Citations

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 Introduction: The role of macrophages in the tumor microenvironment is of increasing interest for tumor progression and invasion. Here we investigated the difference in CD68+ cell density in tissue samples from women with Benign Breast Disease (BBD) and women with no clinical breast disease from the Komen Tissue Bank (KTB) at Indiana University. Method: Archived breast tissue samples from women with BBD and no clinical breast disease were obtained for this age-matched case-control study. BBD samples included cases [later got Breast Cancer (BC)] and controls (did not get BC). Samples were characterized via H&E stain for epithelial proliferation [normal (N), fibrocystic non-proliferative (NP), proliferative (P), or atypia (A)]. CD68+ cell density was obtained by digital image quantitation on a per lobule basis (Total CD68+ cells/mm2). Statistical analysis was performed with mixed effects regression. Results: Among 276 women (median age 53, range 35-79) comprising 92 age-matched triplets, 2508 lobules were evaluated [789 KTB, 839 BBD case, 880 BBD control]. 1407 (56%) lobules were normal and 1101 (44%) were fibrocystic {NP [613 (56%)], P [448 (41%)] and A [40 (4%)]}. Out of 2508 lobules analyzed, 2362 (94%) had CD68+ cells, while 146 (6%) showed zero CD68+ cells [19 (2%) BBD cases, 20 (2%) BBD controls, 107 (14%) KTB, p<0.0001]. KTB samples showed significantly lower (p<0.0001) CD68+ density (median 136 cells/mm2) as compared to BBD cases (median 313 cells/mm2) and BBD controls (median 299 cells/mm2). (See Table) For each lobule type, KTB samples had lower CD68+ density compared with the same lobule type in BBD samples (p<0.0001). Conclusion: CD68+ cells are more frequent in breast lobules from women with BBD compared to normal donors. This finding in premalignant breast tissues is consistent with inflammation as a driver of tumorigenesis. BBD case (n = 92 patients)BBD control (n = 92 patients)KTB(n = 92 patients)n lobulesMedian (IQR)n lobulesMedian (IQR)n lobulesMedian (IQR)Overall839313 (165 - 538)880299 (164 - 527)789136 (55 - 272)By Lobule TypeNormal344310 (177 - 557)434331 (177 - 567)629128 (55 - 255)All non-normal/fibrocystic lobules combined495314 (157 - 527)446274 (152 - 478)160159 (58 - 393)Fibrocystic non-proliferative231319 (153 - 543)263274 (140 - 496)119163 (71 - 359)Fibrocystic proliferative246295 (153 - 496)169277 (153 - 478)33128 (30 - 385)Atypia18484 (313 - 739)14260 (223 - 375)8216 (0 - 493)IQR - Interquartile range (25th percentile - 75th percentile) Citation Format: Muhammad A. Arshad, Daniel W. Visscher, Tanya L. Hoskin, Rushin D. Brahmbhatt, Alvaro Pena Jimenez, Melody L. Stallings Mann, Erin E. Miller, Linda M. Murphy, Jodi M. Carter, Stacey J. Winham, Keith L. Knutson, Derek C. Radisky, Amy C. Degnim. CD68+ immune cells show different infiltration patterns in tissue samples from women with no clinical breast disease and those who have 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 2364. doi:10.1158/1538-7445.AM2015-2364

Abstract Introduction: Increased levels of circulating estrogens are associated with elevated risks of postmenopausal breast cancer. Previously, we showed that levels of sex steroid hormones in breast adipose tissue are positively correlated with circulating hormones. Further evaluation of hormone levels in breast tissues may help to explain the relationship between estrogens and breast cancer risk. Specifically, data suggest that focal breast lesions consisting of macrophages encircling dying adipocytes, referred to as “crown-like structures (CLS)”, are associated with increased aromatase activity, which could increase conversion of androgens to estrogens locally in the breast. Therefore, we assessed whether the presence of CLS affects the correlations between blood and breast adipose tissue hormone levels. Methods: Participants included 84 postmenopausal women with invasive breast cancer in the Polish Breast Cancer Study, a population based case-control study conducted from 2000 to 2003. We previously reported sex steroid hormone levels in cancer-free breast adipose tissue removed as part of breast excisions performed for cancer. Here, using immunohistochemical stains, we evaluated