Degnim, Amy C.

Abstract Purpose: Little is known about the role of the immune system in the earliest stages of breast carcinogenesis. We studied quantitative differences in immune cell types between breast tissues from normal donors and those from women with benign breast disease (BBD). Experimental Design: A breast tissue matched case–control study was created from donors to the Susan G. Komen for the Cure Tissue Bank (KTB) and from women diagnosed with BBD at Mayo Clinic (Rochester, MN) who either subsequently developed cancer (BBD cases) or remained cancer-free (BBD controls). Serial tissue sections underwent immunostaining and digital quantification of cell number per mm2 for CD4+ T cells, CD8+ T cells, CD20+ B cells, and CD68+ macrophages and quantification of positive pixel measure for CD11c (dendritic cells). Results: In 94 age-matched triplets, BBD lobules showed greater densities of CD8+ T cells, CD11c+ dendritic cells, CD20+ B cells, and CD68+ macrophages compared with KTB normals. Relative to BBD controls, BBD cases had lower CD20+ cell density (P = 0.04). Nearly 42% of BBD cases had no CD20+ B cells in evaluated lobules compared with 28% of BBD controls (P = 0.02). The absence of CD20+ cells versus the presence in all lobules showed an adjusted OR of 5.7 (95% confidence interval, 1.4–23.1) for subsequent breast cancer risk. Conclusions: Elevated infiltration of both innate and adaptive immune effectors in BBD tissues suggests an immunogenic microenvironment. The reduced B-cell infiltration in women with later breast cancer suggests a role for B cells in preventing disease progression and as a possible biomarker for breast cancer risk. Clin Cancer Res; 23(14); 3945–52. ©2017 AACR.

Abstract Background: Age-related lobular involution (LI) is a physiological process in which the terminal duct lobular units of the breast regress as a woman ages. Analyses of breast biopsies from women with benign breast disease (BBD) have found that extent of LI is negatively associated with subsequent breast cancer development. Here we assess the natural course of LI within individual women, and the impact of progressive LI on breast cancer risk. Methods: The Mayo Clinic BBD cohort consists of 13,455 women with BBD from 1967-2001. The BBD cohort includes 1115 women who had multiple benign biopsies, 106 of whom had developed breast cancer. Within this multiple biopsy cohort, the progression of the LI process was examined by age at initial biopsy and time between biopsies. The relationship between LI progression and breast cancer risk was assessed using standardized incidence ratios (SIRs) and by Cox proportional hazards analysis. Results: Women who had multiple biopsies were younger age and had a slightly higher family history of breast cancer as compared with the overall BBD cohort. Extent of LI at subsequent biopsy was greater with increasing time between biopsies and for women age 55+ at initial biopsy. Among women with multiple biopsies, there was a significant association of higher breast cancer risk among those with involution stasis (lack of progression, HR 1.63) as compared with those with involution progression, p = 0.036. Conclusions: The multiple biopsy BBD cohort allows for a longitudinal study of the natural progression of LI. The majority of women in the multiple biopsy cohort showed progression of LI status between benign biopsies, and extent of progression was highest for women who were in the perimenopausal age range at initial biopsy. Progression of LI status between initial and subsequent biopsy was associated with decreased breast cancer risk. Citation Format: Derek Radisky, Daniel W. Visscher, Ryan Frank, Robert A. Vierkant, Stacey J. Winham, Melody L. Stallings-Mann, Tanya L. Hoskin, Aziza Nassar, Celine M. Vachon, Lori A. Denison, Lynn C. Hartmann, Marlene H. Frost, Amy C. Degnim. Natural history of age-related lobular involution and impact on 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 LB-364.

