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Background: Black patients with triple negative breast cancer (TNBC) have worse survival outcomes, even after adjusting for stage at diagnosis, income, insurance status and other socioeconomic factors. Little is known regarding anti-tumor immune responses in Black patients and how these differences affect responses to treatment in TNBC. Limited data exists regarding the stromal tumor infiltrating lymphocytes (sTILs, which are strongly prognostic in TNBC) distribution based on race and ethnicity. Here we evaluate the prevalence, distribution, and prognostic impact of sTILs in TNBC by race/ethnicity from 2 prospective clinical trials of adjuvant anthracycline/taxane-based chemotherapy (E2197 and E1199). Methods: Full-face hematoxylin and eosin-stained sections of 481 tumors from ECOG-ACRIN trials E2197 and E1199 were previously evaluated for density of sTILs and shown to be associated with disease-free survival (DFS), distant recurrence-free interval (DRFI), and overall survival (OS) (Adams, et al JCO 2014). Further analyses were undertaken to evaluate the impact of race/ethnicity. Results: The majority of the 481 TNBC were from White patients (82.3%, n=403); with 12.3% (n=59) Black patients, 1.6% (n=14) other (9 Hispanic, 3 Asian, 2 Other), and 0.5% (n=5) unknown race. Age distribution (mean 49.2 for White and 49.2 for Black) and node negative disease (White 68/403 (42%), Black 24/59 (41%)) were similar. However, tumor size ≤2cm was seen more commonly in White patients (34%, 137/403) compared with Black patients (20%, 12/59). Black patients had a higher proportion of high sTILs (≥30%) with 23.7% (14/59) compared to White patients (11.4%, 46/403). The association of continuous stromal TILs with DFS (hazard ratio for a 10-point difference) was 0.84 (95% CI 0.72, 0.98) for White patients and 0.94 (95% CI 0.73, 1.20) for Black patients [159 DFS events for Whites, 26 DFS events for Blacks]. Conclusions: This is the first dataset from prospective clinical trials evaluating sTILs in TNBC in Black patients. Prevalence of high sTILs was greater in Black patients compared to White patients. The association between increasing sTILs and improved invasive disease-free survival across racial/ethnic groups must be investigated in larger datasets. Table 1.Race/EthnicityTotal (n=481)White (n=403)Black (n=59)Other (n=19)Mean age49.049.249.245.6T1 (tumor 2 and Citation Format: Natalie Klar, Robert J Gray, Sylvia Adams, Joseph A Sparano, Lori J Goldstein, Angela M DeMichele, Antonio C Wolff, Nancy E Davidson, George W Sledge, Sunil S Badve. Stromal tumor infiltrating lymphocytes analysis by race and ethnicity in triple negative breast cancers from 2 phase III randomized adjuvant breast cancer trials: ECOG-ACRIN E2197 and E1199 [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-35.

Background: 18F-Fluorodeoxyglucose (FDG) has long been used for measuring tumor glycolytic activity in clinical PET imaging. The FDA recently approved 18F-Fluoroestradiol (FES) (Cerianna) as a PET imaging tracer for characterizing disease in patients with estrogen-receptor positive (ER+) breast cancer. As FES PET enters clinical practice it is important to establish its utility in the full population of hormone-receptor positive patients, including for patients with human epidermal growth factor 2 (HER2)-overexpressing tumors. Patients with HER2-positive metastatic breast cancer have historically been treated with combination cytotoxic and HER2-directed therapy, with the understanding that HER2 is the primary driver in this disease state. This retrospective study examined uptake in matched lesions for both FES and FDG PET and compared activity in patients with HER2 positive versus HER2 negative metastatic breast cancer. Methods: Patients were selected from the UW research database who had a history of biopsy-proven primary ER+ breast cancer as well as FES and FDG PET scans within 30 days. We examined FDG and FES scans and recorded SUVmax in up to 16 matched lesions between the two scans. Patients were also divided by HER2 status (+/-). In addition, a subset of patients who underwent at least 2 paired FDG and FES scans were reviewed. Results: 270 matched FDG and FES scans were analyzed in 216 patients with history of ER+, and HER2+ or HER2- breast cancer who were not part of an ongoing clinical trial. 158 (73%) had ductal disease, 38 (18%) had lobular disease. 183 (85%) had HER2- breast cancer. Of the 33 patients who had HER2+ breast cancer, 28 (85%) had ductal carcinoma. 40 patients underwent serial scans, allowing tracking over multiple timepoints. A total of 1323 metastatic sites were recorded (average = 5/scan (range 1,16)), with the majority (71%) representing bony lesions. No difference in quantitative FES or FDG avidity was observed between soft tissue and osseous sites. FES and FDG SUVmax were similar among patients with either HER2- or HER2+ breast cancer (Table 1). Among 40 patients with multiple paired FDG and FES scans, 26 (65%) had 2 scans while the remaining 14 had 3, with FES avidity remaining stable over time. There was no correlation between FES or FDG scans and HER2 status. Conclusion: In a cohort of ER+, HER2+ and HER2- patients undergoing concurrent FDG and FES PET scans, FDG and FES activity was similar regardless of HER2 status. FES uptake in both HER2- and HER2+ patients and stability over time in serial scans suggest that HER2 does not affect ER density. This suggests that in many patients with so-called “triple positive” disease, endocrine therapy may offer a powerful primary rather than ancillary tool in select patients. FES combined with FDG PET may offer utility in predicting and assessing response to therapy in this patient population. Table 1. FES and FDG uptake in patients with HER2- or HER2+ disease Citation Format: Natasha Hunter, Lanell M. Peterson, David A. Mankoff, Mark Muzi, Delphine Chen, William R. Gwin, Shaveta Vinayak, Nancy E Davidson, Jennifer M. Specht, Hannah Linden. Fluoroestradiol (FES) and Fluorodeoxyglucose (FDG) PET imaging in patients with ER+, HER2+ or HER2- metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-17.

Background: There is growing awareness of the unique etiology, biology, clinical presentation and progression of Invasive lobular breast cancer (ILC), but additional research is needed to assure translation of findings into management and treatment guidelines. We performed a survey to: 1) analyze the landscape of the current understanding of ILC, and 2) identify consensus research questions on ILC. Methods: The IRB-approved survey was developed with input from representatives of three major stakeholder groups - breast cancer clinicians/researchers, laboratory-based researchers, and advocates/patients. We fielded the survey from March to May 2022 using targeted email and via social media. Results: 1,774 participants answered at least one question and 1,310 finished the survey. Participants are from 66 countries from all continents (except Antarctica). Respondents self-identified as clinicians (mostly medical oncologists and surgeons) (N=413), researchers (N=376), and breast cancer patients (1,121), with some belonging to more than one category. 26% of the patients who participated in the survey belong to advocate groups. Only 46% of clinicians reported being confident in describing the differences between ILC and no special type (NST) (invasive ductal) breast cancer. Knowledge of histology was seen as important (73%), affecting their treatment decisions (51%), and refined treatment guidelines would be valuable for patients with ILC in the future (76%). 85% of clinicians have never powered a clinical trial to allow subset analysis for histological subtypes, but the majority would consider it. 88% would participate in a consortium to conduct clinical trials on ILC. The top two most important research questions were: 1) determining mechanisms of endocrine resistance, and, 2) identifying novel therapeutic targets, repurposing existing drugs and progressing them to clinical trials. Of the researchers, 48% reported being confident in describing differences between ILC and NST. They reported that ILCs are inadequately presented in large genomic data sets (52%), and that ILC models are insufficient (42%). Only 13% of respondents have inadequate access to tissue or blood from patients with ILC. The top two most important research questions identified by the laboratory researchers overlapped with those identified by the clinicians, i.e. understanding of endocrine resistance and identifying novel drugs that can be tested in clinical trials. The majority of patients (52%) thought that their health care providers did not explain unique features of ILC, and that in general communication was limited. When asked about top research question, they chose: 1) Improvement of ILC screening/early detection, and, 2) Identifying new and specific imaging tools for ILC. When comparing top priority topics across six research domains, there was a high degree of consistency, especially among clinicians and researcher, but less so when compared with the breast cancer patients (Table 1). Conclusion: In summary, we have gathered timely and representative information from an international community of clinicians, researchers, and patients/advocates that we expect will lay the foundation for a community-informed collaborative research agenda, with the goal of improving the management and personalizing treatment for patients with ILC. Table 1. Ratings by all three stakeholder groups of the most critical and impactful ILC research topics. Top box scores between stakeholder groups were compared using chi-square analysis. Citation Format: Steffi Oesterreich, Leigh Pate, ADRIAN V. LEE, Rachel C. Jankowitz, Patrick Derksen, Rita Mukhtar, Otto Metzger, Matthew J. Sikora, Christopher Li, Christos Sotiriou, Gary Ulaner, Jorge Reis-Filho, Nancy E Davidson, Karen Van Baelen, Laurie Hutcheson, Siobhan Freeney, Flora Migyanka, Claire Turner, Todd Bear, Christine Desmedt. An international survey on invasive lobular breast cancer (ILC) reveals gaps in knowledge and top priority research areas [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-05-10.

Background: It has become increasingly clear that effective treatment of metastatic breast cancer (MBC) requires an in-depth understanding of the molecular differences between primary tumors and metastases. The AURORA US Network was established to collect primary breast cancer-metastasis pairs for multi-platform genomic profiling in order to identify the molecular drivers of metastatic disease. AURORA US has both a retrospective and prospective phase. This is the first report of the retrospective phase. Methods: Archived tissue samples from the primary tumor and at least one distant metastasis were retrospectively collected from 83 MBC patients. Following internal quality assessment, samples from 55 pts, including 105 distinct metastatic lesions, were subject to DNA low pass whole genome and exome sequencing, DNA methylation arrays, and RNA sequencing. Early analyses of these multi-platform data include: DNA methylation, tumor gene expression and microenvironmental signatures, somatic and germline variants, DNA copy number changes, and structural variants between breast primaries and matched metastases. Results: Median age at diagnosis was 49 years (25-76); 32 (58%) were Stage I or II at presentation, 27 (49%) had a family history of breast cancer, and 20 (36%) had a second breast primary. Median disease-free interval before developing MBC was 2 years (range 0-36, 5 patients presented with Stage IV). Median overall survival from initial presentation was 4 years (range 0-37). Median survival after developing MBC was 1 year (range 0-13), with a median of three treatments. Primary tissue samples were banked from 1977-2017 and metastases were banked from 1999-2017. Clinical phenotypes of the primaries included 27 HR+ (49%), 15 triple negative (TNBC, 27%), and 11 HER2+ (20%, 12 missing HER2 status). Intrinsic subtype distribution of the primaries included 17 Basal-like (31%), 17 Luminal A (31%), 7 Normal-like (13%), 5 HER2-enriched (9%), and 1 Luminal B, with 8 pending. All metastases from the Basal-like cases remained Basal-like, while metastases from luminal primaries tended to gain HER2-Enriched subtype features (5/18, p = 0.01). Overall, we identified significant metastasis-enriched alterations in metabolism pathways, an increase in proliferation, and the loss of differentiation signatures and immune infiltrates with progression; the latter being the most pronounced in brain metastases. The most frequent somatic mutations in this cohort were in TP53, NCOR1, and RUNX1. Interestingly, ERBB2, EGFR, and ATM were also mutated in ≥10% of the tumors sequenced. In almost all cases, CpG island hypermethylation was clonally present in the primary tumor and persisted stably in the majority of metastatic lesions. Promoter CpG island hypermethylation was also identified in some metastatic lesions at JAM3, an important cellular adhesion molecule,and this was accompanied by reduced mRNA expression. CONCLUSIONS: Collection of banked primary and metastatic tissue pairs identified a young MBC cohort with a high frequency of breast cancer family history and second breast primaries. Molecular characterization of luminal tumor pairs highlighted acquisition of aggressive traits including increased proliferation and loss of differentiation in the metastases. In contrast, basal-like pairs remained relatively unchanged, except for the loss of immune activation. Ongoing analyses to be presented include clonal heterogeneity and phylogeny, novel metastasis signature discovery, gene fusion, and endogenous retrovirus detection. Citation Format: Tari A King, Minetta C Liu, Marni B McClure, Toshinori Hinoue, Benjamin J Kelly, Chad J Creighton, Jay Bowen, Kristen Leraas, Robyn T Burns, Sara Coppens, Salma Rezk, Amy L Garrett, Justin M Balko, Joel S Parker, Ben H Park, Ian Krop, Carey Anders, Katherine A Hoadley, Julie Gastier-Foster, Mothaffar F Rimawi, Rita Nanda, Nancy U Lin, Claudine Isaacs, P. Kelly Marcom, Anna Maria Storniolo, Fergus J Couch, Elaine R Mardis, Adrian V Lee, Uma Chandran, Peter W Laird, Susan G Hilsenbeck, Larry Norton, Andrea L Richardson, W. Fraser Symmans, Lisa A Carey, Antonio C Wolff, Nancy E Davidson, Charles M Perou, the AURORA US Network. Multiplatform analysis of matched primary and metastatic breast tumors from the AURORA US Network [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 GS3-08.

Purpose of the study: The purpose of this study was to investigate the predictors of objectively-measured sedentary time (ST) among breast cancer (BC) survivors who were 60 days post-treatment and were initiating participation in an intervention to improve diet and physical activity (PA) during the early phase of the COVID-19 pandemic. Methods: Cook and Move for Your Life (CMFYL) was a pilot and feasibility study of stage 0-III BC survivors testing the effects of a remotely-delivered and remotely-assessed nutrition and PA intervention. Women were ≥60 days post-treatment (current endocrine therapy allowed), consumed Results: Among the 84 women included in this analysis who had actigraphy measurements at baseline, the average ST/day was 684±79 minutes. On average, women were 58±10 years in age and most self-identified as non-Hispanic white (87%). The average time since diagnosis at time of enrollment was 4.5 years and 59% of women were receiving endocrine therapy at baseline. Adjusted models show that participants with a college degree had 24.7 (95%CI 2.0, 47.4) more minutes of ST than those with less than a college degree, and for every 1-point increase in PROMIS Physical Function scores participants had 2.5 (95%CI -4.9, -0.2) fewer minutes of ST. Conclusion: In a sample of BC survivors enrolled in a diet and PA intervention, higher level of education and poorer physical function were associated with higher ST during the early phase of the COVID-19 pandemic. These findings provide preliminary insight into factors associated with ST. Future work will investigate how these factors influence change in ST after participation in the CMFYL intervention. Citation Format: Sidney Donzella, Chongzhi Di, Eileen Rillamas-Sun, Hanjie Shen, Sofia Cobos, Yuhan Huang, Katherine A. Guthrie, Rachel Yung, Nancy E. Davidson, Heather Greenlee. Predictors of objectively-measured sedentary time during the COVID-19 pandemic among breast cancer survivors participating in a diet and physical activity intervention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6231.

Purpose: The Cook & Move for Your Life randomized pilot study assessed the feasibility and relative efficacy of two dose levels of a remotely-delivered diet and physical activity (PA) intervention for breast cancer (BC) survivors. Methods: Women with a history of stage 0-III BC who were >60 days post-treatment, ate Results: From December 2019 to January 2021, 74 women were accrued. On average, women were 57.9 years old, 4.8 years post-diagnosis, with body mass index of 29.1 kg/m2. Most were non-Hispanic white (89.2%), 51.4% were diagnosed at stage I, and 40.5% were on endocrine therapy. Questionnaire and biospecimen data collection at 6-months were completed for 93.2% and 83.8% of the sample, respectively. In the low-dose arm (n=36), 94.4% of participants attended the single class, while in the high-dose arm (n=38) 84.2% of participants attended at least 8 of the 12 sessions live or via video archived on the website (mean 9.4 sessions). On average over the 6-month intervention period, participants responded to 71.5% of the text messages, 73.0% wore their Fitbit device ≥50% of the time, and 77.0% accessed the study website. Mean vegetable intake increased by 1 serving per day among women in the high-dose arm and decreased slightly among women in the low-dose arm (P=0.03). Changes in fruit/vegetable intake and MVPA varied little by arm. Blood and stool biomarker analyses are ongoing. Conclusion: We successfully conducted a remotely-delivered diet and PA intervention for BC survivors with high accrual, adherence, and retention during the COVID-19 pandemic. Women in the high-dose arm increased vegetable intake relative to the low-dose arm. Future research will refine and test the intervention in a larger and more diverse study population. Citation Format: Heather Greenlee, Rachel Yung, Sofia Cobos, Eileen Rillamas-Sun, Hanjie Shen, Yuhan Huang, Sidney M. Donzella, Liza Schattenkerk, Kate Ueland, Matthew VanDoren, Samantha Myers, Theresa King, Margarita Santiago-Torres, Chongzhi Di, Neel Dey, Katherine A. Guthrie, Nancy E. Davidson. Cook and Move for Your Life: A pilot study of an online intervention to improve diet and physical activity among breast cancer survivors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT509.

