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Paraffin-embedded tumor samples are valuable in the study of cancer for routine staging, tumor marker analysis, and in retrospective studies to test new prognostic and predictive biomarkers. Their utility in retrospective pharmacogenetic analysis of clinical trials has yet to be evaluated. We set out to establish genotyping methods for relevant genes from archival tumor samples and determine if fixation, processing or somatic changes in the tumor might affect our ability to identify germ-line polymorphisms.To establish the assays, paraffin blocks were made using pellets prepared from eight tumor cell lines. DNA was isolated from viable cells and from sections from these blocks, and genotyped for polymorphisms in CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A5 and MDR1 using conventional PCR-RFLP assays. This demonstrated that fixation and processing did not alter the genotypes obtained (100% concordance). Next, sections were obtained from paraffin-embedded archival breast samples from 10 patients for whom gDNA isolated from peripheral blood was available for comparison. Concordance was complete with the same genotype being obtained for 100% of the samples tested. Attempts to extend these methods for the study of hematoxylin/eosin or immunohistochemically stained sections were not successful since the staining inhibited the PCR reactions. Only 25 of 50 samples were successfully amplified and of those only 14 produced accurate genotypes.Accurate genetic testing for polymorphisms in several genes of pharmacogenetic importance can be obtained from archival paraffin-embedded tumor samples. Thus, pharmacogenetic analysis can be applied to existing cancer therapy trials to test associations between these polymorphisms and treatment response.

Cytochrome p4502C19 (CYP2C19) plays an important role in drug biotransformation and has been shown to be genetically polymorphic. While polymorphisms in the coding region that have large effects on activity are well described, until recently, a lack of knowledge of the promoter sequence has hindered efforts to study it. Genetic variants in the promoter region have not been described and factors that influence its gene expression via promotor regulation remain largely undefined. We have cloned and sequenced 1.8 kb of the human CYP2C19 promoter. This promoter contains a number of putative transcription factor sites, including HepG2-specific factor 1, glucocorticoid response element, estrogen receptor element, constitutive androstane receptor and peroxisome proliferator-activated receptor. Sequencing of DNA obtained from 67 individuals identified eight single nucleotide polymorphisms within this region. No sequence of a known human pregnane X receptor response element was found in this section of the CYP2C19 promoter, despite the known effect of rifampin on the expression of this gene. A plasmid containing the 1.8-kb CYP2C19 promoter coupled to a luciferase reporter gene has been constructed and demonstrated to be functional and sensitive to induction by omeprazole in HuH7 cells. Nested deletions of CYP2C19 promoter were generated and the ability of serial promoter deletion constructs to activate luciferase expression in the HepG2 cell line was analysed. These data make possible future studies to elucidate the molecular mechanisms by which CYP2C19 can be induced in clinical settings and the consequences of genetic variability in its promoter.

