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Background: Age is the main risk factor for developing breast cancer (BC) in women. The increase in incidence with age is dominated by estrogen receptor positive (ER+) BC despite the >90% reduction of estrogen levels during menopause and later life. Recent models suggest the lifetime risk of BC is correlated with the number of stem cell divisions with random mutations arising during each division and driving cancer development. However, only two genes are mutated in more than 20% of BC while several recurrent copy number alterations (CNAs) occur in more than 50%. New models of age-related risk factors need to be developed that incorporate mutations, CNAs, epigenetics, and other biological factors. As data of this type is limited in normal breast tissue, factors correlated with age of diagnosis in ER+ BC were identified to understand which variables changed with age and might impact risk of developing BC. Methods: In silico analysis of public databases was used to identify factors in ER+ primary BC and normal adjacent tissue (NT) that are correlated with age of diagnosis including DNA mutations, CNAs, DNA methylation and gene/protein expression (TCGA n=599ER+,113NT; METABRIC n=1435ER+,144NT). Results: DNA mutations accumulated with age in ER+ BC with the median mutation count below 28 in primary tumors diagnosis in patients 80yr. However, the two most frequently mutated genes (PIK3CA and TP53) showed no significant correlation with age. As previously reported, GATA3 mutation rate was nearly twice as high in younger patients (80yr=0.12). There was little evidence of a general accumulation of CNAs with age, but there were higher rates of gain in 16p (80yr=0.51) and loss of 1p (80yr=0.37) in older patients and lower rates of loss in 6q (80yr=0.24). The most significant correlations with age related to ESR1 including expression of ESR1 mRNA (rho=0.39,P=1.2e-23 Spearman), ERα protein (rho=0.35,P=1.7e-15 Spearman), and demethylation of ESR1 promoter (rho=-0.36,P=1.6e-13 Spearman). The levels of the activated form of ERα (pS118) also correlated with age but to a much lesser extent than total ERα (rho=0.12,P=0.01 Spearman). Demethylation and expression of ESR1 were highly correlated (rho=-0.50,P Conclusions: Analysis of DNA mutations and CNAs fit previous theoretical models with both the result of stochastic processes. In these models, the low impact on fitness from individual mutations allows the accumulation over time while the high fitness costs of CNAs prevent accumulation and are likely acquired in a single catastrophic event. The ability of ER+ BC to progress in the presence of very low postmenopausal estrogen levels may be partly explained by demethylation leading to higher ESR1 expression and maintenance of ERα activation. In theory, changes in expression and demethylation can be targeted to reduce the age-related increase in risk for developing BC. Citation Format: Schuster EF, Dowsett M. Age-related genetic and epigenetic changes in estrogen-receptor positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-05-02.

Genomic profiling has become mainstream for the management of early ER+ breast cancer and has become a favoured approach for research into pathways that govern prognosis and response and resistance to medical therapies.Management: Several commercial tests are available for the assessment of residual risk of recurrence of patients receiving hormonal therapies. We have compared several of these in postmenopausal patients treated with anastrozole or tamoxifen in the ATAC trial, including OncotypeDX (ODX), Prosigna (PAM50) and EndoPredict (EP). While PAM50 and EP provide estimates of risk that by design integrate tumour size and nodal status, estimates from ODX are also improved substantially by integration with clinicopathologic factors. The 3 tests include genes related to proliferation and to dependence on estrogen signalling with the ODX being dominated by the latter and PAM50 by the former. This may explain the better performance of the PAM50 than ODX at cessation of endocrine treatment after 5 years when the outcome for patients with high ER appears to worsen. While the tests have largely been validated in postmenopausal cases they are used in premenopausal cases as well. We found that while individual gene modules varied in expression across the menstrual cycle there was little systemic variability in the overall estimates of risk. Our recent work has indicated that mutations in TP53 add significant prognostic information to that from the commercial profiling tests.Research: We have conducted genomic analyses during presurgical therapy of ER+ breast cancer to determine the key pathways of endocrine resistance using change in Ki67 as the end-point of response. Very few tumours with low levels of ER by IHC ( Citation Format: M Dowsett. Genomic Profiling in Early Stage ER Positive Breast Cancers/Precision Medicine [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PL1.

