ER-positive Tumors Research Articles

Abstract 2109: A novel role for ESRP1 in regulating proliferation in therapy-resistant ER-positive breast cancer

Abstract Epithelial Splicing Regulatory Protein 1 (ESRP1) is a key splicing factor that plays an important role in cancer. We have previously demonstrated that ESRP1 is associated with poor prognosis and tamoxifen resistance in human ER-positive (ER+) breast tumors. Knockdown of ESRP1 decreased the tumor growth in ER+ breast cancer models resistant to endocrine therapy. In this study, we explored the upstream/downstream regulatory mechanisms of ESRP1 in tamoxifen-resistant breast cancer using ESRP1 knockdown model. Probe based-Human Transcriptome Array 2.0 (HTA; Applied Biosystems/Thermo Fisher) was performed using RNAs from control and ESRP1 knockdown cells [tamoxifen-resistant LCC2 cells (2-control) versus LCC2 cells with ESRP1 knockdown (2C3)] in ER+ breast cancer. Transcriptome profiling of ESRP1 in 2C3 knockdown model revealed that CEBP gamma, a transcription factor belonging to the CCAAT/enhancer-binding protein (C/EBP) family, was significantly decreased in ESRP1 knockdown cells (p value=2.33E-06). The expression of other related proteins C/EBP family including C/EBP alpha, C/EBP beta, and C/EBP delta that form homodimers and heterodimers with each other was also assessed. In knockdown cells, C/EBP alpha was increased (p value=0.0022), while two different C/EBP beta transcripts were decreased (p values=0.0010 and 0.03, respectively). C/EBP delta expression was not significantly altered. Using a competitive promoter-binding transcription-factor assay, we measured the transcription activity of C/EBP gamma in the absence and presence of ESRP1 promoter DNA fragment. We identified strong binding of C/EBP gamma to ESRP1 promoter region indicating that it is upstream regulator of ESRP1. CEBP transcription factor family members are important regulators of proliferation via cyclin-dependent kinases (CDKs). We further demonstrated that CDK4/CDK6 inhibitor abemaciclib decreases the expression of ESRP1 and C/EBPG levels significantly (p values= 0.0009 and 0.0019, respectively). In conclusion, for the first time, we identify that C/EBP gamma upregulates ESRP1 expression, which further regulates proliferation through control of expression of CDKs. Citation Format: Yesim Gökmen-Polar, Yuan Gu, Xiaoping Gu, Sunil S. Badve. A novel role for ESRP1 in regulating proliferation in therapy-resistant ER-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2109.

Abstract 26: Development of potent lead compounds in multiple tumor types

Abstract Background: We had earlier reported a novel therapeutic agent, ERX-11, that modulates estrogen receptor coregulator interactions. For lead optimization, we designed, synthesized and tested over 500 analogs of ERX-11 in multiple models of BC. We also tested this library of compounds for activity against other cancer cell lines both in vitro and in vivo and against primary tumors ex vivo. Methods: In vitro activity was tested using Cell titer glo, MTT, and apoptosis assays.The utility of the ERX analogs in treating therapy resistant ER-positive BC was evaluated using models with acquired resistance (Tamoxifen, Letrozole), and engineered models that express ER mutations. Xenografts were used for testing the utility of ERX analogs in vivo. Primary patient tumor derived explants were used for ex vivo testing of ERX analogs. Results: Our screening studies identified several ERX analogs with potent activity against BC cells. Subtle changes in the ERX analogs appear to have significant ramifications on both their potency against ER+ BC cell lines and against other tumors types. Some analogs like ERX-41 were more potent than ERX-11 in their ability to block the proliferation of multiple ER-positive BC cell lines (IC50 ranging from 20-200nM). Other analogs like ERX-208 showed similar activity as ERX-11 against ER-positive BC cell lines (IC50 ranging from 100-500nM) but had potent activity against ovarian cancer cell lines. Through iterative changes, we have identified leads compounds with significant activity against other cancers, including in gliomas, ovarian and pancreatic cancers. Although all these compounds were designed to better target the ligand binding pocket of ER, these active compounds do not all target ER and appear to target other proteins, including other nuclear receptors. Some compounds for example have activity in ER-negative breast cancers. In several xenograft models, including pancreatic cancer and ER-negative, the activity of the compounds have been confirmed by oral administration of the ERX analog in vivo. We have also validated the activity of these analogs in patient-derived explants cultured ex vivoin multiple tumor types. Conclusions: From our studies to develop a more potent ERX-11 lead analog, we have identified multiple analogs with distinct activities against multiple cancers. While the intended target of these analogs was ER, our library of analogs has potent activity against both ER-positive and ER-negative tumors. In collaboration, we are pursuing further leads in multiple cancers to further delineate the mechanism of action of these various analogs. Citation Format: Ganesh V. Raj, Xihui Liu, Dede Ekoue, Jung-Mo Ahn, Ratna Vadlamudi. Development of potent lead compounds in multiple tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 26.