the number of CD68 positive macrophages and the presence of full CLS (i.e., complete adipocyte encirclement) in benign breast tissue sections prepared from surgical pathology specimens containing breast cancer. The relationship between the number of CD68 positive macrophages per unit area of fat and hormone levels was examined using Spearman rank correlations (rs). We assessed potential effect modification of the association between blood and tissue hormone levels by presence or absence of CLS by including an interaction term in linear regression models. Results: The number of CD68 positive macrophages per unit area of fat was not significantly correlated with levels of estrone, estradiol, androstenedione or testosterone measured in blood (rs = 0.001, -0.04, -0.07, and -0.09, respectively; p>0.05) or breast adipose tissue (rs = -0.15, 0.06, -0.06, and -0.08, respectively; p>0.05). Correlations between serum and adipose tissue hormones did not statistically significantly differ between women with CLS (n = 30) compared to those without CLS (n = 54). CLS were found more frequently in breast adipose tissue from overweight (i.e., women with a body mass index (BMI) between 25 and 29.9) (43%) and obese women (BMI >30) (30%) versus normal weight women, with (BMI between 18.5 and 24.9), (27%); (p = 0.04, χ2 test). The number of CD68 positive macrophages per unit area of fat was not significantly related to BMI (p = 0.31). Conclusion: Among postmenopausal women with invasive breast cancer, the presence of CLS was more common among overweight and obese women, but CLS did not influence the correlation between circulating and breast adipose tissue sex steroid hormone levels. Citation Format: Maeve Mullooly, Hannah P. Yang, Roni T. Falk, Sarah Nyante, Renata Cora, Ruth M. Pfeiffer, Derek C. Radisky, Daniel W. Visscher, Lynn C. Hartmann, Amy C. Degnim, Frank Z. Stanczyk, Jonine F. Figueroa, Montserrat Garcia-Closas, Jolanta Lissowska, Melissa T. Troester, Louise A. Brinton, Mark E. Sherman, Gretchen L. Gierach. Investigation of the relationship between crown-like structures and adipose tissue hormone levels among postmenopausal women with 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 2767. doi:10.1158/1538-7445.AM2015-2767

Abstract Distinct subtypes of triple negative (TN) breast cancer have currently been identified by tumor expression profiling and are thought to be influenced by the tumor microenvironment. However, little is known about the relationship between distinguishing histopathologic features of TN tumors, which reflect aspects of both tumor behavior and tumor microenvironment, and molecular TN subtypes. We analyzed whole genome expression profiles of 593 TN tumors from the Triple Negative Breast Cancer Consortium (TNBCC) using the Illumina Whole Genome DASL array. Histopathologic review of mitotic index, grade, trabecular pattern, growth pattern, involution, presence of ductal carcinoma in situ, and degree of lymphoid infiltrate, necrosis, fibrosis, and lobulitis was completed for 310 of these tumors. Clusters were identified both agnostically and using genes associated with these histopathologic features, and subtypes were evaluated for enriched pathways and associations with histopathologic features. Agnostic clustering identified four stable clusters which represented a basal-like (BL) signature, a luminal signature with overexpression of androgen receptor and targets, an immune signature, and a stromal (STR) signature. Overall, the TN tumors tended to have little or no necrosis (91%), high grade (88%), a high mitotic index (80%), fibrosis (77%), an epicentric growth pattern (62%), incomplete lobular involution (55%), trabecular structures (37%), lymphocytic infiltration (25%), adjacent ductal carcinoma in situ (24%), and variable amounts of lobulitis. Using a combined total of 2,776 unique probes individually associated with the ten histopathologic features measured, we identified six distinct TN subtypes, with an additional BL and STR signature compared to the four agnostic subtypes. Our findings suggest that histopathologic features that reflect heterogeneity in tumor cell structure and behavior as well as the tumor microenvironment, particularly the presence of lymphocytic