The persistent risk of recurrence from ER-positive primary breast cancer has been known for many years. It was recently described in detail and in relation to baseline clinicopathologic factors in a meta-analysis by the EBCTCG. This involved nearly 63,000 patients that had been prescribed 5 years of endocrine therapy and had reached the end of that time recurrence-free (Pan et al, NEJM, 2017, 377, 1836). Among other things this revealed that even among patients with T1N0 disease 13% had a distant recurrence between years 5 and 20. Our group have previously created a Clinical Treatment Score (CTS) from nodal status, tumor size, grade, age and type of adjuvant endocrine treatment and validated this for estimating risk of distant recurrence from the time of diagnosis. Drawing on a training dataset of 4,735 patients from the ATAC trial and a validation set of 6,711 patients from the BIG1-98 trial we created the CTS5, a prognostic score of risk from year 5, that features the same factors as CTS other than type of endocrine treatment (Dowsett et al, JCO, 2018, 36, 1941). Of note, only 4/304 patients with one to three positive nodes that were categorized as low risk in the validation set had a distant recurrence between years 5 and 10. The CTS5 calculator can be accessed online at www.cts5-calculator.com. There are several commercially available molecular profiles that estimate the risk of recurrence over years 0-10 after diagnosis in endocrine-treated primary breast cancer. In the TransATAC study some of these, including the EPClin and PAM50-ROR, show similar prognostic information over years 5-10 (after 5 years of endocrine therapy) as they do over years 0-5 (during endocrine therapy). In contrast, while the Oncotype Recurrence Score (RS) showed similar prognostic information to EPClin and PAM50-ROR in years 0-5 its performance in years 5-10 was substantially worse. Examination of the time-dependent prognostic relationship of each of the individual genes and modules in the RS showed that those patients with higher ER signaling had good prognosis before year 5 but poorer after year 5, suggesting that the cessation of endocrine therapy may lead to this poorer post-5 year outcome (Dowsett et al Clin Cancer Res, 2015, 21, 2763). A broad-based assessment of gene expression in the TransATAC sample set found that there was limited potential to identify a prognostic profile that performed substantially better after 5 years (Buus et al, Breast Cancer Res, 2018, 20, 103). One test, the 7-gene Breast Cancer Index, has reported on a relatively small sample set that it can predict the benefit or not from extended adjuvant endocrine therapy. Further validation studies of this are warranted as well as other tests that provide the same predictive information. Citation Format: Dowsett M. Clinical and molecular profiles to identify who is at risk? [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr ES5-2.

In experimental models, human epidermal growth factor receptor-2 (HER-2) amplification leads to estrogen independence and tamoxifen resistance in estrogen receptor (ER)-positive human breast cancer cells. Some but not all reports suggest an association between HER-2 positivity and hormone independence in breast cancer patients. This study aimed to evaluate the antiproliferative effects of endocrine therapy in HER-2-positive/ER-positive primary human breast cancer. The effect on proliferation (Ki67) of hormone therapy was assessed at 2 weeks and/or 12 weeks in biopsies from 115 primary breast cancers with ER-positive tumors. The patients took part in one of 3 neoadjuvant trials of hormonal therapy with a SERM (tamoxifen or idoxifene) or an aromatase inhibitor (anastrozole or vorozole). HER-2 status was assessed by immunocytochemistry and fluorescence in situ hybridization (FISH). Fifteen patients were defined as HER-2 positive by both immunohistochemistry and FISH, with the remaining 100 patients HER-2 negative. Geometric mean Ki67 levels were substantially higher in HER-2-positive than HER-2-negative tumors (27.7% versus 11.5%, respectively; P = 0.003). In HER-2-negative patients, Ki67 was reduced by 62 and 71% at 2 and 12 weeks, respectively (P0.0001 for both), but HER-2-positive patients showed no significant fall. The proportional change in Ki67 was significantly different between HER-2-positive and -negative patients (P = 0.014 at 2 weeks; P = 0.047 at 12 weeks). Mean ER levels were lower in the HER-2-positive patients (P = 0.06) but the change in Ki67 was impeded even in those with high ER. Apoptotic index was reduced by 30% at 2 weeks in the HER-2-negative group. However, there were no statistically significant differences in apoptotic index between the groups. It is concluded that ER-positive/HER-2-positive primary breast carcinomas show an impeded antiproliferative response to endocrine therapy that nonetheless may vary between individual treatments. This together with high baseline proliferation is likely to translate to poor clinical response.

Preliminary studies have suggested that measuring the ability of immunoreactive 67-kDa estrogen receptor (ER) to bind DNA and form in vitro complexes with its cognate estrogen response element (ERE) might serve to identify breast tumors most likely to respond to antiestrogens like tamoxifen. Data from two different surveys of untreated primary breast tumors confirmed that only 67% (74 of 111) of ER-positive tumors express a receptor capable of forming ER-ERE complexes by gel-shift assay, with tumors of lower ER content having significantly reduced ER DNA-binding frequency (56%) relative to those of higher ER content (82%; P = 0.007). In contrast to these untreated tumors, a panel of 41 receptor-positive breast tumors excised after acquiring clinical resistance to tamoxifen during either primary (n = 26) or adjuvant therapy (n = 15) showed a significantly greater ER DNA-binding frequency, with nearly 90% capable of forming ER-ERE complexes (P0.02). To assess experimentally whether ER DNA-binding function is altered during the development of antiestrogen resistance, nude mouse MCF-7 tumor xenografts were analyzed before and after the acquisition of in vivo resistance to either tamoxifen or a pure steroidal antiestrogen, ICI 182,780. Tamoxifen-resistant MCF-7 tumors retained full expression of 67-kDa DNA-binding ER, and despite a markedly reduced ER content in the ICI 182,780-treated tumors, the expressed ER in these antiestrogen-resistant tumors exhibited full ability to form ER-ERE complexes. These findings indicate that breast tumors with acquired antiestrogen resistance continue to express ER of normal size and DNA-binding ability and suggest that the failure of antiestrogens to arrest tumor growth during emergence of clinical resistance results from an altered gene-regulatory mechanism(s) other than ER-ERE complex formation.