BACKGROUNDWomen with atypical hyperplasia (AH) on breast biopsy have a substantially increased risk of breast cancer (BC). Here the BC risk for the extent and subtype of AH is reported for 2 separate cohorts.METHODSAll samples containing AH were included from 2 cohorts of women with benign breast disease (Mayo Clinic and Nashville). Histology review quantified the number of foci of atypical ductal hyperplasia (ADH) and atypical lobular hyperplasia (ALH). The BC risk was stratified for the number of AH foci within AH subtypes.RESULTSThe study included 708 Mayo AH subjects and 466 Nashville AH subjects. In the Mayo cohort, an increasing number of foci of AH was associated with a significant increase in the risk of BC both for ADH (relative risks of 2.61, 5.21, and 6.36 for 1, 2, and ≥3 foci, respectively; P for linear trend = .006) and for ALH (relative risks of 2.56, 3.50, and 6.79 for 1, 2, and ≥3 foci, respectively; P for linear trend = .001). In the Nashville cohort, the relative risks of BC for ADH were 2.70, 5.17, and 15.06 for 1, 2, and ≥3 foci, respectively (P for linear trend < .001); for ALH, the relative risks also increased but not significantly (2.61, 3.48, and 4.02, respectively; P = .148). When the Mayo and Nashville samples were combined, the risk increased significantly for 1, 2, and ≥3 foci: the relative risks were 2.65, 5.19, and 8.94, respectively, for ADH (P < .001) and 2.58, 3.49, and 4.97, respectively, for ALH (P = .001).CONCLUSIONSIn 2 independent cohort studies of benign breast disease, the extent of atypia stratified the long‐term BC risk for ADH and ALH. Cancer 2016;122:2971‐2978. © 2016 American Cancer Society.

BACKGROUNDWomen with benign breast disease (BBD) have an increased risk of developing breast cancer (BC). Nearly 30% of all BCs develop in women with prior BBD. Information regarding features of the expected number of BCs after BBD would enhance individualized surveillance and prevention strategies for these women. In the current study, the authors sought to characterize BCs developing in a large cohort of women with BBD.METHODSThe current study cohort included 13,485 women who underwent breast biopsy for mammographic or palpable concerns between 1967 and 2001. Biopsy slides were reviewed and classified as nonproliferative disease, proliferative disease without atypia, or atypical hyperplasia. BCs were identified by follow‐up questionnaires, medical records, and Tumor Registry data. BC tissues were obtained and reviewed.RESULTSWith median follow‐up of 15.8 years, 1273 women developed BC. The majority of BCs were invasive (81%), of which 61% were ductal, 13% were mixed ductal/lobular, and 14% were lobular. Approximately two‐thirds of the BC cases were intermediate or high grade, and 29% were lymph node positive. Cancer characteristics were similar across the 3 histologic categories of BBD, with a similar frequency of ductal carcinoma in situ, invasive disease, tumor size, time to invasive BC, histologic type of BC, lymph node positivity, and human epidermal growth factor receptor 2 positivity. Women with atypical hyperplasia were found to have a higher frequency of estrogen receptor‐positive BC (91%) compared with women with proliferative disease without atypia (80%) or nonproliferative disease (85%) (P = .02).CONCLUSIONSA substantial percentage of all BCs develop in women with prior BBD. The majority of BCs after BBD are invasive tumors of ductal type, with a substantial number demonstrating lymph node positivity. Of all the BCs in the current study, 84% were estrogen receptor positive. Prevention therapy should be strongly encouraged in higher‐risk women with BBD. Cancer 2016;122:378–385. © 2015 American Cancer Society.

