Absence Of Estrogen Receptors Research Articles

Abstract PS7-86: Molecular determinants of racial disparity guide triple-therapy for triple negative breast cancer

Abstract Background and Aim: Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and HER2 receptor expression. Mortality from TNBC is significantly higher in African American (AA) women in comparison to White American (WA) women (5-year relative survival of only 14% for AA in comparison to 36% for WA) even though the incidence rates are lower in AA women. Irrespective of stage at diagnosis, AA-TNBC is more aggressive with higher metastasis and a poorer survival than WA-TNBC; therefore, it is imperative to understand the molecular determinants that drive aggressive progression of AA-TNBC. Overall aim of this study is to decipher the alterations in the molecular circuitry underlying racial disparity in TNBC progression in AA women compared to WA women. Results: AA-TNBC cells (HCC1806 and HCC1569) exhibited increased growth and higher migration potential; higher expression of stemness factors, increased number of mammospheres and higher CD44+/CD49f+ population in comparison to WA-TNBC (Hs578t, BT549, HCC1937 and HCC1187) cells. We analyzed RNA sequencing data of multiple AA and WA TNBC cell lines to query the status self-renewal pathways (Wnt/βCatenin, GLI1/Shh and YAP-TAZ) and observed significantly higher levels of GLI1 in AA-TNBC cell lines while no significant alterations were observed in other pathway components. Further analysis of TCGA dataset revealed a positive correlation between GLI1 and Notch1 in AA-TNBC with a negative correlation in WA-TNBC. Increased expression of components of GLI1 and Notch1 pathway (SHH, Jagged, cleaved-Notch (NICD), Hes1 and FOXM1) were noted in AA-TNBC compared to WA-TNBC cells. AA-TNBC cells showed increased nuclear localization of GLI1 and NICD as compared to WA-TNBC cells. We observed that GLI1 and NICD co-localize and co-immunoprecipitate in AA-TNBC indicating a direct interaction between these two transcription factors. High expression of GLI1 and Notch1 correlated with poor recurrence free survival in TNBC patients. Analyses of clinical samples revealed higher levels of nuclear NICD and GLI1 in AA-TNBC in comparison to WA-TNBC. Concomitant inhibition of GLI1 and Notch1 using respective small molecule inhibitors, GANT61 and DAPT, along with standard chemotherapeutic agents (Doxorubicin and carboplatin) effectively inhibited AA TNBC tumor growth in mice. Also, ex vivo analyses of tumor cells showed reduced migration and invasion potential as well as downregulation of CD44+/CD49f+ and ADLH1A1+ population in a synergistic manner. Combined treatment with GANT61+ DAPT+ Carboplatin effectively reduced stem cell frequency of AA-TNBC tumors in in vivo limiting dilution assay. Conclusions: In conclusion, these results show that AA-TNBC cells are inherently aggressive with increased growth, migration and stemness potential. We found aberrant activation of GLI1 and Notch pathway and a crosstalk between GLI1 and NICD whose inhibition effectively inhibits AA-TNBC and sensitizes AA-TNBC to standard chemotherapy. Our studies propose a ‘triple drug regimen’ for AA-TNBC tumors based on its molecular circuitry. Citation Format: Sumit Siddharth, Sheetal Parida, Nethaji Muniraj, Shawn M Hercules, Arumugam Nagalingam, Balázs Győrffy, Juliet Daniel, Dipali Sharma. Molecular determinants of racial disparity guide triple-therapy for triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-86.

Neuroprotective Effects of Chrysin Mediated by Estrogenic Receptors Following Cerebral Ischemia and Reperfusion in Male Rats.

Introduction:Ischemic stroke is one of the leading causes of morbidity and mortality worldwide. Neuroprotective strategies were reported to attenuate cognitive deficits after ischemic incidents. Here we studied the neuroprotective potential of chrysin in a rat model of cerebral Ischemia/Reperfusion (I/R) in the presence or absence of Estrogen Receptors (ERs).Methods:Adult male Wistar rats were pretreated with chrysin (CH) (CH; 30 mg/kg; gavage; for 21 consecutive days) alone or with selective ERs antagonists (ERα antagonist MPP; ERβ antagonist PHTPP; IP) or nonselective ERs antagonist (ICI182780; IP). Then, the bilateral common carotid arteries were occluded for 20 min, which was followed by 72 h reperfusion. Subsequently, cognitive performance was evaluated by Morris Water Maze (MWM) and shuttle box tasks, and afterward, their hippocampi were removed for ELISA assays and H&E staining. Oxidative indicators Malondialdehyde (MDA) and Glutathione Peroxidase (GPx), as well as inflammation mediators interleukin (IL)-1β and tumor necrosis factor-alpha (TNFα), were measured using commercial kits.Results:Results of the current study showed that the anti-oxidative and anti-inflammatory properties of CH are possible mechanisms that could improve cognitive deficits and prevent neuronal cell death following I/R (P<0.001). These effects were reversed by ICI182780 (P>0.05). Furthermore, when chrysin was co-treated with ERβ antagonist, PHTPP showed a weak neuroprotective effect in I/R rats. However, these parameters were not significantly different when chrysin was combined with ERα antagonist MPP.Conclusion:Our data confirm that chrysin could potentially serve as a neuroprotective agent against devastating effects of cerebral I/R injury, which may be mediated via its interaction with ERs, especially ERβ.