Changes in estrogen receptor (ER) expression and function may explain the development of tamoxifen resistance in breast cancer. ER expression was measured by an immunohistochemical assay, validated for use in tamoxifen-treated tumors against a biochemical enzyme immunoassay, in 72 paired biopsies taken before treatment and at progression or relapse on tamoxifen. Progesterone receptor (PgR) and pS2 gene expression were also measured immunohistochemically as an indicator of ER function. Overall the frequency of ER expression was reduced from 37 of 72 (51%) pretamoxifen to 21 of 72 (29%) at progression or relapse, with a significant reduction in the quantitative level of ER (P0.0001; Wilcoxon signed rank sum test). Tumors treated with primary tamoxifen that responded but then developed acquired resistance frequently remained ER positive (ER+) at relapse: 16 of 18 (89%) were ER+ pretamoxifen (75% of these expressed either PgR or pS2) and 11 of 18 (61%) were ER+ at relapse (82% continued to express PgR or pS2). In contrast, only 3 of 20 (15%) tumors that progressed on primary tamoxifen with de novo resistance were ER+ pretamoxifen, and all tumors were ER- at progression. At progression, 6 of 20 (30%) of these tumors expressed high levels of PgR (mean H-score, 98) and/or pS2 (mean, 50% cells positive), despite being ER-. In tumors that recurred during adjuvant tamoxifen therapy, including locoregional and metastatic lesions, ER expression was significantly reduced from 18 of 34 (53%) in the original primary tumor to 10 of 34 (29%) at relapse (P = 0.002). PgR expression was likewise significantly reduced in this group (P = 0.001). This study confirms that expression of a functional ER in breast cancer is a strong predictor for primary response to tamoxifen. Although ER was reduced in tamoxifen-resistant tumors overall, the development of acquired resistance was associated with maintained ER expression and function in many tumors, whereas de novo resistance remained related to lack of ER expression. Recurrence during adjuvant tamoxifen was associated with development of an ER/PgR-negative phenotype in some tumors. These data imply that separate mechanisms of resistance may occur in these different clinical subgroups.

Idoxifene, a novel antiestrogen with reduced estrogenic activity when compared to tamoxifen, has been given to 20 women with metastatic breast cancer, 19 of whom had received tamoxifen previously, in doses between 10-60 mg. Idoxifene had an initial half-life of 15 h and a terminal half-life of 23.3 days. At a maintenance dose of 20 mg, a mean steady-state level of 173.5 ng/ml was achieved. Significant falls in luteinizing hormone and follicle-stimulating hormone were seen, but the falls were not dose related. Idoxifene was well tolerated, with 11 patients complaining of mild symptoms similar to those seen with tamoxifen. Fourteen patients continued idoxifene therapy for 1-56 weeks; 4 patients showed stabilization of disease for 6-56 weeks and 2 patients showed a partial response.

The exon 5 deletion splice variant of estrogen receptor (delta 5 ER), which in vitro is constitutively active in the absence of estrogens, may have a role in conferring both tamoxifen resistance and ER-related phenotype in breast cancer. We have investigated the expression of this variant in vivo (at the level of mRNA) in relation to known tamoxifen resistance and expression of the estrogen-regulated genes progesterone receptor (PgR) and pS2. The amount of delta 5 ER mRNA relative to wild type (WT) ER mRNA (% delta 5/WT) was assayed in 70 tamoxifen-resistant and 50 primary breast carcinomas using reverse transcription/PCR. Both WT and delta 5 ER mRNA were detected in the majority of tumors, although delta 5 ER was detected only in the presence of WT ER. Overall no significant difference was seen in % delta 5/WT ER between tamoxifen-resistant and primary control tumors (medians, 13 and 15%, respectively). Tumors in both control and resistant groups which expressed PgR/pS2 in the absence of measurable ER protein (ER- PgR+ and ER- pS2+) had significantly higher delta 5 ER mRNA levels compared with other phenotypes (P0.002). This association with ER-/pS2+ tumors has not been demonstrated previously. In ER+ tumors which expressed pS2, significantly greater delta 5 ER mRNA expression was observed in tamoxifen-resistant compared with control tumors (P = 0.05). A similar although nonsignificant trend was observed in ER+ PgR+ tumors. While delta 5 ER mRNA is unlikely to be responsible for tamoxifen resistance in most breast cancers, elevated delta 5 ER mRNA levels may be important in some tumors, especially those which continue to express high levels of PgR/pS2.

Vorozole is an orally active, nonsteroidal aromatase inhibitor. Twenty-four postmenopausal patients with advanced breast cancer who had relapsed after treatment with tamoxifen received three separate daily doses of vorozole (1, 2.5, and 5 mg) each for 1 month in a randomized, double-blind, phase II study. There was significant suppression (P0.001) of serum estradiol at all three doses (median reduction, 91, 90, and 89%, respectively). There was a significant trend (P = 0.02) for estradiol to be suppressed below the detection limit of the assay (3 pmol/liter) more frequently with an increasing dose of vorozole; 13, 31, and 40% respectively. Estrone and estrone-sulfate levels were likewise reduced at each dose by 52-55% and 64-69%, respectively. There was no significant effect at any dose on aldosterone, testosterone, androstenedione, 17 alpha-hydroxyprogesterone, or thyroid-stimulating hormone levels. A small reduction in cortisol was seen at the 5 mg dose, although the relevance is unclear given that 17 alpha-hydroxyprogesterone levels did not rise. Eight patients (33%) achieved an objective response (2 complete remission, 6 partial remission) with a median response duration of 13 months. Four patients (17%) achieved disease stabilization for more than 6 months. Patients who had responded previously to tamoxifen were more likely to respond to vorozole. There were no significant clinical side effects and the drug was well tolerated. These data suggest that vorozole is a potent and selective oral aromatase inhibitor for use in postmenopausal breast cancer.

