MD Anne C Regierer
email: anne.regierer@charite.de

R Wolters

C Kurzeder

A Wöckel

I Novopashenny

K Possinger

M B Wischnewsky

R Kreienberg

High estrogen receptor expression in early breast cancer: Chemotherapy needed to improve RFS?

Keywords: Adjuvant chemotherapy, early breast cancer, endocrine treatment, estrogen receptor, immunoreactive score, quantitative hormone receptor expression

Background:

One of the most controversial questions in early breast cancer treatment is the need of chemotherapy in patients with estrogen receptor positive disease.

Therefore we analyzed a group of patients with high ER expression to scrutinize the role of chemotherapy in this situation. To gauge the effect of chemotherapy on RFS three treatment modalities were compared: endocrine treatment only, chemoendocrine treatment, and chemotherapy.

Patients and methods:

3971 breast cancer patients whose treatment modalities as well as ER level were known, were included in this retrospective analysis. Their level of ER expression was documented as IRS. A high ER group was defined as ER IRS ≥ 9; primary endpoint was RFS.

Results:

RFS was associated with ER, with the best outcome for strong and the worst result for negative expression. Adjusted to NPI, RFS did not differ between the treatment cohorts of endocrine treatment and chemoendocrine treatment (p=0.828) in the high ER group. Patients with chemotherapy alone fared significantly worse (p=0.003). Even in high risk patients (according to NPI) the chemoendocrine and the endocrine treatment only groups did not differ significantly (HR=1.15; 95% CI (0.56-2.34), p=0.709). Omission of endocrine treatment led to significantly worse outcome (p=0.013).

Conclusions:

RFS was significantly longer in patients with high ER expression than with weak or no ER expression. In the high expression group, there was no significant

difference in RFS between endocrine treatment only and chemoendocrine therapyeven in high risk patients, for whom chemoendocrine treatment is routinely indicated. It seems insufficient for high ER patients to only consider tumor size, nodal status, and grading in order to decide which patient will benefit from adding chemotherapy to endocrine treatment.

Introduction

The St Gallen consensus meeting in 2009 has proposed a treatment selection algorithm for the management of early breast cancer which is fundamentally different from that used in previous years [START_REF] Goldhirsch | Thresholds for therapies: highlights of the St Gallen International Expert Consensus on the primary therapy of early breast cancer 2009[END_REF]. The indications for the three treatment modalitiesendocrine treatment (ET), anti-HER2 therapy and chemotherapyare based on different criteria which need to be analysed separately for individual treatment decisions. One of the most urgent questions concerns the need of chemotherapy in estrogen receptor (ER) positive diseases [START_REF] Montemurro | Hormone receptor-positive early breast cancer: controversies in the use of adjuvant chemotherapy[END_REF][START_REF] Hassett | Chemotherapy use for hormone receptor-positive, lymph node-negative breast cancer[END_REF][START_REF] Thürlimann | Is chemotherapy necessary for premenopausal women with lower-risk node-positive, endocrine responsive breast cancer? 10-year update of International Breast Cancer Study Group Trial 11-93[END_REF].

The additional benefit of chemotherapy on top of ET is controversial and may be influenced by a variety of factors, the most important of which is the endocrine responsiveness of the tumor. The definition of the categories -highly endocrine responsive‖, -incompletely endocrine responsive‖, and -endocrine nonresponsive‖ relies mainly on the level of ER and PgR expression, but other factors such as grading, HER2/neu status, proliferation index, and levels of uPA/PAI1 also play an important role [START_REF] Goldhirsch | Thresholds for therapies: highlights of the St Gallen International Expert Consensus on the primary therapy of early breast cancer 2009[END_REF]. The cut point between the ER and PgR expression levels needed to define high endocrine responsiveness was according to St Gallen 2009 50% of positive tumor cells [START_REF] Goldhirsch | Thresholds for therapies: highlights of the St Gallen International Expert Consensus on the primary therapy of early breast cancer 2009[END_REF], a value chosen more or less arbitrarily.

We hypothesize that patients with highly endocrine responsive tumors may have only a minor or even no additional benefit by adding chemotherapy to ET.

