Normal Terminal Duct Lobular Units Research Articles

Abstract PO3-08-08: Basal-Luminal (BL) Cells in Histologically Normal and Malignant Breast Tissue from BRCA1 and BRCA2 Carriers

Abstract Background Single-cell studies of normal breast tissue in BRCA mutation carriers have led to the identification of a candidate cancer precursor cell, the basal-luminal (BL) cell. The localization of BL cells within normal, pre-malignant and malignant breast tissue architecture is not well characterized. Here, using cyclic immunofluorescence (CyCIF), we quantify and localize BL cells within normal and malignant breast tissues from BRCA1 and BRCA2 carriers. Methods Formalin-fixed paraffin-embedded breast tissue sections from 9 BRCA1 and 9 BRCA2 consented patients treated at Dana Farber Cancer Institute (2001-2019) were retrieved from the clinical archives and subjected to CyCIF for luminal and basal markers including CK5, CK14 and CK19. Using one section/case, cells co-expressing basal and luminal cytokeratins (CK14/CK19 and/or CK5/CK19) were visually identified in invasive carcinoma, ductal carcinoma in situ (DCIS) and in adjacent normal breast tissue. In histologically normal tissue, regions of interest (ROIs) with co-expressing cells were classified as terminal duct lobular units (TDLUs) or isolated ducts. Within each ROI, the quantity of BL cells was scored as low (1-5 cells), moderate (6-10 cells), or high ( >10 cells). Results The 9 BRCA1 tumors included 8 invasive ductal carcinomas/no special type and 1 tubular carcinoma. The 9 BRCA2 tumors included 4 invasive ductal carcinomas/no special type and 5 invasive carcinomas with ductal and lobular features. 496 ROIs were evaluated in histologically normal tissue: 297 from BRCA1 cases and 199 from BRCA2 cases. BL cells co-expressing CK14/CK19 and/or CK5/CK19 were identified in normal TDLUs and isolated ducts in both BRCA1 and BRCA2 cases. BL cells were more frequently identified in BRCA2 cases, with at least one BL cell present in 97/297 ROIs (33%) in BRCA1 cases and in 133/199 ROIs (67%) in BRCA2 cases (p< 0.01). 55% of BRCA2 ROIs had >10 BL cells each versus 42% of BRCA1 ROIs; however, this difference was not significant. Four BRCA1 cases showed a diffuse BL cell phenotype within the invasive carcinoma with either a diffuse or scattered BL cell phenotype within the DCIS. The BL cell phenotype was not seen diffusely in invasive carcinoma or DCIS in the other 14 cases; however individual tumor cells or small cell clusters with the BL cell phenotype were occasionally found. Conclusions BL cells are found in histologically normal TDLUs and isolated ducts in breast tissue from BRCA1 and BRCA2 carriers, with greater prevalence in BRCA2 carriers. The BL cell phenotype is present diffusely within tumor cells in almost half of BRCA1-associated cancers and none of the BRCA2-associated cancers in this small cohort. Further study of this interesting cell population is warranted in efforts to understand early changes in epithelium at high risk for the development of cancer. Citation Format: Rodrigo Fonseca Abreu, Tabata Alves Domingos, Roberto Bonfim Pimenta Peixoto, Ashka Patel, Krishan Taneja, McKenna Moore, Alicia Pollaci, Judy Garber, Nomeda Girnius, Deborah Dillon. Basal-Luminal (BL) Cells in Histologically Normal and Malignant Breast Tissue from BRCA1 and BRCA2 Carriers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-08-08.

Reliability of CD44, CD24, and ALDH1A1 immunohistochemical staining: Pathologist assessment compared to quantitative image analysis.

