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Abstract Background Women with atypical hyperplasia (AH) are at an approximately four-fold increased risk of subsequent breast cancer (BC). Mammographic breast density (MBD) is a well-established risk factor for BC, but its contribution to BC risk in women with AH remains an open question. We previously reported no association between MBD [measured by Wolfe's parenchymal pattern (PP)] and BC risk in a cohort of 147 women with AH. Here, we present results in an expanded cohort of 459 women diagnosed with AH between 1985 and 2001. Methods The Mayo Clinic Benign Breast Disease Cohort includes 13,485 women who had benign core and/or excisional biopsy 1967-2001. Biopsy tissues were reviewed by our study pathologist to determine presence of AH. MBD was available from clinical records starting in 1985, coded as PP (the standard for 1985-1996) or BI-RADS (1997-2001) density criteria. The original four-level PP (N1-fatty, P1-ductal prominence <25% of breast, P2-ductal prominence >25%, DY-dysplasia) and BI-RADS (fatty, scattered densities, heterogeneously dense, extremely dense) measures were re-categorized as low, moderate or high MBD by combining the middle two categories for each. BC events and clinical information were obtained by questionnaires, medical records and the Mayo Clinic Tumor Registry. Women were followed from benign biopsy to date of BC, death or last contact. Standardized incidence ratios (SIRs) were generated overall and within subgroups defined by density measure (PP vs. BI-RADS), number of atypical foci, and BMI by dividing the observed number of BCs by population-based expected values. Cox regression was used to estimate MBD hazard ratios after adjustment for demographic and clinical variables. Results Of the 551 women diagnosed with AH between 1985 and 2001, 459 (83%) had MBD data within 1 year prior to biopsy. Of these, 68 (15%) had low, 221 (48%) had moderate, and 170 (37%) had high MBD, respectively. Over a median follow-up of 11.7 years, 80 BCs were observed. SIRs for breast cancer did not differ significantly across density categories, overall or within any subgroups examined (see Table). Cox regression adjusting for age, BMI and density measure (PP vs. BI-RADS) also failed to identify an association with MBD (p=0.55). Low MBDModerate MBDHigh MBD N / BCsSIR (95% CI)N / BCsSIR (95% CI)N / BCsSIR (95% CI)P-valueOverall68/123.5 (1.8,6.1)221/393.6 (2.5,4.9)170/293.4 (2.3,4.8)0.97MBD Measure PP59/113.6 (1.8,6.5)85/153.0 (1.7,5.0)130/243.3 (2.2,5.0)0.87BI-RADS9/12.7 (0.1,14.7)136/244.0 (2.6,6.0)40/53.4 (1.1,7.9)0.90No. Atypical Foci 147/62.3 (0.9,5.1)123/182.8 (1.7,4.4)96/163.5 (2.0,5.8)0.63214/47.5 (2.0,19.1)58/134.8 (2.6,8.3)41/62.5 (0.9,5.5)0.213+7/26.7 (0.8,24.0)40/84.4 (1.9,8.6)33/74.0 (1.0,8.2)0.83BMI <2525/21.5 (0.2,5.4)75/184.9 (2.9,7.7)101/173.4 (2.0,5.5)0.1625-2919/54.9 (1.6,11.3)68/92.8 (1.3,5.2)36/42.1 (0.6,5.4)0.4530+23/54.8 (1.6,11.2)76/123.0 (1.6,5.3)32/74.0 (1.6,8.2)0.67SIRs compare observed numer of BCs to expected using Iowa SEER data. Analyses account for the effects of age and calendar period. P-value is test of heterogeneity in SIRs across columns. Conclusions We found no evidence of an association between MBD and subsequent BC in women with AH. Citation Format: Vierkant RA, Degnim AC, Hartmann LC, Frank RD, Radisky DC, Visscher DW, Frost MH, Winham SJ, Ghosh K, Vachon CM. No evidence of association between mammographic breast density and risk of breast cancer in women with atypical hyperplasia. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-09-05.