Retrospective analyses of prospective randomised trials [START_REF] Pagani | International Breast Cancer Study Group. Is adjuvant chemotherapy of benefit for postmenopausal women who receive endocrine treatment for highly endocrine-responsive, node-positive breast cancer? International Breast Cancer Study Group Trials VII and 12-93[END_REF][START_REF] Viale | Chemoendocrine Compared With Endocrine Adjuvant Therapies for Node-Negative Breast Cancer: Predictive Value of Centrally Reviewed Expression of Estrogen and Progesterone Receptors-International Breast Cancer Study Group[END_REF][START_REF] Colleoni | Increasing steroid hormone receptors expression defines breast cancer subtypes non responsive to preoperative chemotherapy[END_REF], in which the benefit of chemotherapy decreased with increasing levels of ER/PgR expression have served to a high degree to support our hypothesis. In order to further address this issue we analyzed a group of patients with high ER expression as to the influence of the different treatment modalities of ET alone versus chemoendocrine therapy versus chemotherapy alone. Our cohortincluding 3971 patientsis part of the BRENDA project (breast cancer care under evidence-based guidelines), a large cohort study funded by the German Federal Ministry of Education and Research (BMBF).

Obviously retrospective observational studies have methodological caveats, the main problem being a selection or information bias and known as well as possibly unknown confounding factors [START_REF] Grimes | Bias and causal associations in observational research[END_REF]. While results are irreparably compromised by selection and information biases, confounding factors can be corrected by e.g. multivariate adjustment models. However, analysing -real life‖ cohorts has the advantage that effectiveness is tested in everyday practice with relatively unselected participants and under flexible conditions , whereas explanatory prospective randomized trials test efficacy in a research setting with highly selected participants and under highly controlled conditions [START_REF] Booth | Reflections on Medical Oncology. Twentyfive Years of Clinical Trials in Oncology: Where Have We Come and Where Are We Going?[END_REF][START_REF] Haynes | Can It Work? Does It Work? Is It Worth It? The Testing of Healthcare Interventions Is Evolving[END_REF]. We therefore believe that our results, since they have been derived from a large cohort study will help to answer the crucial question which ER positive patient will benefit from adding chemotherapy to ET.

Patients and Methods

Study Design

Within the BRENDA project (breast cancer care under evidence-based guidelines) funded by the BMBF a large clinical cancer register was established.

Patients were diagnosed between 1992 and 2005 at the Department of Obstetrics and Gynecology, University of Ulm Medical Center, and affiliated breast cancer centers. This analysis included 3971 early breast cancer patients whose treatment modalities as well as ER immunoreactive score (IRS) levels were known. Baseline characteristics are shown in table 2.

The level of HR expression was documented as IRS. IRS is the product of a proportion score and an intensity score (table 1)with a range of 0-12. A high ER group was defined as ER IRS ≥ 9.

Statistical analysis

The primary endpoint is recurrence free survival (RFS). Nominally scaled variables were tabulated in contingency tables and tested for differences in frequency distribution. Variables for location and variance were calculated whenever ordinally scaled and intervally scaled continuous variables were involved. For each group we calculated numbers of cases, means, standard deviations, standard error of mean, minimum, maximum, 95% confidence interval [CI] for the mean, and medians. Levene's test for homogeneity of variance, analysis-of-variance tables and robust tests of the equality of means (medians) were similarly used. Normal distribution was tested using the Kolmogorov-Smirnov (K-S) goodness-of-fit test. In addition we also used the Anderson-Darling test and the Cramer-Smirnov-Von-Mises test, a modification of the K-S test giving more weight to the tails of the distribution. Standard survival analysis using a Kaplan-Meier approach was performed for assessing RFS. Logrank test was used to provide a formal statistical assessment of the differences between treatment arms. Cox proportional hazards model was used to estimate the hazard ratio and confidence intervals. Comparisons of categorical variables between groups were made by using χ 2 tests.

In order to improve the internal validity we checked for selection bias, information bias and confounding factors. Results were adjusted by taking into account all measurable confounders and inhomogeneities in baseline status in the form of multivariate adjustment using the Cox proportional hazard regression method.

Results

We found the following distribution of levels of estrogen expression according to IRS: 27% negative, 7% weak, 26% intermediate, and 40% strong.

As described in table 2, the ER groups differ significantly in the clinical risk factors age, menopausal status, tumor size, nodal status, grading, and HER2/neu.