infiltrate, are critical for TN subtype identification. Further work is needed to validate these signatures and to better understand the immunologic pathways associated with each subtype. Citation Format: Kristen Purrington, Daniel Visscher, Drakoulis Yannoukakos, Jane Carpenter, Heli Nevanlinna, Arto Mannerma, Xianshu Wang, Graham Giles, Wei Zheng, Angela Cox, Hiltrud Brauch, Ute Hamann, Diana Eccles, Celine M. Vachon, Fergus J. Couch. Genes associated with histopathologic features of triple negative breast tumors predict molecular subtypes. [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 1924. doi:10.1158/1538-7445.AM2015-1924

ObjectiveAssess algorithms for linking patients across de‐identified databases without compromising confidentiality.Data Sources/Study SettingHospital discharges from 11 Mayo Clinic hospitals during January 2008–September 2012 (assessment and validation data). Minnesota death certificates and hospital discharges from 2009 to 2012 for entire state (application data).Study DesignCross‐sectional assessment of sensitivity and positive predictive value (PPV) for four linking algorithms tested by identifying readmissions and posthospital mortality on the assessment data with application to statewide data.Data Collection/Extraction MethodsDe‐identified claims included patient gender, birthdate, and zip code. Assessment records were matched with institutional sources containing unique identifiers and the last four digits of Social Security number (SSNL4).Principal FindingsGender, birthdate, and five‐digit zip code identified readmissions with a sensitivity of 98.0 percent and a PPV of 97.7 percent and identified postdischarge mortality with 84.4 percent sensitivity and 98.9 percent PPV. Inclusion of SSNL4 produced nearly perfect identification of readmissions and deaths. When applied statewide, regions bordering states with unavailable hospital discharge data had lower rates.ConclusionAddition of SSNL4 to administrative data, accompanied by appropriate data use and data release policies, can enable trusted repositories to link data with nearly perfect accuracy without compromising patient confidentiality. States maintaining centralized de‐identified databases should add SSNL4 to data specifications.

Abstract INTRODUCTION Next generation sequencing (NGS) of patients has significantly changed our ways to study cancer genomics as it provides precise estimates of gene expression, fusion transcripts, expressed single nucleotide variants (eSNVs), splice variants and copy number variants. The Breast Cancer Genome Guided Therapy (BEAUTY) is an ongoing clinical study in which RNA sequencing (RNAseq) and whole exome sequencing (WES) are performed prior to, during and after neoadjuvant chemotherapy. Here we report the use of these sequencing technologies to investigate gene expression levels and mutational profiles in a triple negative breast cancer (TNBC) patient enrolled in BEAUTY whose disease did not respond to neoadjuvant paclitaxel and anthracycline/cycphosphamide. METHODS Computational approaches were used to integrate WES and RNAseq data obtained before therapy (V1T), after 12 weekly paclitaxel treatments (V2T) and after anthracycline-based regimen at surgical resection (V3T) to study a single patient with persistent TNBC disease after neoadjuvant chemotherapy. RESULTS Using RNA-Seq data, we identified an inter-chromosomal fusion transcript between chromosome 20 and 22 (GNAS-TTC38) that was highly expressed at V1T, V2T and V3T. We also identified intra-chromosomal fusion transcripts that were expressed at two time points, such as fusion transcript (KANSL1-ARL17A) on chromosome 17 for V1T and V2T and fusion transcript (RBM12B-LINC00535) on chromosome 8 for V2T and V3T time points. Several gene expression changes were also observed. Gene expression analysis of V1T, V2T and V3T tumors was performed. Differential temporal gene expression profiles of 9884 genes that were significant and varying at different time points were obtained for pathway analysis. Pathway analysis of 9884 genes identified up regulation and down regulation of several transcription factors with a fold change of 2x or more. When compared to blood, DNA tumor and RNA-Seq data, we identified 81 common somatic