The data on the expression of stem cell markers CD44, CD24, and ALDH1A1 in the breast tissue of cancer-free women is very limited and no previous studies have explored the agreement between pathologist and computational assessments of these markers. We compared the immunohistochemical (IHC) expression assessment for CD44, CD24, and ALDH1A1 by an expert pathologist with the automated image analysis results and assessed the homogeneity of the markers across multiple cores pertaining to each woman. We included 81 cancer-free women (399 cores) with biopsy-confirmed benign breast disease in the Nurses' Health Study (NHS) and NHSII cohorts. IHC was conducted with commercial antibodies [CD44 (Dako, Santa Clara, CA, USA) 1:25 dilution; CD24 (Invitrogen, Waltham, MA, USA) 1:200 dilution and ALDH1A1 (Abcam, Cambridge, United Kingdom) 1:300 dilution]. For each core, the percent positivity was quantified by the pathologist and Definiens Tissue Studio. Correlations between pathologist and computational scores were evaluated with Spearman correlation (for categorical positivity: 0, >0-<1, 1-10, >10-50, and >50%) and sensitivity/specificity (for binary positivity defined with 1 and 10% cut-offs), using the pathologist scores as the gold standard. Expression homogeneity was examined with intra-class correlation (ICC). Analyses were stratified by core [normal terminal duct-lobular units (TDLUs), benign lesions] and tissue type (epithelium, stroma). Spearman correlation between pathologist and Definiens ranged between 0.40-0.64 for stroma and 0.66-0.68 for epithelium in normal TDLUs cores and between 0.24-0.60 for stroma and 0.61-0.64 for epithelium in benign lesions. For stroma, sensitivity and specificity ranged between 0.92-0.95 and 0.24-0.60, respectively, with 1% cut-off and between 0.43-0.88 and 0.73-0.85, respectively, with 10% cut-off. For epithelium, 10% cut-off resulted in better estimates for both sensitivity and specificity. ICC between the cores was strongest for CD44 for both stroma and epithelium in normal TDLUs cores and benign lesions (range 0.74-0.80). ICC for CD24 and ALDH1A ranged between 0.42-0.63 and 0.44-0.55, respectively. Our findings show that computational assessments for CD44, CD24, and ALDH1A1 exhibit variable correlations with manual assessment. These findings support the use of computational platforms for IHC evaluation of stem cell markers in large-scale epidemiologic studies. Pilot studies maybe also needed to determine appropriate cut-offs for defining staining positivity.

A Case Report of HER2-Amplification in the Breast Without Histological Abnormality.

Atypical ductal hyperplasia and low-grade ductal carcinoma in situ (DCIS) are regarded as precursor lesions of estrogen receptor (ER)-positive invasive breast cancer. In HER2-amplified invasive breast cancer, a precursor lesion before DCIS has not yet been described. Here we introduce a case of HER2-positive lobules in the breast without histological abnormality. A 39-year-old premenopausal woman visited a hospital due to identification of microcalcifications on screening mammography. The subsequent biopsy confirmed high-grade DCIS. She underwent wide excision, and a residual high-grade DCIS with a size of 1.4 cm was found. The tumor cells were hormone receptor positive and HER2 negative on immunohistochemical (IHC) staining. Around DCIS, there were terminal duct lobular units (TDLUs) showing strong HER2 positivity on IHC. These HER2-positive lobules were 7 mm away from the high-grade DCIS and was 1.1 mm in size. These HER2-positive lobules showed no histologic abnormality and had no difference compared with the surrounding normal TDLUs. Nevertheless, in addition to showing HER2 overexpression in IHC, HER2 amplification was also identified in HER2 silver in situ hybridization. HER2 amplification plays an important role in breast cancer development, and HER2 amplification is known to occur as an early event in cancer development. There have been studies identifying driver mutations in normal tissues of other organs. These findings suggest that HER2 amplification in the breast without histological abnormality could occur, and the HER2-positive lobules could be "at risk" for transformation into the precursor lesion of HER2-positive breast cancer, although the biological significance of these findings is unclear.

Expression of LGR5 in mammary myoepithelial cells and in triple-negative breast cancers

Lineage tracing in mice indicates that LGR5 is an adult stem cell marker in multiple organs, such as the intestine, stomach, hair follicles, ovary, and mammary glands. Despite many studies exploring the presence of LGR5 cells in human tissues, little is known about its expression profile in either human mammary tissue or pathological lesions. In this study we aim to investigate LGR5 expression in normal, benign, and malignant lesions of the human breast using RNA in situ hybridization. LGR5 expression has not been observed in normal lactiferous ducts and terminal duct lobular units, whereas LGR5-positive cells have been specifically observed in the basal myoepithelium of ducts in the regenerative tissues, ductal carcinoma in situ, and in ducts surrounded by invasive cancer cells. These findings suggest LGR5 marks facultative stem cells that are involved in post injury regeneration instead of homeostatic stem cells. LGR5 positivity was found in 3% (9 of 278 cases) of invasive breast cancers (BC), and it showed positive associations with higher histologic grades (P = 0.001) and T stages (P < 0.001), while having negative correlations with estrogen receptor (P < 0.001) and progesterone receptor (P < 0.001) expression. Remarkably, all LGR5-positive BC, except one, belong to triple-negative BC (TNBC), representing 24% (9 of 38 cases) of all of them. LGR5 histoscores have no correlations with EGFR, CK5/6, Ki-67, or P53 expression. Additionally, no β-catenin nuclear localization was observed in LGR5-positive BC, indicating that canonical Wnt pathway activation is less likely involved in LGR5 expression in BC. Our results demonstrate that LGR5 expression is induced in regenerative conditions in the myoepithelium of human mammary ducts and that its expression is only observed in TNBC subtype among all invasive BC. Further studies regarding the functional and prognostic impact of LGR5 in TNBC are warranted.