Recurrence free survival according to ER groups

As expected, RFS was associated with ER, with best outcome for strong and worst for negative expression (fig. 1). RFS for weak and negative expressions in relation to high expression was significantly worse (weak expression: HR=1.85; 95% CI (1.35-2.54), p<0.001; negative expression: HR=2.29; 95% CI (1.86-2.82), p<0.001; using logrank test). On the other hand, RFS for moderate in relation to high expression was not significantly worse (HR=1.23; 95% CI (0.97-1.56), p=0.084).

Outcome for patients with high ER expression according to adjuvant systemic treatment

We analysed the subgroup of patients with high ER expression (ER+, IRS ≥ 9: n=1595 (40.2%)) with regard to the treatment modalities of chemoendocrine therapy (chemotherapy followed by ET) (n=383 (24.0%)), ET alone (n=892 (55.9%)) and chemotherapy alone (n=51 (3.2%)). 269 pts obtained either no or an unknown drug therapy.

Without adjustment to risk factors, RFS was significantly longer in patients with

ET alone compared to chemoendocrine and chemotherapy alone (chemoendocrine vs. ET: HR=1.74; 95% CI (1.13-2.68), p=0.012; chemotherapy alone vs. ET: HR=3.52; 95% CI (1.95-6.35), p<0.001) (fig. 2A).

After adjustment to standard prognostic factors (Nottingham prognostic index (NPI), menopausal status, and HER2/neu), RFS did not differ between the groups with ET and chemoendocrine treatment (HR=1.05; 95% CI (0.67-1.65), p=0.828). However, patients treated with chemotherapy alone still fared significantly worse (HR=2.48; 95% CI (1.36-4.51), p=0.003) (fig. 2B). The significant differences between the curves without and with adjustment reflect the fact that, due to the retrospective nature of the analysis, the treatment modality depends on the risk factors. Therefore these factors are not balanced between the treatment groups; i.e. patients with more risk factors are more likely to have received chemotherapy than patients with less risk factors.

The correlation that chemotherapy seems not to add any benefit to ET was shown for post-and premenopausal patients to a similar extent.

Nottingham prognostic index (NPI)

The high ER group was further analysed regarding NPI, which combines grading, nodal status, and tumor size. As expected, RFS curves differ significantly between the groups of low, intermediate, and high risk (intermediate vs. low: HR=2.33; 95% CI (1.41-3.84), p=0.001; high vs. low: HR=8.12; 95% CI (4.95-13.32), p<0.001) (fig. 3A).

We then analysed the NPI-groups separately according to treatment modalities.

Even in the high risk group, ET alone showed the longest RFS. Chemoendocrine treatment did not differ significantly, and, as expected, the omission of ET led to significantly worse results (chemoendocrine vs. ET: HR=1.15; 95% CI (0.56-2.34), p=0.709; chemo vs. ET: HR=3.07; 95% CI (1.27-7.40), p=0.013) (fig. 3B). The correlation described above -i.e. that chemotherapy seems to add no further benefit in high ER expression -was also found in the low and intermediate NPI groups.

Influence of the type of endocrine therapy

Tamoxifen or aromatase inhibitors were mainly used as ET. We included all patients who received aromatase inhibitors in this cohort, not distinguishing between upfront or sequential treatment and found no significant difference in the RFS of patients with high ER expression receiving either tamoxifen or aromatase inhibitors (data not shown).

Influence of the type of chemotherapy

Types of chemotherapy used in our cohort included mainly CMF, anthracycline or taxane containing regimens. Consequently we analysed the three main regimen types separately. CMF regimens were given to 25%, 49% received anthracycline containing regimens, and 26% taxane containing regimens. The survival curves showed the same pattern, regardless of the type of chemotherapy in the high ER group (see fig. 4) as was shown for chemotherapy as a whole cohort (fig. 2B). Thus fig. 4 -showing RFS for the high ER group stratified for ET, and taxane-endocrine treatment -can serve as an example for all chemotherapy groups. As described above in fig. 2B in which all chemotherapies are included, the RFS curves for ET only and taxane containing chemotherapy followed by ET are not separated.

