bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P4-07-13: High microRNA-621 level predicts a better response to neoadjuvant chemotherapy in breast cancer patients
Authors: Xue J , Chen Y , Chi Y , Ye X , Wu Z , Wu J
Published: August 2014

Purpose: Weekly paclitaxel plus carboplatin (PCb) regimen is an effective neoadjuvant chemotherapy for breast cancer. However, some patients respond to this regimen poorly and suffer from side effects. In this study, we aimed to define the microRNA (miRNA) biomarker for predicting sensitivity to PCb chemotherapy in breast cancer patients, and to explore the potential molecular mechanisms.

Experimental Design: 81 patients with stages II and III breast cancer, who received four cycles of weekly PCb regimen preoperative chemotherapy, were included. Gene expression profiles from 31 patients was used as training set to determine differentially expressed genes (DEGs) and to predict miRNAs that may be associated with chemotherapeutic sensitivity. The other 50 patients were analyzed as validation set to test the accuracy of DEG identification and miRNAs prediction by quantitative real-time PCR. In parallel, in vitro and in vivo analyses were carried out to determine the potential mechanisms of miRNA-dependent drug sensitivity.

Results: Gene profiling analyses in the training set revealed significant correlation between high expression level of miR-345 and miR-621 and pathologic complete response (pCR) in breast cancer patients. We further validated that the expression level of miR-621 was significantly higher in tumor tissues of pCR patients than that in non-pCR patients (P = 0.01). In breast cancer cell lines, ectopic overexpression of miR-621 promoted apoptosis and increased chemosensitivity to paclitaxel and carboplatin both in vitro and in vivo. The potential miR-621-target genes were determined by TargetScan and miRNA CLIP-seq database, which were further confirmed as DEGs reversely-correlated to miR-621 in breast cancer patient samples. Among those, FBXO11 was verified as one of the direct targets of miR-621 in breast cancer cells, whose expression level negatively associated with chemosensitivity in breast cancer patients. The molecular mechanisms by which miR-621/FBXO11 axis regulates chemosensitivity may involve regulation of p53 transcriptional activity and epithelial-mesenchymal transition (EMT) in breast cancer cells.

Conclusions: Our study revealed a strong correlation between miR-621 expression and chemosensitivity in breast cancers. High level of miR-621 predicts sensitivity to PCb regimen neoadjuvant chemotherapy in breast cancer patients who tend to achieve pCR. The increased chemosensitivity may be mediated by down-regulation of FBXO11 gene, which leads to enhanced p53 transactivity and reduced EMT in breast cancer cells. Therefore, miR-621 may be used as predictive biomarker and the potential therapeutic target in breast cancer treatment.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-13.
Abstract P4-07-18: PDGFRbeta-induced miR-9 is up-regulated in triple negative breast cancer
Authors: Plantamura I , D'Ippolito E , Tessari A , Cresta S , Orlandi R , Moliterni A , Carcangiu ML , De Braud F , Tagliabue E , Iorio MV
Published: August 2014

miR-9 has been described as an oncogenic microRNA associated to a metastatic phenotype and able to induce EMT (epithelial-to-mesenchymal transition) through direct targeting of E-cadherin. However, data available concerning the expression and the role of this microRNA in different subgroups of breast cancer are still not exhaustive.

Evaluating miR-9 expression by Real-Time PCR in a series of 92 breast cancer specimens (35 luminal, 36 HER2, 21 triple negative), we found that this microRNA is increasingly higher in HER2 and Triple Negative versus ER positive patients (fold change 3 and 8 respectively).

Moreover, preliminary analysis of miR-9 expression in correlation with bio-pathological features and clinical data also indicates a trend in association with disease progression.

Triple Negative Breast Cancers represent a very aggressive breast cancer subgroup, still lacking specific markers for an effective targeted therapy; we investigated whether miR-9 might play a role in the biology of this tumor subtype. Preliminary data indicate that miR-9 is activated downstream PDGFRbeta, which represents a crucial player in the aggressive phenotype of Triple Negative Breast Cancer.

In summary, here we show that miR-9 is significantly upregulated in triple negative breast cancer in comparison with other breast cancer subgroups and is activated downstream PDGFRbeta.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-18.
Abstract P4-12-03: Role of primary tumor molecular characteristics in identifying relapses in HER2-positive breast carcinomas adjuvantly treated with trastuzumab
Authors: Triulzi T , De Cecco L , Giussani M , Canevari S , Carcangiu ML , Menard S , Campiglio M , Tagliabue E
Published: August 2014

Trastuzumab, a recombinant humanized monoclonal antibody directed to the HER2 protein, has shown survival benefits in women with HER2-positive breast cancer, and treatment is now FDA-approved in combination with chemotherapy. Nonetheless, some patients relapse after treatment, underscoring the need to identify patients for whom chemotherapy + trastuzumab is adequate versus patients requiring additional drugs. To search for factors predictive of relapse in HER2-positive breast carcinoma patients treated adjuvantly with trastuzumab, we conducted gene expression profiling analysis in 53 cases selected among a cohort of 243 patients (32 of whom relapsed) treated in the clinic with chemotherapy + trastuzumab in our institute during 2006-2009, with median follow-up of 32 months; the 53 cases comprised 23 of the relapsed and 30 of the non-relapsed patients with similar clinico-pathological characteristics (size, lymph node involvement and estrogen receptor positivity) for whom sufficient material was available. RNA extracted from formalin-fixed, paraffin-embedded (FFPE) was profiled using HumanHT12_v4 wgDASL expression BeadChips (Illumina). A Cox's proportional hazard model was used to estimate the association between gene expression and relapse-free survival (RFS), and a multivariate permutation test was used to check the proportion of false discoveries. Analysis identified 330 probes corresponding to 308 unique genes as significantly associated to RFS (a<0.005; permutation test p<0.01), showing an enrichment for gene ontology biological processes such as cell cycle regulation of immune cell proliferation and activation, cytoskeleton organization, ER pathway and ion transport.