Invasive lobular carcinoma (ILC) is the second most common subtype of breast cancer following invasive ductal carcinoma (IDC) and characterized by the loss of E-cadherin–mediated adherens junctions. Despite displaying unique histologic and clinical features, ILC still remains a chronically understudied disease, with limited knowledge gleaned from available laboratory research models. Here we report a comprehensive 2D and 3D phenotypic characterization of four estrogen receptor–positive human ILC cell lines: MDA-MB-134, SUM44, MDA-MB-330, and BCK4. Compared with the IDC cell lines MCF7, T47D, and MDA-MB-231, ultra-low attachment culture conditions revealed remarkable anchorage independence unique to ILC cells, a feature not evident in soft-agar gels. Three-dimensional Collagen I and Matrigel culture indicated a generally loose morphology for ILC cell lines, which exhibited differing preferences for adhesion to extracellular matrix proteins in 2D. Furthermore, ILC cells were limited in their ability to migrate and invade in wound-scratch and transwell assays, with the exception of haptotaxis to Collagen I. Transcriptional comparison of these cell lines confirmed the decreased cell proliferation and E-cadherin–mediated intercellular junctions in ILC while uncovering the induction of novel pathways related to cyclic nucleotide phosphodiesterase activity, ion channels, drug metabolism, and alternative cell adhesion molecules such as N-cadherin, some of which were differentially regulated in ILC versus IDC tumors. Altogether, these studies provide an invaluable resource for the breast cancer research community and facilitate further functional discoveries toward understanding ILC, identifying novel drug targets, and ultimately improving the outcome of patients with ILC. Significance: These findings provide the breast cancer research community with a comprehensive assessment of human invasive lobular carcinoma (ILC) cell line signaling and behavior in various culture conditions, aiding future endeavors to develop therapies and to ultimately improve survival in patients with ILC. Cancer Res; 78(21); 6209–22. ©2018 AACR.

Background: EarlyR is a prognostic gene signature score in ER+ breast cancer (BC) computed from the expression values of ESPL1, SPAG5, MKI67, PLK1 and PGR using a nonlinear mathematical formula. EarlyR has been validated in multiple cohorts profiled on Affymetrix and Illumina microarrays and by RNA-seq. This study sought to assess the prognostic features of EarlyR in a cohort of E2197. Patients and Methods: Illumina DASL assay was used to measure gene expression in FFPE tissue of primary BC from a case-cohort sampling subset of women in E2197 treated with doxorubicin plus docetaxel (AT) or doxorubicin plus cyclophosphamide (AC). ER+ patients received hormone therapy at physician's discretion. After 79.5 months median follow-up, disease-free survival was 85% in both treatment arms. Among patients centrally reviewed with sufficient RNA material for the DASL assay, 319 with ER+ status and assessed for EarlyR are included in the analytic cohort. EarlyR scores and pre-specified risk strata (≤25=low, 26-75=intermediate, >75=high) were computed, while blinded to clinical data. The analysis endpoint was disease-free survival (DFS), defined as the time from randomization to date of invasive BC recurrence or death from any cause within 8 years. Weighted Cox proportional hazards models were used to associate EarlyR score or risk strata with DFS. Variances of the estimated coefficients were adjusted to account for the case-cohort design. Results: The distribution of the EarlyR risk groups was 59% low, 11% intermediate and 30% high risk in this ER+ cohort. The continuous EarlyR score was significantly prognostic of DFS up to 8 years after randomization (p = 0.02). Patients with low EarlyR score (≤ 25) had significantly lower risk of BC recurrence within 8 years (p = 0.031, univariate HR=0.562, 95%CI: 0.334-0.948) compared to those with high EarlyR score (> 75). Analysis within the AC arm showed that patients with low EarlyR score had significantly lower risk of 8-year BC recurrence (p = 0.023, univariate HR=0.392, 95%CI: 0.175-0.878) compared to those with high EarlyR score. Within the AT arm there was no significant difference in 8-year DFS prognosis between any of the EarlyR risk groups. Conclusions: This study confirmed the prognostic significance of EarlyR using FFPE tissue in a cohort of patients treated with AC chemotherapy from E2197. Patients with high EarlyR score who were treated with AC had significantly higher risk of recurrence than low EarlyR score patients treated with AC. On the other hand, prognosis of high EarlyR score AT-treated patients was not significantly lower than the prognosis of low EarlyR score AT-treated patients. Further study in a larger cohort is needed to assess the relative benefits of AC versus AT within the EarlyR high risk group and the EarlyR low risk group. Citation Format: Badve S, Wang V, Willis S, Leyland-Jones B, Gokmen-Polar Y, Shulman L, Martino S, Sparano J, Davidson N, Goldstein L, Buechler S. Independent validation of EarlyR gene signature in E2197: A randomized clinical trial comparing doxorubicin plus docetaxel to doxorubicin plus cyclophosphamide as adjuvant chemotherapy in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-06-08.

BACKGROUND: Metastasis is the major cause of mortality in breast cancer (BrCa) patients. Our understanding of brain metastasis (BrM) is limited, reflected by a lack of effective treatments. We aimed to (1) determine BrCa gene signature differences between primary tumors and matched BrM and (2) uncover BrM-specific alterations that may be clinically actionable. MATERIALS and METHODS: NanoString expression profiling of 127 genes from 5 major prognostic tests (MammaPrint, EndoPredict, PAM50, OncotypeDX, MGI) was performed on 20 patient-matched primary (10 ER-neg, 10 ER-pos) and metastatic brain tumors. Subtype classification was performed using genefu. Protein changes in ER and HER2 (ERBB2) were confirmed by IHC. BrM-specific ERBB2 gains were corroborated in a publicly available dataset of 18 additional patient-matched cases (dbGAP phs000730.v1.p1). To test whether ERBB2 amplification and base pair mutation is metastasis-site specific, we further analyzed an expanded cohort of 7,884 breast tumors enriched for metastatic samples (52%) including liver (16.7%), lung (4.3%), bone (3.6%), and brain (2.0%) using comprehensive hybrid-capture sequencing of ERBB2. RESULTS: 17/20 BrM retained the PAM50 subtype of the primary BrCa. Despite this concordance, 17/20 BrM harbored expression changes (< or > 2-fold) in clinically actionable genes including gains of FGFR4 (30%), FLT1 (20%), AURKA (10%) and loss of ESR1 expression (45%). The most recurrently upregulated gene was ERBB2, showing a >2-fold expression increase in 35% of BrM. 3 of 13 (23.3%) cases originally HER2-negative, and thus HER2-therapy naive, in the primary BrCa were IHC-positive (3+) in the paired BrM with an observed metastasis-specific amplification of the ERBB2 locus. In an independent dataset, 2 of 9 (22.2%) HER2-negative BrCa switched to HER2-positive with one BrM acquiring ERBB2 amplification and the other showing metastastic enrichment of the activating V777L ERBB2 mutation. Analysis of a large cohort of breast tumors (n=7,884) showed that across all organs ERBB2 amplification and/or base pair mutation was similar (p=0.18) between primary (13%) and metastatic disease (12%), however, a strong and significant enrichment was seen for BrM (primary 13% vs BrM 24%, p CONCLUSIONS: Taken together, these results demonstrate that the majority (85%) of patient-matched BrM retain the intrinsic subtype of the primary cancer. However, despite this transcriptional similarity, alterations in clinically actionable genes are common, with BrM acquiring ERBB2 amplifications and/or base pair mutations at a frequency of ∼20%, even in HER2-therapy naive tumors. In a large cohort of primary and metastatic breast cancers, there is also a unique enrichment for ERBB2 alterations in BrM. This study provides a strong rationale to molecularly profile metastatic lesions to both better understand biological mechanisms of metastases and to perhaps refine therapeutic decision-making in advanced cancers. Citation Format: Priedigkeit N, Hartmaier RJ, Chen Y, Vareslija D, Basudan A, Thomas R, Leone JP, Lucas PC, Bhargava R, Hamilton RL, Chmielecki J, Davidson NE, Oesterreich S, Brufsky AM, Young L, Lee AV. Breast cancer brain metastases show limited intrinsic subtype switching, yet exhibit acquired ERBB2 amplifications and activating mutations [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD1-05.

Background: Patients achieving pathologic complete response to neoadjuvant chemotherapy have excellent disease free and overall survival. For patients with residual disease, the residual disease in breast and lymph node (RDBN) method provides useful prognostic information. RDBN is calculated as follows: 0.2*tumor size (in cm)+lymph node status (0-3) + tumor grade (1-3). pCR, low, intermediate and high risk of recurrence categories correspond to RDBN index of 0, 0.1 to 2.9, 3 to Methods: Retrospective review of 614 consecutive patients who underwent neoadjuvant therapy for breast cancer was performed. At our institution, tumor size/volume reduction in the breast is determined using the equation: Estimated % tumor size reduction = [(pre-therapy clinical size – “revised” pathology tumor size)/pre-therapy clinical size]*100. “Revised” pathology tumor size is calculated by multiplying the largest dimension of the gross tumor bed by the invasive tumor cellularity of the tumor bed (in comparison to the pre-therapy core biopsy sample). For example, if a 3 cm tumor bed has only 50% cellularity for invasive cancer (in comparison to pre-therapy core biopsy), the revised tumor size is 1.5 cm. Hence, we were able to use the “revised tumor size” for calculating the modified RDBN index (mRDBN). We also used gross tumor bed size for gross RDBN (gRDBN) to compare with mRDBN. mRDBN and gRDBN could be calculated on 459 of the 514 cases. Chi-Square statistical analysis was performed. Results: Mean follow up was 33.1 months (median 31, range 4-70). The results are shown in Table 1 & 2. Table 1. Overall Recurrence and MortalityRDBN Score Category Overall Recurrence Mortality nn (%)RR95% CI; pn (%)RR95% CI; pmRDBN (n=459)High5829 (50.0)19.63[7.22, 53.40]; p= Table 2; Reclassification of gRDBN categoriesgRDBN mRDBN ReclassificationClassificationnLow (%)Intermed (%)High (%)Low7272 (100)0 (0)0 (0)Intermed1498 (5.4)140 (93.9)1 (0.7)High810 (0)24 (29.6)57 (70.4) Conclusions: Both mRDBN and gRDBN provide prognostic information; however, separation of categories is improved with mRDBN (Table 1). mRDBN reclassified 30% of the high risk-gRDBN patients into intermediate risk category with a recurrence rate of 20%, leaving the 'true' high risk subgroup with a revised recurrence rate of 50% (Table 2). RDBN index also identified a group of low risk patients who have prognosis similar to patients with pCR. Citation Format: Farrugia DJ, Landmann A, McAuliffe PF, Diego EJ, Johnson R, Bonaventura M, Soran A, Dabbs DJ, Clark B, Lembersky BC, Puhalla SL, Brufsky A, Jankowitz R, Davidson NE, Ahrendt GM, Bhargava R. Prognostic significance of a modified residual disease in breast and nodes (mRDBN) algorithm after neoadjuvant therapy for breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-14.

Background: Recent research has shown that collagen fiber arrangement in the tumor microenvironment plays a key role determining breast cancer prognosis. In this work, we present a new quantitative histomorphometric approach to identify and evaluate whether Collagen Fiber Orientation Disorder from Tumor-associated Stroma (CFOD-TS) on digitized H&E slides are associated with disease free survival (DFS) in estrogen receptor positive (ER+) breast cancer patients. Method: 234 and 171 ER+ invasive breast cancer patient’s slides were acquired and digitized from E2197 (training set) and TCGA-BRCA (test set) respectively. Collagen fibers were first segmented out on the digitized H&E slides by a combination of Derivative of Gaussian filters. The CFOD-TS features were then extracted from tumor patches in the whole slide image. A multiple linear regression model (MLR) was then trained in conjunction with the CFOD-TS features distinguishing patients likely to recur from not (>15 year follow-up period) to predict DFS for the non-censored patients. Result: The CFOD-TS discriminated patients (p=0.01) with and without recurrence and was significantly associated with DFS on ECOG 2197. The univariate and multivariate analysis on the training set were summarized in table 1 and table 2. CFOD-TS was also independently prognostic on patients in the TCGA for stratifying patients into low- and high-risk groups with p=0.009 via a log rank test and a hazard ratio (HR) of 3.09 (95% CI =1.33~7.18, p=0.0088). HR between the two risk groups was 7.11 (n=104, 95% CI =1.53~32.94, p=0.01) for stage I / II tumors and 3.03 (n=39, 95% CI =0.79-8.67, p=0.11) for stage III / IV cancers. In a multivariate setting, CFOD-TS (HR -3.7) was more significant than T-stage (HR-2.88) in stratifying risk. (Table2) Table 1: Univariate DFS analysis on the training setP-valueHR(95% CI)Case numberCFDO-TS derived risk groups0.00272.04 (1.28 – 3.26)234low / intermediate Oncotype DX (ODx) vs High ODx0.940.96(0.36 – 2.56)68low ODx vs intermediate / High ODx0.00323.49 (1.52 – 8.01)68grade0.300.82 (0.56 – 1.19)209 Table 2: Multivariate DFS analysis on the training set (n=68) and test set (n = 171)VariableP-valueHR(95% CI)ECOG 2197low ODx vs intermediate / High ODx0.00144.00 (1.71 – 9.34)CFDO-TS derived risk groups: Low vs high-risk0.0192.53 (1.17 – 5.47)TCGAT-stage: T1/T2 vs T3/T40.032.88 (1.10 -7.50)N-stage: N0 vs N1/N2/N30.411.47 (0.59 - 3.70)CFOD-TS derived risk groups: Low vs high-risk0.0033.70 (1.57 - 8.67) Conclusion: Over-expression of CFOD-TS was found to be independently associated with lower likelihood of recurrence and could potentially serve as a prognostic marker of outcome for ER+ breast cancer. Citation Format: Haojia Li, Kaustav Bera, Hannah Gilmore, Nancy E Davidson, Lori J Goldstein, Anant Madabhushi. Histomorphometric measure of disorder of collagen fiber orientation is associated with risk of recurrence in ER+ breast cancers in ECOG-ACRIN E2197 and TCGA-BRCA [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 P5-06-16.

[][1] With the death of Bernard Fisher, MD, on October 16, 2019, at the age of 101, we lost a visionary thinker and medical pioneer in cancer research and care. Fisher, a surgeon and clinical trialist, revolutionized breast cancer treatment, leading trials that transformed breast

DNA structural variations (SVs) are a major source of genetic instability in cancer, but they remain understudied. Large-insert mate-pair sequencing (MPS) is a powerful method designed to detect SVs, even in highly repetitive regions. Using MPS and other methods, we performed a comprehensive analysis of genomic alterations in breast cancer progression. Matched primary/recurrent frozen tumor samples from 6 patients, including two patients from our rapid autopsy program with multiple metastatic tissues (20 total samples; average 5.5 years to recurrence) were examined by multiple large-insert library (3-5, 5-8, 8-12kb) MPS to identify metastatic acquired SVs. This was supplemented with RNAseq (n=15), whole exome sequencing (n=18;~75x), whole genome sequencing (n=3; 40-65x), and SNP arrays. A relatively small fraction (~10%) of somatic single nucleotide variants (SNVs) in the primary tumor were identified in matched metastatic samples, and the majority of metastatic SNVs were not found in the matched primary tumor. This indicates that a rare sub-clone colonizes the metastatic site and evolves extensively before becoming clinically evident. For example, in one patient with an ER+ tumor who initially declined anti-estrogen therapy, the recently described ESR1 Y537S mutation was not present in the primary tumor or in metastatic disease 5 years later. However, after extensive anti-estrogen treatment for metastatic disease, the mutation was identified at rapid autopsy, indicating that this mutation can be acquired even after initial metastatic spread. Chromatin immunoprecipitation assays in metastatic tissue from tumors with mutant ERα show strong enrichment for ERα at classical ERα target genes and we are currently assessing the genome-wide binding pattern of ERα to identify novel binding sites. We observed extensive patient-to-patient variability in the number and types of SVs. In general, the overall patterns of SVs were remarkably similar between matched primary and metastatic samples indicating that these events likely occurred early in tumorigenesis and are stable throughout disease progression. We identified a number of metastatic specific SVs that likely contribute to disease progression. Specifically, in one patient with an ER+ primary tumor treated with adjuvant Tamoxifen, we identified a novel fusion gene between ESR1 (estrogen receptor-α, ERα) and DAB2 (disabled-2) only in a lymph node recurrence. RT-PCR and western blot analysis confirmed that the fusion RNA/protein was expressed/translated only in the recurrent disease. The fusion retains the DNA-binding domain (DBD) and hinge region of ERα while the ligand-binding domain (LBD) is replaced with the majority of DAB2. We hypothesized that this is a functional genetic alteration conferring ligand-independent ERα-mediated signaling and growth. Confirming this, in vitro ERE-Tk-luc reporter assays showed that the ESR1-DAB2 fusion has ligand-independent activity that is 13-290x higher than wild-type ERα. We are currently assessing the genome-wide binding of ESR1-DAB2 and the functional contribution of DAB2 to the fusion protein. This study represents the most comprehensive analysis to date of genomic changes in breast cancer progression and indicates extensive changes occur during metastatic spread. A number of acquired changes likely represent therapeutically targetable metastatic dependencies. Citation Format: Ryan James Hartmaier, Amir Bahreini, Shannon L. Puhalla, Steffi Oesterreich, Aju Mathew, Nancy E. Davidson, Adam M. Brufsky, Adrian V. Lee. Identification of base pair mutations and structural rearrangements acquired in breast cancer metastases including a novel hyperactive ESR1-DAB2 fusion gene in hormone-resistant progression. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-07.