Discussion

It has by now been widely accepted that the merely dichotomous reporting of ER and/or PgR status as positive resp. negative is oversimplified and thus the categories of -highly endocrine responsive‖, -incompletely endocrine responsive‖, and -endocrine nonresponsive‖ have been introduced [START_REF] Goldhirsch | 10th St. Gallen conference. Progress and promise: highlights of the international expert consensus on the primary therapy of early breast cancer 2007[END_REF][START_REF] Goldhirsch | Thresholds for therapies: highlights of the St Gallen International Expert Consensus on the primary therapy of early breast cancer 2009[END_REF].

Although their definition relies mainly on the level of ER and PgR expression, other factors such as grading, HER2/neu status, proliferation index, and levels of uPA/PAI1, also play an important role. Thus the controversy about which HR positive patient will benefit from adding chemotherapy to ET, so important in clinical practice, comes more and more into research focus, too. In this analysis we have tried to define the role of chemotherapy in a group of patients with high ER expression measured by classical immunohistological ways.

As previously described, we found an association between the level of HR expression and outcome in early breast cancer. ER status has been used for decades as a predictive marker for the response to ET in all clinical indications and for all commonly used endocrine substances [START_REF]International Breast Cancer Study Group (IBCSG) (2002) Endocrine responsiveness and tailoring adjuvant therapy for postmenopausal lymph node-negative breast cancer: a randomized trial[END_REF][START_REF] Colleoni | Tamoxifen after adjuvant chemotherapy for premenopausal women with lymph node-positive breast cancer: International Breast Cancer Study Group Trial 13-93[END_REF][START_REF] Hutchins | Randomized, controlled trial of cyclophosphamide, methotrexate, and fluorouracil versus cyclophosphamide, doxorubicin, and fluorouracil with and without tamoxifen for high-risk, node-negative breast cancer: treatment results of Intergroup Protocol INT-0102[END_REF][START_REF] Chia | Fulvestrant: expanding the endocrine treatment options for patients with hormone receptor-positive advanced breast cancer[END_REF]. A moderate prognostic value has been described previously in patients not receiving any type of systemic therapy [START_REF] Harvey | Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer[END_REF].

In this analysis, we have focused on the population of patients with a high expression of ER. The definition of high HR expression is not uniform. The St Gallen experts stated that a count of ≥50% of positive tumor cells should be considered highly endocrine-responsive [START_REF] Goldhirsch | Thresholds for therapies: highlights of the St Gallen International Expert Consensus on the primary therapy of early breast cancer 2009[END_REF]. Other studies derived from the definition of the Allred-Score [START_REF] Elledge | Estrogen receptor (ER) and progesterone receptor (PgR), by ligand-binding assay compared with ER, PgR and pS2, by immuno-histochemistry in predicting response to tamoxifen in metastatic breast cancer: a Southwest Oncology Group Study[END_REF] have defined high expression as ≥33% of positive cells, and still others even as ≥80% [START_REF] Andre | Estrogen receptor expression and efficacy of docetaxel-containing adjuvant chemotherapy in patients with node-positive breast cancer: results from a pooled analysis[END_REF].

The historically accepted scoring systems are using not only the percentage of positive cells but also staining intensity [START_REF] Reiner | Estrogen receptor analysis on biopsies and fine-needle aspirates from human breast carcinoma. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies[END_REF][START_REF] Hammond | American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Immunohistochemical Testing of Estrogen and Progesterone Receptors in Breast Cancer[END_REF]. The Allred score, widely used in the USA, is calculated by summing staining intensity (0-3) and the percentage score (0-5) receiving values from 0-8 [START_REF] Allred | Prognostic and predictive factors in breast cancer by immunohistochemical analysis[END_REF]. The Remmele score, widely used in Germany, is calculated by multiplying staining intensity (0-3) and percentage score (0-4) receiving values from 0-12 [START_REF] Remmele | Vorschlag zur einheitlichen Definierung eines immunreaktiven Scores (IRS) für den immunhistochemischen Östrogenrezeptornachweis (ER-ICA) im Mammakarzinomgewebe[END_REF][START_REF] Remmele | Pathologie Bd 4. Weibliches Genitale; Mamma; Pathologie der Schwangerschaft, der Plazenta und des Neugeborenen[END_REF]. The Remmele Score has two major limitations. Due to the calculation by multiplication valid values are not continuous. This is not in accordance with the fact that HR expression is a continuous biological variable ranging from no expression in any tumor cell to high expression in all tumor cells. The second limitation of the score is that the interpretation was not used uniformly. In the original work neither a cut-off value to define positive vs. negative expression nor a definition of high expression was given [START_REF] Remmele | Vorschlag zur einheitlichen Definierung eines immunreaktiven Scores (IRS) für den immunhistochemischen Östrogenrezeptornachweis (ER-ICA) im Mammakarzinomgewebe[END_REF]. As the interpretations of the results thus differ remarkably, we have used the most common classification (see table 1). An IRS of 9 and higher defining the group of high ER expression in this analysis corresponds to at least 50% positive cells with a strong staining intensity.