Abstract Introduction: CD4+ and CD8+ T cells are important effector cells in the adaptive immune response against cancers. In premalignant proliferative breast tissues, the role of the immune system remains relatively undefined. In this study, we sought to investigate and quantify the presence of CD4 and CD8 expressing immune cells in benign breast tissues and their association with breast cancer risk. Method: Archived breast tissue samples from women with benign breast disease (BBD) or no clinical breast disease [Komen Tissue Bank (KTB)] were obtained for this age-matched case-control study. BBD samples included BBD cases (subsequently developed breast cancer) and BBD controls). Tissue sections were characterized for non-proliferative or proliferative disease and degree of lobular involution. CD4+ and CD8+ cell densities (cells/mm2) were obtained by digital image quantitation for up to 10 individual lobules per sample and a per-sample estimate of cell density was calculated as the median cell density across lobules within a sample. Statistical analysis was performed using Wilcoxon signed-rank and Kruskal-Wallis tests. Results: Among 267 women (median age 52, range 35-75) comprising 89 age-matched triplets, 2417 lobules were evaluated [783 KTB, 804 BBD case, 830 BBD control]. 1372 (57%) lobules were normal and 1045 (43%) were fibrocystic. CD4+ cells were present in 1903 (79%) lobules while CD8+ cells were present in 2214 (92%) lobules. CD4 and CD8 densities showed a moderate positive correlation (r = 0.39) with each other, but overall, lobules showed a lower density of CD4+ than CD8+ cells (mean difference -54 cells/mm2, 95% CI: -68 to -39 cells/mm2). At the per sample level (see Table), BBD controls showed significantly higher levels of both CD4+ (p=0.009) and CD8+ cells (p=0.0001) compared to KTB samples. In contrast, the BBD cases only showed elevated CD8 (p=0.007) and not CD4 (p=0.21) cell infiltrates compared to KTB samples. Comparing BBD cases to BBD controls, BBD cases showed significantly lower CD8 cell density as compared to BBD controls (p=0.002) and cases had lower CD4 cell density but the difference was not significant (p=0.07). Despite these differences in the cell densities, the ratio of CD4:CD8+ cells did not differ significantly across groups (p=0.83). The CD4:CD8+ ratio also did not differ significantly by histologic impression (p=0.74), degree of involution (p=0.27), or age (p=0.32). KTBBBD ControlsBBD CasesMedian (IQR)(n=89)(n=89)(n=89)CD4+ cells/mm240 (17-95)60 (25-124)49 (21-92)CD8+ cells/mm285 (49-131)157 (84-225)134 (80-168)Ratio (CD4:CD8)*0.45 (0.14-0.96)0.41 (0.15-0.81)0.40 (0.19-0.70)*Ratio is missing for n=7 samples (3 KTB, 2 BBD control, 2 BBD case) with undefined ratio due to zero CD8+ cells for all lobules in the sample. Conclusion: Our findings suggest that BBD is associated with increased infiltration of T cells into the breast tissue. Furthermore, these immune responses appear more robust in BBD patients at lower risk of developing breast cancer. Citation Format: Pena Jimenez A, Hoskin TL, Arshad MA, Visscher DW, Brahmbhatt RD, Miller EE, Stallings Mann ML, Carter JM, Murphy LM, Winham SJ, Radisky DC, Denison LA, Knutson KA, Degnim AC. CD4 and CD8 T cell densities are increased in benign breast disease compared to normal breast tissue. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-09.

Abstract Background: Tumor-infiltrating lymphocytes (TIL) have prognostic and potentially predictive significance in the (neo)adjuvant treatment of high-risk breast cancer. However, quantitative TIL measurement is not routinely performed. It is unclear why some tumors attract large quantities of TIL while others do not. We sought to confirm the association between TIL and pathologic complete response rate (pCR) and to further use next generation sequencing (NGS) to identify genes and gene pathways associated with the presence/absence of TIL. Methods: We studied 140 women with high risk stage I-III breast cancer, enrolled in the Breast Cancer Genome Guided Therapy Study (BEAUTY), obtaining serial biopsies for DNA/RNA sequencing and MRI imaging to assess response to neoadjuvant chemotherapy (NAC) with taxane (+/- trastuzumab+/-pertuzumab for HER2+ disease) followed by AC or (F)EC. Diagnostic pre-NAC core needle biopsies and surgical resection specimens post-NAC were available from 110 patients. Stromal TIL were semi-quantitated on a scale of 1-4 (with 1: ≤10/hpf, 2: subtle infiltrate >10/hpf, 3: moderate infiltrate readily visible at low power magnification, 4: dense infiltrate with innumerable lymphocytes). For this analysis, low TIL was defined as scores of 1-2 vs. high defined as 3-4. Using pre-NAC biopsies, RNAseq was performed using the Illumina HiSeq2000 and the Mayo Analysis Pipeline for RNAseq (MAP-Rseq) for quality control, sequence alignment, and gene counts. The quantity of TIL was associated with transcripts across the transcriptome after conditional quantile normalization. Differentially expressed genes were obtained using EdgeR analysis, using a false discovery rate of 0.05, and pathways were evaluated using GAGE methods. Results: The pCR and residual cancer burden (RCB)-0/I rates by stromal TIL status within each molecular subtype are presented in the table. A diverse