Abstract P5-02-02: Artificial intelligence powered predictive analysis of atypical ductal hyperplasia from digitized pathology images

Abstract Background: Approximately 15-25% of patients with atypical ductal hyperplasia (ADH) diagnosed on breast core needle biopsy (CNB) are upgraded to ductal carcinoma in situ (DCIS) or invasive carcinoma (IC) on surgical excision. The reproducible identification of patients with ADH on CNB who are more likely to have upgrades at excision remains elusive. We hypothesized that a machine learning approach could be utilized to train models to recognize ADH on digitized pathology images and to identify cases of ADH more likely to be upgraded to DCIS or IC at excision. The purpose of this study was to determine the accuracy of the machine learning approach to identify ADH. Methods: 726 digitized images of CNB slides derived from 306 cases with a diagnosis of ADH between 11/2004-3/2018 were included in this study. Independent histologic review by two breast pathologists identified slides with and without ADH from each case. 39 board certified pathologists with experience in evaluation of breast biopsies were employed for tissue region annotation on the PathAI research platform (not intended for diagnostic purposes), yielding 14,118 tissue region annotations. Region annotations included ADH, ADH stroma, flat epithelial atypia (FEA), lobular neoplasia (LN), calcifications (Ca), columnar cell change/hyperplasia, sclerosing adenosis, papilloma, normal terminal duct lobular units and other non-atypical breast tissue regions. These annotations were used to train a convolutional neural network (CNN) with 35 layers and approximately 9 million parameters to identify ADH. The data were split into training and testing sets, representing 61.1% and 38.9% of the data respectively. The distribution of cases, images with ADH and cases with upgrade were balanced between the training and testing sets. Results: CNB specimens were assigned labels of “ADH” or “No ADH” based on histologic assessment. AI models were able to predict the diagnosis of ADH with 85% sensitivity (144 of 168 images within the test set) and 69% specificity (78 of 113 images within the test set). The slide-level area under the receiver operator curve (ROC) for this model was 0.84. Conclusions: A deep learning-based classifier showed strong performance for the identification of ADH from whole slide images of H&amp;E stained breast CNBs. With further development, this approach may improve the reproducibility and standardization of the diagnosis of ADH. Future analyses will focus on determining if morphologic features of ADH extracted by the deep learning system can be used to predict upgrade to DCIS and IC. This approach may help stratify patients with ADH on CNB into those who require surgical excision and those who can be followed with active surveillance. Citation Format: Jennifer K. Kerner, Allison Cleary, Suyog Jain, Harsha Pokkalla, Benjamin Glass, Sam Grossmith, Maya Harary, Elizabeth Mittendorf, Andrew H. Beck, Aditya Khosla, Stuart J. Schnitt, Ilan Wapinski, Tari King. Artificial intelligence powered predictive analysis of atypical ductal hyperplasia from digitized pathology images [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-02-02.

Rapid virtual hematoxylin and eosin histology of breast tissue specimens using a compact fluorescence nonlinear microscope

Up to 40% of patients undergoing breast conserving surgery for breast cancer require repeat surgeries due to close to or positive margins. The lengthy processing required for evaluating surgical margins by standard paraffin-embedded histology precludes its use during surgery and therefore, technologies for rapid evaluation of surgical pathology could improve the treatment of breast cancer by reducing the number of surgeries required. We demonstrate real-time histological evaluation of breast cancer surgical specimens by staining specimens with acridine orange (AO) and sulforhodamine 101 (SR101) analogously to hematoxylin and eosin (H&E) and then imaging the specimens with fluorescence nonlinear microscopy (NLM) using a compact femtosecond fiber laser. A video-rate computational light absorption model was used to produce realistic virtual H&E images of tissue in real time and in three dimensions. NLM imaging could be performed to depths of 100 μm below the tissue surface, which is important since many surgical specimens require subsurface evaluation due to contamination artifacts on the tissue surface from electrocautery, surgical ink, or debris from specimen handling. We validate this method by expert review of NLM images compared to formalin-fixed, paraffin-embedded (FFPE) H&E histology. Diagnostically important features such as normal terminal ductal lobular units, fibrous and adipose stromal parenchyma, inflammation, invasive carcinoma, and in situ lobular and ductal carcinoma were present in NLM images associated with pathologies identified on standard FFPE H&E histology. We demonstrate that AO and SR101 were extracted to undetectable levels after FFPE processing and fluorescence in situ hybridization (FISH) HER2 amplification status was unaffected by the NLM imaging protocol. This method potentially enables cost-effective, real-time histological guidance of surgical resections.