We also tested the feasibility of developing a predictive model including the gene expression data. Based on 10-fold cross-validation, our model was able to stratify patients into two groups (high and low risk), with a 4-year RFS probability of 14.5% in high-risk versus 87% in low-risk tumors (HR = 8.33, 95% CI = 3.53-18.18, p<0.0001). To test whether this signature identifies patients with intrinsic poor prognosis independent of trastuzumab treatment, we conducted in silico analysis on a dataset containing 144 HER2-positive breast carcinomas treated with chemotherapy alone; results revealed an inverse association between risk groups and prognosis, with an overall survival probability of 83% in our high-risk group and 63% in the low-risk group (HR = 0.60, 95% CI = 0.37-0.94, p = 0.0249).

Together, our results suggest the usefulness of molecular characteristics of primary tumors as indicators of early relapse in patients treated adjuvantly with trastuzumab and as tools to identify patients for whom additional treatments are required.

Supported by AIRC.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-03.
Abstract P4-12-21: Trastuzumab cardiac toxicity and HER2 polymorphism: A case-control study
Authors: Mailliez A , Dumont A , Bonneterre J
Published: August 2014

Background: Gene polymorphisms of HER2 have been identified. Their impact on the risk of breast cancer, the response and the toxicity of anti HER2 treatments have been studied. According to a small report of Beauclair (2007), the polymorphism at codon 655 of the HER2 gene may represent a risk factor for cardiotoxicity induced by Trastuzumab (T). In this study, we evaluated the incidence of the Val655Ile polymorphism in (neo)adjuvant breast cancer patients (pts) with cardiac toxicity and in matched patients without cardiac toxicity.

Methods: 40 HER2 positive breast cancer pts treated between 2008 and 2011 by (neo)adjuvant sequential chemotherapy plus T developed a cardiac toxicity. Definition of cardiac toxicity was a 10% absolute decrease in LVEF (Left Ventricular Ejection Fraction) or a decrease in LVEF to a threshold value of <50%. 84 pts without cardiac toxicity were matched according to age, received dose of anthracyclin and T, BMI and cardiac risk factors. Polymorphism of Her2 gene in codon 655 was searched by PCR on lymphocyte DNA from blood samples in this population and in the control group. Genomic DNA was extracted from EDTA blood samples (300ml) on the MagNa Pure Compact instrument. The HER2 Ile655Val (rs1163201) polymorphism was detected by sequencing using a 3730 DNA Analyser.

Results: In the cardiac toxicity group 2,5% (1 patient) presented a genotype Val/Val at HER2 codon 655, 40% (16) were heterozygous (Ile) and 57,5% (23) had the wild type. In the control population, the incidence was respectively 3,6% (3 pts), 36,9% (31) and 59,5% (50). No differences were found between the both groups (p = 0,91).

Conclusions: The incidence of mutated, heterogeneous and wild type genotype was similar to the literature. We could not confirm the Val allele at HER2 codon 655 as a risk factor of cardiotoxicity. HER2 polymorphism cannot be used in clinical practice to predict the risk of cardiac toxicity of T.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-21.
Abstract P4-15-09: An exploratory analysis of inactivation of PI3K/AKT/mTOR signaling pathway using neoadjuvant BKM120 in PI3KCA mutated early breast cancer
Authors: Estevez LG , Suarez A , Calvo I , Fernandez-Abad M , Perea S , Hidalgo M
Published: August 2014

Background: The PI3K/AKT/mTOR signaling pathway plays a key role for the growth and survival of breast cancer cells and aberrations such as phosphatidylinositol-3-kinase (PI3KCA) mutations are common. BKM120 is an oral pan-class I PI3KCA inhibitor. The aim of this study was to evaluate neoadjuvant treatment with BKM120 in PI3KCA mutated early breast cancer in order to assess the effect in PI3K/Akt/mTOR signalling pathway.

Methods: Patients (pts) with previously untreated invasive, non-metastatic histologically confirmed breast cancer, with a tumor size ≥ 1,5cm and non-urgent surgical treatment were enrolled. Only women with ER+ / HER2- and PI3KCA gene mutated were eligible. Patients received treatment with BKM120 (100mg/day; administered orally) during 4 weeks. Subsequently, surgery was performed. Inmunohistochemical analysis of pAkt and pS6 (the H-score intensity x%) as well as KRAS mutation were evaluated on tumor tissue at surgery. H score < 150 was related with inactivation of PI3K signalling pathway.

