Endocrine Therapy In Estrogen Receptor Research Articles

Abstract P5-13-02: Neoadjuvant endocrine therapy for estrogen receptor (ER) positive breast cancer: Comprehensive systematic review and meta-analysis

Abstract Background: ER positive tumors are generally highly responsive to endocrine treatment. However, the specific indications for neoadjuvant endocrine therapy, both as monotherapy and in combination with other therapies, in early breast cancer remain unclear. We conducted a comprehensive systematic review and meta-analysis to evaluate the impact of neoadjuvant endocrine therapy on response rate (RR), based on clinical response or imaging, and pathological complete response rate (pCR) for ER positive breast cancer. Methods: Based on QUORUM guidelines, a librarian-led search of PubMed, Ovid, and EMBASE was performed to identify eligible trials published prior to May 15, 2015. Inclusion criteria were prospective, randomized neoadjuvant trials that had at least one arm with neoadjuvant endocrine therapy and reported RR. Pooled odds ratios (ORs), 95% confidence intervals (CI), and p values were estimated for endpoints using the fixed and random effects statistical model. Results: We identified 477 citations initially with 277 remaining after duplications were removed. Twenty trials ultimately met inclusion criteria, with a total sample size of 3,493. The majority of studies (90%) focused on post-menopausal women and compared chemotherapy to endocrine therapy, different durations and types of endocrine therapies, or combinations with growth factor pathway inhibitors. Overall, as compared to chemotherapy, neoadjuvant endocrine therapy had a similar clinical RR (OR 0.93, CI: 0.43-2.02, p = 0.85) and radiological RR (OR 0.73, CI: 0.48-1.09, p = 0.12), but lower rate of toxicity (febrile neutropenia, mucositis). Aromatase inhibitors were associated with significantly higher clinical RR (OR 1.69, CI: 1.36-2.10, p = <0.01) and radiological RR (OR 1.49, CI: 1.18-1.89, p = <0.01), as compared to tamoxifen. Dual combination therapy with growth factor pathway inhibitors (n = 4) was also associated with higher radiological RR (OR 1.59, CI: 1.04-2.438, p = 0.03), but not clinical RR (OR 0.76, CI: 0.541.07-1.07, p = 0.11), as compared to endocrine therapy alone. The incidence of pCR in any arm was low overall (<10%) with resultant low numbers not suitable for inter-group comparisons. Conclusion: Neoadjuvant endocrine therapy is associated with similar response rates as neoadjuvant chemotherapy but with lower toxicity, and neoadjuvant AIs are superior to tamoxifen for ER-positive tumors. Compared to endocrine monotherapy, dual combination therapy may have superior response rates by imaging, but the low number of trials limits strong conclusions. Additional studies and more predictive biomarkers are needed to personalize the optimal neoadjuvant endocrine combination for an individual patient with ER positive breast cancer. Citation Format: Spring L, Gupta A, Reynolds KL, Gadd MA, Isakoff SJ, Ellisen LW, Moy B, Bardia A. Neoadjuvant endocrine therapy for estrogen receptor (ER) positive breast cancer: Comprehensive systematic review and meta-analysis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-13-02.

Cohort Study of Secondary Endocrine Therapy in Metastatic Breast Cancer with a Poor Response to Initial Endocrine Therapy

Background: Several classification models of sensitivity and resistance to endocrine therapies have been proposed for the clinical course of initial endocrine therapy for metastatic breast cancer. However, the efficacy and safety of secondary endocrine therapy in cases with a poor response to the initial endocrine therapy have not been examined. Methods: A multicenter prospective observational study is planned with the objectives of demonstrating the efficacy and safety of secondary endocrine therapy in estrogen receptor (ER)-positive, human epidermal growth factor receptor type2 (HER2)-negative postmenopausal metastatic breast cancer for which initial endocrine therapy did not have a favorable clinical effect (i.e., low sensitivity to initial endocrine therapy). The subjects are patients with breast cancer with low sensitivity to initial endocrine therapy, and are defined as cases with recurrence during 5 years of adjuvant therapy or those with metastatic breast cancer that showed progression within 9 months after initial endocrine therapy. The efficacy and safety of current endocrine therapeutic agents selected by physicians and patients will be examined using outcomes including clinical benefit, progression-free survival, overall survival, time to treatment failure, time to chemotherapy, response, health-related quality of life, and adverse events. Conclusion: Evaluation of the efficacy and safety of secondary endocrine therapy for breast cancer with low sensitivity to initial endocrine therapy will provide information for evidence-based selection of appropriate secondary endocrine therapy. The results will also clarify the remaining clinical issues to be resolved and provide a foundation for planning of future clinical research.

Clinicopathological factors predicting early and late distant recurrence in estrogen receptor-positive, HER2-negative breast cancer.