We have conducted a clinical trial of a novel pure antiestrogen, 7 alpha-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl]estra-1,3,5,(1 0)-triene-3,17 beta-diol (ICI 182780), to assess its tolerance, pharmacokinetics, and short term biological effects in women with primary breast cancer. Fifty-six patients were randomized to either a control group (n = 19), in which they received no preoperative treatment, or a treatment group (n = 37), in which they received daily i.m. injections of ICI 182780 at doses of 6 mg (n = 21) or 18 mg (n = 16) for 7 days prior to primary breast surgery. Serum drug concentrations, gonadotropin levels, and sex hormone-binding globulin levels were measured during the study period by radioimmunoassay. Expression of estrogen receptors (ER), progesterone receptors, the estrogen-induced protein pS2, and the cell proliferation-related antigen Ki67 was determined immunocytochemically in pre- and poststudy tumor samples. Treatment with ICI 182780 caused no serious drug-related adverse events and had no effect on serum gonadotropin or sex hormone-binding globulin levels. Minor adverse events occurred in 5 patients receiving the 6-mg dose and 3 patients receiving the 18-mg dose. The serum concentration of ICI 182780 was dose dependent but showed variation between individuals. There was evidence of an approximately 3-fold drug accumulation over the short treatment period but steady state levels were not reached by the end of the 7 days. In patients with ER-positive tumors, treatment with ICI 182780 was associated with significant reductions in the tumor expression of ER (median ER index, 0.72 before versus 0.02 after treatment; P0.001), progesterone receptor (median progesterone receptor index, 0.50 before versus 0.01 after treatment; P0.05), and Ki67 (median Ki67 labeling index, 3.2 before versus 1.1 after treatment; P0.05). Treatment with ICI 182780 also resulted in a significant reduction in pS2 expression (P0.05) but this appeared unrelated to tumor ER status. In conclusion, ICI 182780 was well tolerated after short term administration and produced demonstrable antiestrogenic effects in human breast tumors in vivo, without showing evidence of agonist activity. These properties identify ICI 182780 as a candidate agent with which to evaluate whether a pure estrogen antagonist offers any additional benefit in the treatment of human breast cancer over conventional nonsteroidal antiestrogens, typified by tamoxifen, which exhibit variable degrees of agonist activity.

A phase I study was performed of CGS 20267, an oral nonsteroidal, highly potent, and selective aromatase inhibitor, in 21 postmenopausal patients with advanced breast cancer. The patients were recruited in 3 successive groups of 7, receiving 0.1, 0.5, and 2.5 mg p.o./day, respectively. All patients had received at least one prior endocrine treatment (range, 1-4), and six patients had received prior chemotherapy. The treatment was very well tolerated, and no toxicity was seen at any of the three doses. There was a statistically significant suppression of estradiol (E2) and estrone (E1) levels by 74% and 79% from baseline levels, respectively (P0.0001). Suppression occurred in all three patient groups, with many patients having serum concentrations of estradiol and estrone, which were below the limit of detection of the assays (3 and 10 pM, respectively), which corresponds to a maximum measurable estrogen suppression of 86%. CGS 20267 had no significant effect on serum levels of follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone, cortisol, 17 alpha-hydroxyprogesterone, androstenedione, and aldosterone. Seven (33%, 95% confidence interval, 15-57%) of the 21 patients have responded to treatment (one complete remission, 6 partial remissions according to criteria of the Union Internationale contre le Cancer), and 6 are still responding to CGS 20267 (duration of response; 4+, 6+, 6+, 9+, 9, 12+, and 12+ months). Five have had stable disease for more than 3 months, and 9 had progressive disease. These results suggest that CGS 20267 is a very potent and specific aromatase inhibitor, and phase II studies are now required to confirm its clinical efficacy.

We have investigated the ability of tamoxifen to regulate members of the transforming growth factor beta (TGF-beta) family in human breast cancers in vivo. Using immunohistochemical techniques, we find that 3 months of tamoxifen treatment causes a consistent induction of extracellular TGF-beta 1 in breast cancer biopsies, compared with matched pretreatment samples from the same patient. The induced TGF-beta is localized between and around stromal fibroblasts and appears to be derived from these cells. Lower levels of TGF-beta 1,-beta 2, and -beta 3 seen in epithelial cells were not altered by tamoxifen treatment. The increased stromal staining of TGF-beta 1 occurred in estrogen receptor-negative as well as estrogen receptor-positive tumors. These results provide in vivo evidence for a novel, estrogen receptor-independent mechanism of action for tamoxifen, involving the stromal induction of a potent growth inhibitor for epithelial cells.