The impact of quantitative measurement of HR expression as a prognostic and predictive test was analysed in various settings [START_REF] Pagani | International Breast Cancer Study Group. Is adjuvant chemotherapy of benefit for postmenopausal women who receive endocrine treatment for highly endocrine-responsive, node-positive breast cancer? International Breast Cancer Study Group Trials VII and 12-93[END_REF][START_REF] Viale | Prognostic and predictive value of centrally reviewed expression of estrogen and progesterone receptors in a randomized trial comparing letrozole and tamoxifen adjuvant therapy for postmenopausal early breast cancer: BIG 1-98[END_REF][START_REF] Dowsett | Relationship between quantitative estrogen and progesterone receptor expression and human epidermal growth factor receptor 2 (HER-2) status with recurrence in the Arimidex, Tamoxifen, Alone or in Combination trial[END_REF][START_REF] Paik | Expression of the 21 genes in the Recurrence Score assay and tamoxifen clinical benefit in the NSABP study B-14 of node negative, estrogen receptor positive breast cancer[END_REF][START_REF] Fisher | National Surgical Adjuvant Breast and Bowel Project randomised clinical trials. Treatment of lymphnode-negative, oestrogen-receptor-positive breast cancer: long-term findings from National Surgical Adjuvant Breast and Bowel Project randomised clinical trials[END_REF][START_REF] Mazouni | Is quantitative oestrogen receptor expression useful in the evaluation of the clinical prognosis? Analysis of a homogeneous series of 797 patients with prospective determination of the ER status using simultaneous EIA and IHC[END_REF]. For example in adjuvant patients DFS was significantly longer in patients with high ER levels (>10% positive cells) than in those with lower (1-10%) and negative (absent) expression [START_REF] Viale | Prognostic and predictive value of centrally reviewed expression of estrogen and progesterone receptors in a randomized trial comparing letrozole and tamoxifen adjuvant therapy for postmenopausal early breast cancer: BIG 1-98[END_REF]. This was also shown in a subgroup of the ATAC-trial population, who showed a significant relationship between ER level and time to recurrence [START_REF] Dowsett | Relationship between quantitative estrogen and progesterone receptor expression and human epidermal growth factor receptor 2 (HER-2) status with recurrence in the Arimidex, Tamoxifen, Alone or in Combination trial[END_REF]. Paik et al [START_REF] Paik | Expression of the 21 genes in the Recurrence Score assay and tamoxifen clinical benefit in the NSABP study B-14 of node negative, estrogen receptor positive breast cancer[END_REF] described a significant test for the interaction of tamoxifen treatment and ER expression indicating that quantitative ER expression predicts benefit from tamoxifen treatment. The largest benefits of tamoxifen were observed with high quantitative ER.

Elledge et al [START_REF] Elledge | Estrogen receptor (ER) and progesterone receptor (PgR), by ligand-binding assay compared with ER, PgR and pS2, by immuno-histochemistry in predicting response to tamoxifen in metastatic breast cancer: a Southwest Oncology Group Study[END_REF] described a significant positive correlation of increasing ER levels and outcome in tamoxifen treated patients with metastatic breast cancer.

Conflicting results were described in a subgroup of patients from the PO25 trial comparing letrozole vs. tamoxifen in advanced breast cancer [START_REF] Henriksen | An ER activity profile including ER, PR, Bcl-2 and IGF-IR may have potential as selection criterion for letrozole or tamoxifen treatment of patients with advanced breast cancer[END_REF]. No correlation was found between the ER expression level and TTP, neither in letrozole nor in tamoxifen treated patients. However, there was a significant effect of PgR expression (negative vs low vs high) on TTP in both treatment arms.