Pathological findings in the endometrium after microwave endometrial ablation

The acceptance of MEA in Japan is well demand due to its outstanding effectiveness and safety. Infrequently, a repeat MEA or hysterectomy is needed for recurrent menorrhagia in case of failure ablation. The reasons of recurrent menorrhagia subsequent MEA treatment are unclear. The objective of current study is to identify the possible causes of menorrhagia repetition following MEA, together with the observation of histological changes in the endometrium due to this treatment compared with normal cycling endometrial tissue. A total of 170 patients, 8 (4.7%) of them carried out hysterectomy after 16.8 months (range, 2–29 months) of MEA treatment. Normal (n = 47) and MEA (n = 8) treated paraffin embedded endometrial tissue were prepared for hematoxylin and eosin (H&E) and immunostaining study to recognize the histological changes in the endometrium as a result of MEA treatment. The histological features observed increased tubal metaplasia (TM) including negative expression of the estrogen receptor (ER) and progesterone receptor (PR) in the endometrium subsequent MEA treatment. Increased TM together with the absence of ER and PR expression might be a reasonable explanation for repetition menorrhagia in cases of failure ablation. Further study is required to clarify the molecular mechanisms of tubal metaplasia and the expression loss of hormone receptor in the endometrium as a result of MEA treatment. Current studies propose that low dose estrogen-progestin may not be effective with recurrent menorrhagia patient’s due to the inadequacy of hormone receptor expression in the endometrium following MEA.

Immunotherapeutic Approaches in Triple-Negative Breast Cancer: State of the Art and Future Perspectives.

Triple-negative breast cancer (TNBC) is characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). It accounts for 15%–20% of all breast cancers and is associated with an aggressive evolution and poor outcomes with the majority of recurrences and deaths occurring in the first 5 years. Chemotherapy remains the mainstay of treatment in the absence of effective targets, but the good understanding of immune tumor microenvironment, the identification of immune-related targets, and the role of tumor-infiltrating lymphocytes (TILs) in TNBC has allowed to develop promising immunotherapeutic strategies for this unique subset of breast cancer. Recently, immunotherapy is being extensively explored in TNBC and clinical trials have shown promising results. In this article, we tried to explain the rationale and mechanisms of targeting the immune system in TNBC, to report the results from recent clinical trials that put immunotherapy as a new standard of care in TNBC in addition to ongoing trials and future directions in the next decade.

Canady cold plasma conversion system treatment: An effective inhibitor of cell viability in breast cancer molecular subtypes

Breast cancer is a heterogenous disease which can be classified into subtypes by the presence or absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 receptor (HER2). Cold atmospheric plasma (CAP) has been shown to be a potential treatment for cancer. In this report, a 92–99% reduction of viability by CAP treatment was achieved across all seven tested breast cancer cell lines (p ≤ 0.05). Increasing treatment duration and power significantly reduced breast cancer cell viability (** f(2,2) ≤ 0.0176, *** f(5,14) ≤ 0.0033). The authors are the first to report that breast cancer sensitivity to CAP is based on receptor status. Cells with identical receptor status showed the least difference in CAP sensitivity (p ≤ 0.05), the difference being 33% between the two ER+/PR+/HER2- cell lines (p ≤ 0.05) and 22–44% between the three TNBC cell lines (p ≤ 0.05). HER2-negative cell lines, irrespective of ER/PR status, also showed ≤ 50% difference in CAP sensitivity (p ≤ 0.05). Moreover, demonstration of ER-/PR-/HER2+ CAP susceptibility and ER+/PR+/HER2+ CAP resistance suggests that ER/PR status is a significant factor in determining CAP sensitivity in HER2-positive cells. Our novel findings on CAP sensitivity will provide insight on how to optimize CAP treatment to better overcome CAP resistance and thus prevent tumor recurrence.

Abstract 3704: MicroRNA-3977 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting FoxM1