Most studies analyzing prognostic factors for late relapse have been performed in postmenopausal women who received tamoxifen or aromatase inhibitors as adjuvant endocrine therapy for estrogen receptor (ER)-positive breast cancer. A total of 223 patients (108 premenopausal and 115 postmenopausal) with early distant recurrence and 149 patients (62 premenopausal and 87 postmenopausal) with late distant recurrence of ER-positive, HER2-negative breast cancer who were given their initial treatment between 2000 and 2004 were registered from nine institutions. For each late recurrence patient, approximately two matched control patients without relapse for more than 10years were selected. Clinicopathological factors and adjuvant therapies were compared among the three groups by menopausal status and age. Factors predicting early recurrence in premenopausal women were large tumor size, high lymph node category and high tumor grade, whereas predictors for late recurrence were large tumor size and high lymph node category. In postmenopausal women under 60years of age, factors predicting early recurrence were bilateral breast cancer, large tumor size, high lymph node category, low PgR expression and high Ki67 labeling index (LI), while predictors for late recurrence were large tumor size and high lymph node category. On the other hand, in postmenopausal women aged 60years or older, factors predicting early recurrence were bilateral breast cancer, large tumor size, high lymph node category, high tumor grade, low ER expression and high Ki67 LI, whereas predictors for late recurrence were high lymph node category, low ER expression and short duration of adjuvant endocrine therapy. Predictors of early and late distant recurrence might differ according to menopausal status and age.

Abstract 2435: FGFR1 is associated with resistance to interaction with estrogen receptor (ER) α endocrine therapy in ER+/FGFR1-amplified breast cancer

Abstract Background: Molecular alterations in the fibroblast growth factor receptor (FGFR) pathway occur in breast cancer. FGFR1 gene amplification is found in ∼10% of ER+ breast cancers, where it is associated with early recurrence on endocrine therapy. FGFR1 amplification has been reported in 30-40% of breast tumors with CCDN1 amplification; co-amplification of these genes is associated with shorter patient survival. We investigated the mechanisms by which FGFR1 amplification confers endocrine resistance. Results: We used ER+ human breast cancer cell lines with (MDA-134, CAMA-1, HCC1500) or without (MCF7, ZR75-1) FGFR1 amplification. In these cells, we investigated the effect of FGFR1 silencing with siRNA or pharmacological inhibition using the small molecule lucitanib on cell growth, ER transcriptional activity and the interaction of FGFR1 and ERα. Both FGFR1 siRNA and treatment with lucitanib reduced ER transcriptional reporter activity and proliferation of ER+/FGFR1 amplified cell lines. Conversely, addition of FGF3 and FGF19 ligands stimulated ER reporter activity in estrogen-free medium, suggesting FGFR activation regulates ER activity. FGFR1 signaling is mainly transduced to the PI3K/AKT and RAS/RAF/MEK/ERK pathways by the adaptor FRS2. Neither siRNA against FRS2 nor pharmacological inhibitors of PI3K (BKM120) and MEK (GSK1120212) reduced ER transcriptional activity in ER+/FGFR1 amplified cells, suggesting a FRS2 independent, FGFR1-ERα direct interaction explaining the inhibitory effect of FGFR1 siRNA and lucitanib on ER function. MDA-134, CAMA-1 and HCC1500 FGFR1 amplified cell lines also harbor CCND1 amplification. Further, nuclear FGFR1 has been shown to regulate target gene expression, including CCND1. Therefore, we next examined if nuclear FGFR1 and cyclin D1 interact with ER. In ER or cyclin D1 antibody pulldowns from whole cell lysates and nuclear extracts of MDA-134, CAMA-1 and HCC1500 cells, ER co-precipitated with FGFR1 and cyclin D1. The associations of ER with FGFR and of ER with cyclin D1 were not inhibited upon blockade of the FGFR1 tyrosine kinase with lucitanib. However, the association of FGFR1 with cyclin D1 was inhibited by lucitanib treatment. Chromatin immunoprecipitation of DNA protein crosslinks in CAMA-1 cells ± estradiol with a FGFR1 antibody showed that FGFR1 binds to different estrogen response elements (ERE) in DNA. This FGFR1-ERE association was abrogated by treatment with lucitanib, suggesting it depended on an active FGFR1 tyrosine kinase. Conclusions: These data suggest FGFR1 binds ER and regulates ligand-independent ER transcriptional activity. This role depends on the FGFR1 kinase activity and may involve its association with cyclin D1. These interactions may explain the endocrine resistance reported in ER+/FGFR1 amplified breast cancers and suggest these tumors should be treated with a combination of antiestrogen and FGFR inhibitors. Citation Format: Luigi Formisano, Christian D. Young, Neil Bhola, Jennifer M. Giltnane, Monica V. Estrada, Carlos L. Arteaga. FGFR1 is associated with resistance to interaction with estrogen receptor (ER) α endocrine therapy in ER+/FGFR1-amplified breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2435. doi:10.1158/1538-7445.AM2015-2435

Abstract 2319: Sulforadex targets breast cancer stem-like cells in patient-derived cells and xenograft tumors