DNA structural variations (SVs) are a major source of genetic instability in cancer, but they remain understudied. Large-insert mate-pair sequencing (MPS) is a powerful method designed to detect SVs, even in highly repetitive regions. Using MPS and other methods, we performed a comprehensive analysis of genomic alterations in breast cancer progression. Matched primary/recurrent frozen tumor samples from 6 patients, including two patients from our rapid autopsy program with multiple metastatic tissues (20 total samples; average 5.5 years to recurrence) were examined by multiple large-insert library (3-5, 5-8, 8-12kb) MPS to identify metastatic acquired SVs. This was supplemented with RNAseq (n=15), whole exome sequencing (n=18;~75x), whole genome sequencing (n=3; 40-65x), and SNP arrays. A relatively small fraction (~10%) of somatic single nucleotide variants (SNVs) in the primary tumor were identified in matched metastatic samples, and the majority of metastatic SNVs were not found in the matched primary tumor. This indicates that a rare sub-clone colonizes the metastatic site and evolves extensively before becoming clinically evident. For example, in one patient with an ER+ tumor who initially declined anti-estrogen therapy, the recently described ESR1 Y537S mutation was not present in the primary tumor or in metastatic disease 5 years later. However, after extensive anti-estrogen treatment for metastatic disease, the mutation was identified at rapid autopsy, indicating that this mutation can be acquired even after initial metastatic spread. Chromatin immunoprecipitation assays in metastatic tissue from tumors with mutant ERα show strong enrichment for ERα at classical ERα target genes and we are currently assessing the genome-wide binding pattern of ERα to identify novel binding sites. We observed extensive patient-to-patient variability in the number and types of SVs. In general, the overall patterns of SVs were remarkably similar between matched primary and metastatic samples indicating that these events likely occurred early in tumorigenesis and are stable throughout disease progression. We identified a number of metastatic specific SVs that likely contribute to disease progression. Specifically, in one patient with an ER+ primary tumor treated with adjuvant Tamoxifen, we identified a novel fusion gene between ESR1 (estrogen receptor-α, ERα) and DAB2 (disabled-2) only in a lymph node recurrence. RT-PCR and western blot analysis confirmed that the fusion RNA/protein was expressed/translated only in the recurrent disease. The fusion retains the DNA-binding domain (DBD) and hinge region of ERα while the ligand-binding domain (LBD) is replaced with the majority of DAB2. We hypothesized that this is a functional genetic alteration conferring ligand-independent ERα-mediated signaling and growth. Confirming this, in vitro ERE-Tk-luc reporter assays showed that the ESR1-DAB2 fusion has ligand-independent activity that is 13-290x higher than wild-type ERα. We are currently assessing the genome-wide binding of ESR1-DAB2 and the functional contribution of DAB2 to the fusion protein. This study represents the most comprehensive analysis to date of genomic changes in breast cancer progression and indicates extensive changes occur during metastatic spread. A number of acquired changes likely represent therapeutically targetable metastatic dependencies. Citation Format: Ryan James Hartmaier, Amir Bahreini, Shannon L. Puhalla, Steffi Oesterreich, Aju Mathew, Nancy E. Davidson, Adam M. Brufsky, Adrian V. Lee. Identification of base pair mutations and structural rearrangements acquired in breast cancer metastases including a novel hyperactive ESR1-DAB2 fusion gene in hormone-resistant progression. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B2-16.

Background It is uncertain if the addition of OFS to adjuvant endocrine therapy with T improves outcomes in premenopausal HR+ BC. The SOFT trial was designed to determine the value of OFS in women who remain premenopausal and are treated with T (OFS question) and also to test whether an AI improves outcome in premenopausal women with HR+ BC treated with OFS (AI question). In the combined analysis of the TEXT and SOFT trials (AI question), the group treated with the AI exemestane+OFS had a significantly better disease-free survival (DFS) than those with T+OFS. This abstract presents the results of the OFS question (n=2,045). Patients and Methods Between November 2003 and January 2011, the SOFT phase 3 trial randomized 3066 premenopausal women with HR+ BC to 5 years of adjuvant endocrine therapy with exemestane+OFS versus T+OFS versus T alone, with OFS initiated by choice of GnRH agonist triptorelin, oophorectomy or ovarian irradiation. Prior chemotherapy was allowed, provided women had confirmed premenopausal estradiol levels within 8 months of completing chemotherapy. The primary end point was DFS which included invasive local, regional, distant and contralateral breast events, second non-breast malignancies and deaths without prior cancer. Because of a lower-than expected overall event rate and to ensure results within a reasonable time-frame, a protocol amendment in 2011 changed the originally event-driven analysis plan. The amendment (1) designated the comparison of T+OFS versus T alone as the primary analysis for SOFT (n=2045), recognizing that the statistical power for the original three pairwise comparisons would be substantially reduced and (2) specified that the primary analysis of T+OFS versus T be undertaken when median follow-up was at least 5 years. The comparison would be tested at the 2-sided 0.05 level with no interim analysis. Based on the projected number of events at the lower rates, the estimated power to detect hazard ratios of 0.75, 0.70, and 0.66 for the comparison would be 52%, 69%, and 80%. Results The SOFT trial completed planned patient enrollment with an international collaboration involving 426 centres from BIG and NABCG led by the International Breast Cancer Study Group (IBCSG). Numbers of patients randomized, randomization strata, and pt age are summarized in the Table. The database lock for the primary analysis of the OFS question will occur in September 2014 and the final analysis will be completed by October 15, 2014. SOFT Patients (%)Tamoxifen alone1021 (33%)Tamoxifen+OFS1024 (33%)Exemestane+OFS1021 (33%)* Prior Chemotherapy54%Lymph node positive35%Median age (% < 40 years)43 years (30%)*Patients randomized to Exemestane+OFS are not part of the current analysis Conclusions We will present the primary analysis of outcomes and toxicities by treatment for women randomized to receive T+OFS versus T and address the value of OFS in addition to T as adjuvant endocrine therapy for premenopausal women with HR+ BC. Citation Format: Aron Goldhirsch, Richard D Gelber, Prudence A Francis, Meredith M Regan, Gini F Fleming, Istvan Lang, Eva M Ciruelos, Meritxell Bellet, Herve Bonnefoi, Miguel A Climent, Lorenzo Pavesi, Harold J Burstein, Silvana Martino, Nancy E Davidson, Charles E Geyer Jr, Barbara A Walley, Robert E Coleman, Pierre Kerbrat, Manuela Rabaglio-Poretti, Alan S Coates. Randomized comparison of adjuvant tamoxifen (T) plus ovarian function suppression (OFS) versus tamoxifen in premenopausal women with hormone receptor-positive (HR+) early breast cancer (BC): Analysis of the SOFT trial [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 S3-08.

Background: The ECOG E5194 study was a prospective trial designed to evaluate surgical excision (lumpectomy) without radiation for selected women with ductal carcinoma in situ (DCIS) of the breast with low risk clinical and pathologic features. Methods: Eligible patients were enrolled on two study cohorts (not randomized): (1) low or intermediate grade DCIS, tumor size < 2.5 cm; or (2) high grade DCIS, tumor size < 1.0 cm. Cohort assignment was based on pathology assessment from the treating institution. Protocol specifications included surgical excision of the DCIS tumor with a minimum negative margin width of at least 3 mm or no tumor on re-excision. Radiation treatment was not allowed. From April 1997 to October 2002, 665 evaluable patients were enrolled through ECOG or NCCTG (561 in Cohort 1; 104 in Cohort 2). Tamoxifen was optional (not randomized) beginning in May 2000, and was given to 30% of the patients. The primary study endpoint was the rate of developing an ipsilateral breast event (IBE), defined as local recurrence of DCIS or invasive carcinoma in the treated breast. The median follow-up was 12.3 years. We have previously reported 7-year results (L. Hughes, J Clin Oncol 27:5319, 2009; median follow-up 6.3 years; 66 IBE’s), and we herein provide 12-year results. Results: Median patient age was 60 years and 58 years for Cohort 1 and Cohort 2, respectively. Tumor size was < 10 mm for 79% and 80% of patients, respectively. The minimum negative margin width was > 5 mm for 64% and 69% of patients, respectively. There were 99 IBE’s, of which 51 (52%) were an invasive IBE. The IBE and invasive IBE rates increased over time in both cohorts (see Table). The 12-year rates of an IBE were 14.4% for Cohort 1 and 24.6% for Cohort 2 (p = 0.003), and for an invasive IBE, 7.5% and 13.4%, respectively (p = 0.08). No difference was seen for the 12-year rates of overall survival (84.0% vs 82.8%; p = .96) or contralateral breast events (6.7% vs 12.0%; p = 0.16). On multivariate analysis, study cohort (hazard ratio = 1.81; p = 0.01) and tumor size (p = 0.01) were statistically significant for an IBE, and study cohort was borderline statistically significant for an invasive IBE (p = 0.08). On central pathology review (75% of cases), neither grade nor comedo necrosis was associated with the risk of an IBE or invasive IBE (all p > 0.15). Salvage treatment at the time of an IBE included mastectomy for 42% (31/74) and 64% (16/25) of the patients, respectively. Conclusions: For these selected patients with favorable DCIS based on clinical and pathologic characteristics treated with surgical excision without radiation, the rates of an IBE and an invasive IBE continued to increase through at least 12 years of follow-up. IBE Rates According To Study Cohort. Cohort 1 (Low or Intermediate Grade)Cohort 2 (High Grade)TimeIBEInvasive IBEIBEInvasive IBEAt 5 years6.0% (4.0%, 8.1%)2.7% (1.3%, 4.1%)15.0% (7.7%, 21.7%)5.3% (0.8%, 9.7%)At 7 years9.5% (7.0%, 12.0%)4.8% (2.9%, 6.6%)18.2% (10.6%, 25.8%)7.6% (2.2%, 13.0%)At 10 years12.5% (9.5%, 15.4%)6.4% (4.2%, 8.6%)24.6% (15.7%, 33.4%)13.4% (5.9%, 20.9%)At 12 years14.4% (11.2%, 17.6%)7.5% (5.1%, 10.0%)24.6% (15.7%, 33.4%)13.4% (5.9%, 20.9%) Citation Format: Lawrence J Solin, Robert Gray, Lorie L Hughes, William C Wood, Mary Ann Lowen, Sunil Badve, Frederick L Baehner, James N Ingle, Edith A Perez, Abram Recht, Joseph Sparano, Kathy Miller, Nancy E Davidson. Local excision without radiation for ductal carcinoma in situ: 12-year results from the ECOG E5194 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 P6-13-01.

Background: More than half of pts with TNBC do not achieve a pCR after NAC, which carries a poor prognosis. It is unclear whether these patients respond to subsequent therapies on relapse. We sought to determine the time to progression (TTP) on subsequent therapies for recurrent disease in TNBC patients who did not achieve pCR to neo-adjuvant therapy. Methods: We retrospectively identified 117 TNBC pts who received NAC from 2009 through 2012 using an electronic database at the Magee Women’s Breast Cancer Program at the University of Pittsburgh. Patient records were then assessed for pts who did not achieve a pCR for time to local or distant recurrence (TTR) and time to progression (TTP) on susbequent therapies. Results: 86 of 117 TNBC pts did not achieve pCR to NAC defined as residual disease present in either the breast and/or axilla. Median follow up was 2 yrs (range 1-4yrs). Out of these 86 pts, 21 pts(25%) had recurrence in form of distant metastasis or local recurrence. Nearly all pts(95%) received anthracycline and taxane based NAC. Median TTR was 19 months (4-29 months). Therapies received in the metastatic setting most commonly included single agent capecitabine, paclitaxel, nab-paclitaxel, and eribulin. The most commonly received doublets included carboplatin with paclitaxel or gemcitabine. Median TTP on systemic chemotherapy was 15 weeks (3 -39 weeks), 6 weeks (1-24 weeks) and 6 weeks (2-9 weeks) in first, second and third line setting, respectively. Greater than 80% pts were able to receive two lines of systemic chemotherapy but Conclusions: Response to subsequent chemotherapeutic regimens in metastatic setting in TNBC pts who do not achieve pCR to anthracycline and taxane based NAC is limited. This highlights the importance of identifying new agents and targeted therapies for this poor prognosis sub-type. Citation Format: Shalu Pahuja, Kandace McGuire, Nancy Davidson, Adam Brufsky, Priya Rastogi, Rachel Jankowitz, Barry Lembersky, Shannon Puhalla. Response to subsequent therapies after failure to achieve pathologic complete response (pCR) after neo-adjuvant chemotherapy (NAC) in patients (pts) with triple negative breast cancer (TNBC) [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 P3-11-10.

Introduction. Invasive lobular carcinoma (ILC) accounts for 10-15% of breast cancer diagnoses and is characterized by a high rate of estrogen and progesterone receptor (ER and PR) expression, low rate of HER2 amplification, and a defining lack of e-cadherin expression. Multiple population-based, observational studies have shown that the risk of ILC is significantly elevated with the intake of combined estrogen/progestin (E+P) hormone replacement therapy (HRT) but not with estrogen-alone (E alone) HRT. In addition, recent studies have revealed that ILCs might respond differently to hormone therapies than IDCs. We sought to understand the influence of exogenous hormone exposure in the form of HRT on biological pathways in ILCs compared to the more common histologic subtype, ER+ invasive ductal carcinoma (IDC). Specimens and Methods. We assessed 297 ER+/HER2- FFPE breast carcinomas, 165 IDCs and 132 ILCs, previously collected from an epidemiological study of HRT use and breast cancer in post-menopausal women. These tumors were characterized for histopathological features and protein marker expression by immunohistochemistry along with the extensive clinical and demographic data, including HRT use, collected in the parent study. Using the Nanostring nCounter assay, we measured expression of ~1000 genes representing canonical cancer-promoting pathways and steroid hormone-associated pathways to determine the relationship between HRT use and pathway alterations in ER+ breast cancer subtypes. Results. From the histopathological assessment, we observed that ILCs had lower overall lymphocyte response than IDCs (p=0.005), but HRT use shifted the lymphocyte response higher than what was observed in ILCs from never/former HRT users (p=0.002). In the gene expression analysis, we found that IDCs and ILCs have distinct expression profiles after adjusting for HRT use, including genes in cell cycle, steroid hormone-response pathways, and cytokine/cytokine receptor interactions, with increased signaling seen in the IL6/12-like family. When comparing gene expression between the HRT-use groups, adjusting for histology, we observed that E+P and E alone are both associated with down-regulation of cell cycle and DNA repair genes compared to the never/former use group. When we focused on expression patterns specific to progestin exposure, (i.e., E+P versus all others), we found decreased expression of genes related to DNA replication initiation and G1/S transition and WNT4. Unsupervised hierarchical clustering revealed additional subgroups within each histologic subtype including a group of low-grade, low-proliferating IDCs enriched for E+P exposure, which clusters with the majority of the ILCs. While many gene expression changes with HRT use are shared across the tumors, we observed alterations that are specific to either ILCs or IDCs, suggesting different steroid hormone-responsive pathways between the subtypes. Conclusions. Our study demonstrates there are potentially specific pathways differentially activated between ILCs and IDCs dependent on the type of hormone exposure. Further, while our study is focused on tumor molecular pathways modulated in response to HRT use, steroid hormone exposure can come from multiple sources, and we propose that the results from this study can provide insight into hormonally-driven breast cancers that develop with excess estrogen and/or progesterone exposure. This could have implications in clinical management of ILCs and IDCs that arise in HRT users. Citation Format: Jamie Guenthoer, Isaac C Jenkins, Beti Thompson, Mei-Tzu C Tang, Nancy E Davidson, Ryan L Ashley, Christopher I Li, Li Hsu, Peggy L Porter. Exogenous steroid hormone exposure and the biology of lobular breast cancer [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 P6-05-03.