Tumor-Associated Macrophages: Oblivious Confederates in Invasive Mammary Carcinoma

The infiltrating margins of carcinomas are associated with the presence of inflammatory cell infiltrate which are an integral part of the tumor microenvironment. Among the inflammatory cells, tumor-associated macrophages (TAMs) play a key role in the tumorigenesis. This study elucidates the density of TAMs in invasive mammary carcinomas and attempts to establish an association with the following pathological variables: tumor size, histological grade, nodal status, hormonal expression status and Her2Neu overexpression. Ninety diagnosed archival cases of invasive mammary carcinomas at a tertiary care center were included. Density of TAMs was assessed by using CD68 which is a pan-macrophage marker by immunohistochemistry on the archival tissue blocks. The density of TAMs (CD68 positive cells) was dichotomized into high (> 50 CD68 positive cells/HPF) and low (< 50 CD68 positive cells/HPF) and compared with the above-mentioned pathological variables using appropriate statistical tests. The density of TAMs was significantly higher around the infiltrating edge of the carcinoma in comparison with the adjoining normal terminal duct lobular units. The density of TAMs was more in the infiltrating edge of the tumor than within the tumor nodule/nests. A higher TAM density showed a significant association in tumors having large tumor size, higher histological grade, nodal metastasis, the absence of ER and PR expression and Her2Neu overexpression (p value < 0.05). TAMs play an important role in tumor progression in invasive mammary carcinomas. This is as a result of the multiple roles enacted by TAMs in the various stages of tumor development starting from tumor growth, invasion, angiogenesis and metastases. Targeted therapy against TAMs has great potential in being important components of future treatment strategies against breast carcinomas.

Tumor-associated macrophages: Oblivious confederates in invasive mammary carcinoma

Abstract Background: The infiltrating margins of carcinomas are associated with presence of inflammatory cell infiltrate which are an integral part of the tumor microenvironment. Amongst the inflammatory cells, Tumor Associated Macrophages (TAMs) play a key role in the tumorigenesis. This study elucidates the density of TAMs in invasive mammary carcinomas and attempts to establish aa association with the following pathological variables: tumor size, histological grade, nodal status, hormonal expression status and Her2Neu overexpression. Materials and Methods: 90 diagnosed archival cases of invasive mammary carcinomas at a tertiary care centre were included. Density of TAMs was assessed by using CD68 which is a pan-macrophage marker by immunohistochemistry on the archival tissue blocks. The density TAMs (CD68 positive cells) was dichotomised into high (&gt;50 CD68 positive cells/HPF) and low (&lt;5050 CD68 positive cells/HPF) and compared with the above mentioned pathological variables using appropriate statistical tests. Results: The density of TAMs was significantly higher around the infiltrating edge of the carcinoma in comparison to the adjoining normal terminal duct lobular units. The density of TAMs was more in the infiltrating edge of the tumor than within the tumor nodule/nests. A higher TAM density showed a significant association in tumors having large tumor size, higher histological grade, nodal metastasis, absence of ER and PR expression and Her2Neu overexpression (p value &lt;0.05). Conclusion: TAMs play an important role in tumor progression in invasive mammary carcinomas. This is as a result of the multiple roles enacted by TAMs in the various stages of tumor development starting from tumor growth, invasion, angiogenesis and metastases. Targeted therapy against TAMs has great potential in the being important components of future treatment strategies against breast carcinomas.

Expression of estrogen receptor, progesterone receptor, and Ki67 in normal breast tissue in relation to subsequent risk of breast cancer.

Although expression of estrogen receptor (ER), progesterone receptor (PR), and cell proliferation marker Ki67 serve as predictive and prognostic factors in breast cancers, little is known about their roles in normal breast tissue. Here in a nested case–control study within the Nurses’ Health Studies (90 cases, 297 controls), we evaluated their expression levels in normal breast epithelium in relation to subsequent breast cancer risk among women with benign breast disease. Tissue microarrays were constructed using cores obtained from benign biopsies containing normal terminal duct lobular units and immunohistochemical stained for these markers. We found PR and Ki67 expression was non-significantly but positively associated with subsequent breast cancer risk, whereas ER expression was non-significantly inversely associated. After stratifying by lesion subtype, Ki67 was significantly associated with higher risk among women with proliferative lesions with atypical hyperplasia. However, given the small sample size, further studies are required to confirm these results.