Our finding that adding chemotherapy to tamoxifen in patients with high HR expression shows no additional benefit corresponds to findings by Viale et al [START_REF] Viale | Chemoendocrine Compared With Endocrine Adjuvant Therapies for Node-Negative Breast Cancer: Predictive Value of Centrally Reviewed Expression of Estrogen and Progesterone Receptors-International Breast Cancer Study Group[END_REF]. Also retrospective analyses of adjuvant and neoadjuvant trials have shown a decreasing chemotherapy efficacy with increasing HR levels [START_REF] Pagani | International Breast Cancer Study Group. Is adjuvant chemotherapy of benefit for postmenopausal women who receive endocrine treatment for highly endocrine-responsive, node-positive breast cancer? International Breast Cancer Study Group Trials VII and 12-93[END_REF][START_REF] Colleoni | Increasing steroid hormone receptors expression defines breast cancer subtypes non responsive to preoperative chemotherapy[END_REF].

In our patient population the group of patients with high expression of ER has not proved an additional benefit from adding chemotherapy to ET. This holds true for all risk groups according to NPI, even for high risk patients. To our knowledge all treatment guidelines recommend chemotherapy to NPI high risk patients. The recent St Gallen recommendations differentiate indications for chemoendocrine therapy in patients with ER positive, HER2 negative disease [START_REF] Goldhirsch | Thresholds for therapies: highlights of the St Gallen International Expert Consensus on the primary therapy of early breast cancer 2009[END_REF]. Certain clinicopathological features such as ER/PgR, grading, proliferation index, nodal status, peritumoral vascular invasion, and tumor size, which together define the categories of -highly endocrine responsive‖, -incompletely endocrine responsive‖, and -endocrine nonresponsive‖, need to be considered for treatment decisions. Our data supports this and might be used as additional information in the controversy over the need for chemotherapy in patients with high HR expression, even in high risk patients.

We also analysed the impact of the type of ET and of chemotherapy. We did not find a significant difference between tamoxifen and AI. For the chemotherapy groups we found the same RFS patterns regardless of the types of chemotherapy. Even for taxane containing regimens, which are widely recognised as the most potent chemotherapy regimens up to now [START_REF] De Laurentiis | Taxane-based combinations as adjuvant chemotherapy of early breast cancer: a metaanalysis of randomized trials[END_REF][START_REF] Bedard | Taxanes: optimizing adjuvant chemotherapy for early-stage breast cancer[END_REF], we did not find an additional benefit compared to ET alone. This is contradictory to the findings that the risk reduction effect due to taxanes was almost identical in HR negative and positive patients [START_REF] Montemurro | Hormone receptor-positive early breast cancer: controversies in the use of adjuvant chemotherapy[END_REF][START_REF] Andre | Estrogen receptor expression and efficacy of docetaxel-containing adjuvant chemotherapy in patients with node-positive breast cancer: results from a pooled analysis[END_REF]. Other adjuvant or preoperative studies however, have found significant differences in the extent of the chemotherapy effect according to HR [32][START_REF] Green | Weekly paclitaxel improves pathologic complete remission in operable breast cancer when compared with paclitaxel once every 3 weeks[END_REF][START_REF] Mazouni | Inclusion of taxanes, particularly weekly paclitaxel, in preoperative chemotherapy improves pathologic complete response rate in estrogen receptor-positive breast cancers[END_REF][START_REF] Von Minckwitz | German Breast Group: In vivo chemosensitivity adapted preoperative chemotherapy in patients with early-stage breast cancer-The GEPARTRIO pilot study[END_REF].