The effect of a number of steroids, growth factors, and peptides on aromatase activity in two estrogen receptor positive breast cancer cell lines (MCF7 and T47D) was investigated. The cells were incubated in Dulbecco's minimum essential medium containing phenol red and 10% fetal calf serum. Pronounced differences in basal aromatase activity and different responses to the addition of experimental agents were found in the two cell lines. Aromatase activity in MCF7 cells was significantly stimulated by phorbol 12,13-diacetate [PDA], dibutyryl cyclic AMP [(Bu)2cAMP], transforming growth factor alpha, and epidermal growth factor individually and PDA and (Bu)2cAMP in combination, while it was inhibited by dexamethasone and unaffected by transforming growth factor beta, fibroblast growth factor, platelet-derived growth factor, prolactin, and tamoxifen. Addition of cortisol to MCF7 cells had no effect on aromatase activity at 1 nM, caused suppression of activity at 10 nM and stimulated activity at 100 nM. Aromatase activity in T47D cells was stimulated by transforming growth factor alpha, epidermal growth factor, platelet-derived growth factor, prolactin, dexamethasone, and cortisol individually and PDA and (Bu)2cAMP in combination. It was unaffected by transforming growth factor beta, PDA, (Bu)2cAMP, and fibroblast growth factor. These findings suggest that aromatase activity is induced by agents which stimulate cyclic AMP-dependent protein kinases [e.g., (Bu)2cAMP] and that this effect is potentiated by factors which stimulate protein kinase C [e.g., PDA]. The effect on aromatase activity of growth factors, the actions of which are believed to be mediated by receptors linked to tyrosine kinase activity, is not as clearly defined, with a factor causing stimulation, inhibition, and no change in activity depending on the tissue concerned. Further insight into these differences will require resolution of the molecular mechanisms that mediate the actions of stimulatory and repressive growth factors on aromatase activity of oestrogen-producing cells.

Aim: To compare SP6 and MIB1 antibodies against Ki67 for use with visual scoring, image analysis and TMA (tissue microarray) analysis.Background: Immunohistochemical detection of Ki67 has been widely used in assessing the proliferation fraction in breast cancer. Neoadjuvant studies revealed that changes in Ki67 predict benefit from endocrine treatment, and that on-treatment Ki67 measurements are better predictors of long-term outcome than pretreatment levels (Dowsett at al, JNCI 2007; 99:167-170). As a result Ki67 is the primary end-point for some pre-surgical trials. MIB-1 has been the preferred Ki67 antibody for over 10 years. A rabbit monoclonal antibody, SP6, has become available with apparently improved performance, e.g. reduced background non-nuclear staining. The importance of Ki67 led us to conduct a systematic series of studies to validate SP6 in comparison to MIB1.Methods: We used TMAs constructed from (i) 177 archival FFPE blocks of primary or locally recurrent breast tumours from women treated with an aromatase inhibitor (AI) for advanced disease (AI TMAs) and (ii) one set of replicate blocks (200 tumours) from the TransATAC program. The TMAs were of 600µm cores. Three replicate TMAs (A, B, C) were used. Staining was performed using DAKO Autostainer and DAKO REAL Kit Detection System. MIB-1 antibody (DAKO) was used at a dilution of 1:40, whereas 1:100 dilution was used for SP6 antibody (Abcam). Stained sections were examined either visually or using the Ariol image analysis system (Genetix Ltd.).Results: There was a strong correlation between the antibodies in AI TMAs scored manually (n=161, r=0.93, p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3048.

Aims: To (1) define the effect of oestrogen deprivation on genes related to hypoxia in oestrogen receptor positive (ER+) breast cancer and (2) identify any link between hypoxia and proliferation.Background: The majority of breast cancer patients are postmenopausal women with ER+ tumours and at some point receive an aromatase inhibitor (AI) as part of their treatment. Hypoxia and proliferation are important factors in the progression of ER+ tumours. Proliferation is profoundly reduced in most ER+ cancers after treatment with AIs[1], however little is known on the effects of AIs on hypoxia.Materials and methods: 81 pre- and 2-week post-treatment core-cut tumor biopsies were obtained from postmenopausal women with ER+ breast cancer who received single agent neoadjuvant anastrozole (AI)[2], and from 20 of these patients after 16 weeks of AI treatment. RNA was extracted and analysed on Illumina 48K microarrays. A hypoxia metagene (MG) was developed by identification of genes whose expression clustered with the expression of classical hypoxia-regulated genes[3]. Genes associated with proliferation were removed from the MG. A proliferation MG was derived by selecting the intersection of proliferation clusters from three public breast cancer datasets.Results: Spearman correlations revealed a strong relationship between the hypoxia and proliferation MGs prior to AI treatment (r =0.61, p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 408.