Results: To date 24 patients has been included in the study (median age, 52,5 years; range 38-69 years;). 13 out of 24 (54%) had PI3KCA mutated and 8 have completed BKM120 treatment. PI3KCA mutation: Exon 20, 4 pts; exon 9, 2 pts; exon 4, 1 pts and exon 20 and 9, 1 pts. One patient discontinued treatment due to adverse event (nausea grade 2). The most relevant adverse events included transitory increase of transaminases. No serious or grade 4 adverse events were observed. Preliminary results of 7 pts showed inactivation of pAkt (H score < 150) in 2 pts with no change in the reminder 5 pts (median H score 300).

Conclusion: Our preliminary data shown that four weeks of BKM120 treatment do not inactivate PI3K/Akt/mTOR signalling pathway, measured throughout pAkt in ER+/HER2-/PI3KCA mutated breast cancer pts. However, we cannot draw any conclusion because the small number of pts included so far. KRAS mutation and pS6 analysis will be presented along with updated results.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-09.
Abstract P4-16-05: Hsp90 inhibition potentiates chemotherapy effectiveness in triple negative breast cancer: Role of glucocorticoid receptor signaling
Authors: Agyeman A , Jun W , Proia D , Lee N , Skor M , Conzen S
Published: August 2014

Background: Few promising molecular targets exist for chemoresistant triple negative breast cancer (TNBC). Our laboratory has previously demonstrated that a subset of TNBCs express high glucocorticoid receptor (GR) levels. In breast epithelial cells, GR transcriptional activity directly upregulates the expression of several anti-apoptotic target genes including serum/glucocorticoid regulated kinase 1 (SGK1). High GR and/or SGK1 expression are both associated with decreased chemotherapy-induced cell death in TNBC models. A meta-analysis of patients with early-stage TNBC found that high primary tumor GR (NR3C1) expression significantly associates with earlier relapse compared to GR-non expressing TNBC. Inhibiting GR/SGK1 activity is therefore a testable strategy for improving TNBC responsiveness to chemotherapy.

Both GR and SGK1 require Hsp90 for their activity; therefore, Hsp90 inhibition is predicted to disrupt both GR and SGK1 cell survival signaling in TNBC. Employing a second-generation Hsp90 inhibitor, ganetespib (Synta Pharmaceuticals), we determined the effectiveness of Hsp90 inhibition on GR/SGK1 activity and chemotherapy sensitivity using in vitro and in vivo TNBC models.

Hypothesis: Hsp90 inhibition will decrease GR and SGK1 activity in TNBC in association with improved chemotherapy effectiveness.

Results: Using TNBC cell lines treated with ganetespib at clinically relevant concentrations, we found that total GR and the relative proportion of phosphorylated Ser211 GR were significantly reduced beginning 2 hours following treatment. Reduced glucocorticoid-induced (SGK1) mRNA expression was also seen when cells were pre-treated with ganetespib. We next used fluorescent microscopy and live cell imaging to measure TNBC cell death over a 96 hour time course following chemotherapy +/- ganetespib therapy. Addition of ganetespib to paclitaxel treatment of MDA-MB-231 cells led to significant in vitro cytotoxic synergy; as hypothesized, this synergy was diminished in GR-depleted MDA-MB-231 cells. Cytotoxic synergy following ganetespib and chemotherapy treatment was also seen in vivo in MDA-MB-231 xenografts. Interestingly, tumor GR expression following Hsp90 inhibitor and paclitaxel treatment was significantly reduced compared to tumors from mice treated with paclitaxel alone. Using GR-depleted or control MDA-MB-231 xenografts, studies are currently underway to further characterize the role of GR/SGK1 activity as a target of Hsp90 inhibition to reverse TNBC chemoresistance.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-16-05.
Abstract P5-01-09: Early tumor antigens discovered in TgMMTV-neu mice may provide targets for early breast cancer diagnosis and prevention
Authors: Stanton SE , Mao J , Ladd J , Rastetter L , Gadd E , Hanash S , Disis ML
Published: August 2014

Breast cancer is immunogenic and breast tumor proteins stimulate both an antibody and a T cell immune response. Identification of cancer related proteins that become immunogenic prior to the clinical development of cancer may allow the use of humoral immunity to identify those “exposed” to the cancer phenotype. Alternatively, these proteins that become aberrant enough to trigger such early immune recognition may be ideal targets for a vaccine aimed at preventing the development of breast cancer. We used the TgMMTV-neu mouse model to discover immunogenic proteins i.e. proteins expressed in pre-invasive breast cancer. The mammary tumors in these mice are genotypically similar to human luminal breast cancer and can be evaluated longitudinally, allowing for collection of pre-diagnostic sera prior to tumor development to use for antigen discovery.