Introduction: Fulvestrant is a selective estrogen receptor down-regulator (SERD). Recent studies have shown that the efficacy of fulvestrant is dose-related. However, at the higher dose (500 mg/month) most cancers develop resistance and progress. We previously reported expression of several markers, including estrogen receptor (ER) and BCL-2, on breast cancer circulating tumor cells (CTC) using CellSearch®. We now report pilot data showing inter-patient heterogeneity of these markers on CTC in patients with known ER positive breast cancer whose disease is progressing on fulvestrant. Methods: We conducted a pilot trial to determine the analytical validity of measuring expression of markers of endocrine sensitivity (ER, BCL-2) or resistance (HER-2, Ki-67) with fluorescent-labeled antibodies using the CellSearch® system. Patients with ER positive metastatic breast cancer (MBC) whose disease was progressing on any type of therapy were eligible after signed informed consent. This report is limited to the subjects who were progressing on fulvestrant. Whole blood (WB) was characterized for CTC counts and each of the four molecular markers using the CXC CellSearch® kit. Results: Of 50 enrolled patients, seven were progressing on fulvestrant. Two patients had no detectable CTC, while five patients had an average of ≥5 CTC/7.5 mL WB. Results are shown in a table below: CTC-ERCTC-BCL-2Patient #Fulvestrant dose (mg/month)Days since last doseN CTC/7.5 mL of WB% of CTC-ER+N CTC/7.5 mL of WB% of CTC-BCL-2+295002880%110%4550028170%170%2250341010%714%850031812%1735%172507728%367% These exploratory data suggest widely different mechanisms of resistance to fulvestrant in different patients with ER positive MBC. In two of the patients (29, 45) treated with 500 mg/month, both CTC-ER and CTC-BCL-2 expression were absent, suggesting no signaling through the ER pathway. We hypothesize either that fulvestrant was actively down-regulating ER, but the cancers had adopted other growth and survival pathways, or that ER negative, hormone-independent clones had evolved. In the other three cases, ER was clearly present with evidence of signaling, based on BCL-2 expression. Two of these patients (2, 17) were on the lower dose of fulvestrant, now considered to be less effective. However, the third (8) was on the higher dose and yet still had evidence of ER signaling. This observation suggests that some patients may benefit from even higher doses of SERD therapy. Conclusions: These pilot results suggest heterogeneous biological or pharmacological mechanisms of resistance to SERD therapy. These data suggest that CTC-ER and CTC-BCL-2 expression could serve as pharmacodynamic monitoring tools for dose escalation of fulvestrant or other SERDs. Further molecular analysis might provide biological bases for resistance to fulvestrant. Supported by Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP). Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD6-4.

Estrogen affects multiple aspects of human physiology, including the normal growth and development of female reproductive tissues, bone integrity, cardiovascular and central nervous system functions, and plays a central role in normal mammary development and breast pathogenesis. It modulates diverse cell signaling pathways, some of which appear to be independent of the known estrogen receptors (ERs). Although many of estrogen's actions can be explained by the nuclear ERs (ER-alpha and ER-beta) functioning as ligand-activated RNA transcription factors, there are numerous rapid biochemical and physiological responses that cannot be explained by the classical genomic effects of estrogen signaling. It has long been postulated that the rapid effects of estrogen are due to a membrane-bound ER, and two recent reports suggest that it is in fact a G-protein-coupled receptor named 'GPR30'.

525 Background: Discontinuation of adjuvant AI therapy due to intolerable symptoms occurs in up to 30% of patients. Predictors of which patients will be unable to tolerate these medications have not been defined. We hypothesized that inherited variants in candidate genes are associated with treatment discontinuation because of AI-associated toxicity. Methods: We prospectively evaluated reasons for treatment discontinuation in women with hormone receptor positive breast cancer initiating adjuvant AI through a multicenter, prospective, randomized clinical trial of exemestane (exe) versus letrozole (let). Using multiple genetic models, we evaluated potential associations between 136 single nucleotide polymorphisms (SNP) in 24 candidate genes, selected a priori, primarily with roles in estrogen metabolism and signaling, and discontinuation of AI therapy because of toxicity. To account for multiple comparisons, statistical significance was defined as p

1system were stored in which CTC were identified by an automated algorithm. Furthermore, this algorithm determined the Her-2 positivity threshold in each sample, using the fluorescence of leukocytes within each sample as internal control. Results: This automated Her-2 assessment method successfully distinguished Her-2 positive from Her-2 negative breast cancer cell lines. We report here for the first time that Her-2 expression of CTC varied greatly between but also within patients, suggesting a possible mechanism of treatment resistance. However, in M1 patients, a threshold of 75% of Her-2 positive CTC in patients with ≥5 CTC showed a relatively low discrepancy rate between the primary tumor and CTC Her-2 status. Applying this threshold, ∼2% of M1 patients with Her-2 negative primary tumors had Her-2 positive CTC status and ∼30% of M1 patients with Her-2 positive primary tumors had Her-2 negative CTC status. Interestingly, no Her-2 discrepancy was observed between CTC and primary tumors in M0 patients. Conclusions: Our results suggest that Her-2 status shift appears mostly at metastatic relapse, and consists generally in Her-2 low expression on CTC isolated from M1 patients with Her-2 positive primary tumors. These findings also demonstrate the feasibility of real-time quantitative and reproducible assessment of treatment targets on CTC, opening a path towards personalized treatment. Disclosure: All authors have declared no conflicts of interest.

