312 women had both normal TDLUs included in the TMAs and evaluable IGF1R staining; 75 subsequently developed breast cancer (cases) and 237 did not (controls). The epithelial cells in the normal TDLUs were scored for both cytoplasmic and membranous staining for IGF1R. Membranous IGF1R expression in normal TDLU epithelial cells was inversely associated with subsequent breast cancer risk (OR= 0.49; 95%CI 0.26-0.93) independent of type of benign breast disease (BBD). In contrast, the presence of cytoplasmic IGF1R expression was positively associated with subsequent risk of breast cancer (OR=2.88, 95% CI 1.60-5.19). Women in which TDLU epithelial cells showed little or no membrane expression of IGF1R but high levels of cytoplasmic IGF1R were at the highest breast cancer risk and were 15 times more likely to develop subsequent breast cancer when compared with women who had little or no membranous or cytoplasmic IGF1R expression in their TDLU epithelium (OR=15.1, 95% CI 3.2-70.2). It has been demonstrated that stimulation of cells with IGF1 results in altered localization of IGF1R expression. Upon stimulation, IGF1R levels on the cell membrane decrease while internalized levels increase. Studies of other receptor tyrosine kinases indicate that chronic stimulation by the ligand results in increased internalization of the receptor. In this study, IGF1R expression patterns in the epithelial cells of normal TDLUs were associated with an increased risk of subsequent breast cancer. The results of this study support the hypothesis that the IGF1 pathway plays an important role in the early development of breast cancer and suggest that there may be opportunities for breast cancer chemoprevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2782.

Background: Terminal duct lobular units (TDLUs) are the anatomical source of most breast cancers. TDLU involution, manifested as atrophy and loss of TDLUs, is associated with reduced breast cancer risk. We hypothesize that breast cancer susceptibility loci are associated with TDLU involution. Accordingly, we evaluated relationships between 25 established breast cancer susceptibility markers and their association with TDLU involution in the Susan G. Komen for the Cure® Tissue Bank (KTB). Methods: Subjects were women ages 18-84 (N=680), who had no personal history of any cancer, were not currently taking menopausal hormones, completed a risk factor questionnaire, provided an Oragene® saliva sample, and donated a 10-guage needle tissue core from the upper outer breast quadrant for research. We genotyped 25 known single nucleotide polymorphisms (SNPs) associated with breast cancer risk using TaqMan® assays. Pathologists masked to risk data classified three features inversely related to TDLU involution using one histological section of breast tissue/subject: number of TDLUs per section (TDLU density), TDLU diameters and number of acini per TDLU. SNPS were compared to metrics of TDLU involution categorized in tertiles: TDLU density (0-1, 2-8, 9+), mean TDLU diameter (53-291, 292-415, 416-1143 microns), and mean number of acini per TDLU (2-10, 11-20, 21+). We estimated age-adjusted per-allele odds ratios (ORs) and 95% confidence intervals (CIs) using ordinal logistic regression models with TDLU measures as the outcome and SNPs as the explanatory variables. Associations with TDLU density were based on up to 680 women with genotype data, while associations with diameter and acini measures were based on up to 469 women with observed TDLUs. Results: The rs3803662 (TOX3/TNRC9) SNP risk allele showed an association with increased TDLU density, larger TDLUs, and more acini in TDLUs (TDLU density per-allele OR=1.29 95%CI=1.04-1.60; TDLU diameter OR=1.32 95%CI=1.00-1.74; and TDLU acini OR=1.34 95%CI=1.02-1.77), consistent with previous observations of significant associations seen for this SNP with mammographic density (a well-established risk factor for breast cancer), breast cancer risk, and survival of breast cancer. In addition, we observed associations for four other SNPs with TDLU density (rs311499, rs10483813, rs2981582, rs3817198), for one other SNP with mean TDLU diameter (rs11249433), and for three other SNPs with mean TDLU acini number (rs704010, rs10941679, rs11249433). Conclusion: These data suggest that breast cancer susceptibility SNPs may be associated with TDLU involution levels, and could represent a mechanism by which they influence breast cancer risk. These findings warrant replication in other studies and provide support for the concept that levels of TDLU involution represent an intermediate marker of breast cancer risk. Citation Format: Jonine D. Figueroa, Gretchen L. Gierach, Ruth Pfeiffer, Stephen Chanock, Louise Brinton, Deesha A. Patel, Daniel Visscher, Carolyn Mies, Stephen Hewitt, Susan Clare, Anna Maria Storniolo, Mark Sherman. Associations between breast cancer susceptibility markers and terminal duct lobular unit involution in normal breast tissues among women from the Susan G. Komen for the Cure® Tissue Bank. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 58.