Recently gene expression profiles have been developed to analyse the individual recurrence risk in order to individualise adjuvant breast cancer treatment [START_REF] Sotiriou | Gene-expression signatures in breast cancer[END_REF][START_REF] Pusztai | Chips to bedside: incorporation of microarray data into clinical practice[END_REF][START_REF] Mook | The 70-gene prognosissignature predicts disease outcome in breast cancer patients with 1-3 positive lymph nodes in an independent validation study[END_REF][START_REF] Bueno-De-Mesquita | Validation of 70gene prognosis signature in node-negative breast cancer[END_REF]. For example, the 21-gene panel [START_REF] Paik | A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer[END_REF] includes mainly genes involved in tumor cell proliferation and hormonal response. The score was developed in node negative, tamoxifen-treated patients [START_REF] Paik | A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer[END_REF] to predict the recurrence risk in these low risk patients. Paik et al then showed that the recurrence score (RS) also predicts the likelihood of benefit from chemotherapy [START_REF] Paik | Gene expression and benefit of chemotherapy in women with node-negative, estrogen receptor-positive breast cancer[END_REF]. Patients with high-RS tumors (i.e. high risk of recurrence) had a large benefit from chemotherapy; patients with low-RS tumors derived minimal if any benefit from chemotherapy. Similar results have recently been published [START_REF] Albain | Breast Cancer Intergroup of North America. Prognostic and predictive value of the 21-gene recurrence score assay in postmenopausal women with node-positive, oestrogen-receptor-positive breast cancer on chemotherapy: a retrospective analysis of a randomised trial[END_REF][START_REF] Goldstein | Prognostic utility of the 21gene assay in hormone receptor-positive operable breast cancer compared with classical clinicopathologic features[END_REF][START_REF] Knauer | The predictive value of the 70-gene signature for adjuvant chemotherapy in early breast cancer[END_REF].

There is a lively debate about the additional predictive value of these multi gene arrays to classical clinical, histopathological, and immunohistological parameters. It has been shown that the combination of classical parameters and RS has increased the prognostic power to define the risk of patients [START_REF] Goldstein | Prognostic utility of the 21gene assay in hormone receptor-positive operable breast cancer compared with classical clinicopathologic features[END_REF][START_REF] Tang | Recurrence risk of node-negative and ER-positive early-stage breast cancer patients by combining recurrence score, pathologic, and clinical information: A meta-analysis approach[END_REF]. We believe that the integration of quantitative HR expression into the treatment decision will help towards optimising this crucial decision.

Our data derives from a clinical cancer register and not from a trial population, with all the inherent advantages and disadvantages. The major problem is the presence of known and possibly unknown confounding factors affecting both treatment and outcome (cp. fig. 2A and2B). Therefore, in order to come as close as possible to the clinically most relevant subject-specific measure of treatment effect, we adjusted our results to the most important prognostic covariates. The three main reasons why we used covariate adjustment methods are the following: First, to correct for imbalances in baseline prognostic covariates; second, to increase power by modelling the variability in outcome explained by relationships with highly prognostic covariates, and third, to obtain treatment effect estimates that would be more closely relevant for individual patients than to an average population [START_REF] Hauck | Should We Adjust for Covariates in Nonlinear Regression Analyses of Randomized Trials?[END_REF][START_REF] Berger | Good research practices for comparative effectiveness research: defining, reporting and interpreting non-randomized studies of treatment effects using secondary[END_REF][48].

The interpretation of observational study data in regard to causality should be carried out with great caution [START_REF] Grimes | Bias and causal associations in observational research[END_REF]. The Hills criteria of causation (strength of association, consistency, specificity, temporality, biological gradient, biological plausibility, coherence with existing knowledge, evidence, and analogy) outline the minimal conditions needed to establish a causal relationship between two items. Since the Hills criteria of causationeven if met, as in our casecannot bring indisputable evidence for or against a cause-and-effect hypothesis and none can be required as a sine qua non, we can only derive associations, and not causality, from our results.

We believe that reviewing the role of chemotherapy in ER high expressing patients is a very important issue, especially since this subgroup is the largest group of patients with HR positive diseases, representing approximately 40% of all breast cancer patients. Our findings of no significant difference between ET and chemoendocrine treatment in ER high expressing patients indicates that using the clinical routine parameters of tumor size, nodal status and grading is not sufficient for the crucial treatment decision of ET vs. chemoendocrine in

Figure legends Fig. 1 :

 legends1 Figure legends

Fig. 2 :

 2 Fig. 2: Recurrence free survival of patients with high expression of ER stratified

Fig. 3 :

 3 Fig. 3: Recurrence free survival of patients with high expression of ER stratified

Fig. 4 :

 4 Fig. 4: Recurrence free survival of patients with high expression of ER stratified

FigureFigure

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Table 1 :