Introduction Only ∼50% of patients (pts) with estrogen receptor (ER) positive (+) metastatic breast cancer (MBC) benefit from endocrine therapy (ET). Circulating Tumor Cells (CTC) are reliably enumerated using CellSearch (Veridex, LLC). High CTC levels predict rapid progression in pts with MBC. We have developed a multi-parameter assay, the CTC-Endocrine Therapy Index (CTC-ETI) using CellSearch that may identify pts with ER+ MBC who are unlikely to benefit from ET and may be better served with chemotherapy. CTC-ETI scoring is based on CTC levels coupled with the relative percent and degree of marker positivity on CTC. We report preliminary results from a feasibility study. Methods CellSearch has 4 fluorescence channels: 3 distinguish CTC from WBC and the 4th “empty” channel was used to measure ER, BCL-2, HER-2, and Ki-67 expression with fluorescent-labeled antibodies. These 4 markers reflect sensitivity (ER, BCL-2) or resistance (HER-2, Ki-67) to ET. Blood from pts with progressive MBC was processed and characterized for CTC counts and each of the 4 markers using the CXC CellSearch kit. Results 34 pts have been accrued. One was ineligible. Ten of 33 pts had low CTC counts ( Conclusions CTC-ETI can be determined in MBC pts using the 4th channel in the CellSearch system. We predict that lower CTC-ETI scores (low or no CTC, or CTC with high CTC ER and BCL-2 and low CTC HER-2 and Ki-67) could be associated with favorable response to ET. Successful completion of the feasibility study will lead to a prospective trial to determine if high CTC-ETI at baseline predicts resistance and rapid progression on ET in pts starting new ET for MBC. Supported by Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP). Disclosure M.C. Connelly: I am an employee of Veridex, LLC. D.A. Chianese: I am employed by Johnson and Johnson who in part funded this research. I am in addition, a shareholder of Johnson and Johnson. D.F. Hayes: I receive research support for laboratory and clinical research from Veridex LLC. All other authors have declared no conflicts of interest.

Introduction. CTC can be reproducibly and reliably enumerated using a commercially-available, automated immunomagnetic system (CellSearch®; Veridex LLC). High levels of CTC predict rapid progression in patients with metastatic breast cancer (MBC). Only ∼ 50% of patients with estrogen receptor (ER) positive MBC benefit from endocrine therapy (ET). Patients with endocrine-refractory MBC are better palliated with chemotherapy. We have developed a multi-parameter assay using CellSearch® that may identify patients with ER positive MBC who are unlikely to benefit from ET and may be better served with chemotherapy. Methods. CellSearch® has four fluorescence channels. Three of these are used to distinguish CTC from WBC (DAPI, anti-cytokeratin, anti-CD45). The 4th “empty” channel was used to measure ER, Bcl-2, HER2, and Ki67 expression with antigen-specific fluorescent-labeled antibodies. These four markers were chosen because of their associations with sensitivity (ER, Bcl-2) or resistance (HER2, Ki-67) to ET. Cultured human breast cancer cells (MCF-7: ER +, BCL-2 +, HER2 -, Ki67 +; MDA-MB-231: ER -, Bcl-2 -, HER2 -, Ki67 +; BT-474: ER +, Bcl-2 +, HER2 +, Ki67 +) were spiked into 7.5 ml human whole blood from normal donors and separated and characterized using the CXC CellSearch® kit. Results. Each cell line stained appropriately for the respective markers, with appropriate negative controls, although staining was heterogeneous, even within a single cell line. Using these preliminary data, we have developed a CTC-ETI in which scores are assigned to the individual categories consisting of cell counts coupled with the relative percent and degree of cell positivity of each marker. We predict that lower scores (low or no CTC, or CTC with high % and intensity of ER and Bcl-2; low HER2 and Ki-67) will be associated with favorable response to ET. Conclusions. CellSearch® can be used to measure the expression of 4 clinically useful and proven breast cancer biomarkers. A clinical trial is underway at U. Michigan to permit calculation of CTC-ETI in patients with MBC, which will lead to a prospective trial to determine if high CTC-ETI predicts resistance and rapid progression on ET. Supported by Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale ™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4154. doi:10.1158/1538-7445.AM2011-4154