eSNVs that were expressed in both V1T and V2T time points and we are in the process of investigating V3T data. We found alterations of key transporter domains (CD225, Coatamer_beta_C, DUF2435, Dynamitin, EI24, GLTP, LMF1, Porin_3, V-ATPase_C) in our V1T and V2T SNV data. Similar to gene expression analysis, we are in the process of obtaining the list of mutations at various time points to identify driver and passenger mutation candidate genes for this specific TNBC patient. CONCLUSIONS Our initial time-series analysis of eSNV, fusion transcripts and gene expression data demonstrate that intensive analysis for individual patients is feasible. Further investigation of drug transporters and transcription regulators may help develop personalized treatment strategies for patients with disease resistant to current regimens. Citation Format: Krishna R. Kalari, Xiaojia Tang, Kevin J. Thompson, Douglas W. Mahoney, Poulami Barman, Jason P. Sinnwell, Hugues Sicotte, Peter Vedell, Steven N. Hart, Travis J. Dockter, Katie N. Jones, Amy L. Conners, Ann M. Moyer, Daniel W. Visscher, Jia Yu, Bowen Gao, Sarah A. McLaughlin, John A. Copland, Alvaro Moreno-Aspitia, Donald W. Northfelt, Richard J. Gray, Vera J. Suman, Jeanette E. Eckel Passow, Jean-Pierre A. Kocher, Eric D. Wieben, Gianrico Farrugia, Cloann G. Schultz, James N. Ingle, Richard Weinshilboum, Matthew P. Goetz, Liewei Wang, Judy C. Boughey. Analysis of sequencing data to identify potential drug targets for an individual newly diagnosed with basal breast cancer who failed to respond to current standard neoadjuvant chemotherapy. [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 4185. doi:10.1158/1538-7445.AM2014-4185

Abstract Introduction: Histologic characteristics of nonmalignant breast tissues (epithelial proliferation and involution) are associated with future breast cancer (BC) risk. We evaluated immune infiltration (lobulitis) in benign breast lobules and its association with features of BC risk. Methods: We analyzed archived nonmalignant breast tissue samples from 81 women in age-matched groups of varying cancer risk: 27 with benign breast disease (BBD) who later developed BC (cases), 27 with BBD without subsequent BC (controls), and 27 normal women without clinical breast disease from the Komen Tissue Bank at Indiana University (KTB). Up to 10 lobules in each sample were characterized by H&E for lobulitis, involution (none, partial, complete), and fibrocystic (FC) status (normal, nonproliferative, proliferative). Lobulitis was defined by H&E staining as lymphocyte nuclei in the intralobular stroma at low magnification and between adjacent acini at high magnification. Data was analyzed by mixed effects logistic regression. Results: Median age was 57 years (range 37-70). Among 81 breast tissue samples, 750 lobules were evaluated: 249 in BBD Cases, 265 in BBD Controls, and 236 in KTB donors. 56% of lobules were normal and 44% were FC, with 87% of FC lobules occurring in BBD samples. Lobulitis was present in 44% of lobules and varied according to subject age and individual lobule features. Younger women were more likely to have lobulitis (p=0.002); among women <50 the median proportion of lobules with lobulitis was 50%, versus 30% among women 50+. Lobulitis was less frequent in lobules with complete involution (37%) compared to partial (74%) or no involution (84%), p<0.0001. Lobulitis was common in normal lobules (52%) and decreased with increasing epithelial abnormality-39% of nonproliferative lobules and 27% of proliferative lobules (p<0.0001). In multivariate analysis including all 3 variables, all 3 remained significantly associated with lobulitis: age (p=0.03), proliferation (p<0.0001), and involution (p=0.0001). Lobules with lobulitis were less frequent in groups of women with increased breast cancer risk-55% of lobules in normal KTB samples, 43% in BBD controls, and 35% in BBD cases (p = 0.02). Conclusion: Lobulitis is common in normal and benign breast tissue lobules but varies with age, involution status, and epithelial abnormality. Further work is needed to understand the role of immune infiltrates in breast cancer risk. Citation Format: Rushin D. Brahmbhatt, Daniel W. Visscher, Tanya L. Hoskin, Derek C. Radisky, Linda M. Murphy, Melody L. Stallings Mann, Erin Miller, Vernon S. Pankratz, Lynn C. Hartmann, Marlene H. Frost, Amy C. Degnim. Immune infiltration of normal and benign breast lobules varies in breast tissues based on cancer risk. [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 1652. doi:10.1158/1538-7445.AM2014-1652