We identified 6 antigens that were present in mice prior to the development of mammary cancer (Pdhx, Otud6b, Stk39, Zpf238, Lgals8, and Vps35). These proteins were associated with inflammation, autoimmunity, and cellular homeostasis. In mouse validation cohorts, detecting IgM and IgG antibody responses against a panel of three “pre-diagnostic” tumor antigens discriminated pre-diagnostic sera from non-transgenic control sera with an AUC of 0.924. We next evaluated samples obtained from the Women's Health Initiative Study and demonstrated women with autoantibodies to the human homologues of these proteins. IgM and IgG autoantibodies to the “pre-diagnostic” antigen panel could discriminate the samples of women who eventually developed breast cancer from matched controls. The discriminatory potential of the pre-diagnostic autoantibodies was enhanced if samples were collected more than 5 months prior to diagnosis (AUC 0.68; CI 0.565-0.787). We questioned whether these antigens, which could predict women who would eventually develop breast cancer, could mediate anti-tumor immunity. When TgMMTV-neu mice (n = 5/group) were vaccinated with the individual antigens, vaccination with Pdhx inhibited tumor growth by 62.1%, Otud6B inhibited tumor growth by 23.5%, and Stk39 inhibited tumor growth by 50.3% as compared to empty vector vaccinated control at 27 weeks (p<0.001 for each of the individual antigens as compared to empty vector). Spontaneous tumorigenesis was inhibited in TgMMTV-neu mice (n = 20 mice/group) vaccinated with a panel of three of the “pre-diagnostic” antigens (Pdhx, Otud6b, and Stk39) inhibited tumor growth by 27.3% by 37 weeks as compared to vector vaccinated mice (p<0.05). These data suggest that the same pre-invasive breast tumor proteins are found in mice and women and vaccines against these pre-invasive breast cancer proteins inhibit tumorigenesis in mice, future studies will address if these antigens elicit T-cell responses in patients with high risk breast lesions.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-09.
Abstract P5-02-02: Specific gene delivery to Her-2+ breast cancer cells using immunoliposomes
Authors: Kullberg M , McCarthy R , Anchordoquy TJ
Published: August 2014

The objective of our research is to develop a liposomal gene delivery system that specifically targets potent therapeutic genes to breast cancers which are positive for the human epidermal growth receptor 2 (Her-2). Current liposomal gene delivery systems predominately utilize cationic lipids, which efficiently bind and deliver plasmid DNA, but also result in nonspecific gene expression in the lungs and liver tissue. We attempt to improve on specificity, by using a two-component delivery system. The first component is a cationic polyethylene glycol (PEG) grafted polylysine which carries the plasmid DNA. The polylysine/DNA particle binds nonspecifically to cells but has limited ability to bypass the endosomal membrane. The second component is an anionic Her-2 targeting liposome which is disulfide bound to the pore forming protein Listeriolysin O (LLO). These liposomes bind with high selectivity to Her-2 positive cells and once internalized into the reducing conditions of an endosome, release LLO which forms pores in the endosomal membrane. Neither component delivers DNA plasmid on its own, but if the polylysine/DNA and LLO/liposome co-localize within an endosome, the DNA is able to access the cytoplasm which leads to gene expression of the plasmid.

Using fluorescent microscopy and a cell panel, we show that labeled polylysine/DNA and LLO/liposomes have a much greater chance of co-localizing in an endosome of Her-2 positive breast cancer cells compared to Her-2 negative cells. In addition, utilizing luciferase plasmid, we demonstrate that this two-component system results in gene expression that is over 1000-fold greater in Her-2 overexpressing cells than in Her-2 negative cells. This is a vast improvement over standard cationic liposome techniques which show no specificity for Her-2 positive cells.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-02-02.
Abstract P5-03-03: Effect of rapamycin on the function and transformation of aged murine mammary stem cells
Authors: Dong Q , Gao H , Wang D , Bandyopadhyay A , Wu A , Huang C , Sun L-Z
Published: August 2014

Age is the number one risk fact for breast cancer with only 5% of all breast cancers occurring in women under 40 years old and age specific incidence of invasive breast cancer increases from less than 1.5% at age 40, to about 3% at age 50 and over 4% by age 70 in American women. Recent research implicated that adult mammary stem cells (MaSCs) might be responsible for the initiation and progression of certain types of breast cancer. Our preliminary study showed that aging is associated with a significant increase of MaSC frequency, but with a functional decline of self-renewal and differentiation as well as increased neoplastic transformation potential. These findings indicate that aged MaSCs might be the precursors of preneoplastic lesions and serve as the cell of origin for malignant transformation in breast tissue. Therefore, intervention of MaSC aging process could be an effective method for cancer prevention. The drug of rapamycin has been shown to extend life span and ameliorate age-related pathologies (e.g., cancer) in murine models, and a recent study suggested that rapamycin's anti-aging effect may partially act through enhancing the function of tissue-specific stem cells. It is unknown whether rapamycin treatment will also enhance the function of aged MaSCs and decrease their transformation potential. In this study, we fed C57BL/6 mice with microencapsulated rapamycin-containing food (14 mg/kg, food designed to deliver ∼2.24 mg of rapamycin per kg body weight/day to achieve about 4 ng/ml of rapamycin per kg body weight/day) or control diet with empty capsules for 2 year (starting at 2-month old) or 5-10 days (starting at 25.5-month old) and then isolated primary mammary cells at 26-month old for MaSC quantification using an in vitro mammosphere formation and 3D-ECM sphere differentiation assay as well as by the in vivo cleared mammary fat pad transplantation assay. Our findings indicate that short-term (5-10 days) or long-term (> 2 year) rapamycin treatment reversed phenotypic changes associated with aged MaSC, which was mainly characterized by decreased luminal-to-basal cell ratio and increased MaSC frequency. Histological analysis of regenerated glands of aged MaSCs derived from control and rapamycin-treated mice showed a significant decrease of early neoplastic transformation potential in rapamycin-treated group. Subsequent in vivo serial transplantation and mating experiments revealed that rapamycin treatment reverted aged MaSCs more resemble to young MaSCs in self-renewal/differentiation function during regeneration and improved lobulo-alveolar differentiation function for lactation. In conclusion, our findings suggest that rapamycin can rejuvenate the function of aged MaSCs as well as reduce their incidence of preneoplastic transformation.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-03.
Abstract P5-04-15: Investigating the role of the AMPK signaling cascade in breast cancer tumor dormancy
Authors: Sullivan KL , Phoenix KN , Fox MM , Kopsiaftis S , Claffey KP
Published: August 2014