Abstract LB-179: Expression of estrogen receptor, progesterone receptor, and ki67 in normal breast tissue and subsequent risk of breast cancer

Abstract 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

Abstract 4682: Standardized measures of lobular involution and subsequent breast cancer risk among women with benign breast disease

Abstract 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

Abstract P5-02-06: Digital quantification of estrogen receptor expression in normal breast in post-menopausal women with breast cancer and association with tumor subtypes

Abstract Background: ER expression in normal breast epithelium (NBR) is higher in women with a history of breast cancer (BC) compared to controls. In adjuvant setting, metanalysis showed effective Tamoxifen treatment was restricted to ER positive cancers. However, it is also known prophylactic oophorectomy (a form of estrogen suppression) significantly reduces the incidence of BC in BRCA1 carriers. This is in contrast with the 80% rate of ER negative tumors in BRCA1 patients. The aim of this study is to quantify ER expression in NBR away from tumor in women with BC and to correlate it with BC subtypes. Methods: 204 consecutive patients were identified for whom NBR away from tumor was available. 27 reduction mammoplasty (RM) tissues from women with no history of BC were used as controls. Tissue microarrays were constructed and slides were stained with ER and scanned using Aperio XT Scan Scope. Normal terminal duct lobular epithelium was manually circled on scanned images and annotations were recorded in separate digital layers. ER staining was quantitated in marked areas of the electronic image using an optimized scoring nuclear IHC algorithm (Aperio technologies, Inc., Vista, CA). Clinical information and tumor characteristics (menopausal status, ER, HER2 expression, grade, size, number of positive nodes, stage) were recorded based on pathology reports and tumor registry data. Results: The mean ER positivity in NBR was 16 ± 12.4 % (range: 0-5-5.7%) for all patients with BC, 20.8±13.9% for postmenopausal (n=74) and 13.7 ±10.9% for peri+ premenopausal (n=170) subgroups. ER positivity was higher in patients with BC compared to those undergoing RM with no history of BC. ER positivity in NBR did not vary by tumor size, positive lymph nodes status, tumor grade, or stage in post-menopausal, and pre + perimenopausal women. Older age at diagnosis was significantly associated (p&amp;lt;0.0001) with ER in NBR. In post- menopausal women ER expression in NBR was significantly higher in patients with ER negative or triple negative tumors. Estrogen receptor expression in normal terminal duct lobular units of post-menopausal women with breast cancer in relation to tumor subtypes. Postmenopausal (N=74) NMean % (SD)P ValueTumor ER Status 0.05 Negative1427.4 (13.9) Positive6019.3 (13.6) Tumor HER2 Status 0.34 Negative5721.8 (14.9) Positive817.7 (5.7) Tumor Triple Negative 0.05 No5319.7 (13.6) Yes1228.5 (14.7) HER2 status was not known in some cases and was excluded from statistical calculations. Conclusion: This computer assisted image analysis study confirms ER expression in NBR increases with age and menopausal status in women with BC. We report, for the first time, a significant association between ER expression in normal breast epithelium with ER negative and triple negative cancers in post-menopausal women. Our study suggests that ER expression in normal epithelium may play a role in development of hormone receptor negative breast cancers. Citation Format: H Evin Gulbahce, Cindy Blair, Carol Sweeney, Rachel Factor, Mohamed Salama. Digital quantification of estrogen receptor expression in normal breast in post-menopausal women with breast cancer and association with tumor subtypes [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-02-06.

Ki-67 expression in sclerosing adenosis and adjacent normal breast terminal ductal lobular units: a nested case–control study from the Mayo Benign Breast Disease Cohort

Sclerosing adenosis (SA) increases risk for invasive breast cancer (BC) 2.1 times relative to that in the general population. Here, we sought to evaluate whether the proliferation marker Ki-67 stratifies risk among women with SA. A nested case-control sample of patients with SA was obtained from the Mayo Clinic Benign Breast Disease Cohort. Ki-67 expression was evaluated in SA lesions and in the adjacent normal terminal duct lobular units (TDLU) in women who did (cases, n=133) or did not (controls, n=239) develop BC. Ki-67 was scored by intensity and number of positively stained cells per one high-power field (magnification, ×40) (40×HPF) for both SA and normal TDLU. Associations of Ki-67 expression with case-control status were assessed using conditional logistic regression. Higher Ki-67 expression was significantly associated with case-control status in both SA (P=0.03) and normal background TDLU (P=0.006). For the SA lesion, >2 average positively stained cells/40×HPF showed an adjusted odds ratio (OR) of 1.9 (95% CI, 1.1-3.4) compared to samples with an average of ≤2 positively stained cells. For background TDLU, lobules with >2 but ≤6 average positively stained cells showed an adjusted OR of 1.3-1.5, whereas those with an average of >6 positively stained cells had an OR of 2.4 (95% CI, 1.1-5.3) compared to those with an average of <2 positively stained cells. Among women with SA, increased Ki-67 expression in either the SA lesion or the normal background TDLU carried an approximately twofold increased odds of subsequent BC as compared to lower Ki-67 expression.

