ER-negative Disease Research Articles

Combined evaluation of the FAS cell surface death receptor and CD8+ tumor infiltrating lymphocytes as a prognostic biomarker in breast cancer.

Multiple studies showed the prognostic capacities of tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC), but not in other subtypes. We evaluated tumor expression of FAS, a key receptor in T-cell mediated apoptosis, as possible explanation for this differential prognostic value of TILs. Furthermore, we evaluated the prognostic relevance of FAS, both as an independent biomarker and in relation to CD8-positive T-cell presence. The study cohort consisted of 667 breast cancer patients treated in the LUMC between 1997 and 2009. FAS expression was determined using immunohistochemistry and the percentage of FAS-positive tumor cells was quantified. Furthermore, the number of CD8-positive infiltrating cells was determined, and its prognostic relevance was associated to FAS-expression using stratified survival analysis. In TNBC, FAS was averagely expressed in 49% of tumor cells, whereas ER-positive subtypes showed an average Fas expression of 16-20%. In the entire cohort, FAS was identified as significant prognostic marker for recurrence (adjusted HR 0.53, 95% CI 0.36-0.77) and borderline significant marker for overall survival (adjusted HR 0.72, 95% CI 0.52-1.01). Upon stratification for FAS-expression, CD8+ TILs were only prognostic at high levels (above median) of FAS expression in ER-negative disease. In summary, FAS was identified as an independent prognostic marker for recurrence free survival in breast cancer, with large variation in expression by receptor subtypes. Interestingly, the prognostic effect of CD8+ TILs in ER-negative disease was only valid for tumors with a high FAS expression.

Comparable Survival between Additional Radiotherapy and Local Surgery in Occult Breast Cancer after Axillary Lymph Node Dissection: A Population-based Analysis.

Purpose: To investigate the clinical value of additional local treatment strategies in occult breast cancer (OBC) after axillary lymph node dissection (ALND).Methods: Patients diagnosed with OBC between 1990 and 2013 were included from the Surveillance, Epidemiology, and End Results registry database. The significant risk factors of cause-specific survival (CSS) and overall survival (OS) were identified using univariate and multivariate Cox regression analyses.Results: We identified 980 patients, including 219 (22.3%), 252 (25.7%), 263 (26.8%), and 246 (25.1%) of patients underwent ALND, ALND + radiotherapy (RT), ALND + surgery (S) (mastectomy or breast-conserving surgery), and ALND + S + RT, respectively. Patients with younger age, diagnosed before 2000, advanced nodal stage, ER-negative disease, and PR-negative disease were more likely to undergo additional local treatment compared with ALND only. The 10-year rate CSS of the ALND only group was 57.2%, while that of the ALND + RT, ALND + S, and ALND + S + RT groups was 78.0%, 81.0%, and 71.5%, respectively (p < 0.001). The 10-year OS rate in the ALND only, ALND + RT, ALND + S, and ALND + S + RT groups was 46.0%, 69.5%, 66.1%, and 67.0%, respectively (p < 0.001). Multivariate analysis indicated that older age, advanced nodal stage, and ALND only were independent risk factors for decreased CSS and OS. CSS and OS among the groups including ALND + RT, ALND + S, and ALND + S + RT were not significantly different.Conclusions: Additional local treatment (local surgery or RT) improves survival outcomes compared with ALND only in OBC after ALND. ALND + RT may be the optimal local treatment for OBC due to no different in survival outcomes and cosmesis is better.

Patterns of Immune Infiltration in Breast Cancer and Their Clinical Implications: A Gene-Expression-Based Retrospective Study.

BackgroundImmune infiltration of breast tumours is associated with clinical outcome. However, past work has not accounted for the diversity of functionally distinct cell types that make up the immune response. The aim of this study was to determine whether differences in the cellular composition of the immune infiltrate in breast tumours influence survival and treatment response, and whether these effects differ by molecular subtype.Methods and FindingsWe applied an established computational approach (CIBERSORT) to bulk gene expression profiles of almost 11,000 tumours to infer the proportions of 22 subsets of immune cells. We investigated associations between each cell type and survival and response to chemotherapy, modelling cellular proportions as quartiles. We found that tumours with little or no immune infiltration were associated with different survival patterns according to oestrogen receptor (ER) status. In ER-negative disease, tumours lacking immune infiltration were associated with the poorest prognosis, whereas in ER-positive disease, they were associated with intermediate prognosis. Of the cell subsets investigated, T regulatory cells and M0 and M2 macrophages emerged as the most strongly associated with poor outcome, regardless of ER status. Among ER-negative tumours, CD8+ T cells (hazard ratio [HR] = 0.89, 95% CI 0.80–0.98; p = 0.02) and activated memory T cells (HR 0.88, 95% CI 0.80–0.97; p = 0.01) were associated with favourable outcome. T follicular helper cells (odds ratio [OR] = 1.34, 95% CI 1.14–1.57; p < 0.001) and memory B cells (OR = 1.18, 95% CI 1.0–1.39; p = 0.04) were associated with pathological complete response to neoadjuvant chemotherapy in ER-negative disease, suggesting a role for humoral immunity in mediating response to cytotoxic therapy. Unsupervised clustering analysis using immune cell proportions revealed eight subgroups of tumours, largely defined by the balance between M0, M1, and M2 macrophages, with distinct survival patterns by ER status and associations with patient age at diagnosis. The main limitations of this study are the use of diverse platforms for measuring gene expression, including some not previously used with CIBERSORT, and the combined analysis of different forms of follow-up across studies.ConclusionsLarge differences in the cellular composition of the immune infiltrate in breast tumours appear to exist, and these differences are likely to be important determinants of both prognosis and response to treatment. In particular, macrophages emerge as a possible target for novel therapies. Detailed analysis of the cellular immune response in tumours has the potential to enhance clinical prediction and to identify candidates for immunotherapy.