spectrum of 1344 genes with differential expression between tumors with high vs. low stromal TIL was identified. The genes with >2.0-fold change (FC) and p<1e-09 included S100A7 (4.49 FC), LCN2 (2.48 FC), and ART3 (2.82 FC) (genes known to be involved in immune regulation), as well as TDRD1 (2.71 FC) (a gene related to ERG [ETS-related gene] expression). In addition, the "regulation of actin cytoskeleton" pathway was upregulated in tumors with high TIL, while the "Hedgehog signaling" and "Wnt signaling" pathways were downregulated. Molecular SubtypeStromal TILspCR rate n (%)RCB-0/I rateLuminal AHigh------Luminal ALow0/9 (0%)0/9 (0%)Luminal BHigh1/9 (11.1%)1/8 (12.5%)Luminal BLow3/24 (12.5%)6/23 (26.1%)ER+/HER2+High3/9 (33.3%)4/9 (44.4%)ER+/HER2+Low1/6 (16.7%)1/6 (16.7%)ER-/HER2+High8/9 (88.9%)7/7 (100%)ER-/HER2+Low4/8 (50.0%)6/8 (75.0%)Triple NegativeHigh10/19 (52.6%)13/19 (68.4%)Triple NegativeLow7/14 (50.0%)9/13 (69.2%) Conclusions: We identified genes and gene pathways associated with high TIL expression in breast tumors prior to NAC that provide insight into the interactions between TIL and tumors. TIL can be easily semi-quantitated on H&E and along with these novel biomarkers, may contribute to the personalization of breast cancer therapy. Citation Format: Moyer AM, Boughey JC, Kalari KR, Suman VJ, McLaughlin SA, Moreno-Aspitia A, Northfelt DW, Gray RJ, Sinnwell JP, Carlson EE, Dockter TJ, Jones KN, Felten SJ, Conners AL, Wieben ED, Ingle JN, Wang L, Weinshilboum RM, Visscher DW, Goetz MP. Differential mRNA expression patterns in breast tumors with high vs. low quantity of stromal tumor–Infiltrating lymphocytes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-05.

Abstract Background: Tumorigenesis involves both genetic and epigenetic changes. Epigenetic alterations are reversible and are promising cancer therapeutic targets. Decitabine (5-aza-2'-deoxycytidine), a DNA methyltransferase inhibitor, is FDA approved for hematological malignancies. However, the effect of decitabine in breast cancer is not completely understood. Previous reports indicated that one decitabine mechanism involves regulation of protein levels for DNMT1, the major DNA methyltransferase that methylates hemimethylated CpG di-nucleotides in DNA. However, the E3 ligase involved in this process has not been identified. Whether decitabine also regulates DNMT3A and 3B in a similar fashion remains unclear. Therefore, our goals were to 1) understand mechanisms underlying decitabine action, 2) test the antitumor activity of decitabine in breast cancer models and 3) identify biomarkers associated with response to decitabine. Methods and Results: Western blots of breast cancer cell lines showed that DNMT1, DNMT3A, and DNMT3B protein levels decreased following decitabine treatment without a reduction in mRNA levels. Bioinformatic analysis of DNA methyltransferase sequences revealed a potential TRAF6 binding motif, and the interaction with TRAF6 (TNF receptor-associated factor 6) was confirmed by IP. TRAF6 functions as an E3 ligase. To determine whether TRAF6 might be the E3 ligase responsible for the degradation of DNMTs after decitabine treatment, we knocked down TRAF6 by RNA interference or knocked out the TRAF6 gene by CRISPR/Cas9. Down regulation of TRAF6 attenuated DNMT ubiquitination and increased DNMT protein levels, suggesting that TRAF6 might mediate proteasome-dependent degradation of all three DNMTs. This was further confirmed by reconstituting the knockout cells with WT and a TRAF6-C70A mutant, followed by assessing DNMT protein levels. Global DNA methylation was also increased after TRAF6 depletion and was confirmed in TRAF6 knock out cells in which DNMT levels were unaffected by decitabine. Cell cytotoxicity and colony forming assays showed that TRAF6 knockout cells were resistant to decitabine, suggesting that a major decitabine mechanism of action is through the regulation of TRAF6 which, in turn, degrades DNMTs, leading to decreased global methylation. Finally, decitabine significantly induced TRAF6 at both mRNA and protein levels, a process that might create positive feedback leading to increased degradation of DNMT proteins upon decitabine treatment. Based on these results, we further hypothesized that levels of the three DNMTs might influence decitabine response. Using 18 breast cancer patient derived xenograft (PDX) models, we found a wide range of DNMT protein levels regardless of ER/HER2 status. DNMT levels in the PDX models were directly associated with sensitivity to decitabine treatment, confirming our hypothesis. Conclusion: Our data showed that decitabine might be an effective agent for treating breast cancer and revealed a novel mechanism underlying decitabine treatment. Baseline DNMT protein levels may serve as a biomarker for predicting decitabine drug response. Citation Format: Yu J, Qin B, Boughey JC, Moyer AM, Visscher DW, Sinnwell JP, Yin P, Thompson KJ, Docter TJ, Kalari KR, Suman VJ, Wieben ED, Felten SJ, Conners AL, Jones KN, McLaughlin SA, Copland JA III, Moreno Aspitia A, Northfelt DW, Gray RJ, Ingle JN, Lou Z, Weinshilboum R, Goetz MP, Wang L. Regulation of DNA methyltransferases via TRAF6 determines breast cancer response to decitabine. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-51.