Abstract 21: Generation and characterization of potent analogues of ERX-11

Abstract Breast cancer (BC) is the most common malignancy in women. Approximately 70% BC are Estrogen receptor (ER) positive, However, most patients develop resistance to current therapies and progress to incurable metastases. We had earlier reported a novel therapeutic agent, ERX-11, that modulates estrogen receptor coregulator interactions. For lead optimization, we designed, synthesized, and tested over 500 analogs of ERX-11 in multiple models of BC. Methods: In vitro activity was tested using cell titer glo, MTT, and apoptosis assays. The utility of the ERX analogs in treating therapy resistant ER-positive BC was evaluated using models with acquired resistance (Tamoxifen, Letrozole), and engineered models that express ER mutations. Xenografts, patient derived xenografts (PDX), and patient derived xenograft explants (PDEx) were used for testing the utility of ERX analogs. Results: Our screening studies identified several ERX analogs with potent activity against BC cells. Subtle changes in the ERX analogs appear to have significant ramifications on both their potency against ER-positive BC cell lines and against other tumors types. Some analogs like ERX-41 were more potent than ERX-11 in their ability to block the proliferation of multiple ER-positive BC cell lines (IC50 ranging from 50-200 nM). Other analogs like ERX-208 showed similar activity as ERX-11 against ER-positive BC cell lines but had potent activity (IC50 ranging from 50 -100 nM) against ovarian cancer cell lines. Through iterative changes, we have identified lead compounds with significant activity against other cancers, including in gliomas, ovarian and pancreatic cancers. Although all these compounds were designed to better target the ligand binding pocket of ER, CRISPR-Cas9 based KO screening revealed that these active compounds do not all target ER and appear to target other proteins, including other nuclear receptors. Some compounds for example have activity in ER-negative BC. In several xenograft and models, including pancreatic cancer and ER-negative BC, the activity of the compounds have been confirmed by oral administration of the ERX analogs in vivo. We have also validated the activity of these analogs using PDX and PDEX models. Conclusions: From our studies to develop a more potent ERX-11 lead analog, we have identified multiple analogs with activities against multiple cancers. While the intended target of these analogs was ER, our library of analogs has potent activity against both ER-positive and ER-negative tumors. In collaboration, we are pursuing further leads in multiple cancers to further delineate the mechanism of action of these various analogs. Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Shi-Hong Ma, Gangadhara Reddy Sareddy, Tae-Kyung Lee, Mei Zhou, Yiliao Liu, Xihui Liu, Dede N. Bahun-Wilson, Kara Kassees, Jung-Mo Ahn, Ganesh V. Raj, Ratna K. Vadlamudi. Generation and characterization of potent analogues of ERX-11 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 21.

Abstract 5072: Targeting the mTOR/TORC Pathway for the prevention of er-negative and triple-negative breast cancer

Abstract Background: Women with “triple-negative breast cancer” (TNBC), are currently treated with chemotherapy, and have a very poor prognosis. TNBC tumors also frequently have p53 and BRCA1 gene mutations. Dysregulation of PI3K-mTOR pathway has been commonly associated with ER-negative breast cancers with poor prognosis. The mTOR inhibitor everolimus is used to treat ER-positive tumors that have become resistant to anti-estrogen therapy. We hypothesized that targeting mTOR may prevent development of ER-negative and BRCA1-mutant breast cancers and asked whether the mTOR inhibitor everolimus exhibited tumor preventive efficacy in several mouse models of breast cancer. Methods: p53-null mammary gland donor mice were transplanted into cleared fat pads of p53 wild-type mice. BRCA/p53-deficient mice: We produced BRCA1 mice by breeding males and females. All mice were separated into two groups 1) Control and 2) everolimus. MMTV-erbB2 and p53 null mammary gland mice were treated with everolimus (5mg/kg, by oral gavage). BRACA1 mice were given 2mg/kg and 5mg/kg 2X a week of everolimus. All of these mice spontaneously developed mammary tumors within 12 months. Mice were observed daily, toxicity and the percentage of tumor free mice were recorded. Tumor incidence and time to tumor formation was visualized using Kaplan- Meier curves and analyzed using the generalized Wilcoxon test. Results: In MMTV-erbB2 mice everolimus reduced tumor incidence and was associated with an increase in median survival time from 240 days to 410 days. At 365 days, when all mice in control group died, only half of the mice treated with everolimus had developed tumors (p=0.0001). Everolimus also reduced tumor incidence in p53 null mammary gland mice. At 420 days, 50% of the control mice developed mammary tumors compare to only 7 % of the everolimus treated mice (p=0.04). Everolimus also reduced tumor incidence in BRCA1 mice. At 262 days, when 13 (out of 18) (72%) of the control mice developed mammary tumors compare to only 7 (out of 18) (38%) in everolimus treated group developed tumors (p=0.02). Long term treatment (>150 days) of everolimus was associated with mild toxicity that includes slight weight loss (<10%) and skin changes (matted hair, skin erythema) in MMTV-erbB2 and p53-null mammary gland models. We have not observed any visible toxicity in BRCA1 mice yet. Results of mammary tissue biomarkers will be presented. Conclusions: The mTOR inhibitor everolimus prevented mammary tumorigenesis in all three mouse models. Our results suggest that everolimus can be an effective cancer preventive drug and that further studies with reduced everolimus dose alone or in combination with other targeted therapies are warranted. In the future, clinical trials of the everolimus should be considered for the prevention of breast cancer in high-risk patients. Supported by NCI PREVENT Cancer Preclinical Drug Development Program (HHSN-2612015000-18I (PB)). Citation Format: Abhijit Mazumdar, Jamal Hill, Yun Zhang, Lakshmi Reddy Bollu, Alejandro Contreras, Michelle Savage, Shizuko Sei, Altaf Mohammed, Powel Brown. Targeting the mTOR/TORC Pathway for the prevention of er-negative and triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5072.

Inflammatory breast cancer: A review from our experience.

Inflammatory Breast Cancer (IBC) is a distinct and rare type of breast cancer accounting for up to 6% of all breast cancer cases in Europe. The aim of this study was to investigate diagnostic methods, treatments, and outcome after IBC in patients treated at a single institution in Denmark. All patients treated for IBC at Aarhus University Hospital between 2000 and 2014 were identified and included in the cohort. Survival was assessed using Kaplan-Meier curves and log-rank statistics. A total of 89 patients were identified with a median follow up of 3.6 years. The overall survival at 5 and 10 years were 41% and 18%, respectively. The disease free survival at 5 and 10 years were 47% and 27%, respectively. Thirty-four percent had distant metastasis at time of diagnosis. Patients with ER positive tumors had a significantly better overall survival than patients with ER negative tumors (p = 0.01). Despite a more aggressive systemic and loco-regional treatment today, IBC is still a very serious disease with a high mortality.

Phosphoproteome Analysis Reveals Estrogen-ER Pathway as a Modulator of mTOR Activity Via DEPTOR.