Abstract Triple negative breast cancer (TNBC) is an aggressive type of breast cancer characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). TNBC is associated with poor prognosis, high rates of relapse and distant metastasis despite surgery and adjuvant chemotherapy. The lack of effective targeted therapies for TNBC requires new therapeutic approaches. The Forkhead box protein M1 (FoxM1) is a member of forkhead superfamily transcription factors that has been associated with normal cell growth and proliferation through cell cycle transition and mitotic spindle control. FoxM1 is also identified as a master regulator of tumor metastasis. Based on analysis of pooled clinical datasets derived from online accessible databases as Breast Cancer Gene-Expression Miner v4.3 and Kaplan-Meier plotter, the expression and prognostic values of FoxM1 increase in TNBC compare with other breast cancer subtypes. Directly binding to the 3'-untranslated region (3' UTR) of FoxM1 mRNAs suppress its high expression in TNBC that leads to inhibition of cell proliferation, motility, and invasion. These results implied that FoxM1 is an essential poor prognostic factor and promising candidate target in the treatment of TNBC. We identified miR-3977 as a potential regulator of FoxM1 by using in silico prediction algorithms, and we investigated its role in TNBC. In this study, we found miR-3977 expression is significantly suppressed in TNBC cell lines and inversely correlated with FoxM1 levels. We demonstrate that miR-3977 directly bound to the 3′ UTR of FoxM1 mRNA and repressed its expression. We also find reduced miR-3977 expression is associated with shorter patient survival in TNBC based on Kaplan-Meier plotter database. Moreover, in vivo delivery of miR-3977 nanoparticles inhibited FoxM1 expression and tumor growth in TNBC xenograft mouse models. In conclusion, we provide the first evidence that miR-3977 functions as a tumor suppressor and gain of miR-3977 expression leads to decrease in FoxM1 expression and inhibits to cell growth, colony formation, invasion, and progression of TNBC. Taken together, our data suggest that miR-3977-based gene therapy is a potential therapeutic strategy in TNBC. Citation Format: Selda Karamil, Nuray Altintas, Bulent Ozpolat. MicroRNA-3977 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting FoxM1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3704.

Abstract LB-222: Aberrant activation of GLI1 and Notch1 contributes to racial disparity in triple-negative breast cancer progression

Abstract Background and Aim: Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and HER2 receptor expression. Mortality from TNBC is significantly higher in African American (AA) women in comparison to European American (EA) women (5-year relative survival of only 14% for AA in comparison to 36% for EA) even though the incidence rates are lower in AA women. Irrespective of stage at diagnosis, AA-TNBC is more aggressive with higher metastasis and a poorer survival than EA-TNBC; therefore, it is imperative to understand the molecular determinants that drive aggressive progression of AA-TNBC. Overall aim of this study is to decipher the alterations in the molecular circuitry underlying racial disparity in TNBC progression in AA women compared to EA women. Results: AA-TNBC cells (HCC1806 and HCC1569) exhibited increased growth and higher migration potential; higher expression of stemness factors, increased number of mammospheres and higher CD44+/CD49f+ population in comparison to EA-TNBC (Hs578t, BT549, HCC1937 and HCC1187) cells. We analyzed RNA sequencing data of multiple AA and EA TNBC cell lines for self-renewal pathways (Wnt/βCatenin, GLI1/Shh and YAP-TAZ) and observed significantly higher levels of GLI1 in AA-TNBC cell lines while no significant alterations were observed in other pathway components. Further analysis of TCGA dataset revealed a positive correlation between GLI1 and Notch1 in AA-TNBC with a negative correlation in EA-TNBC. Increased expression of components of GLI1 and Notch1 pathway (SHH, Jagged, cleaved-Notch (NICD), Hes1 and FOXM1) were noted in AA-TNBC compared to EA-TNBC cells. AA-TNBC cells showed increased nuclear localization of GLI1 and NICD as compared to EA-TNBC cells. We observed that GLI and NICD co-localize in AA-TNBC. High expression of GLI1 and Notch1 correlated with poor overall survival in TNBC patients. AA TNBC Tumor samples show higher expression of GLI1 and NICD in comparison to TNBC tumors from EA patients. Concomitant inhibition of GLI1 and Notch1 using respective small molecule inhibitors, GANT61 and DAPT, along with standard chemotherapeutic agents (Doxorubicin and carboplatin) effectively inhibited AA TNBC growth and progression in mice; growth, proliferation, migration and invasion of ex vivo tumor cells and downregulated CD44+/CD49f+ and ADLH1A1+ population in a synergistic manner. Combined treatment with GANT61+ DAPT+ Carboplatin effectively reduced stem cell frequency of AA-TNBC tumors in in vivo limiting dilution assay. Conclusions: In conclusion, these results show that AA-TNBC cells are inherently aggressive with increased growth, migration and stemness potential. We found aberrant activation of GLI and Notch pathway and a crosstalk between GLI1 and NICD whose inhibition effectively inhibits AA-TNBC and sensitizes AA-TNBC to standard chemotherapy. Citation Format: Sumit Siddharth, Anirudh Saxena, Nethaji Muniraj, Sheetal Parida, Arumugam Nagalingam, Dipali Sharma. Aberrant activation of GLI1 and Notch1 contributes to racial disparity in triple-negative breast cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-222.

Abstract 6233: APG-2575, a clinical stage BCL-2 selective inhibitor, sensitizes estrogen receptor-positive breast cancers to standard therapies in the preclinical models