Introduction: High mammographic breast density (MD) and reduced levels of terminal duct lobular unit (TDLU) involution (the histologic source of most breast cancers) have been associated with increased risk of developing breast cancer. Data relating MD and TDLU involution to breast cancer characteristics and outcomes are sparse. Therefore, we assessed these relationships among women with invasive ER-positive breast cancer. Methods: The analysis focused on women with ER-positive breast cancers who were diagnosed at Kaiser Permanente Northwest (1990-2008) and followed through the end of 2010. Cases comprised of those who died of breast cancer (n = 54) and controls those that did not die of breast cancer (n = 180) over similar follow-up. Three reproducible measures that are inversely related to TDLU involution were evaluated in digitized hematoxilin and eosin stained sections in benign breast tissues surrounding the tumors: TDLU counts per unit area, TDLU span and median number of acini per TDLU. Percentage MD was estimated from digitized mammograms using computer-assisted thresholding software (Cumulus). Univariate associations between TDLU measurements and patient characteristics, tumor size, and disease stage at diagnosis, were calculated using Mann Whitney Wilcoxon rank test. TDLU measurements were related to baseline MD using analysis of covariance models and adjusted for age, body mass index (BMI), tumor size, stage, year of diagnosis and smoking. Results: TDLUs were observed in 95% of cases and 89% of controls. All TDLU measurements declined with age (p&amp;lt;0.001 for each TDLU measurement). Among cases, TDLU measures were not significantly associated with tumor characteristics. Controls with regional spread had greater TDLU span (p = 0.05) and median acini counts per TDLU (p = 0.03) than those with localized disease; these TDLU metrics also showed a borderline significant association with larger tumor size (&amp;gt;2cm) (p = 0.07 and p = 0.06, respectfully). All TDLU measures were associated with MD among controls (TDLU count: p = 0.04; TDLU span: p = 0.06; median acini count per TDLU: p = 0.01), whereas among cases only, TDLU span showed a significant association MD (p = 0.003). Conclusion: Preliminarily, our data suggest that among women with non-fatal ER-positive breast cancers, TDLU involution was associated with localized tumor stage, size and MD, whereas these relationships were less evident among women who died of their disease. Ongoing analyses will determine whether measures of MD and TDLU involution are independent predictors of breast cancer outcomes in this patient population. Citation Format: Maeve Mullooly, Sarah J. Nyante, Ruth M. Pfeiffer, Renata Cora, Jonine D. Figueroa, Robert N. Hoover, Andrew G. Glass, Erin J. Aiello Bowles, Louise A. Brinton, Amy Berrington de Gonzalez, Sherman E. Mark, Gretchen L. Gierach. Relationship between mammographic breast density and measures of terminal duct lobular unit involution among women diagnosed with estrogen receptor positive breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4283.

Background: Terminal duct lobular units (TDLUs) are the structures in the breast that give rise to most breast cancers. Previous work has shown that TDLU involution is inversely associated with TDLU metrics, such as TDLU count/100mm2, TDLU span (μm), and number of acini/TDLU, and that these metrics may be elevated in the background normal breast tissue of women diagnosed with triple-negative (TN) compared with luminal A breast tumors. However, it is unknown if this relationship exists in black women, who have the highest incidence of TN breast cancer as well as the highest overall breast cancer mortality rate. We sought to determine the relationships of quantitative measures of TDLU involution with breast cancer molecular subtype among participants in the Black Women’s Health Study. Methods: We digitized hematoxylin and eosin stained normal adjacent tissues from TN (estrogen receptor negative (ER), progesterone receptor negative, and human epidermal growth factor 2 (HER2) negative; n=67) and luminal A (ER positive and HER2 negative; n=162) breast cancer cases from the Black Women’s Health Study. We used logistic regression to evaluate associations between TDLU metrics and breast cancer subtype (TN vs. luminal A), with adjustment for age and body mass index. We performed ordinal logistic regression to evaluate relationships between population and clinical characteristics and TDLU metrics. Results: Among the 229 breast cancer cases, mean age at diagnosis was 53.7 years; 68.7% of TN and 54.3% of luminal A cases were under 55 years of age. Most women had a body mass index (BMI) &amp;gt;30kg/m2, were parous, did not smoke, and did not have a family history of breast cancer. The odds of TN breast cancer were elevated for the second and third tertiles of TDLU count relative to the first tertile, with odds ratios (95% confidence interval) of 2.89 (1.11, 4.86) and 1.92 (0.93, 4.08), respectively. Similarly, the odds of TN breast cancer increased with increasing tertiles of median TDLU span, with odds ratios of 2.25 (1.06, 4.91) and 2.38 (1.14, 5.15) for the second and third tertiles, respectively, compared to the first tertile. These associations persisted even after adjustment for age and BMI. No association was observed with median acini count/TDLU and TN breast cancer. We also observed significant associations of some breast cancer risk factors with measures of TDLU involution. Higher TDLU count was associated with younger age, more physical activity, lower BMI, current use of oral contraceptives or menopausal hormones, and premenopausal status. Conclusion: The associations of TDLU metrics with breast cancer subtype observed in this population of black women are consistent with previous studies of white and Asian women, with reduced TDLU involution in TN breast cancers compared with luminal A. Further investigation is needed to understand the factors that influence TDLU involution and the mechanisms that mediate TDLU involution and breast cancer subtype. Citation Format: Brittny C Davis Lynn, Renata Cora, Ruth M Pfeiffer, Traci N Bethea, Gary Zirpoli, Julie R Palmer, Gretchen L Gierach. Associations between quantitative measures of TDLU involution and breast tumor molecular subtypes among breast cancer cases in the Black Women’s Health Study [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A080.