Background: Patients with invasive lobular carcinoma (ILC) would be expected to have favorable outcomes compared to patients with invasive ductal carcinoma (IDC) given that ILC is more often hormone receptor-positive (HR+), human epidermal growth factor receptor 2 (HER2)-negative, of lower grade, and displays decreased proliferation markers. Based on our preclinical studies showing differential hormone response in HR+ ILC vs. IDC and on recent studies suggesting differences in endocrine treatment response between patients with ILC vs. IDC, we designed a biomarker-driven, neoadjuvant window trial for newly diagnosed women with HR+, HER2-negative ILC. We hypothesize that Ki67 will be reduced by 85% in the fulvestrant arm compared with 60% and 75% reduction in the tamoxifen and anastrozole arms, respectively, and that Ki67 reduction will correlate with alterations in expression of ER and ER-regulated genes. Differential Ki67 effect will serve as a surrogate for outcome of patients with ILC on endocrine therapy. Trial Design: This multicenter study (NCT02206984) will enroll 150 women with HR+ and HER2-negative ILC. A mandatory research breast tumor biopsy will be performed at baseline. Fifty patients will be randomized to each of three open-label treatment arms for 21 days: fulvestrant (two 250 mg IM injections on both day 1 and day 14), anastrozole (1mg orally daily), or tamoxifen (20 mg orally daily). Biomarkers of response will be assessed on baseline and post-treatment tumor tissue. Patients will proceed to definitive surgery on day 21 after study drug exposure, or they will undergo a second research breast core biopsy if further neoadjuvant treatment is planned. Eligibility Criteria: Eligible patients include postmenopausal women with newly diagnosed, HR+, HER2-negative ILC (excluding pleomorphic subtype) measuring ≥ 1cm, with adequate organ function, ECOG PS ≥ 2, and agreeable to baseline research breast tumor biopsy. Specific Aims: The primary endpoint is percent change from baseline to post-treatment Ki67 values in ILC tissue after 21 days of endocrine treatment. Comparisons across study arms will be made using a general linear model adjusting for institutional effect, with 80% power estimated for pairwise comparisons of log2(% staining) between treatment arms, allowing for 10% attrition. Secondary endpoints include post-therapy Ki67, and change in ER and PR protein expression by IHC. Finally, planned correlative studies include evaluation of gene expression, epigenetic markers, and DNA sequence variants in ILC tissues in an effort to identify biomarkers of endocrine response and putative drivers of endocrine resistance in ILC. Target Accrual: This study will be open to enrollment by August 2015 at the University of Pittsburgh. Additional sites will be opened through the Translational Breast Cancer Research Consortium (TBCRC). We anticipate an accrual rate of 8 patients per month. (Funding from Susan G. Komen® and AstraZeneca). Citation Format: Jankowitz RC, McAuliffe PF, Sikora MJ, Butler L, Ahrendt G, Johnson R, Diego E, Bonaventura M, Puhalla S, Lembersky B, Clark B, Brufsky A, Kurland BF, Davidson NE, Dabbs DJ, Oesterreich S. A neoadjuvant window trial of endocrine response in women with invasive lobular carcinoma. [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 P3-05-14.

BACKGROUND: We previously reported that low 21 gene Recurrence Scores (RS) identify patients with ER-positive, lymph node-positive breast cancer who may not benefit from anthracycline-based adjuvant chemotherapy added to tamoxifen (SWOG-8814A NCI correlative science study; Albain et al. Lancet Oncol 2010). New exploratory and comprehensive quantitative analyses now permit whole transcriptome NGS on residual RNA extracted from FFPE blocks 12-18 years post-fixation. Herein, we report methodology details and feasibility results (see companion abstract, Albain et al., for clinical outcomes correlations). METHODS and RESULTS: Sequencing was carried out in Illumina HiSeq 2000 instruments, yielding 4.2 trillion data points. Messenger RNA expression was quantified using 3rd quartile normalization. Both Library (RNA) and Sequencing Standards showed high quality coverage as measured by median uniquely mapped reads over a 13 month window (168M and 182M, respectively, including duplicate reads). The median absolute deviation (MAD) of the relative log expression (RLE) of mapped reads for the Library and Sequencing Standards was 0.22 and 0.05, respectively. The Library Standard variation was greater than the Sequencing Standard, as library preparation was manual. Of 360 patient samples with sufficient RNA (≥ 100 ng total RNA), 354 (98.3%) were successfully sequenced and included in the final analysis data set. Average library yield was 39 ng/μL. Only 5 libraries failed yield requirements and one library failed expression quality metrics. The median insert length was 120 bp with the first and third quartiles 93 and 152 bp, respectively. After removal of duplicate reads, 82% of reads were uniquely mapped, and the median library size was 8.95M (number of unique mapped reads). Sequences with counts CONCLUSIONS: High quality whole transcriptome NGS is feasible from decades-old clinical trial FFPE specimens that have not been stored in any special fashion. Controlled laboratory, bioinformatics and biostatistics methods, with inclusion of appropriate process controls, ensure robustness and reliability of the NGS process. This in turn results in the discovery and validation of biologically and clinically relevant variations from prior landmark clinical trials. SUPPORT: NCI CA 180888, CA180819, CA180821, CA180820, CA180863; in part, Genomic Health, Inc. Citation Format: Cherbavaz DB, Hayes DF, Qu K, Crager MR, Barlow WR, Goddard AD, Beasley EM, Jeong J, Collin F, Liu M-L, Rae JM, Ravdin PM, Tripathy D, Gralow JR, Livingston RB, Osborne CK, Ingle JN, Pritchard KI, Davidson NE, Carey LA, Sing AP, Baehner FL, Hortobagyi GN, Shak S, Albain KS. Successful whole transcriptome analysis of 25-year-old breast tumor samples from the phase III trial SWOG-8814 by next generation sequencing (NGS): Standardized analytical methods for exploratory and validation studies. [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 P5-07-01.

BACKGROUND: Because there are currently no reliable predictors for progression of ductal carcinoma in situ (DCIS) to invasive disease, nearly all patients receive aggressive therapy, leading to over-treatment in many cases. Few in vitro models for studying DCIS progression have been developed. We report here the successful culture and expansion of primary DCIS from surgical specimens using a conditional reprogramming protocol. MATERIALS AND METHODS: From 2/2014 to 4/2015, patients with percutaneous core needle biopsy demonstrating DCIS were enrolled in a tissue banking protocol after informed consent was received. Under supervision of the surgical pathologist, fresh tissue measuring between 5-15 mm in length was taken from lumpectomy or mastectomy specimens. Tissue was divided such that half was mechanically and enzymatically dissociated and then cultured in medium conditioned by irradiated mouse fibroblasts and supplemented with rho-associated protein kinase (ROCK) inhibitor, and the second half, known as the "mirror image" remained as part of the clinical specimen. RESULTS: Of 49 consented patients, mean age was 59 ± 10 years. 7 were excluded due to final pathology not consistent with DCIS: 4 upstaged to invasive ductal cancer, 2 had microinvasion and 1 showed pleomorphic lobular carcinoma in situ. Of the remaining 42, 9 were failures: 5 tissues were not received in lab and 4 cases were received, but no cells grew in culture. Of the remaining 33 cases of DCIS, 70% (n=23) and 27% (n=9) were nuclear grade 2 and 3 respectively. 91% (n=30) were ER-positive, with H-score ranging between 4 and 300. 19 (58%) were expanded in cell culture for up to two months in culture, and 14 were frozen immediately after mechanical dissociation for future growth. The 19 cell cultures could be cryopreserved and expanded. The cultures are almost exclusively composed of cytokeratin 8- and EpCAM-positive luminal cells and cytokeratin 14-, cytokeratin 5-, and p63-positive basal mammary epithelial cells, suggesting maintenance of heterogeneity in vitro. Furthermore, as assessed by luminal and basal marker expression, these cells retain their cellular identities both in the "conditionally reprogrammed" proliferative state and when conditioned media and ROCK inhibitor were withdrawn. When grown to 100% confluency, the cultures appear to organize into luminal and basal layers as well as luminal compartments surrounded by basal cells. CONCLUSION: Primary cultures of DCIS derived directly from patient tissues may serve as in vitro models for the study of DCIS. Citation Format: McAuliffe PF, Brown DD, Oesterreich S, Lee AV, Johnson RR, McGuire KP, Davidson NE, Brufsky AM, Dabbs DJ. Developing in vitro models of ductal carcinoma in situ from primary 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 P6-08-02.

DNA structural variations (SVs) are a major source of genetic instability in cancer, but they remain understudied. Large-insert mate-pair sequencing (MPS) is a powerful method designed to detect SVs, even in highly repetitive regions. Using MPS and other methods, we performed a comprehensive analysis of genomic alterations in breast cancer progression. Matched primary/recurrent frozen tumor samples from 6 patients, including two patients from our rapid autopsy program with multiple metastatic tissues (20 total samples; average 5.5 years to recurrence) were examined by multiple large-insert library (3-5, 5-8, 8-12kb) MPS to identify metastatic acquired SVs. This was supplemented with RNAseq (n=15), whole exome sequencing (n=18; ∼75x), whole genome sequencing (n=3; 40-65x), and SNP arrays. A relatively small fraction (∼10%) of somatic single nucleotide variants (SNVs) in the primary tumor were identified in matched metastatic samples, and the majority of metastatic SNVs were not found in the matched primary tumor. This indicates that a rare sub-clone colonizes the metastatic site and evolves extensively before becoming clinically evident. For example, in one patient with an ER+ tumor who initially declined anti-estrogen therapy, the recently described ESR1 Y537S mutation was not present in the primary tumor or in metastatic disease 5 years later. However, after extensive anti-estrogen treatment for metastatic disease, the mutation was identified at rapid autopsy, indicating that this mutation can be acquired even after initial metastatic spread. We observed extensive patient-to-patient variability in the number and types of SVs. In general, the overall pattern of SVs was remarkably similar between matched primary and metastatic samples, however, we identified a number of metastatic specific SVs that likely contribute to disease progression. Specifically, in one patient with an ER+ primary tumor treated with adjuvant Tamoxifen, we identified a novel fusion gene between ESR1 (estrogen receptor-α, ERα) and DAB2 (disabled-2) only in a lymph node recurrence. RT-PCR and western blot analysis confirmed that the fusion RNA/protein was expressed/translated only in the recurrent disease. The fusion retains the DNA-binding domain (DBD) and hinge region of ERα while the ligand-binding domain (LBD) is replaced with the majority of DAB2. We hypothesized that this is a functional genetic alteration conferring ligand-independent ERα-mediated signaling and growth. Confirming this, in vitro ERE-Tk-luc reporter assays showed that the ESR1-DAB2 fusion has ligand-independent activity that is 13-290x higher than wild-type ERα. Chromatin immunoprecipitation assays in metastatic tissue from tumors with mutant ERα show strong enrichment for ERα at classical ERα target genes. We are currently assessing the genome-wide binding of ESR1-DAB2 and the functional contribution of DAB2 to the fusion protein. This study represents the most comprehensive analysis to date of genomic changes in breast cancer progression and indicates extensive changes occur during metastatic spread. A number of acquired changes likely represent therapeutically targetable metastatic dependencies. Citation Format: Ryan J Hartmaier, Shannon L Puhalla, Steffi Oesterreich, Amir Bahreini, Nancy E Davidson, Adam M Brufsky, Adrian V Lee. Identification of base pair mutations and structural rearrangements acquired in breast cancer metastases including a novel hyperactive ESR1-DAB2 fusion gene specifically in hormone-resistant recurrence [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 S1-03.

Purpose: The immune system influences breast cancer prognosis. Analysis of the international BIG 02-98 trial suggests that TILs are associated with disease-free survival (DFS) in patients with operable triple negative breast cancer (TNBC, Loi et al, JCO 2013). Here we seek to validate the association of increased lymphocytic infiltration of primary breast tumors with an improved prognosis in TNBCs in two adjuvant Phase III trials conducted by the Eastern Cooperative Group. Methods: “Whole” H&E stained sections of tumors from E2197 and E1199 were evaluated by two independent pathologists blinded to outcome data for density of TILs in intratumoral (iTIL) and stromal compartments (sTIL). Cases from E2197 and E1199 were randomly selected based on TNBC status (E2197 central review, E1199 local determination) and availability of tumor sections. The association of DFS with TIL scores was determined by fitting proportional hazards models stratified on study. Nonrecurrences from E2197 were given a relative weight of 1.43, to reflect that they had been undersampled, and robust variance estimators were used. Results: 500 cases were obtained for which 481 were evaluable (190 for E2197, 291 for E1199). The median age of patients was 49 with 54% being premenopausal, 59% nodal involvement, 66% of tumors being > 2cm and all patients received anthracycline-based chemotherapy. 80% of tumors had at least 10% lymphocytic infiltration in the stroma (range 10-80%) but only 15% of cancers had at least 10% TILs intratumorally (range 10-50%). The lymphocyte-predominant breast cancer (LPBC) phenotype, defined as ≥50% stromal or intratumoral lymphocytic infiltration was seen in 4.4% of TNBC (21/481). At a median follow-up of 10.6 years, 190 DFS events were reported. Higher TIL scores were associated with a better prognosis: for every 10% increment in sTIL, a 14% reduction of risk for recurrence or death was observed (p = 0.02) and for every 10% increment in iTIL, a 28% risk reduction was seen (p = 0.06). Multivariate analysis confirmed sTIL to be an independent prognostic marker of DFS. Hazard Ratio (95% CI)p-valuesTIL (10% increase)0.84 (0.74,0.95)0.005Tumor Size < = 2.0 vs. 2.1-5.0cm0.59 (0.42,0.82)0.002Tumor Size < = 2.0 vs. >5.0cm0.46 (0.27,0.78)0.004Node-neg vs. 1-3 N+0.53 (0.37,0.78)0.001Node-neg vs. >3 N+0.23 (0.14,0.38)60 vs. 24-402.12 (1.29,3.49)0.003Table: Estimated DFS Hazard Ratios, 95% CI and Individual Term P-values for the Multivariate Model for the Linear Effect of the Stromal TIL Score The presence versus the absence of stromal TILs as binary marker was also a strong prognostic marker with a 31% reduction of risk of recurrence or death for the group with sTILs (95% CI 2 to 51, p = 0.04). Conclusion: We confirm in two independent data sets from national randomized clinical trials using contemporary adjuvant chemotherapy that stromal lymphocytic infiltration constitutes a robust prognostic factor in TNBCs. The data provide strong evidence for the incorporation of this feature into routine histopathological assessment as a prognostic factor for patients with TNBCs. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S1-07.