Abstract Sulforadex (SFX) is a novel therapeutic comprising synthetic sulforaphane (SFN) stabilised within α-cyclodextrin. SFN has been studied as an anti-cancer compound for many years however its development has hitherto been hampered due to its inherent instability. Breast cancer stem-like cells (CSCs) have been identified in all molecular subtypes and are believed to be the drivers of breast cancer metastasis. We investigated SFX effects on breast CSC activity using mammosphere formation and aldehyde dehydrogenase (ALDH) activity in patient samples and patient-derived xenograft (PDX) tumours. Mammosphere formation efficiency (MFE) assessed in cells from primary (n = 12) and metastatic (n = 15) samples treated with SFX (5 μM) or vehicle control (water) showed a significant reduction in breast CSC activity following SFX treatment. A reduction greater than 50% in the MFE was observed in both primary (0.52%±0.06 vs 0.19±0.02, p<0.001) and metastatic (0.93%±0.07 vs 0.43±0.04, p<0.001) cells treated with SFX. Importantly, SFX also inhibits breast CSC activity of early (HBCx34) and metastatic (BB3RC31) patient-derived xenograft (PDX) tumours. Using a 14 day in vivo ‘window’ treatment, we observe that SFX (300mg/Kg/day) decreases ALDH+ cells using ALDEFLUOR assay (HBCx34 6.3%±0.4 vs 3%±0.6, p<0.01; BB3RC31 3%±0.6 vs 1±0.2, p<0.05) and MFE (HBCx34 0.64%±0.09 vs 0.35%±0.03, p<0.01; BB3RC31 0.89%±0.06 vs 0.78±0.04). The majority of breast cancers express the estrogen receptor (ER) but these hormone-responsive breast cancers frequently develop resistance to hormonal therapies (e.g. tamoxifen). We have established that CSCs in ER+ BC lack ER expression, representing a potential mechanism of therapeutic resistance. However, SFX targets such surviving CSCs identified by ALDH positivity and mammosphere-initiating capacity following tamoxifen treatment in vivo in ER+ PDX tumours. Combinatorial treatment of tamoxifen (10 mg/kg/day) and SFX (300mg/Kg/day) in a 14 day ‘window’ treatment reduced ALDH+ cells (HBCx34 10%±0.4 vs 4.2%±0.4, p<0.01; BB3RC31 11.3%±1.1 vs 6.9±0.6, p<0.05) and MFE (HBCx34 0.81%±0.07 vs 0.34%±0.02, p<0.01; BB3RC31 0.47%±0.06 vs 0.27±0.04, p<0.05) compared to tamoxifen treatment alone. Mechanistically SFX potently inhibited the canonical Wnt pathway in MCF-7 cells and their endocrine-resistant derivatives and future studies will explore SFX activity on other CSC regulatory pathways. Our data demonstrate that SFX might produce clinically meaningful improvements to endocrine therapy in ER+ BC by reversing CSC mediated resistance. Clinical trials of endocrine therapy in combination with SFX are currently being planned. Citation Format: Bruno M. Simões, Denis Alferez, Rachel Eyre, Kath Spence, Angélica Santiago-Gomez, Iris Tanaka, Bertram Kohler, David Howat, Sacha J. Howell, Robert B. Clarke. Sulforadex targets breast cancer stem-like cells in patient-derived cells and xenograft tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2319. doi:10.1158/1538-7445.AM2015-2319

Effect of mutations in distinct components of the PI3K/AKT/mTOR pathway on sensitivity to endocrine therapy in estrogen receptor (ER)-positive breast cancer.

532 Background: Aberrations of the PI3K/AKT/mTOR pathway are common in ER-positive breast cancer and may be associated with endocrine-resistance. We hypothesized that, since the majority of ER-posi...

P53 accumulation is a strong predictor of recurrence in estrogen receptor‐positive breast cancer patients treated with aromatase inhibitors

Aromatase inhibitors have played a central role in endocrine therapy for estrogen receptor (ER)-positive breast cancer in postmenopausal women. However, factors predictive of the efficacy of aromatase inhibitors, and prognostic factors, both for early and late recurrence in women treated with adjuvant aromatase inhibitors have not been identified. Whole genome analysis identified that a TP53 gene mutation exists in ER-positive breast cancers, although the frequency of TP53 gene mutation in luminal tumors is lower compared with basal-like or human epidermal growth factor receptor type 2 (HER2)-positive breast cancers. We examined expression of p53, as well as ER, progesterone receptor, HER2 and Ki-67 using immunohistochemistry in postmenopausal ER-positive breast cancer patients who were treated with aromatase inhibitors as adjuvant endocrine therapy. There were 53 (21%) tumors that contained 10% or more p53-positive cells. High p53 expression was positively correlated with tumor grade, HER2 score and Ki-67 expression. Significant association was observed between disease-free survival and high p53 expression in multivariate analysis (P < 0.0001). Compared with women without recurrence, women with early recurrence had significantly higher p53 expression (P < 0.0001), as did women with late recurrence (P = 0.037). The present study demonstrates that p53 accumulation is a strong predictor of both early and late recurrence in ER-positive breast cancer patients treated with aromatase inhibitors as adjuvant endocrine therapy. TP53 gene alteration might be a key biological characteristic of ER-positive breast cancer.

Excess mortality in postmenopausal high-risk women who only receive adjuvant endocrine therapy for estrogen receptor positive breast cancer