Breast cancer can recur in patients long after treatment for initial disease. The mechanisms underlying recurrence as well as the characteristics of these dormant tumor cells remain poorly understood. AMPK (AMP-activated protein kinase) is the central metabolic regulator of the cell, and has two catalytic isoforms, a1 and α2. The loss of AMPK, more specifically AMPKα2, is observed in primary breast cancer and its expression could function to limit tumor expansion in vivo. We hypothesize that the expression of AMPKα2 will confer a survival advantage in dormant breast cancer cells by upregulating the p38MAPK signaling axis. In order to investigate breast cancer dormancy mechanisms in vitro, we generated mammospheres using the estrogen-dependent MCF-7 human breast cancer cell line, which has low expression of AMPKα2. Control MCF-7 cells expressing GFP and MCF-7 cells overexpressing the AMPKα1 or AMPKα2 isoforms were cultured as mammospheres and exposed to various metabolic stressors including estrogen, glucose, and serum deprivation. Spheroids were evaluated for size, viability, gene expression at the RNA level by qRT-PCR, and intracellular signaling by immunoblot as well as immunohistochemistry. AMPKα2 expressing mammospheres were larger and more viable than either the GFP or AMPKα1 expressing spheres. In addition, these cells were resistant to serum deprivation, and upregulated members of the p38MAPK signaling axis, a key player in tumor dormancy. To evaluate estrogen deprivation-induced dormancy in vivo, MCF-7 cell lines expressing either GFP or AMPKα2 embedded in a matrigel matrix were implanted in athymic nude mice containing supplemental slow-release estradiol pellets. After one week, estradiol pellets were removed to induce a dormant period for roughly thirty days. Evaluation of matrigel plugs at this time indicated that AMPKα2 expressing cells demonstrated significant survival whereas GFP expressing cells were significantly depleted. Estadiol pellets were then re-implanted for five weeks to determine if the AMPKα2 expressing cells still responded to estradiol supplementation.This resulted in the AMPKα2 expressing tumors rapidly expanding to a maximal size as compared to the GFP expressing tumors. Consistent with our findings in vitro using mammosphere cultures, the AMPKα2 expressing cells had higher levels of phospho-p38MAPK than the GFP expressing cells, as seen by immunofluorescence. We conclude that the expression of AMPKα2 promotes both survival and dormancy in estrogen-sensitive breast tumor cells, thus implying a significant role in breast cancer recurrence.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-15.
Abstract P5-05-06: Menopausal status: An important consideration in determining the biology and prognosis of estrogen receptor-positive and progesterone receptor-negative breast cancer
Authors: Fujisue M , Nishimura R , Nakano M , Tashima R , Nishiyama Y , Osako T , Toyozumi Y , Arima N
Published: August 2014

Background: Transcription of the progesterone receptor (PgR) gene is regulated by estrogen in the breast tissue. PgR loss is considered to be a result in the reduction of estrogen receptor (ER) activity, due to either low circulating estrogen in some older women or a high growth factor signaling. Among the ER-positive breast cancer patients, PR-negative cases frequently have a higher resistance to endocrine therapy, and have a poorer prognosis. Therefore, we examined the clinical significance of ER+/PgR- tumors in luminal/HER2 negative breast cancer and its relationship to menopausal status.

Methods: The sample for this study came from 1791 consecutive patients with HER2 negative primary breast cancer from January 2002 to March 2013. The ER and PgR expressions were evaluated using immunohistochemistry (IHC). 1586cases had ER+/PgR+ tumor, 205cases had ER+/PgR- tumor. The items examined were nuclear grade, Ki-67 index value, TP53, tumor size and number of lymph node involvement. The Ki-67 index value and TP53 were evaluated using IHC and the cut-off values were 20% and 50%, respectively. Recurrence free survival (RFS) and breast cancer-specific survival (BCSS) was calculated using the Kaplan-Meier method and evaluated by the log-rank test or generalized Wicoxon test in stage 1 and 2 breast cancer.

Results: In terms of the distribution of ER and PgR status, ER+/PgR- were frequently seen in postmenopausal patients (13.7% vs 6.8%, respectively). Patients with ER+/PgR-and HER2 negative tumors had significantly smaller tumors, lower Ki-67 values, and a lower nuclear grade in the postmenopausal group compared with those in the premenopausal group. Moreover, RFS trended to be better in the postmenopausal group (p = 0.06), but there was no significant difference. BCSS was significantly higher in the postmenopausal cases (p = 0.001). On the other hand, in patients with ER+/PgR+ tumors, there was no significant difference in RFS and BCSS between the post- and pre-menopausal groups.