ER-positive breast tumors represent ∼70% of all breast cancer cases. Although their treatment with endocrine therapies is effective in the adjuvant or recurrent settings, the development of resistance compromises their effectiveness. The binding of estrogen to ERα, a transcription factor, triggers the regulation of the target genes (genomic pathway). Additionally, a cytoplasmic fraction of estrogen-bound ERα activates oncogenic signaling pathways such as PI3K/AKT/mTOR (nongenomic pathway). The upregulation of the estrogenic and the PI3K/AKT/mTOR signaling pathways are frequently associated with a poor outcome. To better characterize the connection between these two pathways, we performed a phosphoproteome analysis of ER-positive MCF7 breast cancer cells treated with estrogen or estrogen and the mTORC1 inhibitor rapamycin. Many proteins were identified as estrogen-regulated mTORC1 targets and among them, DEPTOR was selected for further characterization. DEPTOR binds to mTOR and inhibits the kinase activity of both mTOR complexes mTORC1 and mTORC2, but mitogen-activated mTOR promotes phosphorylation-mediated DEPTOR degradation. Although estrogen enhances the phosphorylation of DEPTOR by mTORC1, DEPTOR levels increase in estrogen-stimulated cells. We demonstrated that DEPTOR accumulation is the result of estrogen-ERα-mediated transcriptional upregulation of DEPTOR expression. Consequently, the elevated levels of DEPTOR partially counterbalance the estrogen-induced activation of mTORC1 and mTORC2. These results underscore the critical role of estrogen-ERα as a modulator of the PI3K/AKT/mTOR signaling pathway in ER-positive breast cancer cells. Additionally, these studies provide evidence supporting the use of dual PI3K/mTOR or dual mTORC1/2 inhibitors in combination with endocrine therapies as a first-line treatment option for the patients with ER-positive advanced breast cancer.

Prognostic Factors for Luminal B-like Breast Cancer.

This study aimed to examine the prognostic factors of luminal B-like breast cancer. Clinical data of 695 luminal B-like breast cancer patients who had been treated in our hospital during the period of past 4.5 years were collected and analyzed. Estrogen receptor (ER), progesterone receptor (PgR), antigen identified by monoclonal antibody Ki-67 (Ki67) were immunohistochemically detected. Different cutoffs of ER, PgR, and Ki67 were evaluated. Pearson χ2 test was performed to compare categorical parameters. Univariate and multivariate models were used to evaluate predictors of disease free survival (DFS). The results showed that patients who were younger, and had larger tumors, and more positive lymph nodes were more likely to receive neo-adjuvent chemotherapy (NAC). Patients with ER-positive tumors having <10% positive cells received more anthracycline- and taxane-based chemotherapy and less endocrine therapy than those with ER-positive tumors having ≥10% positive cells (P=0.004 and P=0.007, respectively); however, patients with ER-positive tumors having <10% positive cells experienced more recurrence (P<0.001). PgR expression levels were not associated with therapeutic schedule and DFS. Patients with tumor tissue Ki67 score ≥30% received more anthracycline- and taxane-based chemotherapy and had worse DFS than those with tumor tissue Ki67 score <30%. Univariate and multivariate analysis showed that clinical T stage, lymph nodes, ER, Ki67, and HER2 status were independent prognostic factors. In conclusion, ER-positive rate <10% and Ki67 score ≥30%, similar to higher clinical T stage, more metastatic lymph nodes, and HER2 positive status, may indicate a worse prognosis for luminal B-like breast cancer patients. Multi-center prospective trials with larger sample sizes are necessary for the continued perfection of our work.

Estrogen Receptor Positive Breast Cancer with High Expression of Androgen Receptor has Less Cytolytic Activity and Worse Response to Neoadjuvant Chemotherapy but Better Survival

Estrogen receptor (ER) positive breast cancer (BC), the most abundant BC subtype, is notorious for poor response to neoadjuvant chemotherapy (NAC). The androgen receptor (AR) was reported to support estradiol-mediated ER activity in an in vitro system. Recently, ER-positive BC with fewer tumor infiltrating lymphocytes (TILs) was shown to have a better prognosis, opposite to the trend seen with ER-negative BC. We hypothesized that ER-positive BC with high expression of AR will have fewer TILs and an inferior response to NAC, but with a better prognosis. In both TCGA and METABRIC cohorts, AR expression was significantly higher in ER-positive BCs compared to ER-negatives (p < 0.001, p < 0.001, respectively) and it correlated with ER expression (R = 0.630, R = 0.509, respectively). In ER-positive tumors, AR high tumors enriched UV response down (NES = 2.01, p < 0.001), and AR low tumors enriched DNA repair (NES = −2.02, p < 0.001). AR high tumors were significantly associated with procancer regulatory T-cells, and AR low tumors were associated with anticancer immune cells, such as CD4, CD8, and Gamma-Delta T-cells and memory B-cells in ER-positive BC (p < 0.01). Further, cytolytic activity was significantly lower in AR high BC in both cohorts. Finally, AR high tumors had a significantly lower rate of attaining pathological complete response to NAC (GSE22358), but better survival. In conclusion, our results demonstrated that high AR has fewer tumor infiltrating lymphocytes as well as cytolytic activity and an inferior response to NAC, but better survival in ER-positive BC.

Estrogen Receptor Status Oppositely Modifies Breast Cancer Prognosis in BRCA1/BRCA2 Mutation Carriers Versus Non-Carriers.

Breast cancer (BC) prognosis in BRCA1 and BRCA2 mutation carriers has been reported contradictorily, and the significance of variables influencing prognosis in sporadic BC is not established in BC patients with hereditary BRCA1/BRCA2 mutations. In this retrospective cohort study, we analyzed the effect of clinicopathological characteristics on BC prognosis (disease-free survival [DFS] and disease-specific survival [DSS]) in hereditary BRCA1/BRCA2 mutation carriers. We enrolled 234 BRCA1/BRCA2 mutation carriers and 899 non-carriers, of whom 191 carriers and 680 non-carriers, with complete data, were available for survival analyses. We found that patients with ER-positive tumors developed disease recurrence 2.3-times more likely when they carried a BRCA1/BRCA2 mutation (23/60; 38.3% ER-positive carriers vs. 74/445; 16.6% ER-positive non-carriers; p < 0.001). ER-positive mutation carriers also had a 3.4-times higher risk of death due to BC compared with ER-positive non-carriers (13/60; 21.7% vs. 28/445; 6.3%; p < 0.001). Moreover, prognosis in ER-negative BRCA1/BRCA2 mutation carriers was comparable with that in ER-positive non-carriers. Our study demonstrates that ER-positivity worsens BC prognosis in BRCA1/BRCA2 mutation carriers, while prognosis for carriers with ER-negative tumors (including early-onset) is significantly better and comparable with that in ER-positive, older BC non-carriers. These observations indicate that BRCA1/BRCA2 mutation carriers with ER-positive BC represent high-risk patients.