Differential microRNA expression is associated with androgen receptor expression in breast cancer.

The androgen receptor (AR) is frequently expressed in breast cancer; however, its prognostic value remains unclear. AR expression in breast cancer has been associated with improved outcomes in estrogen receptor (ER)-positive breast cancer compared with ER-negative disease. Eliminating AR function in breast cancer is critically important for breast cancer progression. However, the mechanism underlying AR regulation remains poorly understood. The study of microRNAs (miRNAs) has provided important insights into the pathogenesis of hormone-dependent cancer. To determine whether miRNAs function in the AR regulation of breast cancer, the present study performed miRNA expression profiling in AR-positive and -negative breast cancer cell lines. A total of 153 miRNAs were differentially expressed in AR-positive compared with AR-negative breast cancer cells; 52 were upregulated and 101 were downregulated. A number of these have been extensively associated with breast cancer cell functions, including proliferation, invasion and drug-resistance. Furthermore, through pathway enrichment analysis, signaling pathways associated with the prediction targets of the miRNAs were characterized, including the vascular endothelial growth factor and mammalian target of rapamycin signaling pathways. In conclusion, the results of the present study indicated that the expression of miRNAs may be involved in the mechanism underlying AR regulation of breast cancer, and may improve understanding of the role of AR in breast cancer.

Breast cancer in women aging 35 years old and younger: The Egyptian National Cancer Institute (NCI) experience

ObjectiveThe aim is to identify the epidemiological and clinicopathological features associated with young breast cancer (BC) patients and to discuss factors affecting tumor recurrence and DFS. Patients & methodsA retrospective analysis was conducted based on medical records from young females patients aged ≤35 years with pathologically confirmed primary breast cancer treated during 2008–2010 at NCI. Cases with non invasive cancer and non carcinoma histology are excluded. ResultsOf the 5408 cases diagnosed with breast cancer, 554 were young. Four hundred & fifty eight patients representing 9.2% were within our inclusion criteria. Almost half of the patients (45.9%) presented with stage III. Axillary nodes involvement was in 63.9%, 83.3% were grade 2. More than one quarter of tumors was hormone receptors negative (28.8%) & Her2 was over-expressed in 30%. Mastectomy was offered in 72% while conservative breast surgery in 26%, 69.2% received chemotherapy either adjuvant, neoadjuvant or both, 82.5% received adjuvant radiotherapy, 68.6% received hormonal therapy. Metastatic disease developed in 51.3%, with 31% having more than one site of metastases. After a median follow up period of 66 months, the median DFS of patients was 60 months. The median DFS was significantly shorter among patients with positive lymph nodes (P < 0.0001), ER negative disease (P = 0.045) and stage III disease (P < 0.0001). ConclusionBreast cancer in young women is aggressive from the time of diagnosis. Our results provide baseline data of young BC in the Middle East & North Africa region; thus, contributing to future epidemiological and hospital-based researches.

Age- and Tumor Subtype-Specific Breast Cancer Risk Estimates for CHEK2*1100delC Carriers.

CHEK2*1100delC is a well-established breast cancer risk variant that is most prevalent in European populations; however, there are limited data on risk of breast cancer by age and tumor subtype, which limits its usefulness in breast cancer risk prediction. We aimed to generate tumor subtype- and age-specific risk estimates by using data from the Breast Cancer Association Consortium, including 44,777 patients with breast cancer and 42,997 controls from 33 studies genotyped for CHEK2*1100delC. CHEK2*1100delC genotyping was mostly done by a custom Taqman assay. Breast cancer odds ratios (ORs) for CHEK2*1100delC carriers versus noncarriers were estimated by using logistic regression and adjusted for study (categorical) and age. Main analyses included patients with invasive breast cancer from population- and hospital-based studies. Proportions of heterozygous CHEK2*1100delC carriers in controls, in patients with breast cancer from population- and hospital-based studies, and in patients with breast cancer from familial- and clinical genetics center-based studies were 0.5%, 1.3%, and 3.0%, respectively. The estimated OR for invasive breast cancer was 2.26 (95%CI, 1.90 to 2.69; P = 2.3 × 10(-20)). The OR was higher for estrogen receptor (ER)-positive disease (2.55 [95%CI, 2.10 to 3.10; P = 4.9 × 10(-21)]) than it was for ER-negative disease (1.32 [95%CI, 0.93 to 1.88; P = .12]; P interaction = 9.9 × 10(-4)). The OR significantly declined with attained age for breast cancer overall (P = .001) and for ER-positive tumors (P = .001). Estimated cumulative risks for development of ER-positive and ER-negative tumors by age 80 in CHEK2*1100delC carriers were 20% and 3%, respectively, compared with 9% and 2%, respectively, in the general population of the United Kingdom. These CHEK2*1100delC breast cancer risk estimates provide a basis for incorporating CHEK2*1100delC into breast cancer risk prediction models and into guidelines for intensified screening and follow-up.