Previously we have shown that androgens induce the growth of estrogen-dependent, estrogen receptor (ER)-positive breast cancer cell lines via metabolism to estrogen-like steroids, independent of CYP19 aromatase. One pathway involves the 3β-hydroxysteroid dehydrogenase (3β-HSD) mediated metabolism of 5α-dihydrotestosterone (DHT) to 5α-androstane-3β,17β-diol (3βAdiol). 3βAdiol induces the growth of ER+ breast cancer cells by directly activating ER. However, in MCF-7 cells, maximal growth induction by 3βAdiol was only ∼75% of maximal growth induced by 17β-estradiol (E2), suggesting that 3βAdiol acts as a weak or partial agonist of ERα. In this study, we characterize 3βAdiol as a partial agonist of ERα using cell growth assays and an in vitro receptor binding assay. We use cDNA microarrays to examine gene expression induced by the partial agonist 3βAdiol vs. the full agonist E2. We tested if 3βAdiol as a partial agonist could antagonize E2-induced breast cancer cell growth. MCF-7 cells in estrogen-free conditions were treated with 100pM E2 (∼EC90) and increasing concentrations of 3βAdiol. Increasing 3βAdiol suppressed growth induced by E2 to the maximal level induced by 3βAdiol alone. These data confirm that 3βAdiol acts as a partial agonist of ERα. Additionally, we performed an in vitro, florescence polarization-based receptor binding assay to determine the relative affinities of 3βAdiol and E2 for ERα. Based on their ability to compete with a labeled ligand for binding to ERα, 3βAdiol bound ERα with an approximately 10-fold lower affinity than E2. This decreased affinity of 3βAdiol for ERα is consistent with the reduced potency of 3βAdiol versus E2 observed in growth assays. To compare changes in gene expression induced by either the full agonist E2 or partial agonist 3βAdiol, samples were analyzed with Affymetrix HG-U133 Plus 2.0 microarrays. MCF-7 cells were treated for 24 hours with 10nM of steroid or vehicle control. To evaluate overall changes in gene expression induced by the steroids, probe sets were selected that had >2-fold change in expression versus the control array, with at least one sample having an expression value >26. 1273 probe sets were differentially expressed between the E2 and control arrays, and 1318 between the 3βAdiol and control arrays, however, 1030 of these were common to both steroids. Genes in canonical estrogen signaling pathways were similarly regulated by both E2 and 3βAdiol, including GREB1, Bcl-2, TFF1 and CXCL12. Only 45 probe sets were differentially expressed between the E2 and 3βAdiol arrays. These data demonstrate that 3βAdiol is a partial agonist of ERα in our breast cancer cell model. The majority of changes in gene expression are similar between the full and partial agonist. These data suggest that the differential growth induction by the full and partial agonist are likely due to differential levels or time courses of gene induction, rather than the induction of a novel set of genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4593.