Inhibition of insulin-like growth factor 1 signaling synergistically enhances the tumor suppressive role of triptolide in triple-negative breast cancer cells.

Triptolide (TPL) is an active extract from a Chinese herb, which has been used for centuries in China. TPL exhibits numerous bioactivities and pharmacological effects, including antitumor, anti-inflammatory and immunosuppressive activities. However, previous studies have further revealed a multi-target toxicity of TPL, including reproductive toxicity, hepatotoxicity and renal cytotoxicity. To validate the clinical benefit and reduce the risk of TPL application, studies have investigated the combination of TPL with other reagents to allow lower doses and decrease toxicity. The present study reported that TPL and the insulin-like growth factor-1 receptor (IGF1R) inhibitor AG1024synergistically inhibited cell proliferation and induced apoptosis in triple-negative breast cancer cells. Overexpression of B-cell lymphoma 2 partially reversed the TPL and AG1024-induced increase in apoptosis. A similar synergistic effect was observed with a combination of AG1024 and cisplatin, a DNA damage inducer, in MDA-MB-231 cells. These results suggested that inhibition of IGF1R may sensitize triple-negative breast cancer cells to DNA damage inducers. Using publicly available data from The Cancer Genome Atlas, an amplification and gain of copy number of IGF1R was observed in 38% of triple-negative breast tumors (n=82), 26% of estrogen receptor (ER)-negative tumors (n=174) and 10% of ER-positive tumors (n=594). Similarly, a higher alteration frequency of IGF1R was identified in basal-like breast tumors compared with luminal A/B-like breast tumors. Overexpressed proteins associated with these alterations were revealed to be significantly enriched in multiple oncogenic signaling pathways, key transcription factor networks and DNA repair pathways. In summary, the present study suggested that inhibition of IGFR signaling and induction of DNA damage may exhibit synergistic effects for the treatment of triple-negative and ER-negative breast cancer.

GSTM1 polymorphism in Oncotype DX assay is a potential predictive factor for taxane-based neoadjuvant chemotherapy in estrogen receptor-positive Chinese breast cancer patients.

BackgroundInvestigations of the 21-gene Oncotype DX assay in neoadjuvant settings for breast cancer patients are currently underdeveloped. It is still unclear whether any individual gene of the 16 cancer-related genes can be used to predict response to taxane-based neoadjuvant chemotherapy.MethodsHere, we performed TaqMan RT-PCR reactions to profile the expression patterns of these genes in 66 Chinese ER-positive breast cancer patients’ tumor tissues who underwent taxane-based neoadjuvant chemotherapies. Only GSTM1 has distinct non-expression/expression polymorphism, and we observed an almost 2.5-fold increment in the possibility of achieving pathological complete response (pCR) was defined as no histological evidence of residual invasive cancer both in the breast and in the axilla. in GSTM1 non-expression patients. We then analyzed the expression levels of 4 main GST isozymes (GSTA1, GSTT1, GSTP1 and GSTT1) and docetaxel cytotoxicity in various breast cancer cell lines (MCF7, MDA-MB-231, BT474, and SKBR3). Furthermore, we knocked down 4 GST isozymes with siRNA against each isoform in resistant MDA-MB-231. We then observed the changes of docetaxel sensitivity though WST-1 assay, in addition to apoptosis by Hoechst stain and TUNEL assay.ResultsDocetaxel IC50 values ranging from 33.0 nM (MDA-MB-231) to 7.20 µM (resistant MDA-MB-231) revealed a close correlation with the expression levels of 4 main GST isozymes. Significantly, depletion of GSTM1 and GSTA1 but not GSTP1 nor GSTT1, suppressed cell viability by 174% and 155% respectively, and was able to induce obvious apoptosis in resistant MDA-MB-132.ConclusionsTaken together, our results suggest that GSTM1 polymorphism could be useful in the prediction of taxane-based neoadjuvant chemotherapy in ER-positive Chinese breast cancer patients, and that GSTM1 and GSTA1 expression are linked to docetaxel resistance.

Specific allelic variants of SNPs in the MDM2 and MDMX genes are associated with earlier tumor onset and progression in Caucasian breast cancer patients.

BackgroundGenetic factors play a substantial role in breast cancer etiology. Genes encoding proteins that have key functions in the DNA damage response, such as p53 and its inhibitors MDM2 and MDMX, are most likely candidates to harbor allelic variants that influence breast cancer susceptibility. The aim of our study was to comprehensively analyze the impact of SNPs in the TP53, MDM2, and MDMX genes in conjunction with TP53 mutational status regarding the onset and progression of breast cancer.MethodsIn specimen from 815 breast cancer patients, five SNPs within the selected genes were analyzed: TP53 – Arg72Pro (rs1042522), MDM2 – SNP285 (rs2279744), SNP309 (rs117039649); MDMX – SNP31826 (rs1563828), and SNP34091 (rs4245739). Classification of the tumors was evaluated by histomorphology. Subtyping according hormone receptor status, HER2-status and proliferation rate enabled provision of the clinico-pathological surrogate of intrinsic subtypes.ResultsThe homozygous C-allele of MDM2 SNP285 was significantly associated with a younger age-at-diagnosis of 44.2 years, in contrast to G/G- and G/C-patients (62.4, 62.7 yrs., respectively; p = 0.0007; log-Rank-test). In contrast, there was no difference regarding the age-at-diagnosis for patients with the respective genotypes of MDM2 SNP309 (p = 0.799; log-Rank-test). In patients with estrogen receptor (ER)-positive and TP53-mutated tumors, however, the T/T-genotype of the MDM2 SNP309 was significantly associated with an earlier average age-at-diagnosis compared with T/G+G/G-patients (53.5 vs. 68.2 yrs; p = 0.002; log-Rank-test). In the triple-negative subgroup, the G/G-patients had an average age-at-diagnosis of 51 years compared with 63 years for SNP309T carriers (p = 0.004; log-Rank-test) indicating a susceptibility of the G/G genotype for the development of triple negative breast cancer. Patients with the A/A-genotype of MDMX SNP31826 with ER-negative tumors were diagnosed 11 years earlier compared with patients and ER-positive tumors (53.2 vs. 64.4 yrs; p = 0.025, log-Rank-test). Furthermore, in luminal B-like patients (HER2-independent) the C/C-genotype of MDMX SNP34091 was significantly correlated with a decreased event-free survival compared with the A/A-genotype (p < 0.001; log-Rank-test).ConclusionsWe showed that SNPs in the MDM2 and MDMX genes affect at least in part the onset and progression of breast cancer dependent on the ER-status. Our findings provide further evidence for the distinct etiological pathways in ER-negative and ER-positive breast cancers.