Abstract Background Cancer genomic investigations have identified recurrent genomic aberrations critical for cancer initiation, progression, and metastases. However, these investigations are typically performed in isolation, and the effects of treatment on the clonal selection of tumor cells are mostly unknown. We hypothesized that molecular profiling of residual tumors after neoadjuvant chemotherapy (NAC) would identify new drug targets/pathways in patients at high risk for disease recurrence. To better identify clonal populations of resistant breast cancer cells, we utilized DNA content-based flow sorting of nuclei to identify and isolate clonal populations for aCGH and next generation sequencing (NGS) both before and after NAC. Methods The Breast Cancer Genome Guided Therapy Study (BEAUTY) (NCT 02022202) is a prospective study of patients with high-risk breast cancer treated with neoadjuvant 12 weekly paclitaxel (T) +/- trastuzumab followed by 4 cycles of anthracycline based chemotherapy. Tumor tissue from baseline, residual disease from surgery, distant metastases, and patient derived xenografts (PDX) are obtained for cell sorting by DNA ploidy, aCGH, RNA and exome sequencing. Results: 140 patients have been enrolled, 104 have completed surgery and 30 unique PDX have been established corresponding to 26 patients prior to chemotherapy and 4 from residual disease at surgery. Baseline exome and RNA sequencing is complete in 140. Currently, genomic analyses of flow sorted matched baseline, surgical, PDX, and distant disease samples are available in 6 patients. Substantial genomic variation was observed in the surgical sample compared to the primary tumor including gain of oncogenic drivers (EGFR) and loss of negative regulators (ATG5) (Table). The PDX recapitulated these events with excellent fidelity compared to the corresponding human tumor. In patients with TNBC, RNA seq obained from matched samples demonstrated changes in immune related pathways. Evaluation of drug targets/pathways identified in the resistant tumors are ongoing using the PDX and sequencing of the remaining matched baseline/surgical disease will be reported. Clonal changes occurring over time in patients with residual disease or disease recurrence after NACTumor SubtypeResidual Cancer BurdenDisease StatusClonal Aberration Changes (baseline and post NAC)TNBC (AR subtype)3Contralateral lymph node recurrence at 150 days2p25.2 - p25.1 amplicon lost at recurrence; 6p21.32 -p21.31 amplicon lost at recurrenceTNBC (BL 1)3Bone/liver/lymph node recurrence at 135 days5q11.2 amplicon gained at surgery; 12p13.33 - p13.2 amplicon gained at surgeryTNBC (BL 1)3Disease-free at day 1506q21 amplicon lost at sugeryTNBC (BL 2)0Brain recurrence at 390 daysChr 2 chromothripsis at surgeryLuminal B3Progression during chemotherapy7p12.1 - p11.2 amplicon gained at surgeryLuminal HER23Disease-free at day 3609q33.2 amplicon lost at surgery; 15q13.1 - q13.3 amplicon lost at surgery; 15q22.2 - q24.1 amplicon lost at surgery Conclusions: We observed substantial evolutionary changes in residual breast tumors remaining after NAC. Our findings suggest that a comprehensive assessment of the mutational landscape that has evolved during NAC can inform drug development in high risk breast cancer patients. Citation Format: Matthew P Goetz, Michael T Barrett, Krishna R Kalari, Vera J Suman, Sarah A McLaughlin, Alvaro Moreno-Aspitia, Ann M Moyer, Donald W Northfelt, Richard J Gray, Jason Sinnwell, Douglas Mahoney, Poulami Barman, Peter Vedell, Xiaojia Tang, Kevin Thompson, Travis Dockter, Katie Jones, Sara J Felten, Amy Conners, Jeanette Eckel-Passow, Hughes Sicotte, Steven N Hart, Jia Yu, Daniel W Visscher, Eric D Wieben, Cloann Schultz, Minetta C Liu, James N Ingle, Liewei Wang, Richard W Weinshilboum, Judy C Boughey. Impact of neoadjuvant chemotherapy on the clonal composition of breast cancer [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 PD3-3.