The two most common endocrine treatment modalities, anti-estrogen therapy and aromatase inhibitor therapy target estrogen receptor (ER) activation and estrogen (E2) biosynthesis, respectively. Although these therapies are effective in many patients with ER-positive tumors, prospective clinical trials have demonstrated that there exists a portion of patients with breast cancers that are initially responsive to endocrine therapy but subsequently relapse. Because of the lack of perfect correlation between ER and/or progesterone receptor (PR) status and patients who benefit from hormone therapy, there is a need to identify additional protein markers that would improve prediction of hormone response.Gene Regulated by Estrogen in Breast cancer 1 (GREB1) is regulated by estrogen (E2) in breast cancer and may serve as a candidate marker to predict response to endocrine therapy. The focus of this study was to define the significance of GREB1 protein expression in breast cancer and its correlation with estrogen receptor α status and epidermal growth factor receptor 2 (HER2) expression. Based on the query of 16 breast cancer expression array studies in the Oncomine Research database, GREB1 mRNA is expressed at greater levels in breast carcinomas than normal breast tissue (p value: 2.1E-5). In addition, GREB1 mRNA is significantly more commonly expressed in ER-positive breast cancer patients (n=1651) than ER-negative patients (n=670) (P value: 4.2E-5 ∼ 1.1E-34). Finally, GREB1 mRNA expression was observed to be lower in HER2+ breast cancer patients than in HER-2 – patients (p-value: 0.034). These mRNA expression data supported the hypothesis that GREB1 may have potential as a new biomarker for predicting E2-dependent and anti-E2 responsive breast cancers.Using a novel GREB1 monoclonal antibody generated by our laboratory, Immunohistochemical staining (IHC) was performed on a tissue microarrays containing invasive breast carcinomas from a cohort of 142 operable breast cancer patients (104 ER+ and 38 ER- cancers) with paired uninvolved tissue from the same patient. GREB1 protein was detected in both uninvolved normal tissue and ER+ breast cancer, but was absent in ER- tumors. Thus, IHC staining revealed GREB1 protein expression, predominantly localized in the cell nucleus, has a significant correlation with ER status (p-value Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2008.

It has been well established that GREB1 mRNA expression is induced by estrogen in breast cancers and that GREB1 is critically involved in the estrogen-induced growth of breast cancer. To date, all studies of GREB1 have focused on mRNA levels using PCR and array technologies with very little known about GREB1 protein expression in normal and breast cancer cells. The lack of a specific antibody to GREB1 has inhibited protein-based investigations in target cells. Our collaborative research group has generated a novel monoclonal GREB1 antibody (GREB1ab) for research use in Western blotting as well as immunohistochemical (IHC) applications.Our team created a hybridoma expressing a murine monoclonal antibody against a 119 amino acid peptide specific to human GREB1. By Western Blot Analysis, GREB1ab detects a 216 kD protein corresponding to GREB1a in ER+ breast cancer cells expressing GREB1 as well as cells transfected with a GREB1a expression plasmid. GREB1ab specificity was verified using ICI 182,780, an estrogen receptor antagonist to prevent GREB1 induction, as well as silencing siRNA targeting GREB1 mRNA expression. GREB1 protein expression was reduced in MCF-7 cells treated with estrogen plus ICI 182,780 compared with that of estrogen treatment. There was no detectable GREB1 protein expression when GREB1 mRNA is silenced by siRNA at 48 hours. In a time course study, inhibition of GREB1 protein occurred as early as 24 hours and lasted up to 72 hours. Thus, by Western Blot Analysis, the monoclonal GREB1ab is specific for GREB1 and can be employed to detect changes in GREB1 expression in breast cancer cell lines under various experimental conditions.GREB1ab was validated for detection of GREB1 by IHC in breast cancer cell lines and breast tissue microarrays (TMA). IHC staining with GREB1ab revealed GREB1 was strongly expressed in the ERα-positive breast cancer cell line, MCF-7, with weak GREB1 expression in ERα-negative MDA-231 cells. A panel of breast cancer cell lines was screened for endogenous expression of GREB1 protein in a TMA format using IHC. As expected, ER-positive cell lines (n=5) were observed to express GREB1 while ER-negative cell lines (n=11) did not express detectable levels. Using breast cancer tissue whole sections, IHC with the GREB1ab indicated protein expression in ERα positive breast cancer tissue as well as normal breast tissue, with little GREB1 expression in ERα negative breast cancer tissue.The monoclonal GREB1ab is specific for GREB1 protein. This antibody will serve as a tool for investigations focused on the expression, distribution and function of GREB1 in normal breast and breast cancer tissues. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2126.