Abstract Breast cancer is a heterogeneous disease with at least four categories according to the presence or absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. In ER+ breast cancer, anti-estrogen therapies with tamoxifen, or CDK4/6 plus aromatase inhibitors remain the standard endocrine therapy, whereas the combination of CDK4/6-targeted therapy plus fulvestrant (an ER degrader) follows when the disease progresses on the hormonal therapy. However, drug resistance to the current therapies frequently emerges and patients relapse after the initial remission. Developing more effective treatments becomes an urgent need. Among four subsets of breast cancer, ER+ breast cancer exhibits the highest BCL-2 expression (83% compared with 50% for HER2+, 18.5% for basal-like, and 41.4% for marker-null subtypes). Thus, inhibition of BCL-2 which triggers apoptosis of cancer cells may become an effective therapy and synergizes the current therapies. APG-2575 is a BCL-2 selective inhibitor currently in clinical trials in patients with hematologic malignancies. Here, in preclinical xenograft models of ER+ breast cancer in mice, we evaluated whether APG-2575 enhanced the sensitivity to tamoxifen or CDK4/6-targeted (i.e., palbociclib or palbociclib plus fulvestrant) therapy. The results revealed that, in ER+ MCF-7-derived xenografts, double or triple combination treatment with APG-2575 plus tamoxifen, palbociclib, or palbociclib and fulvestrant significantly attenuated tumor progression in comparison with single or double agents treatments without APG-2575. The combination of APG-2575 plus tamoxifen achieved 4/6 partial tumor regression (PR; 67%) and 2/6 complete regression (CR; 33%) in xenograft-bearing mice. The combination with palbociclib resulted in 4/6 stable disease (SD; 67%) and 1/4 CR (25%). The triple combination with palbociclib plus fulvestrant caused 3/4 PR (75%). The combination benefits were further investigated in tamoxifen-resistant models. The triple combination with palbociclib plus fulvestrant demonstrated superior antitumor activity in tamoxifen-resistant ER+ MCF-7 xenografts. In a patient-derived, tamoxifen-resistant, ER+ breast cancer xenograft model, APG-2575 plus palbociclib achieved 100% overall response rate (1/3 CR, 2/3 PR). The results suggest that BCL-2 inhibition substantially improves the current therapies in ER+ breast cancer in the preclinical setting. The mechanisms underlying the synergistic effect of the combinations are currently under investigation and will be included in our presentation. Taken together, we demonstrated that combining APG-2575 with tamoxifen or palbociclib therapies substantially enhances antitumor activity and overcomes tamoxifen resistance in the preclinical models of ER+ breast cancer, suggesting a novel strategy for the clinical development of BCL-2 inhibitors in ER+ breast cancers. Citation Format: Douglas D. Fang, Qiuqiong Tang, Ran Tao, Guoqin Zhai, Qixin Wang, Dajun Yang, Yifan Zhai. APG-2575, a clinical stage BCL-2 selective inhibitor, sensitizes estrogen receptor-positive breast cancers to standard therapies in the preclinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6233.

Epidemiology, Pattern of Recurrence and Survival in Triple-negative Breast Cancer

Background: Breast cancer is the most common cancer in the world. Triple-negative breast cancer (TNBC) characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and Her2neu receptor. This study investigated the epidemiological characteristics and survival in non-metastatic TNBC. Materials and methods: Data from medical records of patients with breast cancer between 20014 and 2018 were retrieved, and patients with TNBC were identified and analyzed for demographic and clinicopathological features. Survival analyses were performed using the Kaplan–Meier method for disease-free survival (DFS) and overall survival (OS). Results: A total of 457 nonmetastatic breast cancer patients were registered at our institute from January 2014 to August 2018, of which 137 were triple-negative breast cancer (TNBC). This accounted for 29.9% of nonmetastatic breast cancer during this period. With the median age of 45 years at diagnosis, the most common presenting complaint was breast lump. The median duration of symptoms was 30 months. The most commonly affected age group was 41-50 years. The majority of the patients were in a locally advanced stage (69.3%) while 30.7% were in the early stage. 29.2% recurrence at 38 months of median follow up. Recurrence was statistically significantly correlating with age ≤ 35 (p= &lt; 0.001), pathological stage (p= &lt; 0.001), nodal status at diagnosis (p= &lt; 0.001), perineural invasion (PNI) (p= &lt; 0.001), number of positive lymph nodes (p= &lt; 0.001). The mean DFS and OS were 43.6 and 46 months respectively. 3-year DFS and OS were 65.5% and 66.2 % respectively. Conclusion: TNBCs are high-grade tumors mostly presented in locally advanced stages and most of the patients are young. TNBCs are clinically aggressive with high risk of metastasis to visceral organs. The survival of TNBCs in the Indian scenario is poor in comparison to Western populations, probably due to racial factors, socioeconomic factors and health care access facility. &#x0D;

Anticancer mechanisms determination of L‐aminoacids compounds serie on MDA‐MB‐231 cell line TNBC