Background. Omission of chemotherapy may affect mortality in postmenopausal high-risk women despite appropriate adjuvant endocrine therapy for estrogen receptor (ER) positive breast cancer. The aim of this study was to determine how all-cause mortality rate in these patients compares to that of the general female population. Furthermore, to identify a subset without excess mortality using clinical and pathological characteristics. Patients and methods. From the population-based database of the Danish Breast Cancer Cooperative Group we included 6529 postmenopausal patients with ER positive high-risk breast cancer who in 1996 through 2004 by nationwide guidelines were allocated to five years of tamoxifen, an aromatase inhibitor (AI) or both in sequence. Multivariate categorical and fractional polynomials (MFP) models were used to construct prognostic subsets by clinicopathologic characteristics. Results. In a multivariate model excess mortality was inversely (p < 0.0001) associated with increasing age at surgery while recurrence-free survival (RFS) was not. Non-adherence to endocrine therapy was associated with excess mortality (p = 0.0008) while treatment with an AI was associated with a less pronounced mortality excess (p = 0.03). A prognostic standard mortality rate (SMR) index (PSI) was built using the regression coefficients obtained in the MFP model, and the same risk factors were used to construct a flowchart algorithm. Both allocated 75% to a group with increased all-cause mortality as compared to the general female population, but the SMR was significantly increased (SMR 1.38; 95% CI 1.16–1.65) in 462 patients who were allocated to low-risk group by the Flowchart algorithm and to a high-risk group by PSI. Conclusion. Only one quarter of postmenopausal ER positive breast cancer patients are free of excess mortality when omitting adjuvant chemotherapy. Patients should be informed about importance of adherence to adjuvant endocrine therapy and inclusion of an AI. A PSI may better guide recommendations regarding adjuvant chemotherapy.

Neoadjuvant single and dual HER2 blockade among patients with localized HER2-positive breast cancer.

147 Background: Dual neoadjuvant HER2 directed therapy is offered only in a clinical trial setting and is not standard of care, but emerging data suggests targeting multiple mechanisms may be more effective. We conducted a comprehensive systematic review and meta-analysis to evaluate the impact of neoadjuvant dual and single agent HER2 blockade on breast conserving surgery (BCS), and on pathological complete response (pCR) for estrogen receptor (ER)+ and ER- tumors, as well as the impact of pCR on disease-free survival (DFS) and overall survival (OS) for HER2+ breast cancer. Methods: MEDLINE, EMBASE, and Cochrane Controlled Clinical Trials Register databases were queried to identify eligible trials. Inclusion criteria were prospective, neoadjuvant trials that had at least one arm with HER2 directed therapy, and reported pCR. Pooled relative risk ratios (RRs) and p values were estimated for endpoints using the random effects statistical model. Results: We identified 36 trials (N = 4130). High pCR rates (&gt; 40%) were seen with anthracycline-based chemotherapy and trastuzumab alone, and non-anthracycline based dual HER2 blockade. The addition of trastuzumab to chemotherapy did not improve BCS rate (RR 1.40, p = 0.15), but significantly increased rates of pCR (RR 1.91, p = 0.0001). Similarly, dual HER2 blockade compared to trastuzumab alone did not improve BCS rate (RR 1.03, p = 0.84), but significantly increased rates of pCR overall (RR 1.39, p &lt; 0.00001), in both ER+ (RR 1.72, p = 0.01) and ER- subsets (RR 1.91, p = 0.0001), with no increase in grade 3/4 toxicity (RR:1.13, p = 0.16). Dual HER-2 blockade without chemotherapy was associated with pCR in a subset (11.2% - 27%) with minimal toxicity (incidence of grade 3/4 toxicity:1-5%). Higher pCR was associated with improved DFS (RR 2.29, p = 0.006) and OS (RR 4.61, p = 0.009). Conclusions: Neither the addition of trastuzumab to chemotherapy, nor the dual-HER2 blockade compared to trastuzumab, improves rates of BCS. However,both significantly improve rates of pCR, which is associated with improved DFS and OS. Dual HER2 blockade,with endocrine therapy for ER+, could potentially lessen or even obviate the use of chemotherapy.

Mammostrat As an Immunohistochemical Multigene Assay for Prediction of Early Relapse Risk in the Tamoxifen Versus Exemestane Adjuvant Multicenter Trial Pathology Study

Some postmenopausal patients with hormone-sensitive early breast cancer remain at high risk of relapse despite endocrine therapy and, in addition, might benefit from adjuvant chemotherapy. The challenge is to prospectively identify such patients. The Mammostrat test uses five immunohistochemical markers to stratify patients regarding recurrence risk and may inform treatment decisions. We tested the efficacy of this panel in the Tamoxifen versus Exemestane Adjuvant Multicenter (TEAM) trial. Pathology blocks from 4,598 TEAM patients were collected, and tissue microarrays (TMAs) were constructed. The cohort was 47% node-positive, and 36% of patients in the cohort were treated with adjuvant chemotherapy. Triplicate 0.6-mm(2) TMA cores were stained, and positivity for p53, HTF9C, CEACAM5, NDRG1, and SLC7A5 was assessed. Cases were assigned a Mammostrat risk score, and distant relapse-free survival (DRFS) and disease-free survival (DFS) were analyzed. In multivariate regression analyses, which were corrected for conventional clinicopathologic markers, Mammostrat provided significant additional information on DRFS after endocrine therapy in estrogen receptor (ER) -positive node-negative patients (n = 1,226) who did not receive chemotherapy (P = .004). Additional analyses in all patients not exposed to chemotherapy, irrespective of nodal status (n = 2,559) and in the entire cohort (n = 3,837) showed Mammostrat scores provided additional information on DRFS in these groups (P = .001 and P < .001, respectively; multivariate analyses). No differences were seen between the two endocrine treatment regimens. The Mammostrat score predicts DRFS for patients treated with exemestane and patients treated with tamoxifen followed by exemestane irrespective of nodal status and chemotherapy. The ability of this test to provide additional outcome data after treatment provides additional evidence of its use in risk stratification of ER-positive postmenopausal patients with breast cancer.