Background: Neoadjuvant endocrine therapy allows biological studies of de novo response and resistance but not of acquired resistance based on clinical endpoints as surgery is usually carried out after around 4 months. Response is associated with a significant fall in Ki67 (proliferation) after 2 weeks treatment. However, at 12/16 weeks, c.15% of tumours show recovery of Ki67. We hypothesized that this recovery reflected rapidly acquired treatment resistance and have assessed its clinical relevance and association with tumour biomarkers.Methods: Ki67, ER, PgR and HER-2 (with FISH for 2+ cases) IHC measurements taken at baseline, 2 weeks and 12/16 weeks during neoadjuvant aromatase inhibitor (AI) treatment were available from three previously reported clinical trials [1-3] for a total of 177 ER+ patients. To allow pooling of data, ER and PgR levels were expressed as Allred scores. Long-term clinical follow-up data were available for 142 patients who received endocrine therapy within the IMPACT trial. Categories of response/resistance based on Ki67 change were created and patients classified accordingly: de novo resistance (50% decrease in Ki67 from baseline to 2 weeks, but overall fall at 12/16 weeks 50% decrease in Ki67 from baseline to 2 weeks and overall fall >50%).Results: Patients defined as showing acquired resistance had significantly worse RFS than those defined as showing maintained response (HR: 3.73, 95%CI: 1.18-11.79, p=0.025) and very similar to those with de novo resistance (HR: 3.66, 95% CI: 1.41-9.50, p=0.008). More HER-2+ cases showed Ki67 recovery than would be expected by chance (acquired resistance: 5/19 HER-2+, maintained response: 5/100 HER-2+; p=0.02). There was a weak suggestion that pre-treatment ER level may be lower for patients showing Ki67 recovery than those with persistent response (p=0.11), but no evidence was seen for an effect of pre-treatment PgR level (p=0.52).Conclusions: Tumours that exhibit an early Ki67 fall followed by Ki67 recovery at 12/16 weeks are associated with poorer RFS than those showing maintained suppression of Ki67 suggesting that Ki67 may act as a valid intermediate marker of acquired resistance. HER-2 positivity is associated with development of early acquired Ki67-based resistance, but other mechanisms for early acquired resistance exist.Supported by The Mary-Jean Mitchell Green Foundation.[1] Dowsett M et al., J Clin Oncol 2005. 23(11): 2477-92[2] Smith IE et al., J Clin Oncol 2007. 25(25): 3816-22[3] Miller WR et al., J Clin Oncol 2009. 27(9): 1382-7 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2005.

Background: Paired biopsies at diagnosis and relapse on tamoxifen (TAM) provide information regarding adaptive changes associated with endocrine resistance. We previously reported that conversion to a HER2+ phenotype occurs infrequently1, however modest increases in HER2 may be relevant in the context of TAM resistance. Also while TAM resistance in general is associated with a fall in mean ER and PgR levels, this masks changes in individual pts.Aim: To determine whether a subset of HER2 negative BC demonstrated modest increases in HER2 by IHC and FISH, and if so whether this correlated with changes in ER and PgR expression in individual pts.Methods: Triplicate TMAs were constructed of paired primary and recurrent tumor samples from pts relapsing after adjuvant TAM. TMAs were stained for ER and HER2, whole sections for PgR. HER2 amplification was assessed by FISH.Results: Paired samples from primary and TAM-resistant recurrences were available on 53 ER+ and/or PgR+ pts. 10% had a HER2+ primary (IHC3+ and FISH+). 19% (10/53) showed increasing HER2 in the relapsed tumor (0 to 1+ in 5 pts, 1+ to 2+ in 2 pts and 0/1+ to 3+ in 3 pts). The 3 pts converting to 3+ also became FISH+. 71% (38/53) had stable or decreasing HER2. DFI was 50.3 months in the subset with stable or decreasing HER2, 40.2 in the subset with increasing HER2, and 35.2 in the HER2+ subset (p=NS). We next compared changes in ER and PgR in the 3 subsets. Among pts with stable or decreasing HER2 (N=38), there was a significant decrease in mean H-scores for both ER (p=0.001) and PgR (p=0.002) at relapse (Fig 1); the majority recurred with reduced ER (76%) or PgR levels (84%). In contrast, among pts with increasing HER2, there was no change in mean ER or PgR levels (Fig 2A); however when considering changes in individual pts, there was a balanced split between tumors relapsing with up- vs. down-regulation in ER/PgR (Fig 2B,C). In the HER2 positive subset, 100% of the pts relapsed with suppressed ER and PgR.Conclusion: In an updated series of TAM-resistant pts, we show that while conversions to HER2 3+/FISH+ remain rare (5%), modest increases in HER2 are more frequent (19%). TAM resistance in general is associated with decreasing ER and PgR. However, among pts with adaptive increases in HER2, TAM resistance is associated with either down- or up-regulation in ER expression and function. This is consistent with data from acquired endocrine resistance models we previously reported2,3.1Gutierrez et al, JCO 20052Pancholi et al, ERC 2008,3 Martin et al, JBC 2003 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 704.