Mortality after breast cancer as a function of time since diagnosis by estrogen receptor status and age at diagnosis.

Our aim was to estimate how long-term mortality following breast cancer diagnosis depends on age at diagnosis, tumor estrogen receptor (ER) status, and the time already survived. We used the population-based Australian Breast Cancer Family Study which followed-up 1,196 women enrolled during 1992-1999 when aged <60 years at diagnosis with a first primary invasive breast cancer, over-sampled for younger ages at diagnosis, for whom tumor pathology features and ER status were measured. There were 375 deaths (median follow-up = 15.7; range = 0.8-21.4, years). We estimated the mortality hazard as a function of time since diagnosis using a flexible parametric survival analysis with ER status a time-dependent covariate. For women with ER-negative tumors compared with those with ER-positive tumors, 5-year mortality was initially higher (p < 0.001), similar if they survived to 5 years (p = 0.4), and lower if they survived to 10 years (p = 0.02). The estimated mortality hazard for ER-negative disease peaked at ~3 years post-diagnosis, thereafter declined with time, and at 7 years post-diagnosis became lower than that for ER-positive disease. This pattern was more pronounced for women diagnosed at younger ages. Mortality was also associated with lymph node count (hazard ratio (HR) per 10 nodes = 2.52 [95% CI:2.11-3.01]) and tumor grade (HR per grade = 1.62 [95% CI:1.34-1.96]). The risk of death following a breast cancer diagnosis differs substantially and qualitatively with diagnosis age, ER status and time survived. For women who survive >7 years, those with ER-negative disease will on average live longer, and more so if younger at diagnosis.

ERα upregulates the expression of long non-coding RNA LINC00472 which suppresses the phosphorylation of NF-κB in breast cancer.

Low expression of long intergenic non-coding RNA LINC00472 in breast cancer is associated with aggressive tumors and unfavorable disease outcomes in multiple clinical datasets, but the reasons for these associations were unknown. To study the mechanisms underlying the lncRNA's connection to breast cancer, we investigated the molecular targets and regulation of LINC00472 in breast cancer cells, and analyzed relevant molecular features in relation to patient survival. Gene expression profiles of breast cancer cells overexpressing LINC00472 were analyzed for its regulatory pathways and downstream targets. Effects of LINC00472 overexpression on cell behaviors were evaluated in vitro and in vivo. Meta-analysis was performed using online datasets and our own study. Analysis of LINC00472 transcriptome revealed ERα upregulation of LINC00472 expression, and an ERα-binding site in the LINC00472 promoter was identified. Evaluation of LINC00472 overexpression also indicated a possible link between LINC00472 and NF-κB. Cell experiments confirmed that LINC00472 suppressed the phosphorylation of p65 and IκBα through binding to IKKβ, inhibiting its phosphorylation. High LINC00472 expression inhibited tumor growth both in vitro and in vivo and suppressed aggressive tumor cell behaviors in vitro. Suppressing LINC00472 expression in ER-positive tumor cells increased cell aggressive behaviors. Tamoxifen treatment of ER-positive cells inhibited ERα and LINC00472 expression and increased p65 and IκBα phosphorylation. Meta-analysis showed that LINC00472 expression were higher in ER-positive than ER-negative tumors and that high expression was associated with better disease outcomes in ER-positive patients. The study demonstrates that ERα upregulates LINC00472 which suppresses the phosphorylation of NF-κB, and suggests that endocrine treatment may lower LINC00472 and increase NF-κB activities, leading to tumor progression and disease recurrence.

Abstract P1-09-10: Molecular and genetic characterisation of contralateral breast cancers in Northern Ireland: Opportunities for personalised surgery

Abstract Contralateral breast cancer (CBC) incidence is reported as 0.4-0.7% per year, equating to an approximate 10-15% 20 year risk after primary cancer diagnosis. Young age at primary diagnosis and a strong family history are consistently cited as significant risk factors for CBC development. Requests for contralateral prophylactic mastectomy (CPM) are rising, despite a lack of evidence that CPM significantly improves breast cancer specific survival. Therefore, a method to stratify CBC prognosis and risk at an individual level is required to allow us to personalise surgical decision-making for individual patients. This study aims to: 1. Explore prognostic factors following CBC diagnosis; 2. Investigate metastatic CBC prevalence by determining clonal relationships between primary and CBC tumours; 3. Assess the rate of germline predisposition gene mutations in a large cohort of Northern Irish CBC cases. A cohort of 403 CBC patients and 1:1 matched unilateral breast cancer controls (matched for age, year of diagnosis, nodal positivity and tumour morphology) were identified from the Northern Ireland Cancer Registry. Archival primary, CBC and normal lymph node tissue from these CBC patients were obtained for somatic and germline sequencing of a custom 94-gene panel, comprising genes most commonly mutated in breast cancer and all known predisposition genes. CBC patients had a significantly increased risk of breast cancer specific mortality when compared with 1:1 matched unilateral breast cancer controls (HR 6.45, CI 4.27-9.77). Within the CBC cohort, a shorter time interval between primary and CBC diagnosis is associated with increased breast cancer mortality, whereby women with &amp;lt;5 years between diagnoses have the poorest survival (p=0.001). In terms of receptor status, the PR status of ER positive primary and CBC tumours differed significantly (p&amp;lt;0.001). 89.1% of ER positive primary tumours were PR positive, whilst PR positivity was only 54.4% in ER positive CBCs. Indeed, 36.6% of patients with an ER+/PR+ primary BC developed an ER+/PR- CBC, after exposure to anti-oestrogen therapy. Targeted next generation sequencing of 42 cases to date has provided evidence of shared somatic mutations suggestive of metastatic clonality between primary and CBC in four cases (9.5%). Germline pathogenic predisposition gene mutations were identified in five (11.9%) patients and a further ten (23.8%) patients were noted to have germline predisposition variants of unknown significance. Breast cancer specific mortality risk was significantly higher in CBC patients compared with matched unilateral cancer controls, in excess of what would be expected from two primary cancer events. This finding suggests that there are as yet unquantified factors contributing to this excess hazard. Poorer outcome with shorter inter-tumour diagnostic intervals, in addition to evidence of primary-CBC clonality in some cases, suggests that metastatic CBC may play a role in excess risk. Additionally, the significant reduction in PR positivity of ER positive CBC tumours after ER+/PR+ primary tumours, could suggest that acquired hormonal resistance following primary anti-oestrogen therapy exposure may contribute to this additional mortality hazard. Citation Format: McIlmunn CG, Bannon FJ, Fitzpatrick D, Ervine A, Boyd C, Cameron I, Harita T, James J, Gonzalez de Castro D, Savage KI, McIntosh SA. Molecular and genetic characterisation of contralateral breast cancers in Northern Ireland: Opportunities for personalised surgery [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 P1-09-10.