The Treatment of Individual Patients Is More Than a Trial

The use of chemotherapy before surgery (neoadjuvant or preoperative approach) is the standard of care in patients with locally advanced and inflammatory breast cancer because a clinical and/or radiological response is achieved in most patients, facilitating optimal surgery and radiation therapy. The success of neoadjuvant chemotherapy for locally advanced breast cancer led to studies investigating this treatment approach among women with operable breast cancer (primary chemotherapy). Many years have gone by since primary/neoadjuvant chemotherapy was first introduced by Gianni Bonadonna in the manage ment of operable breast carcinoma (1), and the National Surgical Adjuvant Breast and Bowel Project-B18 multicenter randomized trial demonstrated the similar therapeutic value between primary and adjuvant treatments (2). Subsequent randomized clinical trials and a meta-analysis of adjuvant vs primary chemotherapy in patients with early-stage operable breast cancer (3) showed no difference in disease-free or overall survival rates. On these bases, if a patient with operable breast cancer is candidate to a systemic therapy on the basis of presurgical, clinical, and immunohistohemical evaluation, this treatment can be administered either in the neoadjuvant or adjuvant setting. The primary systemic therapy (PST) strategy implies an up-front systemic therapy: chemotherapy ± molecular target agents or endocrine therapy followed after 4–6 months by surgery and subsequent adjuvant therapy if needed (ie, endocrine therapy in estrogen receptor–positive tumors). Potential advantages of the PST approach include tumor downstaging increasing the rates of breast conservation and the possibility of monitoring tumor response. Adjuvant therapy, in fact, is a blind approach, whereas PST can test in vivo the sensitivity of cancer cells to the systemic therapy used, offering the opportunity to obtain information of treatment efficacy in any treated patients. These issues notwithstanding, PST are rarely used in clinical routines because in case of operable primary breast cancer, most clinicians usually prefer to adopt surgery followed by adjuvant therapy. Many reasons may justify this approach: 1) there is no formal demonstration that disease response is a surrogate parameter of efficacy; 2) the real advantage of monitoring tumor response dur ing PST is the opportunity to adjust, tailor, individual treatment on the basis of the initial tumor response obtained; however, no prospective trials have demonstrated the efficacy of this approach; 3) perhaps most importantly, the patient frequently prefers the rapid removal of her tumor because the delay in surgical approach can be a cause of anxiety. The identification of patient subsets in which PST may be superior to adjuvant therapy and the validation of surrogate parameters of treatment efficacy are crucial steps toward the implementation of PST in clinical practice (4). Beside its employment in clinical routine, PST is increasingly popular in terms of research perspective where it has an irreplaceable role in the early clinical investigation of new biological drugs. PST studies are extremely useful in translational research to study the impact of systemic treatment on tumor biology in vivo, to assess potential predictive factors, and to allow pharmacodynamic and pharmacogenetic substudies. They may also be very informative in testing synergies of new associations. Furthermore, a new type of translational research has been recently developed for more frequent smaller tumors. In the “biological window” design (5), very similar to a phase 0 trial, the aim is not to downstage surgery, but to make use of the elapsed time between diagnostic biopsy and surgical resection to give new molecular target agents to carry out information on their biological activity. Future clinical trials of PST should be designed with molecular subsets of breast cancer in mind. As previously mentioned, whether tailoring individual treatment according to response to PST is really beneficial for patients still remains to be demon strated but assessing such a strategy will be facilitated in the near future thanks to functional and molecular imaging that can further improve early assessment of tumor response, and, above all, the development of newer therapies, such as antiangiogenic therapies or DNA repair targeting drugs, with the anticipated absence of cross-resistance with anthracyclines and taxanes. Furthermore, molecular analyses of post-chemotherapy surgical residual disease for proliferative factors and predictive factors may also be very informative in the future to select the most appropriate adjuvant treatment.

Alteration of Y-box binding protein-1 expression modifies the response to endocrine therapy in estrogen receptor-positive breast cancer

Y-box binding protein-1 (YB-1) plays an important role in tumor progression and drug resistance. This study examined whether YB-1 is involved in the alteration of response to endocrine therapy in estrogen receptor (ER)-positive breast cancer cells. MCF7 cells that stably expressed YB-1 (MCF7-YB-1) and vector control cells (MCF7-vector) were established. These cells were used to analyze the expression of the factors related to ER and growth factor receptor signaling pathways and responses to antiestrogens (tamoxifen and fulvestrant) and estrogen responsive element (ERE) activity. The effect of knocking down endogenous YB-1 expression was tested in wild-type MCF7 cells. In addition, the expression of YB-1 and the factors related to ER and growth factor receptor signaling pathways were evaluated in clinical breast cancers treated with preoperative chemotherapy. The expression of HER2, AIB1, p-Erk, and c-Myc was increased in MCF7-YB-1 cells. In contrast, knocking down of YB-1 decreased the expression of these factors but increased the expression of ERα in wild-type MCF7 cells. Furthermore, sensitivity to antiestrogens was decreased in the MCF7-YB-1 in comparison to that in MCF7-vector cells. The introduction of YB-1 into MCF7 cells inhibited apoptosis and cell cycle arrest at G1 phase induced by antiestrogens. In MCF7-YB-1 cells, the expression levels of p-Erk and c-Myc were continuously upregulated when cells were treated with either tamoxifen or fulvestrant. The ERE activity was reduced in MCF7-YB-1 cells in comparison to MCF7-vector cells, and the ERE activity in MCF7-YB-1 cells was inhibited by fulvestrant at a lower concentration than that which inhibited the ERE activity in MCF7-vector cells. In ER-positive clinical breast cancers treated with preoperative chemotherapy, significantly more number of specimens that showed increased or positive YB-1 expression after chemotherapy was positive for HER2 expression. These data suggest that alteration of YB-1 may modify the crosstalk between the ER pathway and HER2 pathway in ER-positive breast cancer cells, and consequently, may alter the response to endocrine therapy in ER-positive breast cancer cells.