BackgroundTerminal duct lobular units (TDLUs) are the primary structures from which breast cancers and their precursors arise. Decreased age-related TDLU involution and elevated mammographic density are both correlated and independently associated with increased breast cancer risk, suggesting that these characteristics of breast parenchyma might be linked to a common factor. Given data suggesting that increased circulating levels of insulin-like growth factors (IGFs) factors are related to reduced TDLU involution and increased mammographic density, we assessed these relationships using validated quantitative methods in a cross-sectional study of women with benign breast disease.MethodsSerum IGF-I, IGFBP-3 and IGF-I:IGFBP-3 molar ratios were measured in 228 women, ages 40-64, who underwent diagnostic breast biopsies yielding benign diagnoses at University of Vermont affiliated centers. Biopsies were assessed for three separate measures inversely related to TDLU involution: numbers of TDLUs per unit of tissue area (“TDLU count”), median TDLU diameter (“TDLU span”), and number of acini per TDLU (“acini count”). Regression models, stratified by menopausal status and adjusted for potential confounders, were used to assess the associations of TDLU count, median TDLU span and median acini count per TDLU with tertiles of circulating IGFs. Given that mammographic density is associated with both IGF levels and breast cancer risk, we also stratified these associations by mammographic density.ResultsHigher IGF-I levels among postmenopausal women and an elevated IGF-I:IGFBP-3 ratio among all women were associated with higher TDLU counts, a marker of decreased lobular involution (P-trend = 0.009 and <0.0001, respectively); these associations were strongest among women with elevated mammographic density (P-interaction <0.01). Circulating IGF levels were not significantly associated with TDLU span or acini count per TDLU.ConclusionsThese results suggest that elevated IGF levels may define a sub-group of women with high mammographic density and limited TDLU involution, two markers that have been related to increased breast cancer risk. If confirmed in prospective studies with cancer endpoints, these data may suggest that evaluation of IGF signaling and its downstream effects may have value for risk prediction and suggest strategies for breast cancer chemoprevention through inhibition of the IGF system.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0678-4) contains supplementary material, which is available to authorized users.

Little is known about the effects of many recognized breast cancer risk factors on the normal breast. We hypothesize that risk factors affect the number and morphometry of terminal ductal lobular units (TDLUs), the microanatomical structure from which most breast cancers arise. To assess this hypothesis, we evaluated relationships between breast cancer risk factors and TDLU number, TDLU diameter, and acini per TDLU in breast tissue donated by healthy volunteers. Subjects included 786 healthy women ages 18-84 years without a personal history of any cancer, who provided a risk factor questionnaire and donated a 10-guage needle core from the upper outer breast quadrant. A pathologist, masked to subject data, assessed TDLU number per section, TDLU diameter and acini per TDLU using digitized images of hematoxylin and eosin stained sections of tissue. Seventy-two randomly selected images were examined by two additional pathologists. We generated ordered categorical variables for the number of TDLUs per section (0-1, 2-8, 9+), median TDLU diameter (53-262, 263-377, 378-1375 microns) and median number of acini per TDLU (2-10, 11-20, 21+). We computed Spearman correlations between measures, and intraobserver and interobserver agreement. Associations between breast cancer risk factors and TDLU measures were assessed by estimating odds ratios (OR) and 95% confidence intervals (CI) using ordinal logistic regression models, adjusted for age. TDLU diameter and acini count were highly correlated (rho =0.71); TDLU number per section was less strongly related to the diameter (rho= 0.18) and acini measures (rho=0.20). Inter and intraobserver agreement for TDLU measures were highly correlated (rho range 0.68-0.98). All TDLU measurements were significantly lower among women age 50 years and older compared with younger women. Compared with Caucasians, African American women had more TDLUs per section (ORadj=2.03, 95% CI= 1.06-3.89). Greater numbers of TDLUs were also related to having a family history of two or more relatives with breast cancer compared with none (ORadj=2.01, 