Abstract P6-05-05: Epigenetic regulation of ER through miR-18a shows ECM activation and identifies poor prognostic subtype within ER+ve HER2-ve breast cancer

Abstract Introduction Although endocrine therapy has improved the survival of patients with hormone receptor (HR) positive breast cancer considerably, patients with weaker HR expression tend to have poorer outcomes. There exists significant intra tumoral heterogeneity of ER expression and a recent study suggested that patients with high intra tumoral heterogeneity of ER had an increased long term risk of recurrence (Lindstrom et al JNCI 2018). Multiple reports have supported a role for alterations in microRNA levels having a role in promoting tumor invasion and metastasis. In this study we have explored the epigenetic regulation of ER by microRNA hsa-miR-18a-5p and its role as a prognostic marker in ER positive tumors. Methods 123 surgically excised specimens of ER positive primary breast cancers were analyzed. Samples were segregated into high and low ER positive groups using 50% positivity as the cut-off. Relative abundance of hsa-miR-18a-5p in these samples was assessed using a TaqMan qRT-PCR. hsa-miR-18a-5p was over-expressed in MCF7 cells using a synthetic mimic and downstream changes in gene and protein expression were assessed using q-RT PCR, immunofluorescence and western blot. Migratory and proliferative ability was assessed using wound healing and MTT assay respectively. Microarray based global gene expression analysis of hsa-miR-18a-5p over-expressing cells was performed. Disease free survival analysis was done using Kaplan-Meier survival analysis. Result We estimated the relative abundance of hsa-miR-18a-5p in 123 ER positive primary breast cancers and found the distribution of this miRNA to be highest in the lower ERgroup (p&amp;lt;0.05). hsa-miR-18a-5p also correlated negatively with ESR1 and PGR (p&amp;lt;0.05).To further probe the role of miR-18a-5p in invasion and metastasis, we over-expressed this microRNA using a synthetic mimic in a luminal cell line MCF7. Microarray analysis revealed an increase in the expression of ECM associated genes, and the Cadherin signalling pathway. We observed a decrease in the expression of luminal genes (PGR, TFF1, GREB1; p&amp;lt;0.05), loss of Tff1 and increase in the levels of basal cytokeratin 14 at the protein level in addition to an increase in proliferative rate of upto 35% (p&amp;lt;0.05). Further, we observed a 15 % (p&amp;lt;0.05) increase in the invasive ability by wound healing assay with a significant loss in the levels of E-cadherin protein. In order to study the prognostic importance of hsa-miR-18a-5p, we performed Kaplan-Meier survival analysis and found that stratification of the ER+ve tumor samples by hsa-miR-18a-5p levels produced significant separation of the groups based on disease-free survival (log rank p &amp;lt;0.05). The prognostic value was also validated with multivariate Cox-proportional hazard analysis (Hazard Ratio (HR)of 3.18 (1.07-9.42); p=0.03). Conclusion The results from the over-expression of miR-18a-5p in MCF7 cells support the existence of an epigenetic pathway of repression of the luminal phenotype and increased acquisition of traits associated with basal-like and mesenchymal characteristics. It is possible that the over-expression of miR-18a-5p plays a role in patients with poorer outcomes. Citation Format: Nair MG, Prabhu JS, Korlimarla A, Hari P, Rajarajan S, Kaul R, Alexander A, Raghavan RS, Rakesh S, Correa M, Prasad M, Srinath B, Tirumalai SS. Epigenetic regulation of ER through miR-18a shows ECM activation and identifies poor prognostic subtype within ER+ve HER2-ve breast cancer [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 P6-05-05.

Abstract P2-02-04: CD73 expression regulated by estrogen signaling associates with poor prognosis in estrogen receptor (ER)-positive breast cancer