Breast cancer represents the second most frequently diagnosed neoplasm worldwide of which approximately 15% is subclassified as triple negative breast cancer (TNBC) [1], characterized by the absence of estrogen receptors, progesterone and growth factor Her2/neu. TNBC is considered as one of the most aggressive cancers, due to its null response to current endocrine therapies and trastuzumab [2]. Breast cancer is a pathology associated with aging and unhealthy lifestyles, it has been proven through in vitro studies that MDA‐MB‐231 TNBC cells overexpress HDAC8 as a consequence of epigenetic deregulation mechanisms, linked to the high proliferative capacity and therefore, a high risk of recurrence and low survival [3]. In this context, our work group have evaluated a serie of compounds on the MDA‐MB‐231 cell line, these compounds have been designed, synthesized and evaluated as iHDAC8 by Docking. Therefore, this work presents the cytotoxicity and evaluation of the antitumor effect on the MDA‐MB‐231 cell line of three compounds derived from L‐amino acids, BisLys, GluEt and I1, which contain the isoindolinyl group and I1 which has a carboxybenzoyl group. To evaluate citotoxicity of BisLys, GlutEt and I1 compounds, MDA‐MB‐231 cells were seeded and incubated at 37°C in a 5% CO2 with DMEM F12 medium supplemented with FBS. Once the cells reached 95% confluence they were treated for 24 hours adding 8 different concentrations of each of the compounds; DMEM F12 medium was used as a negative control and SAHA as a positive control. The proliferation and viability of TNBC cells was quantified by the colorimetric for 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Type of cell death whether it was necrosis or apoptosis was determined using Annexine V and propidium iodide by flow cytometry. Finally, the blocked cell cycle progression by those compounds was determined using Cayman 10009349 Kit by flow cytometry. The BisLys, GluEt and I1 compounds showed IC50 values of 0.83, 0.66 and I1 3.42 mM, respectively, and inhibited cell proliferation and viability in a dose‐dependent manner on the MDA‐MB‐231 cell line of TNBC. The GluEt compound induced death mainly by necrosis and the BisLys and I1 compounds by apoptosis. The BisLys, GluEt and I1 compounds induced cell cycle arrest in a dose‐dependent manner at the expense of the subG0/G1 phase. The compounds that induced the greatest apoptosis in MDA‐MB‐231 cells were BisLys and I1 which makes them leading compounds in suppressing the growth of MDA‐MB‐231 TNBC cells.

Regulation and New Treatment Strategies in Breast Cancer

Breast cancer classifications are based on the presence or absence of estrogen receptor and progesterone receptor along with the overexpression or amplification of the Her2 receptor. Although the overall 5-year survival rate of breast cancer patients has increased due to the use of targeted therapies, a subset of patients can acquire resistance over time or are unresponsive when presented in the clinic. Novel therapies focusing on molecular pathways and cell cycle regulation currently being used in the clinic may lead to increased response in this subset of patients.

Abstract LB-301: Integrative genomic analysis identifies distinct mutational, epigenetic and immunological patterns among triple-negative breast cancer subtypes

Abstract Triple-negative breast cancer is an inherently heterogeneous disease defined by the absence of estrogen receptor, progesterone receptor and human epithelial growth factor receptor amplifications. The lack of therapeutically targetable, high-frequency “driver” alterations has impeded development of targeted therapies for TNBC. TNBCs display transcriptional diversity with at least four tumor-intrinsic subtypes that include two basal-like (BL1, BL2), a mesenchymal (M) and a luminal androgen receptor (LAR) subtype. Each subtype displays unique biology and differentially responds to standard-of-care chemotherapy. Therefore, to better understand TNBC genomic alterations within molecular subtypes we have performed an integrative analysis of mutation, copy-number, gene expression, miRNA expression, reverse-phase protein array and methylation of 192 TNBC patients in The Cancer Genome Atlas and drug sensitivity of TNBC cell lines. Gene expression analysis confirmed our previous subtype-specific findings with enrichment of cell cycle, DNA replication response in BL1, Wnt and KRAS signaling BL2, androgen response in LAR and TGF-beta and mesenchymal differentiation in M. Despite having higher mutational burdens and similar copy number alterations, the BL1 and M subtypes display very different patterns of gene expression. These differences likely originate from epigenetic changes, as global methylation analysis demonstrated large hypomethylation across the genomes of M subtype. In contrast, the highly differentiated LAR subtype displayed global patterns of hypermethylation. Mutation analysis revealed enrichment of PIK3CA mutations in LAR tumors, NOTCH1, NOTCH2, NOTCH3 and FBXW7 mutations in M and BL1 tumors and activating MAPK pathway mutations (KRAS, NRAS, BRAF, MAP2K1, MAPK9 and MAP3K13) exclusive to BL2 tumors. Differential copy number analysis identified amplifications specific to the BL1 subtype (AKT3, KDR and KIT), BL2 subtype (TERT, EGFR and KRAS), M subtype (MYCN and WNT5B) and NOTCH pathway (BL1 and M). Reverse-phase protein array analysis confirmed activated MAPK signaling unique to the BL2 subtype and PI3K signaling in LAR. Cell line subtype correlations and published drug sensitivity screens verified LAR sensitivity to AR antagonists and PI3K/AKT/mTORC inhibitors, while BL2 cell models displayed sensitivity to MEK inhibitors. Subtype-specific differences in lymphocyte composition were inferred from gene expression with BL1 subtype displaying the highest CD8 levels. Despite having the greatest level of macrophages, the M subtype had the lowest ratio of M1/M2 polarized macrophages. These data suggest immune checkpoint therapies may be more effective in BL1 subtype and additional strategies targeting the immune suppressive macrophage population necessary for M tumors. Using a comprehensive multi-omics approach we defined the multi-platform genomic landscape of TNBC, identify potential therapeutic strategies and shed light on the evolutionary paths of TNBC. Citation Format: Brian D. Lehmann, Antonio Colaprico, Jianjiao Chen, Lily Wang, Jennifer Pietenpol, Xi Chen. Integrative genomic analysis identifies distinct mutational, epigenetic and immunological patterns among triple-negative breast cancer subtypes [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 LB-301.