Conclusion: The ER+/PgR- tumors were more commonly seen in postmenopausal patients. The biological characteristics of ER+/PgR- tumors were significantly different in terms of the Ki-67 index value, nuclear grade, and the prognosis (BCSS and RFS) between pre- and postmenopausal status. Moreover, the postmenopausal group had a more favorable biology and prognosis than the premenopausal group. Therefore, the findings in this study indicate that menopausal status is related to the biology and prognosis in patients with ER+/PgR- tumors.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-06.
Abstract P5-08-13: Alteration of sensitivity to chemotherapeutic agents in tamoxifen resistant estrogen receptor positive breast cancer
Authors: Watanabe T , Okada T , Oba T , Iesato A , Hanamura T , Kanai T , Maeno K , Ito K-I
Published: August 2014

Background: In the treatment of estrogen receptor (ER) positive recurrent breast cancer, sequential treatment with endocrine or chemotherapy is generally used. However, there is no indicator by which we can select the appropriate chemotherapeutic agent to the endocrine resistant breast cancer in the subsequent treatment. The purpose of this study is to examine whether a preceding endocrine therapy affects the sensitivity to subsequent chemotherapy in ER positive breast cancer cells.

Materials and methods: Three ER positive breast cancer cell lines (T47D, MCF7, BT474) and their tamoxifen (TAM) resistant derivatives (T47D/T, MCF7/T, BT474/T) were established and analyzed for sensitivity to doxorubicin (DOX), 5FU and paclitaxel (PTX). The mRNA levels of factors related to drug sensitivity were analyzed by RT-PCR.

Results: MCF7/T became more sensitive to DOX and 5FU compared with the parental MCF7. In addition, the apoptosis induced by 5FU was significantly increased in MCF7/T compared with the parental MCF7. On the other hand, no difference of sensitivity to chemotherapeutic agents was observed in T47D/T and BT474/T compared with their parental cell lines. In MCF7/T and T47D/T, the expressions of dihydropyrimidine dehydrogenase (DPD) were significantly decreased compared with those in the parental cell lines, while no difference was observed in the expression of thymidine synthase (TS). The expression of thymidine phosphorylase (TP) was decreased in MCF7/T.

Conclusion: Our data demonstrate that the sensitivity to 5FU might be altered in a subset of ER positive breast cancer cells, and suggest a possibility that the sensitivity to the chemotherapeutic agents might be modified by the preceding endocrine therapy in ER positive breast cancer cells. We are analyzing drug sensitivity in tumor-bearing mouse model and the data are going to be presented in the meeting.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-13.
Abstract P5-09-17: Prognostic significance of progesterone receptor in hormonal receptor-positive breast cancer: Association with endocrine resistance
Authors: Bae SY , Lee SK , Lee JH , Lee H , Kim M , Kim J , Kil WH , Lee JE , Nam SJ
Published: August 2014

Background: Endocrine therapy has dramatic improvements of prognosis in patients with hormonal receptor positive breast cancer. However, long-term follow up shows that endocrine resistance is still major clinical concern. Although the value of estrogen receptor (ER) level can predict response to endocrine treatment in hormonal receptor positive breast cancers, few markers are available that can predict response to endocrine treatment. We attempted to identify molecular markers, especially progesterone receptor (PR), associated with endocrine failure in breast cancer.

Methods: We reviewed the medical records of 2513 breast cancer patients who underwent breast surgery and endocrine therapy at Samsung Medical Center between March 2007 and July 2011. Patients were compared according to ER and PR expression and to assess the clinical and biological features of ER+positive/PR-negative breast cancers to understand how PR might be a useful marker of these activities. ER and PR expression accessed using Allred score, the ‘positive” was defined that total score is larger than 2.

Results PR negative tumors were found more frequently in postmenopausal women (post- 11.2% vs. pre- 2.5%, P < 0.001) and PR negative tumors were significantly associated with higher grade, higher level of Ki 67 and more expression of EGFR and CK5/6. In Kaplan-Meier analysis and multivariate analysis, PR negativity was significantly poorer prognostic factor of DFS (HR 3.1, 95% CI 1.2-8.2, P = 0.021) and OS (HR 89.8, 95% CI 3.4-2352.3, P = 0.007). Stratified by the menopausal status, there was not shown significant difference of survival between the PR+ and PR- tumor in postmenopausal women (DFS, HR 2.1, 95% CI 0.4-10.8, P = 0.381; OS, HR 10.6, 95% CI 0.6-171.4, P = 0.096). However, PR negative tumors showed an association with poor outcome in premenopausal women (DFS, HR 7.2, 95% CI 2.5-20.6, P <0.001; OS, HR 24.6, 95% CI 2.2-275.6, P = 0.009).

Conclusion: In HR positive breast cancers, PR negative tumors were found more frequently in elderly, postmenopausal women. However, PR negative tumors are shown more aggressive and poorer survival than PR positive tumors despite of endocrine therapy, especially in premenopausal women.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-17.
Abstract P5-09-20: Development of a novel selective estrogen receptor down-regulator which shows efficacy in pre-clinical models of endocrine resistance
Authors: Weir H , Nardone A , Lawson M , Rowlinson R , Hulse M , Powell S , Callis R , Walker G , Curwen J , Wilson Z , Bradbury R , Rabow Al , de Savi C , Pazolli E , Reavey N , D'Cruz C , Schiff R
Published: August 2014