Abstract Introduction: CD56+ lymphocytes (a defining marker of natural killer cells) have a predominantly cytotoxic phenotype and a hypothetical role in breast tumor immunosurveillance. Here we investigate whether CD56+ staining density varies in nonmalignant breast lobules according to confirmed breast cancer risk factors- age, and histologic features of fibrocystic change and lobular involution. Methods: Archived breast tissue samples were obtained from 94 women with benign breast disease (BBD). The sample set was selected as a nested case-control study (47 cases, 47 controls) from within a large BBD cohort; cases were those who developed breast cancer subsequent to their benign breast biopsy; controls were matched to cases on age at biopsy, year of biopsy, and length of breast cancer free follow-up at least as long as the time-to-cancer in the matched case. Up to 10 representative lobules in each sample were characterized by H&E for fibrocystic changes and the degree of epithelial proliferation (normal, nonproliferative, proliferative, atypia) and for degree of lobular involution (none, partial, complete). Consecutive sections were immunostained for CD56 and hematoxylin. Using digital image analysis, CD56 staining was quantified on a per lobule basis by pixel ratio (PR) (positive stain:total lobule). Statistical analysis was performed using linear mixed effects regression on the rank transformed density to account for correlations among multiple lobules from the same patient in assessing the association of density with lobule characteristics and using conditional logistic regression for association with case-control status. Results: Among 94 women (median age 52, range 35-73), 876 lobules were evaluated: 431 in BBD cases and 445 in BBD controls. Overall, 391 lobules (45%) were normal and the remaining 485 (55%) demonstrated fibrocystic changes. Among fibrocystic lobules, 247 (51%) had nonproliferative changes, 218 (45%) had epithelial proliferation, and 20 (4%) had atypical hyperplasia. Among normal lobules, 61 (16%) had no involution, 139 (36%) had partial involution, and 191 (49%) had complete involution. The vast majority of lobules (861/876, 98%) demonstrated presence of CD56+ cells, while only 15 of 876 lobules in 11 patients had zero CD56+ cells [7 cases (15%) versus 4 controls (8.5%) OR 1.75, p = 0.37]. The median CD56+ PR was 0.55% overall. Fibrocystic lobules as a group showed significantly lower median CD56+ PR compared to normal lobules (0.37% vs 0.85%, p &lt; 0.0001). Among subcategories of fibrocystic lobules, the median CD56+ PR was lower in proliferative lobules (0.29%) compared to non-proliferative (0.42%), but this difference was not statistically significant (p = 0.55). Older women (age &gt; 55) showed the highest CD56+ PR (median of 0.92%), as compared to 0.38% in women 45-55 (p=0.02), and 0.52% in women age &lt;45 (p = 0.26). Among normal lobules, median CD56+ PR was similar between lobules with no involution (0.55%) and partial involution (0.52%) but was considerably higher among lobules with complete involution (1.3%, p = 0.09 and p = 0.05, respectively). In a multivariable model, CD56+ PR differences between fibrocystic and normal lobules remained significant (p = 0.0002) and also remained significant between age groups of &gt; 55 and 45-55 (p = 0.02), while involution was non-significant. Median CD56+ PR was lower in cases (0.43%) compared to controls (0.72%), but this was not statistically significant (p=0.19) in our sample of 47 case-control pairs. Conclusions: Breast lobules with fibrocystic changes show less CD56+ cells, raising a question of possible immune suppression induced by early epithelial abnormalities. These findings encourage further investigation to improve understanding of the immune microenvironment in premalignant breast tissues. Citation Format: Rushin D. Brahmbhatt, Daniel Kerekes, Tanya L. Hoskin, Alvaro Pena, Daniel W. Visscher, Melody L. Stallings-Mann, Derek C. Radisky, Linda M. Murphy, Vernon Shane Pankratz, Keith L. Knutson, Marlene Frost, Amy C. Degnim. CD56+ immune cell infiltration is decreased in benign breast lobules with fibrocystic changes. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B34.