INTRODUCTION: Multigene tests (MGTs) have entered clinical practice and patient management. All MGTs are currently performed in reference laboratories using RNA isolated from a tumor section. The effect of intra-tumor heterogeneity on detection of genes within these MGTs, and the subsequent ability of to predict prognostic risk, has received little attention. To examine this directly, we used nCounter analysis to measure the expression of genes from five MGTs (OncotypeDx, Mammaprint, EndoPredict, PAM50, and Breast Cancer Index) in formalin fixed paraffin embedded tumor sections compared to cores taken from the tumor block. METHODS: We selected 71 estrogen receptor (ER) positive node-negative tumors, all of which had clinical OncotypeDx scoring performed at Genomic Health Inc, and for which ER, PR, HER2 and Ki67 clinical pathological measurements were available. For each patient we cut a 5uM section, and cut a 0.6mm core from a representative part of the tumor. If the tumor had an area of high focal Ki67 (n = 26), low PR (n = 13), or both (n = 5) cores were cut from these areas. In total we processed 181 samples. RNA was isolated using Qiagen RNAeasy kit, and 100ng used for nCounter analysis using a custom codeset incorporating five MGTs (n = 142 genes of which 12 are reference genes). MGTs were normalized to their respective reference genes, and nCounter OncotypeDx scores were scaled to the clinical OncotypeDx score for estimation of risk recurrence. RESULTS: Hierarchical clustering using all of the MGT genes combined (normalized to all reference genes) showed that the majority (61) of tumor samples clustered by patient, indicating greater inter than intra-tumor heterogeneity. However, when individual MGTs were examined alone, the intra-tumor heterogeneity increased. We found high correlation between Oncotype genes expression in the whole section versus representative tumor cores (r = 0.94). However, areas of low Ki67 and high PR staining showed low Ki67 and high PR gene expression, and consequently a slightly higher but not statistically significant recurrence score. For 17/75 (22.6%) patients, Oncotype Dx recurrence scores crossed the boundaries for low, intermediate and high risk. Conclusion: Inter-tumor heterogeneity is greater than intra-tumor heterogeneity for MGTs, and for the majority of patients a core of tumor gives gene expression measurements that are highly similar to a whole tumor section. This suggests that the tumor microenvironment (in our sample set mainly adipocytes) contribute little to the mRNA levels of MGTs. However, regions of high Ki67 or low PR within tumors have slightly higher OncotypeDx recurrence scores. The finding that recurrence scores differ by the tumor region that is sampled is consistent with other recent studies of intra-tumor genetic and transcriptomic heterogeneity, and indicates the potential clinical importance of these observations. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-10.

BACKGROUND: Although individual cancers are driven by heterogeneous processes, cancer mortality has a near universal cause—therapy resistance, recurrence and metastasis to vital organs. Characterizing more advanced tumors has borne valuable insight into cancer progression, yet studies of longitudinally collected breast cancer specimens are scarce given lengthy periods of cancer dormancy. In this study, we aimed to create the most comprehensive characterization of gene expression alterations to date between patient-matched pairs of primary and advanced ER-positive breast cancers. MATERIALS/METHODS: Hybrid-capture RNA-sequencing was performed on 50 patient-matched pairs of primary and advanced ER-positive tumors from various recurrence sites (9 brain, 11 bone, 3 GI, 10 ovary, 17 local). Time to recurrence was up to 14.1 years with a median of 3.4 years. A shared variant analysis confirmed all paired samples were patient-matched. 1,380 cancer-related genes were analyzed for outlier expression fold-changes in matched recurrences versus primary tumors. Pair-specific, outlier fold-change thresholds were defined as Q1/Q3 +/- [1.5 X IQR]; using each pairs' fold-change values across all genes as the distribution. These discrete, longitudinal transcriptional remodeling events (LTREs) were assessed for recurrence across all sites and analyzed for enrichments within specific cohorts (Fisher's exact tests), such as locoregional vs. distant recurrences. To determine if LTREs represent acquired vulnerabilities, ex vivo and in vivo experiments targeting a recurrent, druggable LTRE gain of RET was performed. RESULTS: The majority of advanced cancers were transcriptionally similar to patient-matched primaries with 23 of 33 distant metastases retaining PAM50 assignments of the matched primary—shifts to HER2 (n=4, 12%) or Luminal B (n=5, 15%) subtypes accounted for most metastatic discordances. Despite this intrinsic conservation, remarkably recurrent gene-level LTRE gains and losses were observed in advanced disease. Recurrent LTRE gains included NCAM1 [42%], FGFR4 [40%], IBSP [36%], ROBO2 [36%] and SPP1 [30%]. Notable LTRE losses included RELN [42%] and ESR1 [26%]. NCAM1 LTREs showed the most significant enrichments (p < 0.001) in distant disease (20 of 33, 61%) versus locoregional disease (1 of 17, 6%). A prominent LTRE enriched in brain metastasis (BrM) was RET (p-value = 0.003), expression of which showed outlier gains in 56% of ER-positive BrM. Marked anti-tumor activity was demonstrated with the RET inhibitor cabozantinib in ex vivo explant cultures of patient resected BrMs (n=3) and a BrM patient-derived xenograft. CONCLUSIONS: Taken together, these results demonstrate profound, recurrent and metastatic site-specific LTREs in advanced breast cancers, which may be essential to our understanding of endocrine-therapy resistance and metastasis. Although current emphasis for longitudinal clinical profiling of tumors is on DNA-level alterations, these results suggest LTREs as a compelling, shared mechanism of cancer progression. Given remarkably high recurrence rates of specific LTREs across multiple cohorts, further preclinical and clinical investigations of LTREs are demanded, especially considering some (i.e. FGFR4 and RET) are readily druggable. Citation Format: Priedigkeit N, Vareslija D, Basudan A, Watters RJ, Lucas PC, Davidson NE, Blohmer J-U, Denkert C, Machleidt A, Heppner BI, Brufsky AM, Oesterreich S, Young L, Lee AV. Highly recurrent transcriptional remodeling events in advanced endocrine resistant ER-positive breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS2-03.

Background Obesity, as defined by body mass index (BMI), has been associated with increased recurrence rate, shorter DFS and increased death rates due to breast cancer (BC). Most of the studies to date have examined the relationship of BMI and DFS in patients with hormone receptor positive disease. To our knowledge, BMI and its relationship with outcome in early stage HER2 positive breast cancer has not previously been examined. The N9831 is a large phase III trial testing the role of trastuzumab in the adjuvant setting of high risk patients with early stage HER2+ BC. We hypothesized that the occurrence of overweight and obesity may correlate with outcome. Methods: This analysis presents BMI and its relation to tumor characteristics and DFS in patients (pts) enrolled in the N9831 clinical trial. Pts were categorized as normal weight, overweight or obese using the WHO BMI classification parameters of < 25%, 25–29% and ≥ 30% respectively. For patient characteristics, patients were grouped into non-obese (BMI< 30) and obese (≥ 30) cohorts. DFS was estimated by the Kaplan-Meier method. Comparisons between arms A (chemotherapy alone), B (chemotherapy plus sequential trastuzumab) and C (chemotherapy plus concurrent trastuzumab) were performed using the Cox proportional hazards model, stratified by BMI. Results: Analysis was completed on 3,017 eligible pts. Obese pts were more likely to be older and postmenopausal (p Conclusions: This analysis of data from the N9831 study confirms that obese pts with early stage HER2+ tumors have worse clinical outcome than pts with BMI < 30%. Adjuvant trastuzumab improved clinical outcome regardless of BMI. This study supports weight loss intervention for obese women with early stage HER2+ BC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-02.

Background We have previously reported that TOP2A RNA expression was prognostic in patients with operable estrogen receptor (ER) positive breast cancer treated with anthracycline containing adjuvant chemotherapy plus endocrine therapy in trial E2197, and provided complementary prognostic information in those who had a mid-range Recurrence Score (RS) (Sparano et al. Clin Cancer Res 2009; 15; 7693). The purpose of this analysis was to provide additional evidence regarding this observation. Methods: We evaluated TOP2A RNA expression in 4 independent data sets individually and in a pooled analysis including all data. We also used an algorithm to simulate the Oncotype DX Recurrence Score (SimRS). The analysis included 752 patients with early stage breast cancer that was ER-positive, HER2−negative defined by gene expression. Results are expressed as the hazard ratio (HR) for estimates of the effect of a one standard deviation increase in the value of the log gene expression (x + 1SD vs. x) as a continuous function. Five-year relapse free survival (RFS) rates were also evaluated for patients with a mid-range SimRS for high vs. low TOP2A expression (above vs. below the median, which was prognostic in E2197). The distribution of SimRS was categorized to be concordant with the distribution of actual RS in E2197 (47%, 34%, 19%, respectively for RS 31 - high). Results: The HR for models including TOP2A alone and joint models including TOP2A and SimRS are shown in the table. TOP2A expression was significantly associated with recurrence in all 4 datasets when evaluated individually (HR 1.56, p Conclusions: This analysis provides additional evidence that TOP2A expression provides prognostic information in patients with ER-positive, HER2−negative disease, a population known to have low incidence of TOP2A gene alterations. These findings also suggest that TOP2A expression provides information that is complementary to RS, and may be useful for identifying high risk subjects who have a mid-range RS. These findings require prospective validation in other prospective or prospective-retrospective trials using the actual rather than simulated RS. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-13.

Invasive lobular breast carcinoma (ILC) is the second most common subtype of breast cancer following invasive ductal breast carcinoma, accounting for 10-15% of all cases. Despite the intriguing histological and clinical differences between these two subtypes, ILC has been chronically understudied. Human ILC cell lines and xenografts are regarded to be of limited utility for ILC research due to slow proliferation rates in culture and low reported intake rates. Data on human ILC cell line xenografts remains limited and knowledge of their metastatic capacity is currently lacking. Using Estrogen Receptor alpha (ER)-positive human ILC cell lines labeled with dual bioluminescent and fluorescent reporters, herein we performed a comprehensive in vivo characterization of their growth as primary tumors orthotopically in the mammary fat pad, as well as at secondary sites following spontaneous or experimental metastasis. Primary tumors exhibited the characteristic dyscohesive, single-file growth of ILC cells, faithfully recapitulating the histology of human ILC tumors. In vivo bioluminescence imaging coupled with ex vivo fluorescence analysis revealed spontaneous metastases to the bone, brain, lungs and ovaries, closely mirroring the clinical patterns of ILC tumor dissemination. This was in stark contrast to experimental metastasis, where none of the cell lines colonized the lungs when injected into the tail vein and bone colonization was observed with only one cell line following intracardiac injections. The metastatic lesions harbored the classical histological features of ILC, including single strand growth, loss of E-cadherin-mediated adherens junctions and mislocalization of p120-catenin to the cytoplasm. Importantly, both the primary tumors and the metastases exhibited high ER expression and significant response to the anti-endocrine agent fulvestrant - a selective ER downregulator. Using these models, we will also present data from functional studies validating candidate genetic drivers of ILC disease progression, which are associated with poor recurrence-free survival in patients with ILC. Ongoing work focused on genomic and transcriptional analyses of metastatic lesions and isolated cell lines from these models will pinpoint additional candidate drivers of ILC dissemination. This is the first report of a hormone responsive ER+ metastatic xenograft model faithfully representing unique ILC features such as ovarian metastases, which will serve as a valuable pre-clinical platform for testing novel therapeutics towards translation into the clinic. Citation Format: Tasdemir N, Scott J, Laotche J, Hou W, Bossart EA, Atkinson J, Sflomos G, Sreekumar S, Castro C, Anderson C, Lee AV, Brisken C, Lucas PC, Davidson NE, Oesterreich S. Novel human cell line xenograft models of ERα-positive metastatic invasive lobular breast carcinoma as pre-clinical platforms for validating candidate genetic drivers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-02.

Invasive lobular carcinoma (ILC) is the second most common type of breast cancer following invasive ductal carcinoma (IDC) and accounts for 10-15% of all cases. Unlike the masses or lumps formed by IDCs, ILCs grow as small, dyscohesive cells in a single-file pattern within dense layers of extracellular matrix. Paradoxically, while patients with ILC display favorable prognostic and predictive factors (Estrogen Receptor [ER]+, Progesterone Receptor+, HER2-, low Ki67), they present with more frequent long-term recurrences compared to those with IDC, indicative of endocrine resistance. Thus, there is urgent need to investigate genetic drivers of ILC disease progression in order to develop more effective therapeutic strategies and improve patient outcome. To this end, we used the Nanostring platform to measure the expression of 577 copy number variation-associated genes in 131 primary ILC tumors with long-term clinical data. This analysis identified CTTN (Cortactin; cortical actin binding protein) as a candidate ILC driver that exhibited higher expression in tumors from patients with subsequent recurrent (n=33) versus non-current disease (n=98). We further validated high CTTN mRNA/protein expression in human ILC cell lines, tumors and patient-derived xenografts, and CTTN locus (11q13.3) amplification in clinical ILC metastases to the brain, bone and ovaries. In follow-upin vitro studies, RNAi-mediated inhibition of CTTN diminished the adhesion and haptotaxis of human ILC cell lines to Collagen I, as well as impairing their growth in 3D Collagen I culture. To assess the functional role of CTTN in ILC tumor growth and metastasis, we transplanted control and CTTN knockdown human ILC cell lines into the mammary fat pads and ducts of immuno-compromised mice and monitored disease progression via bioluminescence imaging. While CTTN inhibition did not lead to a substantial impact on measurable tumor burden, both CTTN shRNAs resulted in a significant survival benefit in the fat pad model. Ongoing work is aimed at pinpointing the underlying mechanisms of CTTN's role in mediating growth in 3D Collagen I culture in vitro and disease progression in vivo. Collectively, the results of these studies will advance our understanding of ILC disease mechanisms and serve as a pre-clinical basis for improving the clinical outcome of patients with this understudied subtype of breast cancer. Citation Format: Tasdemir N, Sikora MJ, Zhu L, Levine KM, Scott J, Basudan A, Sflomos G, Sreekumar S, Bossart EA, Elishaev E, Chandran UR, Tseng GC, Jankowitz RC, Dabbs DJ, McAuliffe PF, Brisken C, Davidson NE, Oesterreich S. Investigating cortactin as a genetic driver of disease progression in invasive lobular carcinoma [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-03.

Previous findings suggested that patients (pts) with copy number alterations of MYC (8q24) and centromere 8 (CEN 8) in the setting of HER2-positive breast cancer may be associated with improved outcome to adjuvant trastuzumab. Our tissue microarray (TMA) data suggested that alternate cutpoints for MYC copy number anomalies [of 802 pts, those with MYC:CEN8 ratio ≥1.3 or 30% a priori. Median follow-up was 5.8 years. Results: Of 1220 pts with completed IHC analyses, 557 (46%) were MYC+. MYC+ was associated with a higher rate of hormone receptor positivity (58% vs 48%, chi-sq P Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-08.

Invasive lobular carcinoma (ILC) represents only 5-15% of all breast cancer. However, if it were an independent cancer type, it would rank as the sixth most common cancer in women, as common as non-Hodgkin lymphoma or melanoma. ILC is chronically understudied as a breast cancer subtype, but compared to invasive ductal carcinoma (IDC) ILC is more often HR-positive, HER2-negative, and lower histologic grade. Despite these favorable tumor characteristics, recent studies suggest outcome in ILC is not consistent with these favorable characteristics. We herein describe our single institution experience with ILC. Using cancer registry data, we identified 709 women over the age of 18 (including 85 with recurrences) diagnosed at our institution between 1990 and 2011 with HR-positive, early-stage (I-III), primary ILC and potentially available banked tissue samples. We implemented a case-cohort design to investigate molecular tumor characteristics associated with ILC prognosis. Patients were excluded for a history of synchronous bilateral breast cancer at the time of diagnosis, a prior history of invasive breast cancer, or a diagnosis of non-breast malignancy within 5 years prior to their diagnosis of ILC. Originally, a sample of 200 subjects was selected for further study and molecular characterization. However, tissue acquisition, subject eligibility and verification of recurrences yielded a sample of in total 131 subjects (including 33 with recurrences), and the case-cohort design was recreated for this final sample. All available information regarding patient and tumor characteristics, treatment history, and outcome was collected from medical records. ER, PR, and Ki67 protein (IHC) and RNA (Nanostring analysis) levels were analyzed with respect to outcome of patients. PR H-score, but not ER H-score, was significantly associated with survival in our overall ILC cohort. In the case-cohort analysis there was a weak correlation between ER, PR and Ki67 RNA levels (measured by nanostring) and ER, PR and Ki67 protein levels (measured by IHC) in the ILC tumor samples. After the Nanostring data was normalized, box plots were constructed for ESR1, PR and MKI67 so that a simple t-test could look at gene expression in recurrences and non-recurrences. There was no significant difference in ER expression between non-recurrences and recurrences. The recurrence cases had significantly lower PR expression levels. We will present quantitative ER and PR data, as well as detailed clinical characteristics for this cohort in relation to outcome. Citation Format: Rachel C. Jankowitz, Kevin Levine, Maryam Zamanian, Matthew Sikora, Nilgun Tasdemir, Priscilla McAuliffe, David Dabbs, Nancy Davidson, Brenda Diergaarde, Steffi Oesterreich. Associations between biomarkers and outcome in a patient cohort with invasive lobular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5645. doi:10.1158/1538-7445.AM2017-5645