Aim: To identify whether breast cancer-associated SNP rs2046210 is associated with ERα level in invasive breast tumours.BackgroundA recent genome-wide association study identified SNP rs2046210 at 6q25.1 as having a strong association with breast cancer risk1. The SNP is located 29kb upstream of the first untranslated exon of ESR1 and 180kb upstream of the transcription start site2. Rs2046210 is not in linkage disequilibrium (LD) with two of the most widely studied polymorphisms in ESR1. We test here the hypothesis that rs2046210 may be associated with altered ESR1 expression.MethodsPatients with both leukocyte DNA and invasive breast tumour paraffin blocks available were identified from two of our ongoing tissue collections: Femara Anastrazole Clinical Evaluation (FACE) and the British Breast Cancer (BBC) study. Germline DNA was extracted from bloods using the QIAamp DNA Blood Mini Kit. PCR was performed with primers spanning the SNP site, with a single basepair mismatch in the reverse sequence to generate an HhaI restriction site in the presence of the C allele. PCR product was run on a 3% agarose gel to confirm presence of a single 120bp band. Restriction digest with HhaI was then performed and products run on a 4% Metaphor agarose gel. Genotype was assigned as follows: 97bp band only = wild-type (C/C); 97bp and 120bp band = heterozygote (C/T); 120bp band only = variant (T/T). From corresponding invasive breast tumour, ERα was assessed on 4μm whole sections using clone 6F11 (Vector Labs) and quantified by H-score. Mean H-scores (left and right invasive breast tumours) were used for the BBC group. ERα- samples (H-score≤1.0) were excluded. Analysis was by ANOVA using non-parametric bias-corrected and accelerated 95% bootstrap confidence intervals (2000 replications), with genotype fitted as a score and study as a stratifying co-variate.ResultsH-score (mean) by genotypeStudyWild-typeHeterozygoteVariantFACE (n=280)171.8180.9184.9BBC (n=46)180.0178.9194.1Fitted mean172.6179.7186.7(% samples)(45)(44)(11) Minor allele frequency was 33%, which is similar to the 38% previously reported in patients of European ancestry1. Within FACE, increased ERα was seen with presence of variant SNP. In the BBC group, variant SNP patients had higher ERα than both wild-type and heterozygote. Overall, there was a significant difference in ERα score per genotype group of 7.05, (95% CI 0.7-13.5, p=0.035). This was circa 4% difference in H-score per variant allele.ConclusionThe variant genotype of SNP rs2046210 is associated with increased ERα expression. While the increase contributed by the variant allele is relatively modest, this may partly explain why the SNP is associated with increased breast cancer risk. Similar studies should be conducted in normal breast tissue.1Zheng et al. (2009) Nat. Gen. 41(3): 324-3282Kos et al. (2001) Mol. Endocrinol. 15: 2057-2063 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4138.

Abstract #63 Background: Estrogen exposure is linked to the development and treatment of the majority of breast cancers. High estradiol (E2) levels are associated with elevated breast cancer risk in postmenopausal women and estrogen antagonism or withdrawal reduces tumor cell proliferation of ER+ breast cancers. Within postmenopausal breast tumors, E2 concentrations are 10-20 times higher than in plasma. Intratumoral synthesis is thought to be the principal cause of this gradient and consequently to dominate estrogen signaling in postmenopausal breast cancers. Contrary to this expectation, we have made the novel observation that intratumoral expression of estrogen responsive genes is correlated with plasma E2 levels in two independent datasets. Methods: Pre- and 2wk post-treatment core-cut tumor biopsies were obtained from 104 postmenopausal women with stage I to IIIB ER+ early breast cancer who received single agent neoadjuvant anastrozole1. RNA extracted from biopsies was analysed on Illumina 48K microarrays. Plasma E2 levels were determined by a highly-sensitive RIA before and after the 2wks treatment. Quantitative trait analysis (QTA) by Spearman correlation was used to identify genes whose expression in pre-treatment tumors or changes upon treatment were significantly correlated with pre-treatment plasma E2 levels. Gene set enrichment analysis (GSEA) was performed on the ranked list of correlated genes. Validation was performed in an independent set of 73 ER+ or PR+ breast cancers. Results: QTA revealed that genes whose expression is known to be highly responsive to estrogen were quantitatively associated with plasma E2 levels. For example, TFF1/pS2, GREB1, PDZK1 and PGR were positively correlated with plasma E2 (p Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 63.

Thirty-one postmenopausal women with advanced breast cancer have been treated with the nonsteroidal competitive aromatase inhibitor CGS 16949A at p.o. doses of 0.3, 1, and 2 mg twice a day. All patients were assessed for response. Five patients, all treated with 1 mg twice daily, had objective evidence of response (two complete responses and three partial responses); disease stabilized in 17 patients. Minor side effects were reported by ten patients. Two further patients treated with 2 mg twice a day experienced persistent nausea which improved after dose reduction, and one patient, treated with 0.3 mg twice daily, developed a vasculitic rash requiring discontinuation of CGS 16949A. Estradiol levels measured in 24 patients were significantly suppressed 2 wk after starting CGS 16949A treatment at all doses used. Treatment with 2 mg twice a day lowered estradiol levels to a mean of 29% of pretreatment values which was significantly lower than the corresponding figure of 57% for patients treated with 0.3 mg twice daily. Aldosterone levels were significantly lowered below pretreatment values by the 1- and 2-mg twice daily doses. No clinically apparent cases of adrenocortical insufficiency occurred, although small changes in serum electrolyte levels were noted. The results indicate that CGS 16949A is an effective aromatase inhibitor, requiring further evaluation in the treatment of advanced breast cancer. The optimal dose is likely to be 1 mg twice a day.