With over 70% of breast cancers expressing the estrogen receptor alpha (ERa), treatment with either anti-hormonal therapies that directly block ER function (e.g.Tamoxifen) or therapies that block the production of estrogen itself (e.g. aromatase inhibitors) are key to management of the disease. Despite initial efficacy seen with endocrine therapies, development of either de novo or acquired resistance ultimately limits the use of these agents although the majority of tumours continue to depend on ERα for growth. The ability to down regulate ER is particularly important in the endocrine resistance setting where ER appears to be activated in a ligand independent fashion by other growth factor signaling pathways. Agents such as Fulvestrant have been shown to offer additional benefit in the advanced setting due to its unique mechanism of binding and degradation of the ER receptor by the ubiquitin-proteosome complex. Given its low level of bioavailability and metabolic profile, Fulvestrant has been formulated as an intramuscular injection with 2 × 250mg monthly doses currently being tested clinically. We have sought to identify a novel, non-steroidal ERα antagonist and down-regulator that can be administered orally and could yield improved bioavailability and clinical benefit through enhanced biomarker modulation. We have identified a novel compound that can induce ERα degradation in breast cancer cell lines at picomolar concentrations with a similar half-life to Fulvestrant and is clearly differentiated from Tamoxifen which appears to stabilise ER protein. Due to good oral pharmacokinetic properties of the compound we have seen significant tumour efficacy in both tamoxifen-sensitive and -resistant models of breast cancer in vivo. In an MCF-7 model we have shown >90% reduction in PR levels in a pharmacodynamic study and complete tumour growth inhibition (TGI) at 5mg/kg dose. Fulvestrant in the same model setting was unable to achieve >60% PR inhibition at dose levels 3.8 fold greater than those achieved clinically with 500mg. We have also seen tumour regressions in a pre-clinical long term estrogen deprived (LTED) model, which mimic the aromatase resistance setting, and efficacy correlates with a significant decrease in ERα levels (>60%). This orally bioavailable compound is progressing towards clinical evaluation and shows promise as a new SERD agent for the treatment of ER+ breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-20.
Abstract P5-10-03: Estrogen, obesity and mammary morphology in a middle-aged rat model of menopause
Authors: Liang J , Davis JD , Garcia AN , Wolfe A , Gore AC , Yin W
Published: August 2014

The risk of occurrence of breast cancer changes at menopause in women. Various studies propose an association between mammographic density and adipose tissue distribution in premenopausal and post-menopausal women. There is a positive correlation between mammographic density and visceral adiposity in post-menopausal women, but an inverse correlation of the two in premenopausal women. Similar findings have also been discovered in rodent models. To model the premenopausal and post-menopausal status in women, this study used a middle-aged ovariectomized (OVX) Sprague Dawley rat model, given 17b-estradiol (E2) or vehicle (VEH) capsule, E2-deprived rats. Rats were OVX at 11 mo, and given E2 or VEH for 3 or 6 mo. A subset of rats were initially given E2 or VEH for 3 mo, then switched to the opposite treatment for another 3 mo, to evaluate the importance of E2 timing/duration. Mammary tumorigenesis was monitored through the treatment. Tumors were surgically removed upon detection. Rats were euthanized at 14 months old (3 mo of treatment) or 17 months old (6 mo of treatment). The 4th mammary gland was whole mounted for mammarographic density studies and comparisions. Serum estradiol, progesterone and prolactin concentrations were measured. Our results showed: 1) Body weight and visceral fat significantly decreased in E2 compared to VEH treated rats. 2) Following 3 or 6 mo E2 treatment, 3/54 and 5/72 rats, respectively, developed palpable mammary tumors. By contrast, 0/54 and 1/72 rats developed palpable tumors following 3 or 6 mo VEH treatment. In the group given 3 mo VEH then switched to E2 for 3 mo, 2/18 rats developed tumors. 1/18 rats developed tumors in the group given 3 mo of E2 then switched to VEH for 3 mo. 3) Mammary gland whole mounts showed increased mammographic density and decreased fat pad in E2 treated group. 4) Serum prolactin concentration significantly increased in E2 compared to VEH treated rats at euthanization. These results suggest timing and duration of hormone treatments have differing effects on mammary morphology and tumorigenesis.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-10-03.
Abstract P5-11-04: Post-translational modification of the cell-fate factor Dachshund determines p53 binding and signaling modules in breast cancer
Authors: Pestell RG , Chen K , Wu K , Gormley M , Ertel A , Zhang W , Zhou J , DiSante G , Li Z , Rui H , Quong AA , McMahon SB , Deng H , Lisanti MP , Wang C
Published: August 2014

Breast cancer is a leading form of cancer in the world. Initially cloned as a dominant inhibitor of the hyperactive EGFR, Ellipse, in Drosophila, the mammalian DACH1 regulates expression of target genes in part through interacting with DNA-binding transcription factors (c-Jun, Smads, Six, ERα), and in part through intrinsic DNA-sequence specific binding to Forkhead binding sites. The Drosophila dac gene is a key member of the retinal determination gene network (RDGN), which also includes eyes absent (eya), ey, twin of eyeless (toy), teashirt (tsh) and sin oculis (so), that specifies eye tissue identity.