Abstract 1952: Tumor microenvironment targeted Rosella nanoparticle for the detection of triple negative breast cancer by multispectral optoacoustic tomography

Abstract Purpose: Recent advances in the nanotechnology and molecular imaging provides an excellent opportunity for development of delivery vehicles and imaging probes to improve real-time assessment and early detection of breast cancer progression.The absence of estrogen receptor (ER), progesterone receptor (PR), or HER-2 genes, represents a major clinical challenge for triple negative breast cancer (TNBC). Because of the absence of reliable markers, there is an unmet clinical need for developing efficient methods to identify TNBC. We have developed an acidic pH targeted Rosella nanoparticles that can actively release IR780 dye into orthotopically implanted TNBC tumors to improve tumor detection using multispectral optoacoustic imaging technology (MSOT). Methods: The Rosella nanoparticles are a mesoporous silica base with wormhole pore architecture containing a chitosan gatekeeper and V3 pHLIP targeting peptide. The particles were synthesized using the sol-gel method and characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The Rosella particles were loaded with propidium Iodide (PI) or IR780 infrared imaging dye to evaluate pH-sensitive cargo release. Female athymic mice were implanted with MDA-MB-468 breast cancer cells by the mammary fat pad injection. Once the tumor reaches 3mm in size, athymic mice were intravenously injected with ROSELLA nanoparticles carrying IR780 dye and were imaged with MSOT. Results: We have synthesized the Rosella nanoparticle with wormhole pores that is 27 nm diameter and can encapsulate the IR780 imaging probes for the detection of the TNBC. The Rosella particle contains a chitosan gatekeeper which can degrade upon contact with acidic pH tumor to prevent off-target release and is further sensitized to pH using V3 peptide. The Rosella nanoparticles can detect acidic tumor microenvironment and can penetrate inside the tumor cells. As a model of TNBC, we have injected MDA-MB-468 cells into the mammary fat pad of the female athymic mice to develop breast tumors. Once the tumor reached 3mm in size, we have intravenously delivered ROSELLA nanoparticles to the athymic mice with TNBC tumors. Our results were demonstrated that the intravenous injection of the ROSELLA particles could detect the orthotopically implanted TNBC tumors (p&amp;lt;0.0001,n=5). Conclusion: most aggressive subtypes of breast cancer, TNBC has a poor response for majority of the FDA-approved breast cancer drugs. To overcome these limitations, the distinct nanoformulations with potent imaging technology will enable to develop tumor-penetrating nanoparticles to more effectively deliver chemotherapeutics or imaging agents with least off-target effects. Future translation of these technologies has a high clinical impact concerning our current treatment options for TNBC patients. Citation Format: Abhilash Samykutty, Molly McNally, Alexandra Thomas, William Grizzle, Lacey R. McNally. Tumor microenvironment targeted Rosella nanoparticle for the detection of triple negative breast cancer by multispectral optoacoustic tomography [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 1952.

Abstract 3468: Aberrant activation of gli1 and notch1 contributes to racial disparity in triple negative breast cancer progression

Abstract Background and Aim: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor and HER2 expression. Mortality from TNBC is significantly higher in African American (AA) women compared to European American (EA) women (5-year relative survival of 14% for AA compared to 36% for EA). Irrespective of stage at diagnosis, AA-TNBC is aggressive with higher metastasis and poorer survival than EA-TNBC; hence, it is imperative to understand the molecular determinants that drive aggressive progression of AA-TNBC. Overall this study aims to decipher the alterations in the molecular circuitry underlying racial disparity in TNBC progression. Results: AA-TNBC cells (HCC1806 and HCC1569) exhibited increased growth and higher migration potential in comparison to EA-TNBC (Hs578t, BT549, HCC1937 and HCC1187) cells. AA-TNBC cells also exhibited higher expression of stemness factors, increased number of mammospheres and higher CD44+/CD49f+ population. To decipher the molecular mechanism underlying these functional differences, we analyzed RNA sequencing data of multiple AA and EA TNBC cell lines for self-renewal pathways and observed significantly higher levels of GLI1 in AA-TNBC cell lines while no significant alterations were observed in other pathway components. Further analysis of TCGA dataset revealed a positive correlation between GLI1 and Notch1 in AA-TNBC (Pearson coefficient = 0.308303) with a negative correlation in EA-TNBC (Pearson coefficient = -0.06695). Immunoblot analyses showed increased expression of components of GLI1 and Notch1 pathway (SHH, Jagged, NICD, Hes1 and FOXM1) in AA-TNBC compared to EA-TNBC cells. AA-TNBC cells showed increased nuclear localization of GLI1 and NICD as compared to EA-TNBC cells. We observed that GLI and NICD co-localize in AA-TNBC cells and their interaction was confirmed using co-immunoprecipitation assays. High expression of GLI1 and Notch1 correlated with poor overall survival in TNBC patients. Concomitant inhibition of GLI1 and Notch1 using respective small molecule inhibitors, GANT61 and DAPT, along with standard chemotherapeutic agents (Doxorubicin and Carboplatin) effectively inhibited AA TNBC growth and progression in mice; proliferation, migration and invasion of ex vivo tumor cells and downregulated CD44+/CD49f+ and ALDH1+ population in a synergistic manner. Combined treatment with GANT61+ DAPT+ Carboplatin effectively reduced stem cell frequency of AA-TNBC tumors in in vivo limiting dilution assay. Conclusions: In conclusion, these results show that AA-TNBC cells are inherently aggressive with increased growth, migration and stemness potential. We found aberrant activation of GLI and Notch pathway and a crosstalk between GLI1 and NICD whose inhibition effectively inhibits AA-TNBC and sensitizes AA-TNBC to standard chemotherapy. Citation Format: Sumit Siddharth, Nethaji Muniraj, Sheetal Parida, Arumugam Nagalingam, Dipali Sharma. Aberrant activation of gli1 and notch1 contributes to racial disparity in triple negative breast cancer progression [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 3468.