Abstract 2662: Interrogation of the landscape of long noncoding RNAs in breast cancer to identify an ER-regulated predictor of tamoxifen resistance

Abstract Background: We previously performed a bioinformatics-based analysis on 7,256 RNA sequencing libraries from tumors, normal tissues, and cell lines to delineate the landscape of long noncoding RNAs (lncRNAs) in the human transcriptome. This analysis identified 58,648 lncRNAs, including over 45,000 novel transcripts (Iyer and Niknafs et al, Nature Genetics, 2015). We now interrogate this lncRNA compendium to identify top candidate estrogen receptor (ER)-associated lncRNAs in breast cancer and characterize their association with disease progression. Methods: To identify and prioritize lncRNAs that were differentially expressed in cancer vs normal tissue, and in ER-positive vs ER-negative disease, we performed differential expression testing on over 1000 RNA sequencing libraries including breast cancer and normal tissue samples from the The Cancer Genome Atlas (TCGA) project. The effect of the top prioritized lncRNA on cancer phenotypes was studied via proliferation, colony formation, invasion and tamoxifen resistance assays in MCF7 and T47D cell lines, and via mouse xenograft studies. The mechanism by which this lncRNA promotes tumor progression was investigated by identifying its top protein interactors and its subdomains responsible for function, and then studying the effects of disrupting function of this lncRNA on cancer phenotypes. Results: Differential expression analyses nominated BRCAT431 as the top overexpressed ER-regulated lncRNA in breast cancer. In vitro experiments demonstrate that shRNA-mediated knockdown of BRCAT431 significantly decreased proliferation, colony formation, and invasion (by &amp;gt;50% in most assays). Tamoxifen resistance was associated with significantly increased BRCAT431 levels in both MCF7 and T47D cells, and knockdown of BRCAT431 reversed tamoxifen resistance. Xenograft studies demonstrate that knockdown of BRCAT431 also significantly decreased xenograft growth and tumor invasion by &amp;gt;50%. RNA pulldown followed by mass spectrometry identified the RNA binding protein hnRNPL as a key protein interacting with BRCAT431. Deletion studies identified a 27 base-pair region of BRCAT431 necessary for its interaction with hnRNPL, and loss of this region abrogated BRCAT431-induced invasion. Finally, guilt-by-association studies demonstrate a strong association between BRCAT431 overexpression and tumor grade, recurrence, and metastases. Conclusion: In this study, we develop the largest reported compendium of breast cancer lncRNAs. We prioritize BRCAT431 as the top lncRNA upregulated in ER-positive breast cancers, and demonstrate that it confers aggressive oncogenic phenotypes in vitro and in vivo. We identify a novel mechanism by which this lncRNA functions through interaction with hnRNPL. Our results suggest that by promoting tamoxifen resistance, this lncRNA increases the clinical risk of recurrence and metastases in breast cancer. Citation Format: Yashar Niknafs, Sumin Han, Teng Ma, James Rae, Arul Chinnaiyan, Felix Feng. Interrogation of the landscape of long noncoding RNAs in breast cancer to identify an ER-regulated predictor of tamoxifen resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2662.

Abstract 2272: Association of G-protein estrogen receptor (GPER) in primary breast cancer in Caucasian and Black women by tumor immunophenotype and menopausal status

Abstract Background: Assignment of breast cancer therapies is largely defined by immunohistochemical analyses that evaluate steroid hormone and growth factor receptor status in primary tumor biopsies. Patients whose tumors are devoid of these receptors have fewer treatment options, and poor survival. This outcome is more commonly measured among Black versus Caucasian patients, and in patients with intact ovarian function. The G-protein coupled estrogen receptor, GPER, holds significance for breast cancer biology and treatment as 17β-estradiol and ER antagonists trigger the release of epidermal growth factor (EGF)-ligands and GPER associates directly with clinicopathological factors that predict advanced disease. Experimental design: GPER was measured by immunohistochemical analysis and a semi-quantitative scoring method in 521 primary breast tumors obtained from the NCI Cooperative Breast Cancer Tissue Resource and the Nashville Breast Health Study, including tumor specimens derived from 389 Caucasian and 108 Black patients. GPER was correlated with disease progression variables and evaluated by race, menopausal status and tumor immunophenotype. Results: GPER is expressed in more than two-thirds of invasive breast carcinomas in this study with no difference observed between Caucasian and Black women (p = 0.69). Coexpression of GPER and ER was measured in less than half of all tumors (48%). Additionally, GPER is commonly expressed in ER-negative tumors (107/171; 62%) and in triple negative breast cancer (TNBC) (42/56; 75%) and did not differ by race for this newly defined measure of estrogen responsiveness (p = 0.42). Moreover, while ER-negative tumors are more common in pre- (79/153; 52%) versus postmenopausal women (92/336; 27%), GPER expression is not influenced by menopausal status. GPER is positively associated with tumor size (particularly in premenopausal women) in both studies, and with frank metastases at first diagnosis in the NCI population. No significant association was seen with nodal invasion. Survival data in NBHS patients found that women who died of breast cancer are more likely to have GPER-positive tumors than survivors. Conclusion: GPER is expressed in the majority of invasive breast tumors (&amp;gt; 60%) at diagnosis and is commonly retained in patients with ER-negative or triple-negative disease regardless of race or menopause. Unlike ER, which varies inversely with variables that predict advanced disease, GPER is positively associated with these same prognostic parameters. These data support GPER as an independent marker of estrogen responsiveness and breast cancer progression. Most importantly, our results suggest that GPER represents a promising therapeutic target since its expression is constant across multiple types of invasive breast cancer, including patients with TNBC and in tumors from pre- and postmenopausal women. Citation Format: Sandra L. Deming-Halverson, Carl Graeber, Jason Machan, Edmond Sabo, Wei Zheng, Edward J. Filardo. Association of G-protein estrogen receptor (GPER) in primary breast cancer in Caucasian and Black women by tumor immunophenotype and menopausal status. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2272.

An intergenic risk locus containing an enhancer deletion in 2q35 modulates breast cancer risk by deregulating IGFBP5 expression.