4-Hydroxyandrostenedione (4-OHA), a potent new aromatase inhibitor, was given i.m. (500-1000 mg) to 58 patients with advanced postmenopausal breast cancer. Of 52 assessable patients 14 responded (27%), in 10 (19%) the disease stabilized, and in 28 (54%) the disease progressed. Sterile abscesses occurred at the injection site in 6 patients and painful lumps were found in a further 3 patients. Two patients developed allergic-type reactions and 4 developed lethargy, suspected to be treatment induced. Plasma estradiol levels were suppressed from a mean of 7.2 +/- 0.8 (SE) pg/ml before treatment to 2.6 +/- 0.2, 2.7 +/- 0.2, and 2.8 +/- 0.3 pg/ml after 1, 2, and greater than 4 months, respectively, of treatment and remained suppressed in patients whose disease relapsed. No significant fall in estrone levels was seen. Similarly, dehydroepiandrosterone sulfate, sex hormone binding globulin, and gonadotrophin levels were unaltered after 6 months of treatment. Plasma 4-OHA levels were measured in a radioimmunoassay for androstenedione after chromatographic separation of 4-OHA from androstenedione. Drug concentrations ranged from 0.7 to 23.2 (7.8 +/- 1.1) ng/ml after 2 months on treatment. 4-OHA is an effective drug in the management of postmenopausal patients with breast cancer and does not produce notable systemic side effects.

Intratumoral aromatase activity (AA) was measured in 145 samples of human primary breast carcinoma using the tritiated water release assay which quantifies the tritium lost to water during the aromatization of 1 beta-[3H]androstenedione to estrone. Significant AA was detected in 91/145 (63%) tumors. The possibility of a relationship between AA and a variety of clinical prognostic factors such as estrogen receptors, menopausal status, site, size, and histological grade of tumor was investigated. Possible relationship with time to relapse, overall survival, and survival of patients after relapse were also studied to determine whether intratumoral AA itself was of any prognostic value. There was no relationship between AA and tumor size, site, nodal status, menopausal status or estrogen receptors. However there was a significant correlation between AA and histological grade with an excess of AA-positive tumors having high grade (P = 0.03). There was no significant relationship between AA and overall survival (P greater than 0.1), but there was a marginal inverse correlation between AA and time to relapse (P less than 0.1). A statistically significant correlation was found between AA and survival of patients after relapse (P less than 0.05).

4-Hydroxyandrostenedione (CGP32349; 4-OHA) is a clinically effective treatment for advanced postmenopausal breast cancer by both the parenteral and p.o. routes, as a result of its inhibition of aromatase and consequent suppression of plasma estrogen levels. Thirty patients were randomized to treatment with 250 mg 4-OHA orally once, twice, and 4 times daily for 2 weeks and 29 of these plus a further 11 patients were then randomized to treatment with 250 or 500 mg i.m. every 2 weeks to determine the optimal dose for each route according to the suppression of serum estradiol levels. There was no significant difference between the 3 oral doses in their suppression of estradiol levels indicating that the maximum required p.o. dose of 4-OHA is probably 250 mg daily. Suppression by the parenteral dose of 250 mg every 2 weeks was marginally suboptimal but clinical considerations of response and tolerability indicate this as the optimal dose for i.m. injection. 4-OHA had no effect on serum levels of androstenedione, testosterone, or 5 alpha-dihydrotestosterone when given by either route but p.o. treatment with 4 doses of 250 mg daily reduced sex hormone-binding globulin levels by a mean of 34%. Serum levels of estrone as measured by gas chromatography-mass spectrometry were suppressed to approximately 40% of baseline by parenteral treatment. The half-life of 4-OHA p.o. was approximately 3 h, whereas the apparent half-life of injected drug was between 5 and 10 days after a more rapid clearance during the first 4 days after injection.

4-Hydroxyandrostenedione (4-OHA) is a potent inhibitor of estrogen production by aromatase and causes suppression of plasma estradiol levels and disease regression in postmenopausal breast cancer patients. Groups of patients were given p.o. or parenteral 4-OHA, and plasma estradiol and 4-OHA levels were measured to enable the delineation of the minimal effective dose and optimal therapeutic regimen. A single injection of 500 mg i.m. suppressed estradiol levels to a mean 36.3 +/- 3.3% (SE) (n = 14) of base line after 4 to 7 days and maintained this suppression in six of seven patients for greater than 14 days. The half-life of 4-OHA was approximately 8 days, and when the level had fallen to less than 3 ng/ml, estradiol levels began to rise. Similar suppression was achieved by a single i.m. injection of 125 mg of 4-OHA and by 500 mg of 4-OHA p.o. daily after 1 wk, but escape from suppression was more rapid.

A group of 122 postmenopausal patients with histologically proven node-positive primary breast cancer have been randomized to receive aminoglutethimide-hydrocortisone or placebo aminoglutethimide-placebo hydrocortisone for 2 years. Median follow-up is 17 months. In general, treatment was well tolerated, but 15 patients required a reduction in the dose of aminoglutethimide, and of these four patients were unable to continue therapy due to side effects. Primary staging, incidence of extensive node involvement, and estrogen receptor were similar in the treatment and control arms. Dehydroepiandrosterone sulfate (DHA-S) and estrone were measured in a subgroup of patients, and significant suppression of DHA-S levels throughout the duration of the treatment period as seen in patients receiving the active drug. No significant suppression of either DHA-S or estrone levels was seen in the controls. Patients were monitored for metastases by serial liver function tests, carcinoembryonic antigen, and chest X-rays, and of 26 relapsing patients only three patients were not detected by this screen. We conclude that adjuvant aminoglutethimide is moderately well tolerated. It is capable of suppressing DHA-S throughout 2 years of treatment. A further 280 patients will be entered into the study to assess the survival benefit for those taking aminoglutethimide-hydrocortisone.