Absence of estrogen receptor is associated with worse oncologic outcome in patients who were received neoadjuvant chemotherapy for breast cancer

Neoadjuvant chemotherapy (NAC) is a standard treatment for locally advanced breast cancer, especially for HER2-positive or triple negative breast cancer which shows good response to chemotherapy. However, because a result of biomarkers is, occasionally, changed after NAC, the treatment strategy should be differently applied for patients with locally advanced breast cancer. We compared the results of biomarkers before and after NAC to evaluate the association with disease prognosis and oncologic results. Fifty-seven patients with locally advanced breast cancer underwent NAC and the immunohistochemical (IHC) staining results were compared between before and after NAC. And the association between oncologic outcomes and biomarkers was analyzed. Negative status of estrogen receptor (ER) was associated with locoregional recurrence and distant metastasis both before and after NAC (p=0.021, 0.019; p=0.018, 0.036). And the negative status of progesterone receptor (PR) and triple negative status before neoadjuvant chemotherapy were also associated with death and distant metastasis, respectively. However, the changes of biomarkers after NAC in breast cancer were not directly associated with any oncologic outcomes. The absence of ER in breast cancer before and after NAC would be a significant prognostic factor for local recurrence and distant metastasis. Therefore, the absence of ER should be considered as important factor in determining the treatment strategy.

Prevalence of Triple Negative Breast Cancers from a Hospital-Based Cancer Registry in a Tertiary Care Hospital in Kerala, South India

Triple-negative breast cancer (TNBC) is defined by the absence of estrogen receptor (ER), progesterone receptor (PR) and over expression of Human epidermal growth factor receptor 2 (Her2neu). They are typically high-grade tumors and occur in young females. Our study is to determine the prevalence of triple negative breast cancers from a hospital-based cancer registry in the year 2016. This is a retrospective, descriptive study where secondary data analysis was done, by obtaining data from the hospital-based immunohistochemistry (IHC) register. The Estrogen receptor, Progesterone receptor and Her2neu receptor status of the breast cancers which presented to the hospital and the referral cases received for IHC in the year 2016 was obtained from the register which totaled to 335 cases. The type of breast carcinoma was also noted. In our study the mean age of breast cancer patients was 56.2±12.387 years. There was 331 (98.8%) females among the study population. The most prevalent type of breast cancer was invasive ductal carcinoma which was 330 (98.5%). We also found out the prevalence of mono negative, bi negative and triple negative breast cancers. The prevalence of triple negative breast cancer was 58 (17.31%). There was no statistically significant difference found between prevalence of triple negative breast cancer and age. Our study showed that the prevalence of triple negative receptor breast was 17.31% among the study population. This study would serve as a baseline study for further research to open up new therapeutic possibilities.

Triple-negative breast cancer: Pattern of recurrence and survival outcomes

Abstract Introduction: Triple-negative breast cancer (TNBC) is a subtype of breast cancer which is defined as the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 overexpression by immunohistochemistry. As the survival data on TNBC in the Indian population are scant, this study was done to analyze the clinicopathological features and clinical outcomes of TNBC patients. Materials and Methods: Data from medical records of patients with breast cancer between 2009 and 2014 were retrieved, and patients with TNBC were identified and analyzed for demographic and clinicopathological features. Survival analyses were performed using the Kaplan–Meier method for disease-free survival (DFS) and overall survival (OS). Results: A total of 1024 breast cancer patients were registered at our institute during the study period, of which 198 were TNBCs accounting for 19.3% of all breast cancers. Median age at the diagnosis was 50 years (range, 22–78 years). Lymph nodal positivity in TNBC was associated with larger tumor size (P = 0.003) and higher tumor grade (P = 0.01). At a median follow-up of 48 months (range, 12–88), 36 (19.1%) patients had recurrence of the disease, whereas 28 (14%) patients were lost to follow-up. Lung (52.7%) was the most common site of recurrence followed by bone (25%) and brain (11.1%). Three-year DFS and OS were 63.2% and 65.6%, respectively. On univariate analysis, nodal status, size of tumor, and lymphovascular invasion were found to have a significant impact on OS and DFS. On multivariate analysis, only nodal status was significant for DFS and OS (P &lt; 0.001 and P = 0.001, respectively). Conclusions: TNBCs have a rapid clinical course, and early recurrences are common inspite of timely medical intervention which reflects the aggressive tumor biology. This warrants further studies on intensification of chemotherapy and identification and development of targeted therapy aimed at decreasing recurrences and improving survival in this patient population.