Breast cancer is the most diagnosed malignancy and the second leading cause of cancer mortality in females. Previous association studies have identified variants on 2q35 associated with the risk of breast cancer. To identify functional susceptibility loci for breast cancer, we interrogated the 2q35 gene desert for chromatin architecture and functional variation correlated with gene expression. We report a novel intergenic breast cancer risk locus containing an enhancer copy number variation (enCNV; deletion) located approximately 400Kb upstream to IGFBP5, which overlaps an intergenic ERα-bound enhancer that loops to the IGFBP5 promoter. The enCNV is correlated with modified ERα binding and monoallelic-repression of IGFBP5 following oestrogen treatment. We investigated the association of enCNV genotype with breast cancer in 1,182 cases and 1,362 controls, and replicate our findings in an independent set of 62,533 cases and 60,966 controls from 41 case control studies and 11 GWAS. We report a dose-dependent inverse association of 2q35 enCNV genotype (percopy OR = 0.68 95%CI 0.55-0.83, P = 0.0002; replication OR = 0.77 95% CI 0.73-0.82, P = 2.1 × 10-19) and identify 13 additional linked variants (r2 > 0.8) in the 20Kb linkage block containing the enCNV (P = 3.2 × 10-15 - 5.6 × 10-17). These associations were independent of previously reported 2q35 variants, rs13387042/rs4442975 and rs16857609, and were stronger for ER-positive than ER-negative disease. Together, these results suggest that 2q35 breast cancer risk loci may be mediating their effect through IGFBP5.

Cost effectiveness and tolerability of dose dense versus weekly paclitaxel chemotherapy in patients with early breast cancer: A real-world comparison.

e18264 Background: Paclitaxel (PAC) regimens used in early breast cancer include 3-weekly, 2-weekly dose-dense (dd) and weekly regimens. Both dd x4 and weekly x12 regimens are superior to the 3-weekly x4 regimen, particularly in ER-negative disease. We set out to compare whether either regimen is superior to the other in terms of cost-effectiveness and tolerability. Methods: We conducted a retrospective case-control study in patients receiving adjuvant PAC for early breast cancer between 2010 and 2015. Patients who received weekly x12 PAC were matched to patients receiving dd x4 PAC by age and year of diagnosis. Data on costs were collected including costs of dayward visits, chemotherapy administration, hospitalization and pegfilgrastim (if used). Data on tolerability included dose delays, hospitalizations and early cessation of planned PAC therapy (with or without alternative chemotherapy). T-test was used to analyze continuous data and chi-squared test was used to analyze categorical data. Results: 38 patients were included in the analysis. Dayward costs were higher for weekly chemotherapy (mean cost €12,259 vs €6371, P < 0.0001), while pegfilgrastim costs were lower as expected in this group (mean cost €108 vs €4003, P < 0.0001). Total costs incurred during the treatment course were not significantly different (mean cost €13,322 vs €11,576, P = 0.23). There were no significant differences in the rate of hospitalization (3 vs 4) and dose delays (4 vs 3). The weekly group were more likely to stop or change treatment without completing the planned number of PAC cycles (0 vs 6 patients, P = 0.008). Conclusions: Weekly and dd PAC regimens are associated with similar overall treatment costs in this real-world setting. Patients receiving weekly chemotherapy are more likely to discontinue therapy early and/or switch to a different drug/regimen. In the absence of evidence for superior efficacy of one regimen over another, we favor the dose dense regimen.

Identification of four novel susceptibility loci for oestrogen receptor negative breast cancer.

Common variants in 94 loci have been associated with breast cancer including 15 loci with genome-wide significant associations (P<5 × 10−8) with oestrogen receptor (ER)-negative breast cancer and BRCA1-associated breast cancer risk. In this study, to identify new ER-negative susceptibility loci, we performed a meta-analysis of 11 genome-wide association studies (GWAS) consisting of 4,939 ER-negative cases and 14,352 controls, combined with 7,333 ER-negative cases and 42,468 controls and 15,252 BRCA1 mutation carriers genotyped on the iCOGS array. We identify four previously unidentified loci including two loci at 13q22 near KLF5, a 2p23.2 locus near WDR43 and a 2q33 locus near PPIL3 that display genome-wide significant associations with ER-negative breast cancer. In addition, 19 known breast cancer risk loci have genome-wide significant associations and 40 had moderate associations (P<0.05) with ER-negative disease. Using functional and eQTL studies we implicate TRMT61B and WDR43 at 2p23.2 and PPIL3 at 2q33 in ER-negative breast cancer aetiology. All ER-negative loci combined account for ∼11% of familial relative risk for ER-negative disease and may contribute to improved ER-negative and BRCA1 breast cancer risk prediction.

Role of Patient and Disease Factors in Adjuvant Systemic Therapy Decision Making for Early-Stage, Operable Breast Cancer: American Society of Clinical Oncology Endorsement of Cancer Care Ontario Guideline Recommendations.