Background. Several studies aimed at correlating genetic variants in the enzymes responsible for tamoxifen metabolism and disposition with therapeutic outcomes have provided equivocal and conflicting data. The IMPACT trial compared the preoperative used of tamoxifen with anastrozole alone or in combination in postmenopausal breast cancer patients and provided strong evidence that tamoxifen-induced changes in the nuclear proliferation marker Ki67 can act as a surrogate marker of tamoxifen response. An advantage of this study design is that all patients contribute data to the end-point analyses which included changes in Ki67 and objective clinical response as defined by the WHO criteria. We therefore genotyped patients from IMPACT for variants in the enzyme (cytochrome p450 2D6 (CYP2D6)) responsible for the conversion of tamoxifen to its potent anti-estrogenic metabolite endoxifen, as well as the enzyme responsible for the detoxification and inactivation of endoxifen via conjugation (UDP-glucuronosyltransferase 2B7 (UGT2B7).Results: Depending on their CYP2D6 variant genotype, patients were assigned an 'activity score' based on predicted allele activities from 0 (no activity) to 2 (high activity). Patients were also tested for the UGT2B7*2 variant, which has lower enzymatic activity and is therefore correlated with higher serum endoxifen concentrations. Of the 135 patient samples available for genotype determinations, 123 and 134 we evaluable for CYP2D6 and UGT2B7, respectively. There was no evidence of an association between CYP2D6 score, as determined by genotype alone, with suppression of Ki67 after 2 or 12 weeks, or with objective clinical response after 16 weeks of therapy in the tamoxifen alone, anastrozole alone or combination groups. Furthermore, this lack of association was not altered by adjustment of the CYP2D6 score for concomitant medications, known to inhibit CYP2D6 and to correlate with lower serum endoxifen concentrations. Lastly, while a statistical test for a potential interaction between CYP2D6 genotype and UGT2B7 genotype was negative, there was a marginally statistically significant association between UGT2B7 genotype alone with suppression of Ki67 after 2 weeks in the tamoxifen only group.Conclusion: Although this was a small study, these data do not support the hypothesis that patients must have active CYP2D6 in order to benefit from tamoxifen therapy. Larger more definitive studies are underway to test the effects of drug metabolizing enzymes on anti-estrogen therapies. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2011.

Abstract #1043 Approximately 1/3 of hormone dependent breast cancers do not respond to tamoxifen treatment. The fact that these tumors are intrinsically resistant suggests that underlying genetic factors are responsible for this phenotype. The two major types of genetic variation are single nucleotide polymorphisms (SNPs) and copy number variations (CNVs). SNPs are the most common form of genetic alteration with an estimated 10 million SNPs across the genome and likely influence both disease susceptibility and response to therapy. Traditionally, researchers have attempted to identify SNPs associated with a clinical outcome in a “top-down” approach by conducting linkage and SNP association studies. However, here we report on a “bottom-up” approach whereby SNPs are first tested in vitro for a functional effect. This awards two benefits over a “top-down” approach: 1) identification of functional SNPs and 2) detailed characterization of the SNP so that the clinical association studies can be designed accordingly. Tamoxifen acts by blocking estradiol (E2) from binding to the estrogen receptor (ERα) and by recruiting corepressors, which then actively repress gene transcription. The critical role of corepressors, especially SMRT, in the proper action of tamoxifen makes them likely to be involved in tamoxifen resistance. Thus, we used a “bottom-up” approach to identify SNPs in ERα corepressors which affect the ability of tamoxifen to suppress ERα function. Here we report the detailed in vitro characterization of a number of SNPs in SMRT (NCoR2) with respect to altered ERα function in the presence of tamoxifen. Further, these SNPs are currently being genotyped so that associations can be made in a population of women receiving tamoxifen as either preventative or adjuvant therapy in which extensive surrogate markers for ERα function were collected during the 1-year follow (hot flash rate, hot flash intensity, lipid profiles, and bone mineral density). Through the discovery of common polymorphisms affecting Tamoxifen response, we hope to identify new diagnostic criteria to predict which patients will respond to anti-estrogen therapy and which will be better candidates for another line of therapy, such as aromatase inhibitors. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1043.