95% CI=0.91-4.46); and for parous compared with nulliparous women (ORadj=1.61, 95% CI=1.16-2.25). In contrast, parity was associated with reduced median number of acini per TDLU (ORadj=0.60, 95% CI = 0.40-0.90) and median diameter (ORadj=0.64, 95% CI = 0.42-0.96) compared with nulliparity. Our data suggest that TDLUs are more frequent among women with a positive family history of breast cancer or African Americans. Parity, an established protective factor for breast cancer risk, was related to increased numbers of TDLUs, but fewer acini per TDLU, suggesting that TDLUs may increase with pregnancy and then involute. Our data, along with published results linking TDLU involution with a reduction in breast cancer risk, suggest TDLU morphometry may reflect early biologic changes marking breast cancer susceptibility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4465. doi:1538-7445.AM2012-4465

&lt;div&gt;Abstract&lt;p&gt;Delayed terminal duct lobular unit (TDLU) involution is associated with elevated mammographic breast density (MD). Both are independent breast cancer risk factors among women with benign breast disease (BBD). Prior digital analyses of normal breast tissues revealed that epithelial nuclear density (END) and TDLU involution are inversely correlated. Accordingly, we examined associations of END, TDLU involution, and MD in BBD clinical biopsies. This study included digitized images of 262 representative image-guided hematoxylin and eosin–stained biopsies from 224 women diagnosed with BBD, enrolled within the cross-sectional BREAST-Stamp project that were visually assessed for TDLU involution (TDLU count/100 mm&lt;sup&gt;2&lt;/sup&gt;, median TDLU span and median acini count per TDLU). A digital algorithm estimated nuclei count per unit epithelial area, or END. Single X-ray absorptiometry of prebiopsy ipsilateral craniocaudal digital mammograms measured global and localized MD surrounding the biopsy region. Adjusted ordinal logistic regression models assessed relationships between tertiles of TDLU and END measures. Analysis of covariance examined mean differences in MD across END tertiles. TDLU measures were positively associated with increasing END tertiles [TDLU count/100 mm&lt;sup&gt;2&lt;/sup&gt;, OR&lt;sub&gt;T3vsT1&lt;/sub&gt;: 3.42, 95% confidence interval (CI), 1.87–6.28; acini count/TDLU&lt;sub&gt;T3vsT1&lt;/sub&gt;, OR: 2.40, 95% CI, 1.39–4.15]. END was significantly associated with localized, but not, global MD. Relationships were most apparent among patients with nonproliferative BBD. These findings suggest that quantitative END reflects different but complementary information to the histologic information captured by visual TDLU and radiologic MD measures and merits continued evaluation in assessing cellularity of breast parenchyma to understand the etiology of BBD.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Delayed terminal duct lobular unit (TDLU) involution is associated with elevated mammographic breast density (MD). Both are independent breast cancer risk factors among women with benign breast disease (BBD). Prior digital analyses of normal breast tissues revealed that epithelial nuclear density (END) and TDLU involution are inversely correlated. Accordingly, we examined associations of END, TDLU involution, and MD in BBD clinical biopsies. This study included digitized images of 262 representative image-guided hematoxylin and eosin–stained biopsies from 224 women diagnosed with BBD, enrolled within the cross-sectional BREAST-Stamp project that were visually assessed for TDLU involution (TDLU count/100 mm&lt;sup&gt;2&lt;/sup&gt;, median TDLU span and median acini count per TDLU). A digital algorithm estimated nuclei count per unit epithelial area, or END. Single X-ray absorptiometry of prebiopsy ipsilateral craniocaudal digital mammograms measured global and localized MD surrounding the biopsy region. Adjusted ordinal logistic regression models assessed relationships between tertiles of TDLU and END measures. Analysis of covariance examined mean differences in MD across END tertiles. TDLU measures were positively associated with increasing END tertiles [TDLU count/100 mm&lt;sup&gt;2&lt;/sup&gt;, OR&lt;sub&gt;T3vsT1&lt;/sub&gt;: 3.42, 95% confidence interval (CI), 1.87–6.28; acini count/TDLU&lt;sub&gt;T3vsT1&lt;/sub&gt;, OR: 2.40, 95% CI, 1.39–4.15]. END was significantly associated with localized, but not, global MD. Relationships were most apparent among patients with nonproliferative BBD. These findings suggest that quantitative END reflects different but complementary information to the histologic information captured by visual TDLU and radiologic MD measures and merits continued evaluation in assessing cellularity of breast parenchyma to understand the etiology of BBD.&lt;/p&gt;&lt;/div&gt;