An American Society of Clinical Oncology (ASCO) panel considered the Cancer Care Ontario (CCO) recommendations on the role of patient and disease factors in selecting adjuvant therapy for women with early-stage breast cancer for endorsement. ASCO staff reviewed the CCO guideline for methodologic rigor, and an ASCO panel of content experts reviewed the content of the recommendations. For making decisions regarding adjuvant therapy, nodal status, tumor size, estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2) status, tumor grade, and lymphovascular invasion are relevant; Oncotype DX score and Adjuvant! Online may be used as risk stratification tools; and age, menopausal status, and medical comorbidities should be considered. Chemotherapy should be considered for patients with positive lymph nodes, ER-negative disease, HER2-positive disease, Adjuvant! Online mortality greater than 10%, grade 3 lymph node-negative tumors (T > 5 mm), triple-negative (ER-negative, PgR-negative, HER2-negative) tumors, lymphovascular invasion positivity, or estimated distant relapse risk of greater than 15% at 10 years based on Oncotype DX recurrence score (RS). Chemotherapy may not be beneficial or required for small node-negative tumors (T < 5 mm) without high-risk features or for patients with HER2-negative, strongly ER-positive, and PgR-positive cancer with micrometastatic nodal disease, T less than 5 mm, or Oncotype DX RS with an estimated distant relapse risk of less than 15% at 10 years. The ASCO panel endorses the recommendations with minor suggested revisions and highlights three areas that warrant further consideration: tumor histology and adjuvant therapy recommendations, risk stratification tools and proposed Oncotype DX RS thresholds to guide decisions about chemotherapy, and patient factors in decision making.

Abstract B19: Interrogation of the landscape of long noncoding RNAs in breast cancer to identify an ER-regulated predictor of tamoxifen resistance

Abstract Background: We have previously performed a bioinformatics-based analysis on 7,256 RNA sequencing libraries from tumors, normal tissues, and cell lines to delineate the landscape of long noncoding RNAs (lncRNAs) in the human transcriptome. This analysis identified 58,648 lncRNAs, including over 45,000 novel transcripts (Iyer and Niknafs et al, Nature Genetics, 2015). We now interrogate this lncRNA compendium to identify top candidate estrogen receptor (ER)-associated lncRNAs in breast cancer and characterize their association with disease progression. Methods: To identify and prioritize lncRNAs that were differentially expressed in cancer vs normal tissue, and in ER-positive vs ER-negative disease, we performed differential expression testing using a previously described non-parametric tool for RNA-seq count data called Sample Set Enrichment Analysis (SSEA) on over 1000 RNA sequencing libraries including breast cancer and normal tissue samples from the The Cancer Genome Atlas (TCGA) project. The effect of the top prioritized lncRNA on cancer phenotypes was studied via proliferation, colony formation, invasion and tamoxifen resistance assays in MCF7 and T47D cell lines, and via mouse xenograft studies in vivo. The mechanism by which this lncRNA promotes tumor progression was investigated by identifying its top protein interactors and its subdomains responsible for function, and then studying the effects of disrupting function of this lncRNA on cancer phenotypes. Finally, in a “guilt-by-association” study, we developed a signature of 150 protein coding genes most strongly associated with our lncRNA of interest, and investigated the association of this signature with tumor grade, recurrence, and metastases using Oncomine analyses. Results: Differential expression analyses on over 1000 TCGA RNA-seq samples nominated Breast Cancer Associated Transcript (BRCAT431) as the top overexpressed ER-regulated lncRNA in breast cancer. In vitro experiments demonstrate that shRNA-mediated knockdown of BRCAT431 resulted in significantly decreased proliferation, colony formation, and invasion (by &amp;gt;50% in most assays). Tamoxifen resistance was associated with significantly increased BRCAT431 levels in both MCF7 and T47D cells, and knockdown of BRCAT431 reversed tamoxifen resistance. In vivo xenograft studies demonstrate that knockdown of BRCAT431 also significantly decreased xenograft growth and tumor invasion by &amp;gt;50%. RNA pulldown followed by mass spectrometry identified the RNA binding protein hnRNPL as a key protein interacting with BRCAT431. Deletion studies identified a 27 base-pair region of BRCAT431 necessary for its interaction with hnRNPL, and loss of this region abrogated BRCAT431-induced invasion. Finally, guilt-by-association studies demonstrate a strong association between BRCAT431 overexpression and tumor grade, recurrence, and metastases. Conclusion: In this study, we develop the largest reported compendium of breast cancer lncRNAs. We prioritize BRCAT431 as the top lncRNA upregulated in ER-positive breast cancers, and demonstrate that it confers aggressive oncogenic phenotypes in vitro and in vivo. We identify a novel mechanism by which this lncRNA functions through interaction with hnRNPL. Our results suggest that by promoting tamoxifen resistance, this lncRNA increases the clinical risk of recurrence and metastases in breast cancer. Citation Format: Yashar S. Niknafs, Sumin Han, Teng Ma, Chao Zhang, Matthew K. Iyer, Rohit Malik, Anton Poliakov, Corey Speers, Daniel Hayes, James Rae, Felix Y. Feng, Arul M. Chinnaiyan. Interrogation of the landscape of long noncoding RNAs in breast cancer to identify an ER-regulated predictor of tamoxifen resistance. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B19.

Abstract P5-15-03: Bisphosphonate use and breast cancer recurrence risk in the QUILT cohort