Abstract #3049 Background: Estrogen receptors (ER) and associated factors, including progesterone receptors (PgR), modulate estrogenic effects both in normal tissue and in hormone receptor (HR)-positive breast cancers. ERα expression occurs in the majority of breast cancers, although the level of overexpression varies between tumors. Multiple single nucleotide polymorphisms (SNPs) in the genes that encode ER, termed ESR1 (ERα) and ESR2 (ERβ), have been shown to affect phenotypic outcomes, including breast cancer risk, bone mineral density, hot flashes, and lipid levels. We hypothesized that SNPs in genes that encode ER may affect level of ER overexpression. Methods: DNA was extracted from white blood cells from 100 subjects with HR-positive breast cancer enrolled in a tamoxifen observational study. SNPs in ESR1 (XbaI: rs#9340799, PvuII: rs#2234693) and ESR2 (01: rs#1256049; 02: rs#4986938) were determined from germline DNA using TaqMan. ER overexpression in paraffin-embedded formalin-fixed tumor specimens was quantified with immunohistochemistry (IHC) of tumor specimens on slides (Ventana Image Analysis System) and with RT-PCR using the Oncotype DX breast cancer assay (Genomic Health, Inc). Gene expression of other ER-associated genes (PgR, BCL2, and SCUBE2) was determined using RT-PCR (Oncotype DX). Expression of ER and associated genes across ER genotype was performed using the Kruskal-Wallis test. For all analyses, a p value of Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3049.

504 Background: CYP2D6 genetic variants and concurrent administration of potent inhibitors (Inh) of the enzyme markedly reduce the plasma concentrations of endoxifen (Jin. JNCI 2005;97:30–9). We have shown that genetic variation in CYP2D6 is associated with a higher risk of relapse in Tam treated patients (pts) (Goetz. JCO 2005;23:9312–18), but the impact of CYP2D6 inhibition on Tam clinical outcome is unknown. Methods: Using the North Central Cancer Treatment Group 89–30–52 adjuvant Tam trial (1989–1994), we evaluated the effect of co-administration of CYP2D6 Inh and CYP2D6*4 genotype on relapse in pts receiving tam alone. Patient charts were reviewed at each randomizing site to record the following known CYP2D6 Inh: fluoxetine (F), paroxetine (P), sertraline (S), cimetidine (C), amiodarone (A), doxepin (D), ticlopidine (T) and haloperidol (H). Poor metabolizers (PM) were defined as pts who were CYP2D6 *4/*4 and/or known to receive a CYP2D6 Inh. Intermediate metabolizers (IM) were defined as WT/*4 without CYP2D6 Inh, and extensive metabolizers (EM) as WT/WT without CYP2D6 Inh. The association between CYP2D6 status and Tam clinical outcome was determined using the log-rank test. Multivariate Cox modeling was performed using traditional prognostic factors. Results: CYP2D6*4 genotype was obtained in 190 of 256 eligible pts, with a CYP2D6*4/*4 genotype frequency of 6.8% as described previously (Goetz JCO 2005). Medication history was available in 171/256 eligible pts and in 143/190 pts with CYP2D6 genotype. Eleven of 173 patients were concomitantly administered these CYP2D6 Inh: F(2), P(2), S(2), H(1), C(4), and D(1). The median duration of use was 2.5 yrs. Twenty-four pts met criteria for PM [13 by *4/*4 genotype and 11 by CYP2D6 Inh (7 WT/WT, 1 WT/*4, and 3 unknown genotype)], 21 for IM, and 102 for EM. Compared with either IM or EM, PMs had significantly worse time to recurrence and disease free survival in both the univariate (HR 2.5, p=0.02; and 2.2, p=0.01) and multivariate (HR 2.7, p=0.01; and HR 2.2 p=.02) analyses utilizing tumor size, nodal status, and tumor grade. Conclusion: Our data indicate that CYP2D6 status can be used to identify pts who should receive tam therapy and tam treated pts should not be co-administered potent CYP2D6 inhibitors. No significant financial relationships to disclose.