Invasive lobular carcinoma (ILC) is the second most common type of breast cancer following invasive ductal carcinoma (IDC) and accounts for 10-15% of all cases. Unlike the masses or lumps formed by IDCs, ILCs grow as small, dis-cohesive cells in a single-file pattern within dense layers of extracellular matrix. Paradoxically, while patients with ILC display favorable prognostic and predictive factors (ER+, PR+, HER2-, low Ki67), their long-term response to endocrine therapy is worse than patients with IDC. Despite such histological and clinical differences, ILC has remained a gravely understudied subtype of breast cancer. Thus, there is urgent need to investigate molecular drivers of ILC progression in order to develop more effective therapeutic strategies and improve patient outcome. To this end, we used the Nanostring platform to measure the expression of 577 copy number variation-associated genes in 131 primary ILC tumors and identified two candidate drivers that exhibit higher expression in tumors from patients with recurrent versus non-current disease: CTTN (cortical actin binding protein) and QSOX1 (quiescin sulfhydryl oxidase). In follow-up studies, we observed high CTTN and QSOX1 expression in human ILC cell lines, tumors and patient-derived xenografts. In addition, QSOX1 expression was further increased in ILC ovarian metastases compared to their matched primary tumors. In functional in vitro studies, RNAi-mediated inhibition of CTTN led to decreased adhesion and haptotaxis to Collagen I, while QSOX1 knockdown diminished the growth of human ILC cell lines. We are currently assessing the in vivo therapeutic utility of CTTN and QSOX1 inhibition in tumor growth and metastasis using human ILC cell line and patient-derived xenograft mouse models. This study greatly advances our understanding of ILC disease mechanisms and identifies novel therapeutic targets towards improving the clinical outcome of patients with this understudied subtype of breast cancer. Citation Format: Nilgun Tasdemir, Matthew Sikora, Zhu Li, Kevin Levine, Ahmed Basudan, Soumya Luthra, Esther Elishaev, Uma Chandran, George C. Tseng, Rachel Jankowitz, David J. Dabbs, Priscilla McAuliffe, Nancy E. Davidson, Steffi Oesterreich. Investigating drivers of disease progression in invasive lobular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2841. doi:10.1158/1538-7445.AM2017-2841

Background: Epigenetic modifiers have been emerging as new players in breast cancer development. FAD-dependent histone demethylases have been proved to play important roles in regulating breast tumor initiation and development. While the role of histone lysine-specific demethylase 1 (LSD1) in breast cancer progression has been well characterized, the activity of its homolog, lysine-specific demethylase 2 (LSD2), in breast cancer is still a riddle that needs to be uncovered. Methods: To explore the precise role of LSD2 in breast cancer, we overexpressed LSD2 protein in MDA-MB-231 cells (LSD2-OE). The phenotypic effect of LSD2-OE on MM231 cells was characterized by proliferation assay, soft agar assay (3D growth), trans-well cell migration and invasion assay and mammosphere formation assay. To extend stem cell-like features study of LSD2 overexpressed MM231 cells, we examined CD44+/CD24- subpopulation by flow cytometry and the expression of several stem cell markers by qPCR and western blot. In addition, we also investigated the potential altered phenotypes of LSD2 knockdown in MM231 cells via RNAi. Results: Results from Oncomine database show that LSD2 expression level is greatly increased in invasive ductal carcinoma (IDC) vs. non-invasive breast cancers. The cellular proliferation assay revealed that LSD2-OE promoted MM231 cell growth. Similarly, LSD2 overexpressed MM231 cells were capable of forming large colonies in soft agar. Trans-well cell migration and invasion assay results indicated LSD2-OE significantly decreased both migration and invasion in MM231 cells. Mammosphere formation assay results showed LSD2 overexpression increased the number of primary and tertiary mammospheres in MM231 cells. Consistent with increase of mammosphere forming efficiency, NANOG and SOX2 were increased by LSD2 overexpression at the protein level. However, LSD2-OE significantly decreased CD44+/CD24- subpopulation. Moreover, siRNA mediated LSD2 depletion significantly decreased cell growth in multiple breast cancer cell lines without altering migration and invasion in MM231 cells. Finally, no obvious phenotypic changes were observed in MM231 cells expressing stable LSD2 shRNA. Conclusion: The multifaceted effects of LSD2 on breast cancer phenotypes may reflect the profound influence of LSD2 activity on chromatin structure and gene expression in the breast cancer genome. Our studies suggest that overexpression of LSD2 facilitates growth and confers stem cell-like traits to breast cancer cells, and transient LSD2 inhibition by siRNA hinders breast cancer cell growth. These results suggest that LSD2 might be a valuable therapeutic target in breast cancer. Citation Format: Lin Chen, Shauna N. Vasilatos, Ye Qin, Steffi Oesterreich, Nancy E. Davidson, Yi Huang. New insights into the roles of histone lysine-specific demethylase 2 (LSD2) in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1385. doi:10.1158/1538-7445.AM2017-1385

Metastatic breast cancer is often intractable due to its inherent ability to overcome current therapies. Genomic alterations are frequently responsible for therapeutic resistance. To better understand genomic mechanisms of acquired resistance in breast cancer we undertook a detailed characterization of single nucleotide variation (SNV) and structural variation (SV) in paired primary-metastasis metachronous tumors from 6 breast cancer patients (median time to recurrence 7.3 years). In ER-positive recurrent tumors treated with endocrine therapies, we identified multiple metastatic-acquired variants in ESR1 including a novel constitutively active, ligand-independent ESR1-DAB2 gene fusion. Importantly, this fusion resulted from a breakpoint in intron 4, retaining the DNA-binding domain but eliminating the ligand-binding domain (LBD), concordant to a similar fusion reported previously in a xenograft model. Hybrid capture based genomic profiling from >7,800 breast cancers identified similar exon/intron 4 fusions in 5 tumors with direct paired-read evidence. Using a novel copy number shift detection strategy, 58 additional tumors showed indirect evidence of a rearrangement at exon 4 based on a novel copy number shift detection strategy. ESR1 fusion and copy number shift positive tumors are strongly enriched in metastatic disease (78%; p Citation Format: Ryan J. Hartmaier, Nolan Priedigkeit, Laurie Gay, Michael E. Goldberg, James Suh, Siraj Ali, Jeffery Ross, Michaela Tsai, Barbara Haley, Julio Peguero, Rena D. Callahan, Irina Sachelarie, John Cho, Amir Bahreini, Shannon L. Puhalla, Steffi Oesterreich, Aju Mathew, Peter C. Lucas, Nancy E. Davidson, Adam M. Brufsky, Philip J. Stephens, Juliann Chmielecki, Adrian V. Lee. Comprehensive genomic analysis of metastatic breast cancers reveals ESR1 fusions as a recurrent mechanism of endocrine therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 421. doi:10.1158/1538-7445.AM2017-421

Background: We have previously reported the results of surgical excision without irradiation for selected patients with DCIS in ECOG E5194, where the 5-year rates of local recurrence varied with age, grade, and lesion size (Hughes et al. J Clin Oncol 27:5319, 2009). New methods are needed to provide more accurate and reproducible assessment of recurrence risk. Methods: ECOG E5194 included 670 eligible patients with DCIS treated with surgical excision (≥ 3 mm negative margins) without irradiation, 228 of whom received tamoxifen. Patients had low or intermediate grade DCIS ≤ 2.5 cm, or high grade DCIS ≤ 1 cm. The Oncotype DX® assay was performed by quantitative RT-PCR using formalin fixed paraffin embedded tumor specimens from 327 patients (49% of the parent study). Recurrence Score® (RS) was calculated using the published algorithm. A new, prespecified DCIS Score™ was designed to predict recurrence using an optimized gene expression algorithm. The primary objective was to determine whether there was a significant association between the risk of an ipsilateral breast event (IBE) and the continuous DCIS Score in Cox models. 46 patients had an IBE (defined as ipsilateral local recurrence of DCIS [n=20] or invasive cancer [n=26]). Median follow-up was 8.8 years. Results: The 10-year IBE rates were 15.4% for low/intermediate grade DCIS and 15.1% for high-grade DCIS (as determined by central pathology review), and for invasive IBE, 5.6% and 9.8%, respectively. Comparison between local and expert grading showed substantial disagreement. Continuous DCIS Score was significantly associated with IBE (HR 2.34 per 50 units; 95% CI 1.15, 4.59; p=0.02) when adjusted for tamoxifen use (prespecified primary analysis) and with invasive IBE (HR 3.73; CI 1.34, 9.82; p=0.01). DCIS Score was significantly associated with outcome when evaluated by the prespecified risk groups (see Table). Similar results were observed with and without adjustment for tamoxifen use or for negative margin width. Features associated with IBE in multivariate models included menopausal status (HR 0.49; 95% CI 0.27, 0.90; p=0.02), tumor size (HR 1.52 per 5 mm; 95% CI 1.11, 2.01; p=0.01), and continuous DCIS Score (HR 2.41; 95% CI 1.15, 4.89; p=0.02). The standard RS, which is calculated using thresholding of many genes unlike the DCIS Score, was not associated with IBE or invasive IBE (p > 0.6). Conclusions: We have prospectively validated a multigene assay that quantifies recurrence risk and complements traditional clinical and pathologic factors in selected patients with DCIS treated with surgical excision without irradiation. The DCIS Score provides a new clinical tool for individualized selection of treatment for patients with DCIS. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S4-6.

Background: Metastasis is the primary cause of death in ESBC. We have shown in mouse models that a subpopulation of tumor cells expressing invasive Mena isoforms stream, form microanatomic structures (“TMEM”) with endothelial cells and macrophages, intravasate into the circulation at TMEM sites, and metastasize (Harney et al. Cancer Discovery, 2015). Further, TMEM sites (“MetaSites”) are identifiable in human ESBC, and “MetaSite score” [MS] is positively associated with distant recurrence in HR+/HER2- ESBC independent of clinicopathologic features, including IHC4 (Rohan et al. JNCI 2014). Here we determined the association between MS and recurrence in an independent ESBC cohort (E2197; NCT00003519). Methods: We evaluated primary tumors from 600 patients (median followup 14.8 years) with ESBC (weighted % = 50% T1, 54% N0, 46% N1) treated with surgery and 4 cycles of adjuvant chemotherapy (AC or AT) and endocrine therapy. Grade, ER, PR, and HER2, and Oncotype DX Recurrence Score (RS) were evaluated in central labs (Badve et al. JCO 2008), and MS was determined in a CLIA-certified lab using an analytically validated, fully automated digital pathology/image analysis method that identifies Mena expressing tumor cells in direct contact with CD68+ macrophages and CD31+ endothelial cells (ie, “TMEMs”, or “MetaSites”). The objectives were to determine the association between MS and distant relapse free interval (DRFI) and relapse free interval (RFI). Kaplan-Meier survival curves were used to estimate time-to-event distributions. Cox proportional hazards models were used to assess hazard ratio associated with MS while controlling for covariates, and allowing time-varying association with MS. Both Kaplan-Meier and Cox regression methods addressed stratified sampling by incorporating proper weights. All analyses were performed in R 3.2.3. Results: MS ranged from 0-199; the weighted mean MS was lower in HR+/HER2- than TN (16.1 vs. 23.8, p=0.001) and HER2+ disease (26.2, p=0.003). MS was not associated with T or N status, and correlated poorly with RS (r=0.29). Proportional hazards models revealed a significant positive association between continuous MS and DRFI (p=0.001) and RFI (p=0.00006) in HR+/HER2- disease in years 0-5 (and by MS tertiles for DRFI [p=0.04] and RFI [p=0.01]), but not after year 5 or in TN or HER2+ disease. Proportional hazards models including clinical covariates (N0 vs. N1; T1 vs. T2; high vs. int. vs. low grade) also revealed significant positive associations for continuous MS with RFI (p=0.04), and borderline association with DRFI (p=0.08). Similar findings for MS (RFI p=0.05;DRFI p=0.10) were noted in a joint model including categorical RS (30). Conclusions: MS, a novel metastasis biomarker reflecting interaction between streaming and metastasizing tumor cells and microenvironment, provides prognostic information complementary to classical clinicopathologic features and RS in HR+/HER2- ESBC. Further evaluation is warranted in order to identify patients at highest risk of recurrence within 5 years most likely to benefit from adjuvant chemotherapy or novel therapies. (Supported by BCRF and NCI CA21115, CA180794, CA23318, CA66636, CA180820). Citation Format: Sparano JA, Gray R, Oktay MH, Entenberg D, Rohan T, Xue X, Donovan M, Peterson M, Shuber A, Hamilton D, D'Alfonso T, Goldstein LJ, Gerlter F, Davidson N, Condeelis J, Jones J. Tumor microenvironment of metastasis (TMEM) score is associated with early distant recurrence in hormone receptor (HR) positive, HER2-negative early stage breast cancer (ESBC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S4-04.

BACKGROUND: Unique genes and pathways were identified for prognosis on tamoxifen (T, 5 yrs) and prediction on CAF-T vs T in S8814 using whole transcriptome RNA-Seq from archival FFPE tissue. (Albain, et al; Cherbavaz, et al; SABCS 2015) Discovery was robust for early DFS events but sparse for late. The aims of this new analysis were to 1) utilize a new endpoint BCSE for gene discovery of late events, prognosis and prediction and 2) add intronic counts to the previous exonic results to define whole genes impacting on late BCSE. METHODS: Charts of patients (pts) on CAF-T (212) vs T (142) were reviewed to define the BCSE endpoint (local/regional, contralateral, distant). Deaths without BC were treated as competing risks. BCSE models (including metagenes) of late prognosis and prediction used cumulative incidence functions. Consolidated intronic regions counts within genes were added to exonic regions counts. Using these “whole gene” (WG) counts, association of gene expression with time to BCSE was assessed by Cox regression. A multiple WG score (MWGS) for BCSE prognosis beyond 5 yrs (to 12.5 yrs) was constructed and evaluated for 1-3 and 4+ node (N) groups. False discovery rate was controlled at 10%. RESULTS: More exons and WG were discovered for prognosis on T alone over 12.5 yrs with the BCSE endpoint than DFS. For prognosis of late BCSE after 5 yrs, more genes were discovered using WG (n=111) than by exons (n=9). There were significantly fewer genes for late BCSE on CAF-T (8, WG; 0, exons). The functions of WG prognostic for late BCSE were: cell cycle/proliferation-26 genes, chromosome segregation/mitotic spindle-22, DNA repair/maintenance-10, transcriptional/translational control-5, cell adhesion/migration-4, immune-3, diverse/unknown-32 and growth factor/hormone receptor signaling-9 (this group was only found by WGs, not exons). Of these 111 WG, a MWGS prognostic for late BCSE on T used 57 previously discovered genes pre-specified for this analysis. Probability of BCSE beyond 5 yrs for low vs high MWGS was 8% vs 21% in N1-3+ and 17% vs 42% in N4+. Late prognosis on T differed by low vs high risk defined in a metagene model: cumulative BCSE at year 10 was 0% vs 47% (low vs high risk, p=0.001). Prediction of 10-yr incidence of BCSE varied by risk level by treatment in a metagene model: low risk- CAF-T=47%, T=0% (p=0.045); high risk- CAF-T=35%, T=45% (p=0.027). CONCLUSIONS: Gene discovery for prognosis of late BCSE is enhanced with a novel WG transcriptome expression approach. Use of chemotherapy (CT) before T significantly attenuated gene discovery, so that molecular tools for decisions on extending endocrine therapy (ET) may not be reliable in a setting of prior CT. Some pts on ET for 5 yrs may not require either longer ET or CT, given a N+ cohort was defined with no BCSE observed over 12.5 yrs. For prediction of CT benefit, CAF-T appeared to be inferior to T in a low risk metagene model for BCSE. In sum, these results add more evidence that ET alone may be sufficient (perhaps better) in select N+ settings. Validation in SWOG S1007 (RxPONDER) is planned. SUPPORT: NCI CA180888, 180819, 180821, 180820, 180863; in part, Genomic Health, Inc. Citation Format: Albain KS, Crager MR, Barlow WE, Baehner FL, Bergamaschi A, Rae JM, Ravdin PM, Tripathy D, Gralow JR, Livingston RB, Osborne CK, Ingle JN, Pritchard KI, Davidson NE, Carey LA, Cherbavaz DB, Sing AP, Shak S, Hortobagyi GN, Hayes DF. Discovery of molecular predictors of late breast cancer specific events (BCSE) in ER+, node+ breast cancer – new transcriptome expression whole gene analysis of the phase III adjuvant trial SWOG S8814 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD7-07.