Relationship of CYP2D6 status and toremifene metabolism.

e13068 Background: Tamoxifen (TAM) is widely used as endocrine therapy for estrogen receptor (ER) positive breast cancer despite a growing controversy regarding its effectiveness in patients with compromised cytochrome P450 2D6 (CYP2D6) activity. Since it is metabolized to active metabolites via CYP2D6, legitimate concerns have been raised over concurrent use of TAM and drugs that are potent inhibitors of CYP2D6 (e.g., SSRI antidepressants). Toremifene (TOR) is a structural analog of TAM that is approved for the treatment of metastatic breast cancer in postmenopausal women with ER positive or unknown tumors. TOR is primarily metabolized by CYP3A4, while CYP2D6 is thought to play a minor role in its metabolism. We examined the role of individual CYP isoforms in the metabolism of TOR and TAM to N-desmethyl (NDM), 4-hydroxy (4-OH) and 4-OH-N-desmethyl (4-OH-NDM) metabolites. Methods: The in vitro metabolism of TOR and TAM was investigated using human liver microsomes and recombinant CYP proteins. Samples were analyzed by LC-MS/MS. Results: N-demethylation is the primary metabolic pathway for both TOR and TAM. Similar levels of N-desmethyl metabolites were formed for both compounds. N-demethylation occurred by CYP2D6, 3A4 and 1A1 but not appreciably by the other six CYP isoforms tested. Specific inhibitors of CYP2D6 and 3A4 activity blocked N-demethylation. The 4‐OH and 4-OH-NDM metabolites of TAM, but not TOR, were observed when the parent drugs were incubated with the CYP2D6 isoenzyme. The formation of both 4-OH-tamoxifen and 4-OH-NDM-tamoxifen (endoxifen) were significantly inhibited by 2D6 inhibitors, including the SSRI antidepressant paroxetine. These results were corroborated by studies using human liver microsomes from poor, intermediate and extensive CYP2D6 metabolizers. Conclusions: Tamoxifen, but not toremifene, is a substrate for 4-hydroxylation by CYP2D6 in human liver microsomes. The metabolism of toremifene is not markedly affected by potent CYP2D6 inhibitors in human liver microsomes. These results suggest that clinical benefit in subjects taking toremifene to treat metastatic breast cancer would not be subject to allelic variation in CYP2D6 status or affected by co-administration of CYP2D6-inhibiting medications.

High estrogen receptor expression and low Ki67 expression are associated with improved time to progression during first-line endocrine therapy with aromatase inhibitors in breast cancer

Aromatase inhibitors have played a central role in endocrine therapy for estrogen receptor (ER)-positive breast cancer in postmenopausal women. However, many breast cancer patients with tumors expressing ER are unresponsive to aromatase inhibitors, and all patients with advanced disease eventually develop resistance to the therapy. Twenty-one postmenopausal women with Stage II to IV breast cancer were treated with aromatase inhibitors as first-line endocrine therapy without surgery. Expression levels of ER, progesterone receptor, HER2 and Ki67 were examined by immunohistochemistry, and correlations between response and duration of the therapy and these levels were analyzed. Patients whose tumors contained two thirds or more ER-positive cells effectively responded to aromatase inhibitors (P=0.006) and displayed longer time to progression during first-line endocrine therapy (P=0.003) and longer time to endocrine therapy failure (P=0.02). Patients whose tumors showed less than 15% Ki67 labeling index also displayed longer time to progression (P=0.003). High ER expression and low Ki67 expression were associated with improved time to progression with aromatase inhibitors as first-line endocrine therapy. Our findings will be helpful when endocrine therapy is planned in either early stage or advanced breast cancer.

Pegylated liposomal doxorubicin in combination with low-dose metronomic cyclophosphamide as preoperative treatment for patients with locally advanced breast cancer

Aim To evaluate the role of pegylated liposomal doxorubicin with low-dose metronomic cyclophosphamide as primary systemic treatment in locally advanced breast cancer. Patients and Methods The activity and safety of intravenous pegylated liposomal doxorubicin 20 mg sqm −1 biweekly for eight courses in combination with metronomic cyclophosphamide 50 mg day −1 orally were evaluated in 29 patients with locally advanced breast cancer who were not suitable to receive a standard chemotherapy due to age or co-morbidities or who asked for a regimen with low incidence of toxic effects irrespective of age. Results The rate of breast-conserving surgery was 44.8%. Eighteen patients (62.1%) achieved a partial response (including one pathological complete response), 10 (34.5%) a stable disease and one patient experienced a progressive disease. Treatment was well tolerated, with no grade 4 toxicities, and with grade 3 skin toxicity in three patients and hand–foot syndrome in four patients. Conclusion The regimen was well tolerated but with limited activity in the preoperative setting. Other options (e.g., endocrine therapy in estrogen receptor -positive disease) should be considered in locally advanced breast cancer patients who are not suitable to receive a standard chemotherapy.