Abstract 4991: Identifying a highly-aggressive DCIS subgroup by studying intra-individual DCIS heterogeneity among invasive breast cancer patients

Abstract Background: Detection rates for ductal carcinoma in situ (DCIS) have dramatically increased because of the widespread use of mammographic screening. The heterogeneity among multiple DCIS lesions within the same patient, also diagnosed with invasive ductal carcinoma (IDC), has not been well evaluated, leaving clinical and research implications of this intra-individual heterogeneity of DCIS yet to be explored. In the study presented here, we tested the hypothesis that classification of heterogeneous DCIS based on adjacent IDC and normal terminal duct lobular units (TDLU) in patients concurrently diagnosed with IDC and DCIS helps to identify DCIS subgroups which have different degrees of “aggressiveness”. Experimental Designs and Results: Ten DCIS lesions from each of 36 patients concurrently diagnosed with DCIS and IDC were randomly selected and scored with IHC biomarkers. Our results showed that expression of PR, HER2, Ki-67 and p16 varied significantly in DCIS lesions from a single patient (P &amp;lt; 0.05 for PR; P &amp;lt; 1 × 10-8 for HER2, Ki-67 and p16). In addition, seventy-two percent of the individuals had heterogeneous expression in at least 2/6 markers (P &amp;lt; 10-18, vs. hypothesized homogeneity baseline, 10%). Importantly, by comparing the expression of promising DCIS risk biomarkers (Ki-67, p53 and p16) among different DCIS subgroups, we found that DCIS lesions in subgroup IIb, which had the same molecular subtypes as the adjacent IDC but not the same subtypes as the adjacent TDLU, had a higher Ki-67 index (P &amp;lt; 10-7), a higher chance of positive p53 staining (P = 0.02), and less p16 staining (P = 0.08) than those in type I DCIS lesions with different molecular subtypes from the adjacent IDC. Further molecular characterization by microarray analysis identified that 246 annotated coding and non-coding genes were differentially expressed between DCIS subgroups Type I and IIb [FC (fold change)&amp;gt;2.0, P&amp;lt;0.05]. A majority of genes were upregulated in type IIb DCIS and more than 50% of genes are cancer-relevant (P &amp;lt; 10-8∼-2). Importantly, a group of differentially expressed genes in subgroup IIb including CD44, IFIT1, STAT1, MLH1, PARP9 and BRIP1 were confirmed by qPCR validation and likely play important roles in DCIS progression based on previous findings, which further support that Type IIb DCIS may represent a highly aggressive subgroup. Conclusions: By using a systematic approach, our results clearly demonstrate that intra-individual heterogeneity in DCIS is very common in patients concurrently diagnosed with IDC. Our novel findings of a DCIS subpopulation with high-aggressiveness will provide a new paradigm for mechanistic studies of breast tumor progression and also have broad implications for prevention research as heterogeneous pre-invasive lesions are present in many other cancer types. This work was kindly supported by the Susan G. Komen for the Cure (KG100274), and Eileen Stein Jacoby Fund. Citation Format: Zhengyu Jiang, Dana Pape-Zambito, Karthik Devarajan, Hong Wu, Carolyn Slater, Lauren Dolinsky, Mary Daly, Xiaowei Chen. Identifying a highly-aggressive DCIS subgroup by studying intra-individual DCIS heterogeneity among invasive breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4991. doi:10.1158/1538-7445.AM2014-4991

Epithelial and stromal microRNA signatures of columnar cell hyperplasia linking Let-7c to precancerous and cancerous breast cancer cell proliferation.

Columnar cell hyperplasia (CCH) is the earliest histologically identifiable breast lesion linked to cancer progression and is characterized by increased proliferation, decreased apoptosis and elevated oestrogen receptor α (ERα) expression. The mechanisms underlying the initiation of these lesions have not been clarified but might involve early and fundamental changes in cancer progression. MiRNAs are key regulators of several biological processes, acting by influencing the post-transcriptional regulation of numerous targets, thus making miRNAs potential candidates in cancer initiation. Here we have defined novel epithelial as well as stromal miRNA signatures from columnar cell hyperplasia lesions compared to normal terminal duct lobular units by using microdissection and miRNA microarrays. Let-7c were among the identified downregulated epithelial miRNAs and its functions were delineated in unique CCH derived cells and breast cancer cell line MCF-7 suggesting anti-proliferative traits potentially due to effects on Myb and ERα. MiR-132 was upregulated in the stroma surrounding CCH compared to stoma surrounding normal terminal duct lobular units (TDLUs), and overexpression of miR-132 in immortalized fibroblasts and in fibroblasts co-cultured with epithelial CCH cells caused substantial expression changes of genes involved in metabolism, DNA damage and cell motility. The miRNA signatures identified in CCH indicate early changes in the epithelial and stromal compartment of CCH and could represent early key alterations in breast cancer progression that potentially could be targeted in novel prevention or treatment schedules.