BACKGROUND: In SWOG-8814A, pts with ER+ node+ breast cancer and low 21 gene recurrence scores (RS) had good prognosis and no CAF benefit, but high RS predicted longer survival from CAF followed by T (CAF-T) vs T (Albain, Lancet Oncol 2010). The aims of SWOG-8814B were to identify novel genes and networks for 1) prognosis of early and late relapse and 2) prediction of CAF benefit, using whole transcriptome expression analysis with next generation RNA sequencing (NGS). METHODS: Stored RNA previously extracted for SWOG-8814A (T, CAF-T arms; T, 5 yrs) was analyzed for RNA/library yield (see companion abstract Cherbavaz et al. for methods). Genes were sequenced and expression of mRNA species was related to disease-free survival (DFS) using Cox proportional hazards. Discovery analyses controlled false discovery rate (FDR) at 10%. Genes were identified for prognosis on T and prediction on CAF-T vs T. Networks of genes/pathways were explored. Early (0-5 yrs) and late (5-13+ yrs) time periods were studied. Gene Ontology, Cytoscape, pathway and hierarchical clustering were used for functional gene and metagene analyses. RESULTS: Of 367 samples, 354 (96%; 142 T, 212 CAF-T; 141 DFS events) had sufficient RNA/library yield, with 20,101 genes sequenced. For prognosis on T, there were 2327 and 568 genes discovered in early and all-yrs follow-up, with only 9 genes prognostic after 5 yrs. Prognosis analyses for residual risk after CAF-T were uninformative. Functional mapping found that genes prognostic for worse DFS were enriched for proliferation (G2M, M-phase), cellular metabolism, DNA repair, stress response and EMT; whereas, those with better DFS involved transcription regulation/repression via zinc finger proteins. Hierarchical clustering (T arm) found significant DFS prognostic metagene signatures for ER-related genes, immune response, ECM/stroma, chromatin remodeling-transcription factor activity and TGFb pathway. All varied for early vs late DFS events. For example, low ER/high stroma expression signatures correlated with high proliferation gene expression and were strongly associated with early events (standardized [st] HR 2.94, p CONCLUSIONS: Unique genes, clusters and pathways were identified by NGS of archival material in ER+ N+ breast cancer, including previously unreported signatures. While ER, stroma and proliferation-related signatures were associated with early prognosis, proliferation best predicted worse DFS after 5 yrs. NGS of the primary tumor is most informative for early events in pts with only 5 years of T, with few genes selecting only for late relapse. If validated, these signatures may identify pts with excellent DFS despite positive nodes for endocrine therapy alone as well as others for whom chemotherapy and/or biologics are also required . SUPPORT: NCI CA 180888, 180819, 180821, 180820, 180863; in part, Genomic Health, Inc. Citation Format: Albain KS, Crager MR, Barlow WE, Baehner FL, Bergamaschi A, Rae JM, Ravdin PM, Tripathy D, Gralow JR, Livingston RB, Osborne CK, Ingle JN, Pritchard KI, Davidson NE, Carey LA, Cherbavaz DB, Sing AP, Shak S, Hortobagyi GN, Hayes DF. Molecular predictors of outcome on adjuvant CAF plus tamoxifen (T) vs T in postmenopausal patients (pts) with ER+, node+ breast cancer – Transcriptome expression analysis of the phase III trial SWOG-8814. [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 S3-02.

Estrogen receptors (ER) are expressed in ∼65% of human breast cancer. Cumulative data from clinical trials and retrospective analyses suggest that some chemotherapeutic agents may be less effective in patients with ER-positive (ER+) tumors than those with ER-negative (ER−) tumors. Paclitaxel is an active agent used in breast cancer chemotherapy. To investigate the possible influence of ER on the therapeutic efficacy of paclitaxel and its underlying mechanism, we established several isogenic ER+ cell lines by stable transfection of ERα expression vectors into ER− breast cancer BCap37 cells. We showed that 17-β estradiol significantly reduces the overall cytotoxicity of paclitaxel in BCap37-expressing ERα but has no influence on the ER− parental cells. Further analyses indicate that expression of ERα in BCap37 cells mainly interferes with paclitaxel-induced apoptotic cell death, without affecting paclitaxel-induced microtubule bundling and mitotic arrest. Moreover, we found that the addition of ICI 182,780 (Fulvestrant), a selective ER down-regulator, could completely reverse the resistance of ER+ BCap37 cells to paclitaxel. These findings showed that ERα-mediated breast tumor cell resistance to paclitaxel was through selective inhibition of paclitaxel-induced tumor cell apoptosis. Additionally, the combination of ICI 182,780 also sensitizes MCF-7 and T47D cell lines to the treatment of paclitaxel, which further confirmed the correlation between ERα and drug resistance in ER+ tumor cells. The results obtained from this study provide useful information for understanding ER-mediated resistance to paclitaxel and possibly other antineoplastic agents. [Cancer Res 2007;67(11):5337–44]

Our previous work has shown that HDAC5 knockdown led to significant increase of H3K4me2 in the TNBC cell line, MDA-MB-231, suggesting a potential role of HDAC5 in regulating LSD1 activity in breast cancer. In this study, we demonstrated that HDAC5 physically interacts with LSD1 in MDA-MB-231 cells. Overexpression of HDAC5 increased LSD1 protein level through a reduction of LSD1 polyubiquitination. siRNA-mediated knockdown of HDAC5 mRNA, but not other class II HDACs or LSD1-containing complex members (HDAC1, HDAC2 and CoREST), significantly decreased LSD1 protein expression and half-life. HDAC5 overexpression reduced levels of H3K4me1/2, suggesting that HDAC5 acts as a positive regulator of LSD1-mediated H3K4 demethylation activity. Enhanced proliferation mediated by HDAC5 overexpression in MDA-MB-231 cells was diminished by LSD1-KD, suggesting a critical role of LSD1 in regulating oncogenic activity of HDAC5. We also observed that high-grade tumorigenic MCF10A-CA1a cells (triple negative) expressed higher levels of LSD1 and HDAC5 compared with parental counterpart MCF10A cells. By gain- and loss-of-function studies using this TNBC progression model, we observed that HDAC5 possesses a critical oncogenic function in driving TNBC development by blocking LSD1 protein degradation and re-shaping epigenetic landscape. Several HDAC inhibitors were tested for their ability to inhibit LSD1 and HDAC5 interaction. Treatment with sulforaphane, a natural HDAC inhibitor found in cruciferous vegetables, blocked the association of HDAC5 and LSD1 through down-regulation of HDAC5 mRNA and protein expression that led to LSD1 protein destabilization. Sulforaphane treatment also re-expressed a unique subset of tumor suppressor genes (RGS16, BIK, PCDH1, CDKN1a, ING1, etc), which are abnormally silenced in TNBC cells, but are expressed in other breast cancer subtypes and normal breast cells. Addition of a novel LSD1 inhibitor, HCI-2509, significantly promoted antineoplastic efficacy of sulforaphane in breast cancer cells, and TNBC cells were more susceptible than other subtypes to the combination therapy. In an in vivo study, while treatment with either sulforaphane or HCI-2509 alone each displayed significant anti-tumor effects against MDA-MB-231 xenografts, combined treatment with both compounds exhibited synergistic inhibitory effect on tumor growth in the MDA-MB-231 xenografts. Taken together, these results suggest that HDAC5 promotes LSD1 protein stabilization, leading to a dysregulated chromatin landscape which functions as an antibraking system in TNBC development. Crosstalk between LSD1 and HDAC5 may represent a novel and effective therapeutic target for this devastating disease. (Supported by DOD W81XWH-14-1-0237/W81XWH-14-1-0238, Breast Cancer Research Foundation, and UPCI Core Grant NIH P30CA047904) Citation Format: Chunyu Cao, Shauna Vasilatos, Steffi Oesterreich, Nancy E. Davidson, Yi Huang. Functional crosstalk between histone deacetylase 5 (HDAC5) and lysine-specific demethylase 1 (LSD1) as a novel therapeutic target in triple-negative breast cancer cells. [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 3838. doi:10.1158/1538-7445.AM2015-3838

Background: We had previously reported after a median followup of 5.3 years that disease free survival (DFS) was improved with either adjuvant weekly paclitaxel (hazard ratio [HR] 0.73, p=0.0006) or every 3 week docetaxel (HR 0.77, p=0.02) compared with every 3 week paclitaxel (N Eng J Med 2008; 358; 1663), and that obesity and black race were independently associated with inferior outcomes in estrogen receptor (ER)-positive disease after a longer followup (Cancer 2012: 118: 5937 & JNCI 2012; 104: 406). We now report updated results after a median followup of 12.1 years. Methods: Eligibility included axillary lymph node positive or high-risk (tumor at least 2 cm) node-negative breast cancer. All patients received 4 cycles of AC (doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2) every 3 weeks, followed by either: (1) paclitaxel 175 mg/m2 every 3 weeks x 4 (P3), (2) paclitaxel 80 mg/m2 weekly x 12 (P1), (3) docetaxel 100 mg/m2 every 3 weeks x 4 (D3), or (4) docetaxel 35 mg/m2 weekly x 12 (D1). Patients with ER-positive breast cancer also received endocrine therapy for at least 5 years, including either tamoxifen and/or an aromatase inhibitor. The primary comparisons included taxane (P vs. D) and schedule (every 3 weeks vs. weekly), and the primary endpoint was DFS, defined as local, regional, and/or distant relapse, second primary breast cancer, or death without recurrence. Results: A total of 4954 eligible patients were accrued between October 1999 and January 2002. At the time of this analysis, 90% of surviving patients were followed for at least 10 years, and there were substantially more DFS events (1639 vs. 1048) and deaths (1283 vs. 686) than the original report. For the primary comparison, there remains no significant difference in DFS by taxane (p=0.33) or schedule (p=0.88), although there was a significant interaction between taxane and schedule (p Conclusions: Improvements in DFS observed at 10 years for the P1 and D3 arms compared with the P3 arm are qualitatively similar but quantitatively less than at 5 years, and the effects on OS are marginal in the overall population. The relative effectiveness of weekly paclitaxel and every 3 week docetaxel may vary by subtype. Citation Format: Joseph A Sparano, Fengmin Zhao, Silvana Martino, Jennifer Ligibel, Thomas Saphner, Antonio C Wolff, George W Sledge Jr, Edith A Perez, William C Wood, Nancy E Davidson. Ten year update of E1199: Phase III study of doxorubicin-cyclophosphamide followed by paclitaxel or docetaxel given every 3 weeks or weekly in patients with axillary node-positive or high-risk node-negative 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 S3-03.

Breast tumors expressing estrogen receptor-α (ER) respond well to therapeutic strategies using selective ER modulators, such as tamoxifen. However, ∼30% of invasive breast cancers are hormone independent because they lack ER expression due to hypermethylation of ER promoter. Treatment of ER-negative breast cancer cells with demethylating agents [5-aza-2′-deoxycytidine (5-aza-dC)] and histone deacetylase (HDAC) inhibitors (trichostatin A) leads to expression of ER mRNA and functional protein. Here, we examined whether epigenetically reactivated ER is a target for tamoxifen therapy. Following treatment with trichostatin A and 5-aza-dC, the formerly unresponsive ER-negative MDA-MB-231 breast cancer cells became responsive to tamoxifen. Tamoxifen-mediated inhibition of cell growth in these cells is mediated at least in part by the tamoxifen-bound ER. Tamoxifen-bound reactivated ER induces transcriptional repression at estrogen-responsive genes by ordered recruitment of multiple distinct chromatin-modifying complexes. Using chromatin immunoprecipitation, we show recruitment of two different corepressor complexes to ER-responsive promoters in a mutually exclusive and sequential manner: the nuclear receptor corepressor-HDAC3 complex followed by nucleosome remodeling and histone deacetylation complex. The mechanistic insight provided by this study might help in designing therapeutic strategies directed toward epigenetic mechanisms in the prevention or treatment of breast cancer. (Cancer Res 2006; 66(12): 6370-8)

Modulation of estrogen depurinating DNA adduct by sulforaphane or KEAP1 knockdown in human breast epithelial cells Sulforaphane (SFN) is a potent inducer of detoxication enzymes such as NAD(P)H:quinone oxidoreductase (NQO1) and glutathione-S-transferases (GST) via the Kelch-like ECH-associated protein 1 (Keap1) - Nuclear Factor- E2-related factor (Nrf2) signaling pathway. NQO1 reduces the carcinogenic estrogen metabolite, catechol estrogen-3,4-quinone (CE-3,4-Q), while GSTs detoxify it through nucleophilic addition. CE-3,4-Q can bind with DNA to form depurinating DNA adducts. Thus, SFN, a isothiocyanate found in broccoli, may alter estrogen metabolism to protect against estrogen-mediated carcinogenesis. MCF10A cells were treated with either vehicle or SFN (10 μM) and either estradiol (E2) or 4-OHE2 (10 μM). NQO1 activity increased 3.2-fold by SFN treatment compared to vehicle. Estrogen metabolites and depurinating DNA adducts in the cell culture medium were partially purified by solid phase extraction and then analyzed by UHPLC-MS/MS. Following E2 treatment, 4-OHE1/2-1-N3Ade and 4-OHE1/2-1-N7Gua adducts were reduced by 60% in SFN treatment; 4-OHE1/2- glutathione conjugates increased 1.9 while 4-OCH3E1/2 increased 3.0 fold with SFN. Following treatment with the proximate metabolite 4-OHE2, 4-OHE1/2-1-N3Ade and 4-OHE 1/2-1-N7Gua adducts also decreased 60% in SFN treated cells compared to vehicle; while 4-OHE1/2-glutathione-conjugates increased 5.0-fold and 4-OCH3E1/2 levels were 3.4-fold higher. To constitutively enhance the expression of Nrf2-regulated genes including NQO1, cells were treated with either scrambled or siKEAP1 RNA and E2 or 4-OHE2. NQO1 activity increased 2.2-fold with siKEAP1 treatment. Following E2 treatment, 4-OHE1/2-1-N3Ade and 4-OHE1/2-1-N7Gua adducts dropped 70% in siKEAP1 treated cells compared to scrambled; 4-OHE1/2-glutathione conjugates increased 1.3-fold; however, 4-OCH3E1/2 decreased 50% with siKEAP1 treatment. Following 4-OHE2 treatment, the 4-OHE1/2-1-N3Ade and 4-OHE 1/2-1-N7Gua adducts declined 60% in siKEAP1 treated cells compared to scrambled; 4-OHE1/2-glutathione conjugates increased 1.4-fold while 4-OCH3E1/2 declined 60% with siKEAP1 treatment. SFN or siKEAP1 have similar effects on up-regulating NQO1 transcripts, protein expression and activity levels and on diminution of depurinating estrogen DNA adducts following E2 or 4-OHE2 challenge. However, these pharmacologic and genetic approaches have different effects on COMT. siKEAP1 down-regulates COMT expression, which inhibits 4-OCH3E1 but not 4-CH3E2 formation whilst SFN elevates COMT expression and subsequently levels of 4-OCH3E1/2. Thus, activation of the Nrf2 pathway may account for some but not all of the protective effects of SFN against estrogen-mediated DNA damage. Supported by DOD BCRP Postdoctoral Fellowship103928. Citation Format: Li Yang, Muhammad Zahid, Eleanor G. Rogan, Ercole L. Cavalieri, James D, Yager, Kala Visvanathan, John Groopman, Nancy E. Davidson, Thomas W. Kensler. Modulation of estrogen depurinating DNA adduct by sulforaphane or KEAP1 knockdown in human breast epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3679. doi:10.1158/1538-7445.AM2013-3679