Abstract Background: Both HR deficiency and BRCA mutation status predict response to platinum-based therapy and BRCA mutation status predicts docetaxel resistance. However, the association of either biomarker with response to the individual elements of either AC or taxanes (T) is unknown since T is commonly given concomitantly with or after anthracyclines (A). We evaluated the association of HRD and BRCA mutation status with response to neoadjuvant weekly T followed by AC or (F)EC in high-risk breast cancer. Methods: We studied 140 high risk Stage I-III breast cancer patients (pts), enrolled in the breast cancer genome guided therapy study (BEAUTY), obtaining biopsies for DNA/RNA sequencing and MRI imaging to assess response to neoadjuvant weekly T (+trastuzumab+/-pertuzumab for HER2+ disease) followed by AC or (F)EC. Germline BRCA status and HR status of tumor samples (Myriad laboratories) were obtained. HR deficient tumor was defined as HRD score ≥42 or BRCA mutation. MRI response by changes in tumor size after 12 weeks of T was classified by WHO criteria. pCR was defined as ypT0/Tis ypN0. Both MRI response after T and pCR (after T and AC) were examined in terms of germline BRCA mutation (gBRCAmut vs. gBRCAwt) and tumor HR deficiency. Results: Of 140 pts enrolled, 8 withdrew consent and 2 carboplatin treated pts were excluded. Germline data were available for 124/130 pts. 12 patients had BRCA deleterious germline mutations (4 BRCA1, 8 BRCA2). MRI partial (PR)/complete response (CR) rate to T was 47.3% (95% CI: 37.8-57.0%) in the BRCAwt group and 66.7% (95% CI: 34.9-90.1%) in the BRCAmut group. No MRI CR's were observed in BRCA1 mut pts. In contrast, pCR rate was 50% in the 12 gBRCAmut pts (95% CI: 21.1-78.9%) and 31.3% in the 112 gBRCAwt pts (95% CI: 22.8-40.7%). HR deficiency status has thus far been determined for 74 pts: 26 pts have HD deficient tumors: 18 TNBC, 5 Luminal B, 2 ER-/HER2+; and 1 ER+/HER2+. Determination of HR deficiency is ongoing and will be reported for the full cohort in terms of 12 week MRI response to T and pCR to T+AC. HR deficientMolecular Subtypeyes (%)no (%)TBD (%)Luminal A0/112/11 (18.2)9/11 (81.8)Luminal B5/37 (13.5)13/37 (35.1)19/37 (51.3)Luminal NOS0/21/2 (50)1/2 (50)ER+/Her2+1/17 (5.8)14/17 (82.4)2/17 (11.8)ER-/Her2+2/20 (10)11/20 (55)7/20 (35)Triple Negative18/43 (41.9)6/43 (18.6)17/43 (39.5)germline BRCA statusMRI partial response after T (%)MRI complete response after T (%)pCR after T&AC (%)BRCA11/4 (25)0/42/4 (50)BRCA25/8 (62.5)2/8 (25)4/8 (50)BRCAwt35/112 (31.3)18/112 (16.1)35/112 (31.3) Conclusion: In the setting of neoadjuvant weekly T followed by AC, pCR rates were non-significantly higher in pts with BRCA1 mutations. While we observed no overall association between BRCA mutation status and response rates to taxanes; nearly all MRI responses to taxanes (partial and complete) were observed in the BRCA2 group. Prospective studies are needed to validate these findings and to determine whether BRCA status can be used to select therapy. HR deficiency is uncommon in luminal A and HER2+, frequent in TNBC, and the association of HRD with both MRI response to taxanes and pCR will be reported at the meeting. Citation Format: Boughey JC, Kalari KR, Suman VJ, McLaughlin SA, Moreno Aspitia A, Moyer AM, Northfelt DW, Gray RJ, Vedell PT, Tang X, Dockter TJ, Jones KN, Felten SJ, Conners AL, Hart SN, Visscher DW, Wieben ED, Ingle JN, Hartman A-R, Timms K, Elkin E, Jones J, Wang L, Weinshilboum RW, Goetz MP. Role of germline BRCA status and tumor homologous recombination (HR) deficiency in response to neoadjuvant weekly paclitaxel followed by anthracycline-based chemotherapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-29.

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 < 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 > 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 <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 > 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 <1%, 1-25%, 26-50%, 51-75%, >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 < 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 < 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.