Identifying a highly-aggressive DCIS subgroup by studying intra-individual DCIS heterogeneity among invasive breast cancer patients.

The heterogeneity among multiple ductal carcinoma in situ (DCIS) lesions within the same patient also diagnosed with invasive ductal carcinoma (IDC) has not been well evaluated, leaving research implications of intra-individual DCIS heterogeneity yet to be explored. In this study formalin-fixed paraffin embedded sections from 36 patients concurrently diagnosed with DCIS and IDC were evaluated by immunohistochemistry. Ten DCIS lesions from each patient were then randomly selected and scored. Our results showed that expression of PR, HER2, Ki-67, and p16 varied significantly within DCIS lesions from a single patient (P<0.05 for PR; P<1×10−8 for HER2, Ki-67 and p16). In addition, seventy-two percent of the individuals had heterogeneous expression of at least 2/6 markers. Importantly, by evaluating the expression of promising DCIS risk biomarkers (Ki-67, p53 and p16) among different DCIS subgroups classified by comparing DCIS molecular subtypes with those of adjacent normal terminal duct lobular units (TDLU) and IDC, our results suggest the existence of a highly-aggressive DCIS subgroup, which had the same molecular subtype as the adjacent IDC but not the same subtype as the adjacent normal TDLU. By using a systematic approach, our results clearly demonstrate that intra-individual heterogeneity in DCIS is very common in patients concurrently diagnosed with IDC. Our novel findings of a DCIS subpopulation with aggressive characteristics will provide a new paradigm for mechanistic studies of breast tumor progression and also have broad implications for prevention research as heterogeneous pre-invasive lesions are present in many other cancer types.

Estrogen receptor and progesterone receptor expression in normal terminal duct lobular units surrounding invasive breast cancer

Molecular and morphological alterations related to carcinogenesis have been found in terminal duct lobular units (TDLUs), the microscopic structures from which most breast cancer precursors and cancers develop, and therefore, analysis of these structures may reveal early changes in breast carcinogenesis and etiologic heterogeneity. Accordingly, we evaluated relationships of breast cancer risk factors and tumor pathology to estrogen receptor (ER) and progesterone receptor (PR) expression in TDLUs surrounding breast cancers. We analyzed 270 breast cancer cases included in a population-based breast cancer case-control study conducted in Poland. TDLUs were mapped in relation to breast cancer: within the same block as the tumor (TDLU-T), proximal to tumor (TDLU-PT), or distant from (TDLU-DT). ER/PR was quantitated using image analysis of immunohistochemically stained TDLUs prepared as tissue microarrays. In surgical specimens containing ER-positive breast cancers, ER and PR levels were significantly higher in breast cancer cells than in normal TDLUs, and higher in TDLU-T than in TDLU-DT or TDLU-PT, which showed similar results. Analyses combining DT-/PT TDLUs within subjects demonstrated that ER levels were significantly lower in premenopausal women versus postmenopausal women (odds ratio [OR]=0.38, 95% confidence interval [CI]=0.19, 0.76, P=0.0064) and among recent or current menopausal hormone therapy users compared with never users (OR=0.14, 95% CI=0.046-0.43, P (trend)=0.0006). Compared with premenopausal women, TDLUs of postmenopausal women showed lower levels of PR (OR=0.90, 95% CI=0.83-0.97, P (trend)=0.007). ER and PR expression in TDLUs was associated with epidermal growth factor receptor (EGFR) expression in invasive tumors (P=0.019 for ER and P=0.03 for PR), but not with other tumor features. Our data suggest that TDLUs near breast cancers reflect field effects, whereas those at a distance demonstrate influences of breast cancer risk factors on at-risk breast tissue. Analyses of mapped TDLUs may provide information about the sequence of molecular changes occurring in breast carcinogenesis.

Abstract 3993: Stromal and epithelial microRNA signatures of early breast lesions linked to cancer progression