Several lines of evidence suggest DACH1 may function as a tumor suppressor. Clinical studies have demonstrated a correlation between poor prognosis and reduced expression of the cell-fate determination factor DACH1 in breast cancer, and loss of DACH1 expression has been observed in prostate and endometrial cancer. DACH1 inhibits breast cancer tumor metastasis and reduces breast cancer stem cell expansion via Sox2/Nanog. Although these studies suggest DACH1 may function as a tumor suppressor, the molecular mechanisms remain poorly defined. Herein, endogenous DACH1 co-localized with p53 in a nuclear, extranucleolar compartment and bound to p53 in human breast cancer cell lines, p53 and DACH1 bound common genes in ChIP-Seq. Full inhibition of breast cancer contact-independent growth by DACH1 required p53. The p53 breast cancer mutants R248Q and R273H, evaded DACH1 binding. DACH1 phosphorylation at serine residue (S439) inhibited p53 binding and phosphorylation at p53 amino-terminal sites (S15, S20) enhanced DACH1 binding. DACH1 binding to p53 was inhibited by NAD-dependent deacetylation via DACH1 K628. DACH1 repressed p21CIP1 and induced RAD51, an association found in basal breast cancer. DACH1 inhibits breast cancer cellular growth in an NAD and p53 dependent manner through direct protein-protein association.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-11-04.
Abstract P5-11-09: Crk II adaptor proteins as positive regulators of HER3 signaling in breast cancer
Authors: Dhupkar P , Zhao H , Mukherjee S , Mujoo K , Zhang N , An Z
Published: August 2014

HER3 (ErbB3), which is a member of the human epidermal growth factor receptor (EGFR) family has emerged as a key oncogene in cancer progression. HER3 overexpression has been observed in lung, prostate, melanomas and colon cancers and is associated with poor patient prognosis. HER3 has been known to play a role in resistance mechanisms towards EGFR tyrosine kinase inhibitors like Gefitinib and anti-HER2 antibodies like Trastuzumab in lung and breast cancers respectively. Upon ligand (neuregulin) binding, HER3 heterodimerizes with its partner receptors, inducing phosphorylation of its tyrosine residues in the c-terminal tail, which mediate HER3-dependent signaling. By binding to the HER3 c-terminal tail, Src homology 2 domain (SH2) or phosphotyrosine-binding (PTB) proteins may regulate the neuregulin-induced signaling. Here, we used a SH2 profiling approach to discover novel SH2 adaptor proteins interacting with HER3 and found that the SH2 adaptor protein Crk II (C10 regulator of kinase II) interacts strongly with phosphotyrosines in CHO cells overexpressing HER3 with or without neuregulin stimulation. Crk II and HER3 are critical in proliferation, migration and invasion of breast cancer cells. In this study, we investigate the protein-protein interaction of Crk II and HER3 in breast cancer. Our preliminary protein-protein interaction data confirmed that Crk II interacts with HER3 in MCF-7, MDA-MB-453 and MDA-MB-468 breast cancer cells. We hypothesize that Crk II affects HER3-induced signaling, thus affecting growth and migration in breast cancer cells.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-11-09.
Abstract P5-13-03: Factors associated with prophylactic mastectomy among BRCA-positive patients with no personal history of breast cancer
Authors: Elsayegh N , Gutierrez-Barrera A , Baum G , Muse K , Jackson M , Woodson A , Jessica P , Kuerer H , Litton J , Arun B
Published: August 2014

Background: The rate of prophylactic mastectomy (PM) has recently increased1. A deleterious mutation in the BRCA1 or BRCA 2 genes is among the major reasons why patients pursue PM 2, 3. Women with BRCA1 or BRCA2 gene mutations have up to an 87% risk to develop an invasive breast cancer (BC), and up to 45% risk for ovarian cancer (OC)2, 3. Most studies evaluating predictors of PM in BRCA mutation carriers are performed among women with breast cancer; however, accurate predictors for PM among unaffected BRCA mutation carriers are less defined. In a single institution study we aimed to evaluate predictors of PM among BRCA carriers with no personal history of breast cancer.

Method: One hundred seventy seven women with no personal history of BC, who tested positive for a BRCA1 or BRCA 2 germline mutation, were included in the study. Patients’ characteristics were obtained from a prospectively maintained research database under an IRB approved protocol at UT MD Anderson Cancer Center. Univariate analyses using chi-square and logistic regression analysis were used to determine predictive factors associated with PM. The patient characteristics examined included age, martial and educational status, bilateral salphingo-oophorectomy (BSO), family history of 1st and 2nd degree relatives with breast (BC) and ovarian cancer (OC), race, and BRCA genetic test result.

Results: Out of the 177 BRCA1 and BRCA 2 positive patients, 51 (29%) elected for PM. The average age for the cohort was 44 years (range 23-91). The majority were Caucasian (81%), and married (72%) with a college degree (64%). One hundred sixty-four (92%) patients had 1st and 2nd degree relatives with BC, 93 (53%) had 1st and 2nd degree relatives with OC, and 85 (48%) had undergone BSO. A logistic regression model was run to identify factors associated with undergoing PM, including family history of OC, family history of BC, BSO and age. Patients with a family history of OC were 2.5 times more likely than those without to have had a PM (p = 0.0125), and patients who had a BSO were 0.137 times less likely to have had a PM (p <.0001). Only 12 patients did not have a family history of BC and none of those patients had a PM, so an Odds Ratio could not be calculated. However, it was determined through the Fisher's exact test that patients with a family history of BC were more likely to undergo a PM (p = 0.0198).

Conclusion: The rate of PM in our cohort was slightly lower than expected. Factors associated with PM included a family history of BC and OC. Interestingly, having had a BSO was associated with lower likelihood of undergoing PM (menopausal status will be further evaluated); possibly due to the knowledge that BSO reduces breast cancer risk.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-13-03.