Abstract Background: Women with breast cancer are often prescribed bisphosphonates (BP) for treatment or prevention of osteoporosis. Clinical trials designed to assess the effect of BP on risk of recurrence in women with breast cancer have produced conflicting results. Several randomized studies and a recent meta-analysis suggested that a protective effect of BP is limited to postmenopausal women. Current data have not definitively addressed the utility of BP in patients with ER-positive vs. ER-negative disease. Methods: We examined use of BP for non-cancer therapy in the QUILT cohort study. The analysis included 1813 women diagnosed with invasive breast cancer at ages 45 to 79 in 1993-1999. Data collection was via medical record review, in-person interview and the Cancer Surveillance System, a population-based registry in the NCI SEER program. Cox proportional hazards regression methods were used to calculate hazard ratios (HR) and 95% confidence intervals (CI) for the association between BP use and outcome. Analyses were adjusted for age and year of diagnosis. Results: Mean age at diagnosis of study participants was 64.2 years. Eighty-four percent were postmenopausal at diagnosis; 86% had estrogen receptor positive disease; 16.7% used a BP after diagnosis of breast cancer. Among women with any BP use, 91% had a diagnosis of osteopenia or osteoporosis. Mean duration of BP use after BC diagnosis was 35.2 months; 90% of BP users took alendronate. Ever use (2+ months) of BP was associated with a significant decreased risk of any breast cancer event (local recurrence, distant recurrence, or second primary breast cancer; HR 0.65; 95% CI 0.47-0.90). Within users, there was a trend toward lower risk with longer duration of use (p=0.096, see Table 1). Association between BP use and any breast cancer event (by duration of use)Duration of bisphosphonate useNo event n (%)Any event n (%)Hazard ratio (95% CI)None/Less than 1 mo1070 (70.8)441 (29.2)1.00 (ref)2 mo to &amp;lt;1 yr40 (76.9)12 (23.1)0.82 (0.46-1.45)1 yr to &amp;lt;3 yrs89 (82.4)19 (17.6)0.76 (0.48-1.20)3+ yrs131 (92.3)11 (7.7)0.44 (0.24-0.81) Though the number of events was smaller, similar effects were seen for each event type (distant recurrence: HR=0.62, 95% CI 0.37-1.06; local recurrence: HR=0.62, 95% CI 0.26-1.44; second primary breast cancer: HR= 0.75, 95% CI 0.47-1.18). Stratification by menopausal status showed a decreased risk of recurrence with BP use for both pre or peri- and postmenopausal women (pre/perimenopausal: HR = 0.34, 95% CI 0.12-0.94; postmenopausal: HR=0.72, 95% CI 0.51-1.01). BP use was protective in both women with ER positive (HR=0.64, 95% CI 0.0.45-0.91) and ER negative (HR=0.46, 95% CI 0.16-1.27) disease. Conclusion: In this study, we observed a decreased risk of locoregional recurrence, distant recurrence, and second primary breast cancer with BP use after a diagnosis of invasive breast cancer. This effect was seen in pre and postmenopausal women, and for both ER positive and ER negative disease. Women in this cohort were prescribed primarily oral BP for treatment or prevention of osteoporosis. Additional prospective studies of adjuvant BP use should focus on women with low bone density regardless of menopausal status, and should include patients with both ER-positive and ER-negative disease. Citation Format: Korde LA, Doody DR, Malone KE. Bisphosphonate use and breast cancer recurrence risk in the QUILT cohort. [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 P5-15-03.

Abstract P4-13-24: Impact of genomic medicine on clinical decision making in patients with advanced breast cancer at two academic medical centers

Abstract Background: A deeper molecular understanding of cancer biology has led to the development of therapies targeting driver mutations. Genomic profiling of tumors is commercially available and has become integrated into many clinical practices as part of a paradigm shift towards personalized care of cancer patients. The current impact of genomic profiling on clinical decision making for patients with advanced breast cancer is not well described. Methods: Patients with metastatic breast cancer (mBC) who had tumors submitted for commercial genomic analysis from 2013-2015 were identified consecutively at two large academic cancer centers with genomic basket trials open for the majority of the collection period. Demographics, tumor pathology, clinical, and treatment histories were extracted through medical chart review as per an IRB approved protocol. Data from genomic analysis reports was extracted including number and type of mutations, FDA approved therapies and clinical trials available. Genomic analysis was determined to have impacted clinical decision making if the next line of therapy was influenced either by accrual to clinical trial, or a decision to prescribe an FDA-approved therapy. The most frequent somatic mutations and their relative frequencies were determined. Results: A total of 82 patients with mBC who had undergone commercially available genomic testing were identified. The median age was 49 (range: 29-70). 42 patients (51%) had ER-positive HER2-negative disease, 33 (40%) had ER-negative HER2-negative disease, 4 (5%) had ER-negative HER2-positive disease and 3 (4%) had ER-positive HER2-positive disease. The median number of lines of therapy received prior to genomic profiling was 2 (range 0-15). Genomic analysis reports suggested that 61 (74%) of these patients had at least one FDA approved medication available for at least one somatic mutation, and 79 (96%) had at least one clinical trial available (39 (46%) in the same state, 11 (13%) in the same institution). Genomic testing influenced management in 8 patients (10%), with 6 patients (7%) experiencing a change in next line of therapy attributable to genomic information. In 74 patients (90%), genomic testing results did not affect clinical decision-making. The most frequently observed somatic mutations included: TP53, PI3KCA, MYC, CCDN1, FGF, ZNF703, GATA3, ARID1A, MCL1, PTEN, MYST3, and BRCA1. Conclusions: Genomic testing did not affect management in the vast majority of mBC patients treated at two major academic cancer centers. Furthermore, the most identified mutated genes found were not targetable. The real world clinical utility of genomic analysis remains limited in breast cancer but may influence clinical decision making in a minority of patients. Citation Format: Kruse ML, Santa-Maria CA, Raska P, Swoboda A, Jain S, Sohal D, Moore H, Budd GT, Abraham J, Montero AJ. Impact of genomic medicine on clinical decision making in patients with advanced breast cancer at two academic medical centers. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-13-24.