Abstract Introduction: CD73, a cell surface enzyme, catalyzes the generation of adenosine from ATP and ADP in the tumor microenvironment along with CD39. Accumulated extracellular adenosine functions as immune-suppressor, and also binds to adenosine receptors which promotes angiogenesis and cell proliferation that results in accelerate cancer progression. However, the clinical significance and molecular function of CD73 expression in breast cancer remains unclear. Methods: Utilizing publicly available breast cancer cohorts of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), clinical significance as well as underlying mechanisms were investigated. Molecular experiments were carried out in MCF7 cells, ER-positive breast cancer cell line, to investigate the role of estrogen signaling on CD73/CD39 expression. Results: In treatment naïve TCGA cohort, CD73 expression level was significantly lower in ER-positive breast cancers compared to ER-negative tumors. Higher CD73 expression was associated with worse overall survival in whole cohort (p=0.021) and ER-positive tumors (p=0.003), but not in ER-negative tumors. Gene Set Enrichment Analysis revealed that estrogen response gene sets (Early; NES=-1.57, p=0.043, Late; NES=-1.61, p=0.021) were significantly enriched in CD73 low expressing ER-positive tumors, suggesting estrogen signaling may repress CD73 expression. To test this hypothesis, we analyzed the expression of CD73 and CD39 in MCF7 cells treated with estrogen, tamoxifen or both. Our data revealed that estrogen treatment suppressed CD73 and CD39 expression, whereas tamoxifen treatment enhanced expression of the genes. These findings suggest that CD73 and CD39 gene expression is suppressed by estrogen signaling, whereas binding of ER antagonists such as tamoxifen can remove the repressive effect on gene expression. On the other hand, epithelial-mesenchymal transition (EMT) (Normalized Enrichment Score; NES=2.41, p&amp;lt;0.001) and angiogenesis (NES=2.33, p&amp;lt;0.001) gene sets were significantly enriched in CD73 high expressing ER-positive tumors. CIBERSORT, which is an algorithm to estimate infiltrating immune cells by gene expression, demonstrated that CD73 high expressing ER-positive tumors have less infiltrating CD8-positive T cells, memory B cells and plasma cells, implying that CD73 high expressing tumors have immune suppressive environment, which is in agreement with the notion that CD73 high tumors are immunosuppressive. Finally, we found that CD73 expression was significantly elevated post-chemotherapy compared to tumors prior to the treatment (p=0.007), and CD73 high expression patients showed worse relapse-free survival in neoadjuvant chemotherapy patients cohort (p=0.003). Conclusion: Molecular studies revealed that CD73 expression is regulated by estrogen signaling. Increased expression of CD73 significantly correlates with worse outcomes in ER-positive breast cancer patients. This may be due to upregulated pro-metastatic gene signatures such as EMT and angiogenesis as well as less infiltration of anti-cancer immune cells by adenosine generated by CD73 in the tumor microenvironment. Our data reveals an intriguing mechanism which may be responsible for recurrence and metastasis of ER-positive breast cancer. Citation Format: Katsuta E, Anand V, Yan L, Dasgupta S, Takabe K. CD73 expression regulated by estrogen signaling associates with poor prognosis in estrogen receptor (ER)-positive breast cancer [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 P2-02-04.

Abstract P5-04-13: Splicing factor ESRP1 controls ER-positive breast cancer progression by altering metabolic pathway genes

Abstract Background Epithelial Splicing Regulatory Protein (ESRP1)is a key splicing factor that regulates Epithelial-to-Mesenchymal Transition (EMT) splicing program. Our previous study demonstrated that high levels of ESRP1 are associated with poor prognosis in human ER-positive (ER+) breast tumors in an independent manner of EMT process. We next explored the potential mechanisms that contribute to the role ESRP1 in endocrine therapy-resistant breast cancer. Methods Probe based-Human Transcriptome Array 2.0 (HTA; Applied Biosystems/Thermo Fisher) was performed using RNAs from control and ESRP1 knockdown cells (LCC2 versus 2C3 ESRP1 and LCC9 versus 9C2 ESRP1) of endocrine resistant breast cancer. Functional enrichment analyses were performed using the DAVID functional annotation tool (http://david.abcc.ncifcrf.gov/). To confirm the functional importance of ESRP1 on regulation of cellular metabolism, we have performed experiments that analyze the metabolic substrate flux in response to ESRP1 knockdown in resistant cells (The Seahorse XFp Cell Energy Phenotype Assay). Differentially expressed genes were validated using Western blotting assay. Results Transcriptome profiling of ESRP1in 2C3 and 9C2 knockdown models revealed differentially expressed genesusing HTA 2.0 platform. In LCC2 versus 2C3 ESRP1 knockdown, expression of 1186 genes (1263 transcripts) have been altered significantly, while 413 genes (432 transcripts) have been significantly regulated in LCC9 versus 9C2 ESRP1 knockdown with FDR&amp;lt;0.1 Of these significant genes, 34 downregulated and 68 upregulated (102 genes total) were shared by both 2C3 and 9C2 ESRP1 knockdowns. Using the DAVID Functional Annotation Clustering Tool, we identified the biological processes altered significantly in response to ESRP1 knockdown. The most significant annotation clusters downregulated in ESRP1 knockdown consists of fatty acid metabolism/lipid metabolism (SCD, ACACA, FASN, ACAT2, PLCH1, and HPGD), and oxireductase processes (SCD, PHGDH, FASN, DHTKD1 and HPGD. We confirmed the altered metabolic function using the Seahorse analyzer. These analyses confirmed that ESRP1 knockdown altered the glycolysis rate (extracellular acidification rate; ECAR) in both tamoxifen-resistant and fulvestrant-resistant models. In addition, ESRP1 knockdown decreased the mitochondrial respiration in tamoxifen-resistant cells, but not fulvestrant resistant cells. Using Western blotting, we validated the altered levels of fatty acid synthase (FASN) and Stearoyl-CoA desaturase 1 (SCD1), key enzymes in fatty acid metabolism. Phosphoglycerate Dehydrogenase (PHGDH), a poor prognosis marker in cancers including breast cancer, was also downregulated in response to ESRP1 knockdown. Taken together, we have demonstrated a novel functional impact of ESRP1 on the regulation of tumor growth at the functional and molecular level independent of EMT. Conclusions For the first time, we show the role of ESRP1 in altering the cellular metabolism thereby contributing to tumor growth. The study provides a molecular evidence for the role of altered metabolism in determining adverse prognosis of ER+ breast cancer via the control of ESRP1, a splicing factor. Further studies to determine the therapeutic value are underway. Citation Format: Gokmen-Polar Y, Gu Y, Gu X, Badve SS. Splicing factor ESRP1 controls ER-positive breast cancer progression by altering metabolic pathway genes [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 P5-04-13.