Abstract Background: Mammographic breast density (MBD) reflects the proportion of fibroglandular tissue in the breast, and is an established risk factor for breast cancer. Benign breast disease (BBD) is also a known risk factor for breast cancer. We previously showed an association between lobular involution (physiologic atrophy) and MBD in a large cohort study of women with BBD. We performed a comprehensive evaluation of the association of histologic features of BBD with MBD. Methods: The Mayo BBD cohort includes women, 18 to 85 years, who were diagnosed with BBD at Mayo Clinic in Rochester, MN between 01/01/1967 and 12/31/1991. For this study, we included women who had a mammogram within 6 months of BBD diagnosis, and diagnosed with BBD between 1985 and 1991 when parenchymal pattern of breast density was clinically recorded. Risk factor information such as age, and body mass index (BMI) was collected from medical records. Parenchymal pattern, assessed clinically and previously used in multiple studies of MBD and risk, classified breast density based on extent and type of density, into four categories: N1 (non-dense, no ducts visible); P1 (ductal prominence occupying <25% breast); P2 (prominent ductal pattern occupying >25% of the breast; DY (homogenous plaque-like areas of density). All assessments of benign breast tissue were performed by expert breast pathologists blinded to both MBD and BBD reports. Histologic characteristics included overall impression (non-proliferative disease (NPD), proliferative disease without atypia (PDWA), and atypical hyperplasia (AH)), proportion of normal lobules that were involuted (no (0%), partial (1 to 74%), or complete (≥75%)), type of AH (ADH or ALH), ductal and lobular hyperplasia, calcifications, sclerosing adenosis, columnar alteration, cyst, fibroadenoma, marked fibrosis, intra-ductal papilloma, radial scar (number and size), duct ectasia, and mucocele like tumors. Associations of parenchymal pattern with BBD characteristics were examined using multicategorical nominal logistic regression models. We first examined associations after adjustment for age and BMI. All variables statistically significant (p<0.05) in these models were then simultaneously included in a fully-adjusted model. Results: Of 2,257 women in the study, 14% were <40 years old, 52.7% between 40- 59 years, and 32.9% were ≥60 years. In this sample, 55.2% of women had NPD, 36% had PDWA, and AH in 8.8%. MBD was classified as N1 in 21.8%, P1 in 12.6%, P2 in 23.7% and DY in 41.9%. Age- and BMI-adjusted analyses showed that there was an association of parenchymal pattern with overall impression, lobular involution, ductal hyperplasia, sclerosing adenosis, columnar alteration, cyst, duct ectasia and fibrosis. Multivariate analyses found that women of younger age (p<0.001) and with lower BMI (p<0.001), presence of fibrosis (p<0.001) and no involution (p=0.016) were more likely to have P2 or DY parenchymal pattern than women without those characteristics. Conclusion: Among women with benign breast disease, higher MBD is associated with younger age, lower BMI, fibrosis, and lack of lobular involution. These findings, concordant with other studies of MBD, provide insight into underlying mechanisms by which MBD and BBD contribute to breast cancer risk. Citation Format: Karthik Ghosh, Robert A Vierkant, Ryan D Frank, Daniel W Visscher, Vernon S Pankratz, Christopher G Scott, Derek C Radisky, Marlene H Frost, Lynn C Hartmann, Amy C Degnim, Celine M Vachon. Association between mammographic breast density and histologic features of benign breast disease [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 P6-10-14.