IMMEDIATE AND LONG-TERM OUTCOMES OF DRUG TREATMENT IN PATIENTS WITH METASTATIC TRIPLE NEGATIVE BREAST CANCER

Triple-negative breast cancer (TNBC) comprises 12–20 % of all breast cancers. TNBC is defined by the absence of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2) expression. TNBC is a heterogeneous disease, with an aggressive clinical feature, a higher risk of both local and distant visceral and / or brain metastases. Recurrence usually develops between 1 and 3 years after the initial diagnosis and most deaths occur within 5 years. Epidemiologic studies illustrate a high prevalence of triple-negative breast cancers among young women. Triple-negative breast cancer is also more likely to occur in women that carry a BRCA mutation, especially if they are diagnosed at a young age. Cytotoxic chemotherapy remains the mainstay treatment for TNBC because there are currently no specific targets for treatment options (hormone receptors or HER-2 amplification). Chemotherapy combined with targeted agents including DNA repair with PARP inhibitors, EGFR inhibitors, anti-angiogenic agents and a Chk1 inhibitor produced modest improvement in response rate and overall survival. Nevertheless there’s no common standards for treatment such patients with metastatic TNBC. Progress in the development of new regimens and combination of drug treatment agents for patient with generalized TNBC remains an important challenge that could lead to improvement immediate and long-term outcomes.

PO-294 Role of fatty acid amide hydrolase (FAAH) in breast development and cancer

IntroductionThere are three major stages of breast development – embryonic, pubertal, and reproductive. During this complex developmental cycling, the epithelial compartment undergoes several rounds of proliferation, remodelling and cell death to eventually form an organised and functional mammary tree. The very nature of the pathways controlling these processes makes them susceptible to promote tumorigenic processes and ultimately lead to breast carcinoma.Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that hydrolyzes the endocannabinoid anandamide and related amidated signalling lipids. Here, we aimed at analysing the expression FAAH in breast physio-pathology.Material and methodsThe expression of FAAH was analysed by immunohistochemistry in a tissue microarray (TMA) containing ≈600 human breast cancer samples and in public microarrays data bases. Cell lines derived from different breast cancer subtypes have been analysed for FAAH expression and the effect of a Knock-out (CRISPR) and overexpression of FAAH examined. FACS and immunofluorescence were also performed to further assess the identity of cell populations. FAAH effects on breast CSC activity was examinated using mammosphere formation and aldehyde dehydrogenase (ALDH) activity assays.Results and discussionsWe have found that FAAH shows remarkable variations in expression within the human breast epithelial hierarchy. Particularly, FAAH expression is restricted to a certain subset of mature cells in normal breast tissue (i.e. luminal cells), with negligible levels being detected in the progenitor/stem cell subset, In line with this idea, pharmacological inhibition of FAAH with URB597 in the mammary stem cell line HC11 delayed differentiation to mature epithelial cells. In addition, we observed significant differences in duct formation between FAAH KO and WT mice during pubertal development.Our results also correlate low FAAH expression with more undifferentiated phenotypes, high histologic grade, absence of oestrogen receptor and triple-negative phenotype in breast cancer. Consistent with this, we found low FAAH mRNA levels associated with metastasis and poor prognosis gene signatures. Finally, pharmacoinhibition and knocking-out of FAAH in breast cancer lines, enriched the cultures in cancer stem cells.ConclusionTogether, our data suggest that FAAH plays a role in breast cell differentiation, both in normal development and in oncologic contexts.

Evaluation of the Expression Level and Hormone Receptor Association of miR-126 in Breast Cancer.

Breast cancer is a major cause of cancer-related death in women worldwide. miRNAs are new players of breast tumorigenesis, used as diagnostic and prognostic biomarkers. Among various miRNAs, miR-126 has been proposed to have a tumor suppressive role in HER2 positive cancer. However, to have a better understanding of its role, further validation is required. The aim of this study was evaluating miR-126 expression level in breast cancer tissues and investigating its potential association with HER2, estrogen and progesterone receptors. miR-126 expression level was measured in 108 specimens including 78 malignant and 30 normal samples using RT-qPCR. The outcome was statistically analyzed. In silico studies were performed to find the potential mechanism of action, through which miR-126 imposes its function. Down-regulation of miR-126 was observed in tumor samples, as compared to the matched normal tissues. Down-regulation of miR-126 was also associated significantly with the absence of estrogen receptor in malignant samples. No association between miR-126 expression and HER2 status was observed. Our in silico analyses showed the possible role of Crk, PI3K and Ras proto-oncogenes in breast cancer tumorigenesis. miR-126 is significantly down-regulated in breast cancer tissues. Statistically, it showed no correlation with HER2 positivity. However, the association between lower miR-126 and estrogen receptor negativity was observed.