Abstract P1-07-04: Etiologic heterogeneity in breast cancer across quantitative levels of estrogen receptor expression

Abstract Introduction: The clinical classification of breast cancer into at least four intrinsic subtypes has advanced targeted therapy and improved prognosis. However, for etiological classification, it has been proposed that breast cancer is comprised of just two main subtypes: basal-like and non-basal-like. Evidence for these two etiologic subtypes emerges strongly from bimodal age frequency distributions at diagnosis. In the absence of RNA-based intrinsic subtyping, estrogen receptor (ER) expression is a useful surrogate for these two distinct etiologic classes. Using data from the population-based Carolina Breast Cancer Study (CBCS), we examined evidence for a two-component (ER-positive vs. ER-negative) mixture model for breast cancer biologic/etiologic heterogeneity. Methods: Automated digital scoring of ER expression was performed on immunohistochemistry-stained tissue microarrays comprising 1,920 invasive breast cancer cases from CBCS. Clinical classification of ER status has changed over time as new data have emerged regarding optimal treatment-relevant thresholds, but optimal etiologic thresholds have not been established. Therefore, we considered ER status as a quantitative, categorical variable with cut points of &amp;lt;1% (ER-negative) vs. ≥1% (ER-positive), with ER-positive cases further categorized as highly positive (≥80-100%), intermediate (≥40-&amp;lt;80%), low (≥10-&amp;lt;40%) or borderline (≥1-&amp;lt;10%). Smoothed age frequency distributions at diagnosis (i.e., density plots) were constructed and logistic regression adjusted for age and race was conducted to assess associations between patient and tumor characteristics and level of ER positivity. Results: As expected for etiologically-distinct entities, ER-negative and highly ER-positive tumors showed predominantly unimodal early-onset and late-onset age distributions at diagnosis with peak frequencies near ages 50 and 70 years, respectively. However, tumors with low and intermediate positivity showed bimodal patterns, consistent with a mixture of two main subtypes. Consistent with these age distribution patterns, young age (&amp;lt;40 years) at diagnosis was associated with an elevated odds ratio (OR) for low positive (OR 1.8; 95% CI 1.1-2.9), borderline (OR 1.9; 95% CI 1.1-3.3) and ER-negative disease (OR 2.2; 95% CI 1.5-3.2). Relative to highly ER-positive tumors, low ER-positive tumors were more likely to be node-positive (OR 1.4; 95% CI 1.0-1.9), higher grade (combined grade III; OR 1.9; 95% CI 1.3-2.9), and were more likely to harbor a p53 mutation (OR 2.0; 95% CI 1.2-3.5). Conclusions: While etiologic differences between dichotomized ER-negative and ER-positive breast cancer categories have been well described in many epidemiologic studies, differences in breast cancer etiology across quantitative levels of ER expression have not been so well-characterized. In this study, we report that ER-positive tumors with low positivity share etiologic features of ER-negative tumors, including young age at diagnosis and aggressive tumor characteristics. These data provide additional support for a two-component breast cancer mixture model, with quantitative level of ER positivity reflecting the relative distributions of ER-positive and ER-negative tumor populations. Citation Format: Allott EH, Tse C-K, Carey L, Anderson WF, Olshan AF, Troester MA. Etiologic heterogeneity in breast cancer across quantitative levels of estrogen receptor expression. [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 P1-07-04.

Abstract OT2-01-07: A phase 2 open label, multi-center, multinational study investigating the efficacy and safety of GTx-024 on advanced, androgen receptor-positive triple negative breast cancer (AR+ TNBC)

Abstract Background: The androgen receptor (AR) is the most highly expressed steroid receptor in breast cancer (BC) with expression seen in up to 95% of estrogen receptor positive (ER+) and up to 50% of ER negative disease. Historically, steroidal androgens exhibited virilizing side effects, thus limiting clinical use. In TNBC, the expression of AR and androgen synthesizing enzymes is associated with lower proliferation, lower tumor grade, better overall survival, and more favorable clinical outcomes as compared to those patients with TNBC not expressing AR. Data from two trials targeting AR in TNBC indicates low level but encouraging clinical activity. A non-steroidal, tissue-selective, AR modulator (SARM), such as GTx-024, offers a targeted approach of AR activation in AR+ TNBC without virilization or estrogenic effects. Trial Design: Open label, multicenter, multinational, Phase 2 study for the treatment of advanced or metastatic TNBC. Subjects will receive GTx-024, 18 mg orally (PO) daily, continued until evidence of disease progression or toxicity. Subjects whose tumors demonstrate Clinical Benefit (CB) will be treated on study drug until progression. Eligibility Criteria: Inclusion: Female, ≥18 years old, confirmed AR+ (≥10% staining) TNBC (confirmed by medical history), up to 1-2 prior chemotherapy regimens, available archived tumor tissue, measurable or bone-only disease, ECOG 0 or 1, with prior toxicities from chemotherapy resolved. Exclusion: Life expectancy &amp;lt;4 months, uncontrolled CNS metastases, radiotherapy ≤14 days prior to enrollment, active hepatitis, HIV positive, prior treatment with anti-androgens, testosterone or testosterone-like agents, or estrogens or megesterol acetate, or prior treatment for a different cancer (other than BC or non-melanoma carcinoma of the skin) within the past 2 year Specific Aims: The primary aim is to measure the proportion of AR+ subjects with CB at 16 weeks; defined as subjects with a complete response (CR), partial response (PR), or stable disease (SD); per modified RECIST 1.1. Secondary endpoints include: objective response rate, progression free survival, and time to progression. Statistical Methods: Simon's two-stage (optimal) design will be used to assess primary efficacy, requiring up to 41 evaluable subjects; i.e., subjects with centrally confirmed AR+ who receive at least one dose of study drug. The first stage will be assessed among the first 21 evaluable subjects. If at least 2/21 achieve CB per modified RECIST 1.1 at 16 weeks, then the trial will proceed to the second stage of recruitment of up to a total of 41 subjects in the evaluable subset of the Full Analysis Set. Otherwise, the trial will be discontinued for lack of efficacy. The trial will test for an unacceptably low CBR of ≤5% versus a CBR more consistent with ≥20%. Target Accrual: Up to 55 subjects will be enrolled. Trial Information: www.gtxinc.com Citation Format: Rugo H, Overmoyer B, Schwartzberg L, Palmieri C, Taylor R, Hancock M, Small S, Johnston MA. A phase 2 open label, multi-center, multinational study investigating the efficacy and safety of GTx-024 on advanced, androgen receptor-positive triple negative breast cancer (AR+ TNBC). [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 OT2-01-07.