Abstract Introduction: Current models to predict breast cancer risk do not differentiate risk for estrogen receptor (ER) positive and negative breast cancer (BC), despite growing evidence that these tumors are biologically very different. We hypothesized that women with ER+ BC cancers have different clinical risk factors and histologic findings on prior benign breast biopsies than those with ER- BC. Methods: After IRB approval, we examined associations of age at benign biopsy and histologic features of the benign biopsy with ER status of incident BCs within the Mayo Benign Breast Disease Cohort. Benign biopsy slides were reviewed for extent of lobular involution and degree of epithelial proliferation by a single breast pathologist blinded to BC events. Invasive BCs occurring within 15 years after benign biopsy were classified as ER+ if ER staining was >1%. BC case-only associations with ER status were evaluated using multivariate logistic regression. Full-cohort hazard ratios (HR) and 95% confidence intervals (CI) for risk of ER-specific subtypes were estimated using Cox proportion hazards regression. Results: Among 13,410 women undergoing a benign breast biopsy from 1967-2001, 656 invasive BCs (459 ER+, 106 ER, 106 unknown) occurred within 15 years. Women who developed ER+ and ER- BCs were similar in age at the time of their prior benign breast biopsy (p=0.34). Although benign biopsies in women who later developed ER+ BC were more likely to show complete involution (23% vs 15% for ER- BC), this was not statistically significant (p=0.06). However, the degree of epithelial proliferation was significantly associated with ER status of later BCs (p=0.001), with ER+ BCs more likely than ER- BCs to have had a prior biopsy with atypical hyperplasia (16% vs 8%), and ER+ BCs less likely than ER- BCs to have had a prior biopsy with proliferative disease without atypia (33% vs 52%); this association remained after multivariate adjustment (p=0.003). We further pursued the association of epithelial proliferation with differential risk of ER+ and ER- BC in our overall cohort of 13,410 women (Table 1). Compared to women with non-proliferative disease, women with proliferative disease +/- atypia had ∼2-fold hazard ratios for ER- BC, whereas hazard ratios for ER+ BC were higher in women with atypical hyperplasia (∼4-fold) compared to proliferative disease. Hazard Ratio of ER+ and ER- BC within 15 years of BBD based upon degree of epithelial proliferation at benign biopsyCancer TypeGroupNN EventsHazard Ratio (95% CI)p-valueInvasive ER- ≤15 yearsNP8478421.00 (ref)<0.0001 PD4229542.63 (1.76, 3.93) AH70382.60 (1.22, 5.54) Invasive ER+ ≤15 yearsNP84782321.00 (ref)<0.0001 PD42291511.33 (1.09, 1.64) AH703744.41 (3.39, 5.73) NP=nonproliferative disease, PD=proliferative disease without atypia; AH=atypical hyperplasia Conclusion: ER+ and ER- breast cancers appear to have different features on prior benign breast biopsy, with atypical hyperplasia showing increased risk for both types of breast cancer, but a greater risk for ER+ tumors. Citation Format: Amy C Degnim, Derek C Radisky, Robert A Vierkant, Ryan D Frank, Marlene H Frost, Vernon S Pankratz, Celine M Vachon, Tanya L Hoskin, Julie M Cunningham, Chen Wang, Jean-Pierre Kocher, Teresa M Allers, Joanne L Johnson, Tina J Hieken, Karthik Ghosh, Lynn C Hartmann, Daniel W Visscher. Histologic features of benign breast biopsy tissue and association with ER positive and ER negative breast cancer in the Mayo BBD cohort study [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 P6-10-06.