535 Background: Progesterone receptor (PR) is a direct downstream target of activated estrogen receptor (ER) and its expression is a marker of estrogen signaling within breast tumors. Studies suggest the absence of PR predicts the likelihood of distant recurrence in patients treated with hormonal therapies such as tamoxifen, but better clinical measures are needed. Methods: Through analysis of public mRNA expression profiling datasets, we identified 36 genes with the following expression patterns similar to PR: (1) induced by 17β-estradiol (E2) in estrogen receptor (ER)-positive breast cancer cell lines in vitro, (2) under-expressed in ER-negative compared to ER-positive breast tumors, and (3) correlated with PR expression in ER-positive tumors. The average expression of these 36 genes was used as a “risk index” for assessing disease-specific survival in two independent tumor profile datasets of 60 and 67 patients treated with tamoxifen (these data not having been used to initially select the 36 genes), with a high risk group in each dataset defined as those with the bottom 25% of risk index values. Results: The Kaplan-Meier estimates of the rates of distant recurrence at 10 years in the low-risk and high-risk groups were 24% and 64% in the one dataset and 32% and 72% in the other dataset. In both validation datasets, the rate in the low-risk group was significantly lower than that in the high-risk group (P=0.008 and P=0.006 by log-rank, respectively), whereas patient groups defined by histologically-assigned PR status did not show significant risk differences (P=0.70 and P=0.94, respectively). In a univariate Cox model, the risk index combined across both validation datasets provided significant predictive power (P=0.03). Conclusions: Through RT-PCR assay of a larger independent cohort of breast tumor samples, we are currently validating the prognostic value of individual genes within the 36-gene signature. No significant financial relationships to disclose.

Background Estrogen plays a central role in breast cancer pathogenesis. Although many studies have characterized the estrogen regulation of genes using in vitro cell culture models by global mRNA expression profiling, it is not clear whether these genes are similarly regulated in vivo or how they might be coordinately expressed in primary human tumors.

Recently, we have shown that endoxifen and 4OHTAM are equipotent inhibitors of estrogen action, with respect to estrogen receptor (ER) binding, proliferation, and inhibition of pS2 and PR gene expression. Since estrogens and anti-estrogens have effects on the expression of many additional genes, we tested the effects of endoxifen and 4OHTAM on genome-wide profiling in ER-positive MCF-7 cell lines by cDNA microarray analysis using the Affymetrix HG-U133A GeneChip. Cells were treated for 24 hrs with vehicle, 17β-estradiol (E2) 10−10M, 4OHTAM 10−7M, endoxifen 10−7M, E2+4OHTAM, E2+endoxifen, and E2+4OHTAM+endoxifen. E2 treatment resulted in up- or down-regulation of ~213 genes which showed greater than 2-fold changes from the vehicle-treated group. 4OHTAM and endoxifen changed the expression of 58 and 52 genes respectively when added to the E2 treatment. Of these genes, 49 (84.5%) and 46 (88.5%) were estrogen-regulated genes. 4OHTAM and endoxifen had overlapping effects on 32 genes, but also brought about distinct patterns of gene regulation (26 vs. 20 genes were changed by only 4OHTAM or endoxifen, resp.). Among the 32 genes coregulated by both 4OHTAM and endoxifen, there was a significant correlation between the fold-effects brought about by these two drugs (R2=0.964, P

Estrogen signaling plays an essential role in breast cancer progression, and estrogen receptor (ER) status has long been a marker of hormone responsiveness. However, ER status alone has been an incomplete predictor of endocrine therapy, as some ER+ tumors, nevertheless, have poor prognosis. Here we sought to use expression profiling of ER+ breast cancer cells to screen for a robust estrogen-regulated gene signature that may serve as a better indicator of cancer outcome. We identified 532 estrogen-induced genes and further developed a 73-gene signature that best separated a training set of 286 primary breast carcinomas into prognostic subtypes by stepwise cross-validation. Notably, this signature predicts clinical outcome in over 10 patient cohorts as well as their respective ER+ subcohorts. Further, this signature separates patients who have received endocrine therapy into two prognostic subgroups, suggesting its specificity as a measure of estrogen signaling, and thus hormone sensitivity. The 73-gene signature also provides additional predictive value for patient survival, independent of other clinical parameters, and outperforms other previously reported molecular outcome signatures. Taken together, these data demonstrate the power of using cell culture systems to screen for robust gene signatures of clinical relevance.