Abstract Introduction: Estrogen receptor beta (ERβ), which is highly expressed in benign breast epithelium, is postulated to function as a tumor suppressor of breast cancer. Atypias of the breast increase the lifetime risk of breast cancer four-fold. However, little is known about ERβ expression in association with atypias or whether ERβ plays a role in mitigating breast cancer risk in high-risk individuals. We therefore undertook this study to examine the relationship of ERβ expression in atypias and adjacent normal lobules with the risk of subsequent breast cancer. Methods: We studied women in a well-characterized benign breast disease cohort with atypical ductal hyperplasia (ADH) or atypical lobular hyperplasia (ALH) diagnosed from 1967 to 1991. Nuclear and cytoplasmic ERβ expression was assessed via immunohistochemistry in both the atypical hyperplasia epithelium (atypia) and background normal lobules for percent (scores 0-4 for &lt;1%, 1-25%, 26-50%, 51-75%, &gt;75%, respectively) and intensity (scores 0-3 for negative, weak, intermediate, strong, respectively) using a highly specific ERβ monoclonal antibody (PPG5/10). For nuclear staining, an ERβ sum score (percent + intensity, range 0-7) was created and grouped as low (0-2), moderate (3-5) or high (6-7). Competing risks regression was used to assess the association of ERβ expression with future breast cancer risk while also accounting for the competing risk of death from other causes. Results: 171 women, median age 56, were studied (79 ADH and 92 ALH). Median follow-up was 15 years overall, and 36 women developed breast cancer at a median of 13 years after their atypia biopsy. ERβ expression was lower in the atypia versus normal lobules whether evaluated by nuclear percent stained, nuclear intensity or cytoplasmic intensity (all p &lt; 0.001). For nuclear staining specifically, ERβ expression in the atypia was low in 44 (26%), moderate in 117 (68%) and high in only 10 (6%) based on the sum score. In contrast, ERβ was higher in the normal lobules with a majority showing high expression (n = 96, 56%), moderate expression in 74 (43%) and low expression in only 1 (0.6%). Lower ERβ nuclear expression in the atypia was associated with increased breast cancer risk, whether evaluated by percent staining, intensity or sum score; the same was true for ERβ nuclear expression in the background normal lobules. Low ERβ expression in the atypia (sum score 0-2) was associated with a 2-fold increased risk of subsequent breast cancer compared to atypia with moderate-high expression (sum score 3-7), HR 2.0, 95% CI: 1.02-3.8, p = 0.04. Lower ERβ expression in the normal lobules was also associated with an increased risk of future breast cancer: HR = 2.5 (95% CI: 1.3-5.1, p = 0.009) for low-moderate versus high expression. Conclusions: Increased ERβ expression in normal and atypical lobules was associated with a significantly decreased risk of breast cancer in our high-risk patient cohort. These data suggest that in women with atypical hyperplasia, ERβ expression is protective against the future development of breast cancer. ERβ may serve as a useful biomarker to further stratify breast cancer risk in women with benign breast disease. Citation Format: Tina J. Hieken, Jodi M. Carter, John R. Hawse, IV, Tanya L. Hoskin, Marlene Frost, Lynn C. Hartmann, Derek C. Radisky, Daniel W. Visscher, Vernon S. Pankratz, Amy C. Degnim. ERβ expression and breast cancer risk prediction for women with atypias. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr A03.

Abstract Estrogen receptor (ER) β is highly expressed in normal breast epithelium and a putative tumor suppressor. Atypical hyperplasia substantially increases breast cancer risk, but identification of biomarkers to further improve risk stratification is needed. We evaluated ERβ expression in breast tissues from women with atypical hyperplasia and association with subsequent breast cancer risk. ERβ expression was examined by immunohistochemistry in a well-characterized 171-women cohort with atypical hyperplasia diagnosed 1967–1991. Nuclear ERβ percent and intensity was scored in the atypia and adjacent normal lobules. An ERβ sum score (percent + intensity) was calculated and grouped as low, moderate, or high. Competing risks regression was used to assess associations of ERβ expression with breast cancer risk. After 15-year median follow-up, 36 women developed breast cancer. ERβ expression was lower in atypia lobules in than normal lobules, by percent staining and intensity (both P &lt; 0.001). Higher ERβ expression in the atypia or normal lobules, evaluated by percent staining, intensity or sum score, decreased the risk of subsequent breast cancer by 2-fold (P = 0.04) and 2.5-fold (P = 0.006). High normal lobule ERβ expression conferred the strongest protective effect in premenopausal women: the 20-year cumulative incidence of breast cancer was 0% for women younger than 45 years with high versus 31% for low–moderate ERβ expression (P = 0.0008). High ERβ expression was associated with a significantly decreased risk of breast cancer in women with atypical hyperplasia. These data suggest that ERβ may be a useful biomarker for risk stratification and a novel therapeutic target for breast cancer risk reduction. Cancer Prev Res; 8(11); 1084–92. ©2015 AACR.

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