Abstract P1-09-05: Clinical and genomic characteristics of borderline ER-positive breast cancers

Abstract Background: Though current guidelines classify breast tumors with ≥1% estrogen receptor (ER) positivity by immunohistochemistry as ER positive, “borderline” tumors expressing ≥1 – &amp;lt;10% positivity have biological similarities to ER negative tumors in prior studies. We sought to describe molecular features of ER-borderline breast cancer in a diverse cohort of incident cases and identify characteristics associated with high-risk borderline tumors. Methods: We used the Carolina Breast Cancer Study, a large population-based study that oversampled young and black women in 44 counties of North Carolina, to study characteristics of ER-borderline cases compared to other ER-positive and ER-negative tumors. Gene expression of ESR1, PAM50 subtype, and risk of recurrence score (ROR-PT, calculated from subtype, proliferation level, and tumor size; categorized as low, medium, high) were quantified using RNA counting methods, with finite mixture modeling to establish a cutoff for ESR1-high vs. -low tumors. The relative frequency differences of clinical and genomic features of borderline vs. positive/negative tumors were estimated using linear regression, adjusted for age and race. Recurrence risk was evaluated by ER status, with and without receipt of endocrine therapy using Kaplan-Meier curves and Cox proportional hazards models. Results: Of 2,859 eligible patients, 8% (n=217) were ER borderline, 26% (n=757) were ER negative and 66% were ER positive. Compared to other ER positive tumors (which were 5% basal-like, 89% luminal, 8% high ROR-PT, 16% ESR1-low), borderlines (40% basal-like, 42% luminal, 26% high ROR-PT, 71% ESR1-low) had a higher relative frequency of basal-like subtype (+37.7%, 95% CI 27.1, 48.4), high ROR-PT (+52.4%, 95% CI 36.8, 68.0), and ESR1-low status (+26.3%, 95% CI 20.2, 32.5). In log-rank analysis, recurrence risk in borderlines was not significantly different from ER negatives. However, after adjusting for patient and tumor factors, recurrence risk in borderlines treated without endocrine therapy was the highest of all subtypes (HR 2.9, 95% CI 1.6, 5.0) and persisted after further adjusting for ROR-PT. Results are summarized in Table 1. Frequency of high ROR-PT was 22% among white women with borderline tumors but was significantly more common among black women with borderline tumors (+26.0%, 95% CI 7.1, 45.0). Among borderlines, high grade was associated with basal-like subtype (+43.4%, 95% CI 18.7, 68.0; reference = non-basal subtype, 52% high grade). Table 1:Recurrence-free survival by ER subtype and endocrine therapy receipt Hazard Ratio (HR) Model 1HR Model 2ER PositiveREFREFER Borderline, no endocrine therapy2.9 (1.6, 5.0)2.3 (1.1, 4.9)ER Borderline, endocrine therapy1.5 (0.7, 3.2)1.5 (0.6, 3.7)ER Negative1.8 (1.3, 2.6)1.5 (0.9, 2.4)Adjusted for (1) age, race, stage, grade, year of diagnosis, (2) variables in (1) and ROR-PT Conclusion: Consistent with prior studies, borderline tumors demonstrated characteristics that were intermediate between other ER positive and negative tumors, with heterogeneity of genomic features. Given the higher recurrence risk among borderlines, identifying aggressive borderline tumors is important. Black women and those with high tumor grade may benefit from further genomic assessment to guide treatment decisions. Citation Format: Benefield HC, Allott EH, Sun X, Reeder-Hayes KE, Troester MA. Clinical and genomic characteristics of borderline ER-positive breast cancers [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 P1-09-05.

Abstract P3-10-22: BluePrint molecular subtyping versus HER2 assessment by immunohistochemistry and FISH in the real-world diagnostic setting

Abstract Background: Both the Aphinity and ExteNET trials for anti-HER2 targeted agents were challenged in showing significant benefit of added HER2-targeted treatment. The findings suggested biological heterogeneity in HER2+ cancers, not entirely identified by IHC/FISH, requiring more nuanced biomarkers to clearly identify patient subsets who may derive benefit from new HER2-targeted agents. We have previously shown that BluePrint (80-gene) molecular subtyping reclassifies nearly half of HER2+/ER+ patients to Luminal-type with differential neoadjuvant treatment response (Whitworth, Ann Surg Oncol 2014; Whitworth, ASCO 2018). Here, we evaluated the reclassification rate in the real-world diagnostic setting. Methods: Physicians regularly provide pathology reports to Agendia, Inc for samples which are processed for MammaPrint (70-gene signature) and BluePrint molecular assays as part of routine diagnostic care. For this analysis, 4986 sequentially available pathology reports (submitted between October 2016 to October 2017) were reviewed; HER2 and ER IHC results were captured. The molecular subtype was compared to IHC/FISH status. Results: HER2 IHC/FISH results were available for 1568 samples. Of those, 85% (1330/1568) were HER2-nonamplified, 10% (153/1568) were HER2-equivocal, and 5% (85/1568) were HER2-amplified by IHC/FISH. Of the HER2-nonamplified tumors, BluePrint reclassified 0.1% (2/1330) as HER2-type. Of the HER2-equivocal tumors, none were HER2-type by BluePrint; 91% (139/153) were Luminal-type and 9% (14/153) were Basal-type. Of the HER2-amplified tumors, 15% (13/85) were dominant HER2-type, 79% (67/85) were dominant Luminal-type, and 6% (5/85) were Basal-type. Conclusions: In this set of tumors identified as HER2-amplified by IHC/FISH, BluePrint reclassified 85% of tumors to non-HER2 molecular subtypes, mostly Luminal-type for ER-positive tumors and Basal-type for ER-negative tumors. Moreover, BluePrint gave clarity where IHC/FISH could not, classifying all HER2-equivocal tumors to non-HER2 subtypes. Additional therapeutic options should be explored for HER2+/ER+ BluePrint Luminal-type patients who have observed much lower pCR rates versus BluePrint HER2-type patients (12% vs. 51%, respectively; Lee, AACR 2018). HER2 IHC/FISH vs BluePrint SubtypeHER2 IHC/FISH StatusHER2-type Luminal-type Basal-type Total IHC ER-IHC ER+IHC ER-IHC ER+IHC ER-IHC ER+ Nonamplified (ER-Unknown, n=28)118120353361330Equivocal (ER-Unknown, n=6) 2131104153Amplified (ER-Unknown, n=2)112 664 85Total21310140067401568 Citation Format: Treece T, Audeh W, Navarro F, Wei J. BluePrint molecular subtyping versus HER2 assessment by immunohistochemistry and FISH in the real-world diagnostic setting [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 P3-10-22.