Annual Hazard Rates of Recurrence for Breast Cancer During 24 Years of Follow-Up: Results From the International Breast Cancer Study Group Trials I to V.

Predicting the pattern of recurrence can aid in the development of targeted surveillance and treatment strategies. We identified patient populations that remain at risk for an event at a median follow-up of 24 years from the diagnosis of operable breast cancer. International Breast Cancer Study Group clinical trials I to V randomly assigned 4,105 patients between 1978 and 1985. Annualized hazards were estimated for breast cancer-free interval (primary end point), disease-free survival, and overall survival. For the entire group, the annualized hazard of recurrence was highest during the first 5 years (10.4%), with a peak between years 1 and 2 (15.2%). During the first 5 years, patients with estrogen receptor (ER)--positive disease had a lower annualized hazard compared with those with ER-negative disease (9.9% v 11.5%; P = .01). However, beyond 5 years, patients with ER-positive disease had higher hazards (5 to 10 years: 5.4% v 3.3%; 10 to 15 years: 2.9% v 1.3%; 15 to 20 years: 2.8% v 1.2%; and 20 to 25 years: 1.3% v 1.4%; P < .001). Among patients with ER-positive disease, annualized hazards of recurrence remained elevated and fairly stable beyond 10 years, even for those with no axillary involvement (2.0%, 2.1%, and 1.1% for years 10 to 15, 15 to 20, and 20 to 25, respectively) and for those with one to three positive nodes (3.0%, 3.5%, and 1.5%, respectively). Patients with ER-positive breast cancer maintain a significant recurrence rate during extended follow up. Strategies for follow up and treatments to prevent recurrences may be most efficiently applied and studied in patients with ER-positive disease followed for a long period of time.

Associations of Breast Cancer Risk Prediction Tools With Tumor Characteristics and Metastasis.

The association between established risk factors for breast cancer and subtypes or prognosis of the disease is not well known. We analyzed whether the Tyrer-Cuzick-predicted 10-year breast cancer risk score (TCRS), mammographic density (MD), and a 77-single nucleotide polymorphism polygenic risk score (PRS) were associated with breast cancer tumor prognosticators and risk of distant metastasis. We used a case-only design in a population-based cohort of 5,500 Swedish patients with breast cancer. Logistic and multinomial logistic regression of outcomes, estrogen receptor (ER) status, lymph node involvement, tumor size, and grade was performed with TCRS, PRS, and percent MD as exposures. Cox proportional hazard models were used to estimate hazard ratios (HRs) of distant metastasis. Women at high risk for breast cancer based on PRS and/or TCRS were significantly more likely to be diagnosed with favorable prognosticators, such as ER-positive and low-grade tumors. In contrast, PRS weighted on ER-negative disease was associated with ER-negative tumors. When stratifying by age, the associations of TCRS with favorable prognosticators were restricted to women younger than age 50. Women scoring high in both TCRS and PRS had a lower risk of distant metastasis (HR, 0.69; 95% CI, 0.49 to 0.98). MD was not associated with any of the examined prognosticators. Women at high risk for breast cancer based on genetic and lifestyle factors were significantly more likely to be diagnosed with breast cancers with a favorable prognosis. Better knowledge of subtype-specific risk factors could be vital for the success of prevention programs aimed at lowering mortality.

Androgen receptor CAG repeat length and estrogen receptor status in postmenopausal breast cancer prognosis.

The influence of the androgen receptor (AR) CAG repeat polymorphism on breast cancer is controversial. We investigated the combined effects of CAG repeat length and estrogen receptor (ER) status on prognosis in 355 postmenopausal women with primary breast cancer. CAG repeat length was determined by the HUMARA test. Relapse-free survival (RFS) and overall survival (OS) according to the X-weighted CAG repeat biallelic mean (XWBM) were investigated by univariate and multivariate analysis. XWBM was not associated with RFS or OS, but a significant interaction between XWBM and ER status (p = 0.002) was found for OS. ER-negative patients with median XWBM &lt;20 showed lower OS than ER-negative/XWBM ≥20 patients (HR = 0.270; 95% Cl: 0.073-0.999). ER-negative/XWBM &lt;20 patients also had significantly lower OS than ER-positive women, irrespective of CAG repeat length (p&lt;0.001). Accordingly, estimated OS was lowest in ER-negative patients with XWBM &lt;20 (OS: 0.63, 95% CI: 0.41-0.79) and highest in ER-positive patients with XWBM &lt;20 (OS: 0.95, 95% CI: 0.90-0.97). Our data suggest that short CAG repeat length is associated with increased risk of death in ER-negative disease but is related to better survival when ER is expressed. These findings are in agreement with the hypothesis that AR may stimulate or inhibit breast cancer growth depending on ER status, AR transactivation, and the endocrine-metabolic environment of breast tumors. Evaluation of CAG repeat length together with ER status could help improve the estimation of the risk of death, with possible implications for the optimization of standard breast cancer treatment and implementation of prevention strategies.