Background: In preclinical breast cancer models, combination epigenetic therapy with a DNA methyltransferase inhibitor (DNMTI) and a histone deacetylase inhibitor (HDACI) yield superior estrogen receptor (ER) re-expression and greater restoration of tamoxifen responsiveness than either agent alone. We conducted a multicenter phase II clinical trial to evaluate the DNMTI 5-azacitidine (5-AZA) and the HDACI entinostat in women with advanced breast cancer. Methods: Women with advanced HER2-negative, either triple-negative (TN; ER/progesterone receptor [PR]/HER2-negative) or hormone-resistant breast cancer received 5-AZA 40 mg/m2 (SQ, days 1-5, 8-10) and entinostat 7 mg (PO, days 3,10) every 28 days. Primary endpoint: objective response rate (ORR) in each group. Secondary endpoints: safety, tolerability, survival, clinical benefit rate. Exploratory endpoints: pharmacokinetics, pharmacogenetics, change in candidate gene re-expression/methylation in circulating DNA and mandatory tumor samples. Patients are offered ongoing study therapy at progression with addition of hormonal therapy (optional continuation phase). Sample size: Simon two-stage design with interim analysis after 13 patients per cohort (1st stage). If ≥1 response, accrual will continue for total of 27 per cohort (2nd stage). Null hypothesis: ORR at most 5% against alternative hypothesis that is at least 20% with type I error 4% and power 90%. Preclinical TN/ ER-positive xenograft studies assessing 5-AZA impact were also performed. Results: Thirteen evaluable patients were enrolled in 1st stage of TN cohort. Median age was 47 (31-67), median prior chemotherapies 3 (1-5), 77% white/33% black, 77% visceral disease. Median cycles received 2 (1-4). Therapy was well tolerated, most common grade 3/4 treatment related adverse events leucopenia and neutropenia (23% each). No responses observed following 1st stage and this cohort was closed. Median 1.5 additional cycles (optional continuation phase) received by 4 patients with no responses to date. Exposure to 5-AZA (Cmax=1134±1670ng/mL; AUCINF=939±724 ng*h/mL) was slightly higher than previous studies, entinostat (Cmin=0.78±0.65ng/mL) was similar. Hormone-resistant cohort proceeded to 2nd stage as 1 partial response observed. Final results will be reported once accrual complete. Ongoing preclinical studies suggest that ER-positive is more sensitive than TN breast cancer to 5-AZA. Conclusion: Combination epigenetic therapy with agents, dose and schedule described was well tolerated but not associated with clinical activity in advanced TN breast cancer. Correlative analyses will be presented at meeting. Promising preclinical findings suggest epigenetic therapy may be efficacious in ER-positive breast cancer. Citation Format: Roisin M. Connolly, Rachel C. Jankowitz, Cynthia A. Zahnow, Zhe Zhang, Michelle A. Rudek, Stacie C. Jeter, Shannon Slater, Penny Powers, Antonio C. Wolff, John Fetting, Adam M. Brufsky, Richard Piekarz, Nita Ahuja, George Somlo, Augustin Garcia, Steven Baylin, Nancy E. Davidson, Vered Stearns. A phase 2 study investigating the safety, efficacy and surrogate biomarkers of response of 5-azacitidine (5-AZA) andentinostat (MS-275) in patients with triple-negative advanced breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4666. doi:10.1158/1538-7445.AM2013-4666

Triple negative breast cancer (TNBC) is the most aggressive type of breast tumor and one of the major clinical hurdles encountered in the development of specific treatment for this disease is lack of effective targeted therapy. Our recent studies demonstrate that lysine-specific demethylase 1 (LSD1) and histone deacetylases (HDACs) closely interact in controlling gene expression and growth of TNBC cells. However the underlying mechanisms are largely unknown. Here we show that knockdown of LSD1 expression (LSD1-KD) via RNA interference (RNAi) inhibits mRNA levels of most HDAC isozymes in a TNBC cell line, MDA-MB-231. HDAC5-targeted RNAi induces the most significant accumulation of H3K4me2, a specific substrate of LSD1, implicating a necessity for HDAC5 in LSD1 function. Combined treatment with LSD1 inhibitor, pargyline, and HDAC inhibitor, vorinostat, leads to superior growth inhibition and apoptosis in TNBC cells, but exhibits additive or antagonistic effect on growth inhibition in non-TNBC counterparts as well as non-tumorigenic mammary epithelial cells. Moreover, LSD1-KD augments vorinostat-induced re-expression of a subset of aberrantly silenced genes such as NR4A1, PCDH1, RGS16, BIK, and E-Cadherin whose re-expression may be tumor suppressive in TNBC cells, but not in other breast cancer subtypes. The re-expression of these genes is associated with increased H3K4me2 and acetyl-H3K9 marks at gene promoters, indicative of active chromatin. Integrative analysis of a panel of 36 human breast cancer cell lines of distinct subtypes shows that the expression of these genes is more profoundly suppressed in TNBC cells compared with other breast cancer subtypes. Genome-wide microarray study in MDA-MB-231 cells identifies a group of tumor suppressor genes (CDKN1A, CDKN1C, CRABP2, TNFAIP3, TP53TG1 and ING1) whose expression is induced by vorinostat and significantly enhanced by LSD1-KD. We also show that concurrent depletion of RGS16 by siRNA reduces overall cytotoxicity of vorinostat and blocks the re-expression of E-Cadherin, CDKN1C and ING1 in LSD1 deficient MDA-MB-231 cells. Furthermore, co-treatment with RGS16 siRNA reverses the down-regulation of NF-KB expression induced by combined inhibition of LSD1 and HDACs, suggesting a crucial role of RGS16 in controlling key apoptotic pathways in response to combination therapy in TNBC cells. Taken together, these results provide novel mechanistic insight into how LSD1 and HDACs modulate each other to enhance therapeutic efficacy of vorinostat in TNBC; and also suggest that inhibition of LSD1-HDACs axis-mediated histone modifications can be a potential therapeutic strategy to improve treatment of TNBC. This work is funded by: Breast Cancer Research Foundation, UPCI CCSG Shared Facilities & the Samuel and Emma Winters Foundation. Citation Format: Shauna Vasilatos, Tiffany A. Katz, Steffi Oesterreich, Nancy E. Davidson, Yi Huang. Targeting LSD1-HDACs crosstalk as a potential therapeutic strategy for triple negative breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 673. doi:10.1158/1538-7445.AM2013-673

Background: Preplanned combined analysis of the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-31 trial and North Central Cancer Treatment Group (NCCTG) (Alliance) N9831 Trial compared adjuvant chemotherapy with or without trastuzumab in women with HER2-positive breast cancer. The initial results were reported in 2005 and updated in early 2011 Methods: 4045 patients with HER2-positive operable breast cancer were enrolled to receive doxorubicin plus cyclophosphamide followed by paclitaxel with or without trastuzumab in both trials. The definitive analysis for OS will occur when 710 events have been reported. Results: Median time on study is 8 years. The required number of events have occurred for the definitive analysis for OS to be conducted by the end of September 2012. Updated analyses of disease free survival and related subgroups will also be presented. Supported by NCI grants U10-CA-12027, -69651, -37377, -69974, -44066, U24-CA-114732, CA25224; Breast Cancer Research Foundation; Genentech 35–03 Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S5-5.

Background: Epidermal Growth Factor Receptor (EGFR, HER1) is known to heterodimerize with HER2 and may impact the effectiveness of trastuzumab. Historically, EGFR has been difficult to measure; thus there is no evidence regarding its effects on trastuzumab clinical therapy. Here we use a new, standardized, quantitative immunofluorescence assay and a novel EGFR antibody to evaluate the correlation between EGFR expression and clinical outcome in a cohort from the North Central Cancer Treatment Group (NCCTG, Alliance) N9831 trial. Methods: Of the 3505 women in the N9831 trial, tissue microarrays were constructed that allowed analysis of 1,444 patients randomly assigned to receive chemotherapy alone (Arm A), sequential trastuzumab after chemotherapy (Arm B) or concurrent trastuzumab with chemotherapy (Arm C). Fluorescence-based, standardized measurement of EGFR was done using the rabbit monoclonal EGFR antibody D38B1 on the AQUA platform (HistoRx). EGFR expression levels were analyzed as a continuous variable and dichotomized using recursive partitioning to define an optimal cut point. The cut point was validated on an independent retrospective cohort comprised of 130 patients with HER2-positive metastatic breast cancer who received trastuzumab. Results: EGFR assessed as a continuous variable shows an association with disease free survival (DFS) in Arm C (p = 0.002) but not in Arms A or B. Dichotomizing EGFR expression shows that high level expression is associated with worse outcome (HR = 2.2; 95% CI 1.33–3.59, p = 0.002) in arm C. Five year DFS within the low level EGFR expression group was 71%, 74% and 90% for Arms A, B and C, respectively (log-rank p-value=0.02) and within the high level EGFR expression (n = 213) group was 62%, 76% and 74% respectively (log-rank p-value 0.22). As validation, the same cut-point was used to define high EGFR expression in a retrospective metastatic breast cancer cohort. The median progression free survival difference between EGFR high group and EGFR low group was 6.1 months (p = 0.0063) and the hazard ratio of high EGFR was 1.92 (p = 0.0073). Conclusions: High expression of EGFR by the AQUA platform appears to be associated with decreased benefit from adjuvant concurrent (not sequential) trastuzumab and shorter PFS in the metastatic setting. The biological underpinning for these observations is being considered. Since there are other treatment options for HER2-driven tumors, this marker, once validated, could help select patients for alternative or additive therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S5-4.

The dysregulation of histone deacetylases (HDACs), which frequently leads to the silencing of gene expression, is linked to breast cancer progression. Studies in recent years have focused on reversing these changes in gene expression through inhibition of histone deacetylases (HDACs), and HDAC inhibitors (HDACi) have emerged as a potential new treatment option for cancer. However the current HDACi are less effective as monotherapy against solid tumors, highlighting the need to develop rational combinations of chemotherapeutic agents for the treatment of solid tumors. Our recent work has shown that combined inhibition of LSD1 and HDACs in breast cancer cell lines leads to re-expression of a unique subset of abnormally silenced genes and enhanced apoptosis in triple negative breast cancer (TNBC) cells, suggesting that crosstalk between lysine-specific demethylase 1 (LSD1) and HDACs is a novel and important epigenetic mechanism for aberrant gene silencing. These data also suggest that inhibition of LSD1 in combination with HDACi might enhance the therapeutic efficacy of HDAC inhibitors, and thus improve breast cancer treatment. Our further investigation showed that TNBC cells are overall more sensitive to combined treatment with LSD1 and HDAC inhibitors than other subtypes of breast tumors, or normal breast cells. LSD1-knockdown (KD) by shRNA sensitized TNBC MDA-MB-231 cells to HDACi-induced growth inhibition and apoptosis, and resulted in a striking synergistic mRNA induction of important growth control and apoptosis-related genes such as ERα, E-Cadherin, NR4A1, PCDH1, RGS16, BIK, CDKN1C, CRABP2, ING1, SQSTM1, TP53TG1, etc. In contrast, LSD1-KD exerted only minor effect on SAHA-induced gene re-expression in hormone receptor-positive or HER2 positive counterparts. Chromatin immunoprecipitation showed that re-expression of silenced genes was accompanied by concurrent increase of active chromatin marks H3K4me2 and AcetylH3K9 at gene promoters. ShRNA-mediated silencing of LSD1 significantly suppressed the mRNA expression of most of the class I (1-3, 8), II (6, 7, 10) and IV (11) HDAC isozymes in TNBC cells, but exerted marginal effect on transcription activities of HDAC isozymes in other subtypes of breast cancer cells. Moreover, siRNA-mediated silencing of the specific HDAC isozymes led to increase of H3K4me2 level in MDA-MB-231 cells. Taken together, our studies suggest that orchestrated interplay between LSD1 and HDACs is an important epigenetic signature contributing to aberrant gene silencing, and combination therapy targeting the crosstalk between histone demethylation and deacetylation may represent a novel therapeutic approach for aggressive TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1044. doi:1538-7445.AM2012-1044

Epigenetic gene silencing plays a critical role in breast tumor aggression. Our previous studies have shown that abnormal activities of histone lysine demethylases (KDMs) are associated with aberrant gene expression in breast cancer development. However, the precise molecular mechanisms underlying the activity of KDMs in chromatin remodeling and regulation of gene transcription in breast cancer are still elusive. Recently, a novel mammalian histone demethylase LSD2 (also known as KDM1B or AOF1) was identified. We have shown that LSD2 possesses the activity to demethylate H3K4 in breast cancer cells. This clearly suggests the existence of a more sophisticated FAD-dependent histone demethylase family whose members play a role in chromatin remodeling and transcription regulation in breast cancer. Here we demonstrated that inhibition of LSD2 mRNA expression by shRNA suppressed migratory behavior in human breast cancer MDA-MB-231 cells. RNAi -mediated LSD2 deficiency led to a significant re-expression of ERα mRNA, in ER negative breast cancer cells and upregulated ER-mediated transcription activity as evidenced by ERE luciferase assay. Microarray studies indicated a unique subset of genes whose expression was significantly changed by LSD2 KD in MDA-MB-231 cells. The genes identified are extensively involved in regulation of tumor cell proliferation, metastasis, cell signaling, transcription regulation, and chromatin remodeling. For example, a panel of the upregulated genes potentially plays a role in cell adhesion and cell death such as CLDN1 & 11, CDH11, CASP5 & 10, PDCD4, and NCR3. In addition, expression of several tumor suppressor genes such as ERBB2IP, MTSS1, and S100A2 were up-regulated by LSD2 KD in MDA-MB-231 cells. Moreover, treating LSD2 KD cells with the low doses of DNMT inhibitor, 5-aza-2-deoxycitidine (DAC) led to a robust reexpression of ERα and CASP5 mRNA in MDA-MB-231 cells. Treatment with the HDAC inhibitor vorinostat in LSD2 KD MDA-MB-231 cells also lead to the reexpression of ERα as well as other genes of interest such as CASP5, ESRRG, and MTSS1. Further simultaneous treatment of LSD2 KD cells with DAC and vorinostat produced a more robust transcriptional re-activation of ERα, CASP5, and MTSS1 mRNA expression. Finally, LSD2 KD MDA-MB-231 cells exhibited increased sensitivity to growth inhibition induced by combination therapy of vorinostat and DAC. Taken together, these data implicate an important role for LSD2 in mediating expression of epigenetically silenced genes in breast cancer cells and suggest that combined inhibition of LSD2 with other epigenetic targets could have the potential to improve the treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1052. doi:1538-7445.AM2012-1052

Background: Epigenetic alterations in the genome, including abnormal DNA methylation and histone hypoacetylation, initiate and promote cancerous changes via several mechanisms, including inactivation of tumor suppressor genes. Preclinical investigations in breast cancer suggest that use of epigenetic modifiers results in re-expression of aberrantly silenced genes and proteins that represent important therapeutic targets (e.g. estrogen receptor alpha, ER). Combination therapy with a DNA methyltransferase inhibitor (DNMTI) and a histone deacetylase inhibitor (HDACI) has yielded superior ER reexpression and greater restoration of tamoxifen responsiveness than with HDACI alone. We hypothesize that clinically tolerable doses of the DNMTI 5-azacitidine (5-AZA) and the HDACI entinostat may not only effect changes in DNA methylation and gene expression, but also yield objective disease responses in women with advanced breast cancer. Trial design: This multicenter phase II study (NCT01349959) is enrolling patients with advanced human epidermal growth factor receptor 2 (HER2)-negative breast cancer with triple negative (ER/progesterone receptor [PR]/HER2−negative, Cohort A) or hormone-resistant (Cohort B) disease. Patients will receive 5-AZA 40 mg/m2 subcutaneously days 1–5 and 8–10 and entinostat 7 mg orally days 3 and 10 every 28 days. Because of the potential for re-expression of the ER with epigenetic agents, patients will be offered continuation of 5-AZA and entinostat at progression with the addition of hormonal therapy (investigator discretion). Mandatory tumor biopsies will be performed at baseline and after 8 weeks of therapy to evaluate correlative biomarkers. Eligibility Criteria: Eligible patients must be ≥ 18 years, have measurable locally advanced/metastatic triple-negative (at least one prior chemotherapy received adjuvant/metastatic setting) or hormone-resistant (must have received two prior hormonal agents and one prior chemotherapy) disease, adequate organ function and ECOG PS ≤ 2. Specific Aims: 1. Objective response rate (ORR) by RECIST 1.1 criteria. 2. Safety and tolerability 3. Progression-free survival, overall survival and clinical benefit rate. 4. Safety and toxicity data, feasibility and response rate where hormonal therapy is added to the combination under investigation at the time of progressive disease. 5. Pharmacokinetics, cytidine deaminase, changes from baseline of candidate gene methylation and expression in circulating deoxyribonucleic acid (DNA) and malignant tissue. Statistical Methods: Using a two-stage three-outcome design to assess the efficacy of the combination, a maximum of 30 patients (requiring 27 evaluable) will be accrued to each cohort unless undue toxicity is encountered for a maximum sample size of 60 patients. The study design tests the null hypothesis that the ORR is at most 5% against the alternative hypothesis that it is at least 20% with a type I error of 4% and power of 90%. Present and Targeted Accrual: This study has just opened to patient enrollment. We anticipate a rapid accrual of 60 patients within 1 year.br](Funding from Stand Up to Cancer and CTEP). Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT3-01-06.