Abstract Columnar cell hyperplasia (CCH) is considered to originate in normal terminal duct lobular units (TDLUs) and is potentially the first histologically identifiable alteration in the development of breast cancer. These lesions have been characterized to have a higher proliferation rate but the underlying mechanisms have not been studied. In order to detect early changes in breast cancer progression we therefore investigated microRNA (miRNA) expression profiles in TDLUs and CCH. Epithelial cells and surrounding stroma of TDLUs and CCH from formalin-fixed paraffin embedded tissue from four patients with high risk of developing breast cancer were laser capture microdissected and RNA was extracted. MiRNA expression profiles were obtained using a qRT-PCR-based array analyzing 667 human miRNAs. Interestingly, array analysis indicated substantial downregulation of several miRNAs in both the epithelial and the stromal compartments of CCH. MiRNA array analysis of paired samples from specimens with both TDLUs and CCH resulted in significant (p&amp;lt;0.05) decreased levels of 15 miRNAs in epithelial cells of CCH (miR-532-3p, miR-92a, miR-886-3p, let-7c, miR-150, miR-27a, miR-491-5p, miR-130a, miR-365, miR-886-5p, miR-204, miR-135a*, miR-494, miR-29c, miR-768-3p) and downregulation of 18 miRNAs in the stroma (miR-195, miR-296-5p, miR-99a, miR-100, miR-224, let-7c, miR-19b, miR-423-5p, miR-26a, miR-511, miR-128, miR-203, miR-30b, miR-374a, miR-374b, miR-29b, let-7e, miR-10a). We next downregulated miR-27a and miR-92a in a CCH derived cell line and observed a significantly increased proliferation. Opposite effect was observed when the miRs were overexpressed altogether suggesting a proliferative inhibitory function for the selected miRNAs. The miRNA signatures indicate concordant early changes in the epithelial and stromal compartment and the findings could be useful in identifying early key alterations in breast cancer progression that potentially could be targeted in novel prevention or treatment schedules. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3993. doi:10.1158/1538-7445.AM2011-3993

Expression of IGF1R in normal breast tissue and subsequent risk of breast cancer

The growth hormone and insulin-like growth factor (IGF) axis plays an essential role in the growth and development of the mammary gland. IGF1 and IGF1 receptor (IGF1R) may also play a role in the early transformation of mammary cells. Using a nested case-control design, the association between IGF1R expression in normal breast tissue from benign biopsies and subsequent risk of breast cancer was examined in patients enrolled in the Nurses' Health Study. The tissue microarrays (TMAs) containing normal terminal duct lobular units (TDLUs) from benign breast biopsies were constructed. Immunostains for IGF1R were performed on sections cut from the TMAs. A total of 312 women had evaluable IGF1R staining in normal TDLUs; 75 subsequently developed breast cancer (cases) and 237 did not (controls). The epithelial cells in the normal TDLUs were scored for both cytoplasmic and membrane staining for IGF1R. Cytoplasmic IGF1R expression was positively associated with subsequent risk of breast cancer (OR = 2.47, 95% CI 1.41-4.33). Women having TDLU epithelial cells showed little or no membrane expression of IGF1R, but those with 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 membrane or cytoplasmic IGF1R expression in their TDLU epithelial cells (OR = 15.9, 95% CI 3.6-69.8). In this study, it was demonstrated that IGF1R expression patterns in epithelial cells of normal TDLUs in benign breast biopsies were associated with an increased risk of subsequent breast cancer. Further studies to confirm these findings are necessary.

Abstract P3-09-06: microRNA Signature of Columnar Cell Hyperplasia from Patients with High Risk of Breast Cancer

Abstract Background: Columnar cell hyperplasia (CCH) is considered to originate in normal terminal duct lobular units (TDLUs) and is potentially the first histologically identifiable alteration in the development of breast cancer. In order to detect early pathological changes in breast tissue, which could reveal the underlying mechanism to cancer progression, we investigated the microRNA (miRNA) expression profile in TDLUs and CCH. Material and Methods: Epithelial cells and surrounding stroma of TDLUs and CCH from formalin-fixed paraffin embedded tissue (FFPE) from patients with high risk of developing breast cancer were laser capture microdissected and RNA was extracted. MiRNA expression profiles were obtained using a qRT-PCR-based array analyzing 667 human miRNAs. Results: Increased levels of ERα were present in the epithelial cells of CCH as previously reported. Immunohistochemical staining for the stromal activation marker smooth muscle actin (SMA) was negative in fibroblasts surrounding both TDLUs and CCH, suggesting lack of major activation of the stroma. Interestingly, array analysis revealed changes in miRNA expression pattern in both epithelial and stromal compartments. Correlation analyses of the array results indicated a high reproducibility (r2:0.82-0.94) supporting stable miRNA profiles in TDLUa and CCH. MiRNA array analysis of paired samples from specimens with both TDLUs and CCH resulted in significant (P&amp;lt;0.05) decreased levels of 15 miRNAs in epithelial cells of CCH and downregulation of 18 miRNAs in the stroma. Examples of altered miRNAs in the epithelial cells are miR-532-3p, miR-886-3p, let-7c, miR-491-5p, miR-365, and example miRNAs in the stroma are miR-195, miR-99a, miR-224, miR-19b, miR-423-5p. Further validation of a selection of miRNAs in a patient material using in situ hybridization is currently being performed to verify the array results. Discussion: This data will potentially link these miRNAs to the development of TDLUs towards CCH and possibly lead to the discovery of new markers for precancerous conditions and potentially new therapeutic targets. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-09-06.