Delayed terminal duct lobular unit (TDLU) involution is associated with elevated mammographic breast density (MD). Both are independent breast cancer risk factors among women with benign breast disease (BBD). Prior digital analyses of normal breast tissues revealed that epithelial nuclear density (END) and TDLU involution are inversely correlated. Accordingly, we examined associations of END, TDLU involution, and MD in BBD clinical biopsies. This study included digitized images of 262 representative image-guided hematoxylin and eosin-stained biopsies from 224 women diagnosed with BBD, enrolled within the cross-sectional BREAST-Stamp project that were visually assessed for TDLU involution (TDLU count/100 mm2, median TDLU span and median acini count per TDLU). A digital algorithm estimated nuclei count per unit epithelial area, or END. Single X-ray absorptiometry of prebiopsy ipsilateral craniocaudal digital mammograms measured global and localized MD surrounding the biopsy region. Adjusted ordinal logistic regression models assessed relationships between tertiles of TDLU and END measures. Analysis of covariance examined mean differences in MD across END tertiles. TDLU measures were positively associated with increasing END tertiles [TDLU count/100 mm2, ORT3vsT1: 3.42, 95% confidence interval (CI), 1.87-6.28; acini count/TDLUT3vsT1, OR: 2.40, 95% CI, 1.39-4.15]. END was significantly associated with localized, but not, global MD. Relationships were most apparent among patients with nonproliferative BBD. These findings suggest that quantitative END reflects different but complementary information to the histologic information captured by visual TDLU and radiologic MD measures and merits continued evaluation in assessing cellularity of breast parenchyma to understand the etiology of BBD.

Introduction: Terminal duct lobular unit (TDLU) involution, an age-related physiological process, is characterized by the reduction of size and numbers. Reduced involution is associated with higher mammographic density, a strong breast cancer risk factor. Both factors predict risk of developing breast cancer among women with benign disease. Given that prior studies have been conducted mainly in White women, we compared the extent of TDLU involution among Western and Asian breast cancer cases to describe potential racial heterogeneity in breast cancer etiology. Method: We obtained three TDLU involution metrics (count/100 mm2, mean span, and mean acini count/TDLU; all inversely correlated with TDLU involution) measured in benign breast tissue sections from 379 Chinese (254 luminal A and 125 core basal phenotype [CBP] cases) and 476 Polish (407 luminal A and 69 CBP) breast cancer cases. Polytomous logistic regression was performed using tertiles of TDLU measures as ordinal dependent variable and race as an independent variable. Covariates included age, body mass index, and parity. Analyses were performed separately by age group (&amp;lt;50, ≥50) and subtype (luminal A and CBP). Results: Among luminal A cases, Chinese had significantly greater TDLU count compared with Polish in both age groups (ORtrend= 11.0; 95% CI=2.80-43.2; Ptrend=0.001 for age&amp;lt;50; ORtrend= 12.1; 95% CI=5.12-28.7; Ptrend=1E-08 for age≥50). In addition, Chinese were more likely to have greater mean span and acini count among younger cases (ORtrend=2.5; 95% CI=1.44-4.28; Ptrend=0.001 for mean span and ORtrend=7.4; 95% CI=4.13-13.4; Ptrend=3E-11 for acini count) but not among older cases. Among CBP cases, Chinese were more likely to have greater acini count compared to Polish only among younger women (ORtrend=2.6; 95% CI=1.12-6.95; Ptrend=0.03 for age&amp;lt;50). Conclusion: We found Chinese were more likely to have reduced TDLU involution compared to Polish cases after accounting for potential confounders, with greater differences for luminal A and younger women. Studies of TDLU involution in diverse populations are needed to confirm and understand whether population difference in TDLU involution metrics are related to age at breast cancer onset and risks for specific tumor subtypes. Citation Format: Hyuna Sung, Changyuan Guo, Jennifer Guida, Shan Zheng, Erni Li, Jing Li, Nan Hu, Joseph Deng, Montserrat Garcia-Closas, Jonine Figueroa, Mark Sherman, Gretchen Gierach, Ning Lu, Xiaohong R. Yang. Racial variation in terminal duct lobular unit (TDLU) involution in Chinese and Polish breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5290. doi:10.1158/1538-7445.AM2017-5290