Abstract P4-02-20: Plasticity of the ER and HER2 Signaling Networks Results in Compensatory Escape Pathways Contributing to Treatment Resistance

Abstract Bidirectional crosstalk between the estrogen receptor (ER) and HER2 has been demonstrated in preclinical model systems. We have previously shown in HER2+ breast cancer cells with low ER that potent HER2- targeted therapy results in ER reactivation that functions as an escape pathway to evade therapeutic inhibition. Here we investigated the role of HER pathway components in resistance to endocrine therapies using UACC812 cells, which are endogenously amplified for HER2. Endocrine resistant (R) derivatives of the UACC812 line were developed through long-term (&amp;gt;6 months) culturing with estrogen deprivation (ED) alone or with 4-OH-tamoxifen (Tam, 10-7M). Endocrine treatment [continued ED, estrogen (E2, 10-9M), Tam (10-7M), or fulvestrant (Ful, 10-7M)] of parental cells slowed cell growth compared to estrogen treatment (P&amp;lt;0.01). EDR and TamR derivatives treated with any of the endocrine therapies, however, exhibited the same growth rates as with estrogen, validating the completely resistant phenotype of our clones. By immunoblot analysis, the resistant derivatives, TamR and EDR, showed a striking and complete loss of ER expression and its downstream gene products including PR, IGF-1R, and IRS-1. In contrast, both resistant lines exhibited significant upregulation of HER pathway components including marked increases in total and phosphorylated (p) EGFR, further upregulation of pHER2, and marked increases in the downstream signaling moieties pMAPK, pAkt, pFAK, pSrc, and pFOXO3a. In contrast, levels of pHER3 were markedly reduced at resistance. Since our in vitro data suggested that ER and HER2 compensate for inhibition of each other's pathways, we next examined whether simultaneous inhibition of both pathways can circumvent the development of resistance in an in vivo xenograft model. Athymic nude mice bearing UACC812 xenografts, established in the presence of E2 supplementation, were randomized to continued E2, endocrine therapies alone (ED, Tam, or Ful), anti-HER2 alone [lapatinib (L), trastuzumab (T), or L+T], or the combination of endocrine and anti-HER2 therapies (ED with L, T, or L+T). We found that tumors were stimulated by E2, as well as by Tam (as a mechanism of de novo resistance), while still being inhibited, though only temporarily (2-3 months), by ED and Ful. Anti-HER2 therapy alone only slowed down (L or T) or stabilized (L+T) tumor growth. In contrast, in the presence of both ED and anti-HER2 therapies, most tumors completely regressed. However, only the potent L+T plus ED regimen achieved continuous long term (&amp;gt;200 days) complete regression in all mice. Our results further illustrate the plasticity of the ER and HER2 pathways and provide additional support for the role of the HER network, especially EGFR, in resistance to endocrine therapy in ER+/HER2+ breast cancer. The data emphasize the need for complete HER blockade for optimal therapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-20.

Cell density is a critical determinant of aromatase expression in adipose stromal cells

Obesity is associated with an increased risk of breast cancer among postmenopausal women. This is at least partly due to excessive estrogen production in adipose tissue of obese women. Aromatase, the key enzyme in estrogen biosynthesis, is an important target in endocrine therapy for estrogen receptor (ER)-positive postmenopausal breast cancer. In this study we show that high confluency of human adipose stromal cells (ASCs) cultured in vitro can significantly stimulate aromatase gene expression and reduce the expression of breast tumor suppressor BRCA1 and members of the NR4A orphan nuclear family. Furthermore, small interfering RNA (siRNA)-mediated knockdown of Nurr1, a member of the NR4A family, substantially increased aromatase expression. Lastly, we found that the cell density-triggered inducibility of aromatase expression varies in ASCs isolated from different disease-free individuals. Our finding highlights the impact of increased cell number on estrogen biosynthesis as in the case of excessive adiposity.

Predictors of response to exemestane as primary endocrine therapy in estrogen receptor–positive breast cancer

Endocrine therapy is the most important treatment of choice for estrogen receptor (ER)-positive breast cancer. Potential mechanisms for resistance to endocrine therapy involve ER-coregulatory proteins and cross-talk between ER and other growth factor-signaling networks. However, the factors and pathways responsible for endocrine therapy resistance, particularly resistance to aromatase inhibitors, have not been clearly established. Sixteen postmenopausal patients with ERalpha-positive primary breast cancer were treated daily with 25 mg of exemestane (an aromatase inhibitor) for 6 months. Expressions of ERalpha, ERbeta, progesterone receptor (PgR), androgen receptor (AR), amplified in breast cancer 1 (AIB1), aromatase, epidermal growth factor receptor, human epidermal growth factor receptor type 2, Ki67, cyclin D1, p53, Bcl2, signal transducer and activator of transcription 5 (Stat5), and insulin-like growth factor binding protein 5 (IGFBP5), and phosphorylations of ERalpha serine (Ser) 118, ERalpha Ser167, Akt Ser473, and p44/42 MAPK threonine (Thr) 202/tyrosine (Tyr) 204, were examined by immunohistochemistry on pretreatment tumor biopsies and post-treatment surgical specimens. Analyses were made to test for correlations with response to exemestane. Of the 16 patients, seven responded and nine retained stable disease. High-level expression of AIB1 and phosphorylation of Akt Ser473 were significantly associated with a better response to exemestane, suggesting that these factors could be considered as predictors of exemestane response. Expressions of ERalpha, ERbeta, PgR, aromatase, Ki67, cyclin D1, and p53, and phosphorylations of ERalpha Ser118, ERalpha Ser167, and p44/42 MAPK Thr202/Tyr204, were decreased, whereas expressions of Stat5 and IGFBP5 were increased in post-treatment specimens compared to the values in pretreatment biopsies. Thus, the analysis of factors involved in the estrogen-dependent growth-signaling pathways may be useful in identifying patients responsive to exemestane.

The Alternative Noncoding Exons 1 of Aromatase (Cyp19) Gene Modulate Gene Expression in a Posttranscriptional Manner

Aromatase (Cyp19) is a key enzyme in estrogen biosynthesis and an important target in endocrine therapy for estrogen receptor (ER)-positive postmenopausal breast cancer. Aromatase transcription is driven by multiple tissue-specific promoters, which result in the production of various mRNA transcripts that contain an alternative noncoding exon 1 followed by a common protein-coding region. Transcriptional activity of these promoters is the only known determinant for aromatase protein abundance in a given tissue or cellular context. To determine whether aromatase expression could be influenced by additional regulatory mechanisms, we used a common heterologous promoter to drive the expression of multiple aromatase cDNA sequences that differ only by the alternative exon 1 sequence. These expression vectors gave rise to vastly different levels of aromatase mRNA and protein in multiple cell lines examined. Furthermore, the relative abundance of several mRNA variants did not correlate with that of the corresponding protein product. The variation in mRNA and protein levels is most likely due to a negative effect of certain alternative exons 1 on RNA stability and protein translation. Deletional analyses indicate that the 5' regions of the adipose tissue-specific exons I.3 and I.4 contain the cis-acting elements responsible for modulation of aromatase levels. Thus, our work uncovers an important role of the alternative exons 1 in posttranscriptional regulation of aromatase gene expression.

Modulation of hormone therapy resistance by CDK2-PELP1 axis.

Abstract Abstract #3022 Background: The estrogen receptor (ER) plays a central role in the progression of breast cancer. Current endocrine therapy for ER+ve breast cancer involves modulating ER-pathway using Tamoxifen, and blocking peripheral estrogen (E2) synthesis by Aromatase inhibitors. Despite the positive effects, de novo and/or acquired resistance to endocrine therapies frequently occur. Although mechanisms for hormonal therapy resistance remains elusive, most downstream events in these pathways converge upon modulation of cell cycle regulatory proteins including upregulation of Cyclin E and A, along with activation of Cyclin Dependent Kinase 2 (CDK2). ER signaling complexes are known to recruit various co-regulatory proteins and recent evidences suggest that deregulated expression, localization and activity of ER coregulators also plays vital role in hormonal resistance. In this study, we found that CDK signaling regulates ER coregulator PELP1 function via phosphorylation leading to hormonal resistance.&amp;#x2028; Methods: Significance of CDK2 axis in the therapy resistance was tested using breast cancer models cells that acquired resistance to endocrine therapy and by using chemical inhibitors that block CDK2 activity. Immunoprecipitation, and confocal analysis was used to confirm protein-protein interactions. In vitro kinase and ortho-phosphate labeling assays were used to test CDK2 phosphorylation of PELP1. Utilizing Breast cancer model cells that express PELP1 mutants that cannot be phosphorylated by CDK2, we examined the significance of PELP1 phosphorylation in cell cycle progression. Using PELP1siRNA nanoparticles, we tested the effect of PELP1 knockdown in hormone therapy resistance.&amp;#x2028; Results: ER coregulator PELP1 interacts with CDK2 upon E2 stimulation. In vitro kinase assays using both purified CDK2/CyclinE and CDK2/CyclinA complexes showed that full length PELP1 is a potential substrate of CDK2. PELP1 exhibited phosphorylation at the time points that corresponds to CDK2 activation in MCF7 cells. PELP1 overexpression increases E2F luciferase activity while PELP1 mutants that lack CDK2 sites failed to enhance the E2F activity. CDK2 mediated phosphorylation of PELP1 is important for PELP1 regulation of E2F and ER target genes. Combination therapies using PELP1 siRNA nano particles or Roscovitine along with tamoxifen or letrozole, sensitized the therapy resistance cells for endocrine therapy.&amp;#x2028; Conclusions: We have identified ER coregulator PELP1 as a novel substrate of CDK2. Because CDK2 activity is deregulated in breast tumors and implicated in therapy resistance, our findings suggests that CDK2-PELP1 axis deregulation may contribute therapy resistance. Combinatorial therapeutic strategy using Roscovitine along with PELP1 siRNA nanoparticles will provide new therapeutic opportunity to increase the sensitivity of hormone resistant cells to Tamoxifen and Letrozole therapy. These studies were supported by DOD breast cancer grant BC083207. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3022.