 1 Calculation of IRS: Proportion and intensity score

	Tables
			proportion
	proportion	staining intensity	score (PS)/ intensity score
			(IS)
	0%	negative	0
	< 10 %	weak	1
	10 % -50 % intermediate	2
	51 % -80 % strong	3
	> 80 %	-	4
	Interpretation (PS * IS)
	0-1	negative
	2-3	positive: weak
	4-8	positive: moderate
	9-12	positive: strong

Table 2 :

 2 Baseline characteristics

					ER-Expression		Chi-Square
				negative	weak or moderate	high
			Total	(0-1)	(2-8)	(9-12)
			N	N	N	N
			(%)	(%)	(%)	(%)	(Sig)
	Patients		3971 (100) 1079 (27.2)	1297 (32.6)	1595 (40.2)
	Age	Median	61	57	58	65
		(Mean; std)	(60.5; 13.1)	(57.1;13.4)	(58.3;12.7)	(64.6; 12.1)
		Range	22-101	28-95	24-101	22-97
	Age categories <35	59 (1.5)	24 (2.2)	22 (1.7)	13 (0.8)
		35-55	1365 (34.4)	483 (44.8)	543 (41.9)	339 (21.3) < 0.001
		56-75	1975 (49.7)	456 (42.3)	598 (46.1)	921 (57.7)
		>75	572 (14.4)	116 (10.8)	134 (10.3)	322 (20.2)
	Menopausal	unknown	185 (4.7)	55 (5.1)	79 (6.1)	51 (3.2)
	status
		premenopausal	903 (22.7)	344 (31.9)	369 (28.5)	190 (11.9) < 0.001
		postmenopausal	2883 (72.6)	680 (63.0)	849 (65.5)	1354 (84.9)
	Tumor size	T1	2024 (52.8)	480 (46.6)	705 (56.0)	839 (54.4)
		T2	1377 (36.0)	433 (42.0)	411 (32.7)	533 (34.6) < 0.001
		T3	178 (4.6)	60 (5.8)	50 (4.0)	68 (4.4)
		T4	251 (6.6)	57 (5.5)	92 (7.3)	102 (6.6)
	Nodal status	unknown	79 (2.0)	24 (2.2)	20 (1.5)	35 (2.2)
		nodal negative	2264 (57.3)	629 (58.5)	735 (56.8)	900 (56.8)	0.008
		1-3	882 (22.3)	201 (18.7)	310 (24.0)	371 (23.4)
		4-10	486 (12.3)	136 (12.7)	150 (11.6)	200 (12.6)
		>10	242 (6.1)	85 (7.9)	78 (6.0)	79 (5.0)
	Grading	unknown	45 (1.1)	22 (2.0)	15 (1.2)	8 (0.5)
		1	268 (6.7)	40 (3.7)	111 (8.6)	117 (7.3) < 0.001
		2	2271 (57.2)	336 (31.1)	814 (62.8)	1121 (70.3)
		3	1387 (34.9)	681 (63.1)	357 (27.5)	349 (21.9)
	HER2/neu	unknown	614 (15.5)	175(16.2)	220 (17.0)	219 (13.7)

Funding

This study has been supported by the German Federal Ministry of Education and Research (BMBF) (grant number: 01ZP0505).


Purpose: mTOR plays a key role in tumor cell cycle control, proliferation and survival. RAD001 (everolimus) is a novel macrolide that inhibits mTOR and thus downstream signaling pathways.

Experimental Design: 31 post-menopausal women with early breast cancer were given 5mg RAD001 once daily for 14 days prior to surgery. Biopsies were taken at diagnosis and at surgery (post 14 days of treatment) and assessed for immunohistochemical changes in proliferation (Ki67), apoptosis (active caspase-3), p-AKT (s473), p-S6 (s235/236 and s240/244), p-mTOR (s2448), ER, and PR.

Results: Five patients did not complete the two week treatment period due to adverse events. All adverse events were grade 1 or 2 (NCIC-CTC scale).

RAD001 treatment significantly decreased proliferation (geometric mean reduction 74% from baseline (p = 0.019)), particularly in HER-2 positive tumors. High Ki67 pre-treatment correlated with reduction in Ki67, an increase in apoptosis, a reduction in p-Akt (cytoplasmic) and reduction in p-mTOR following treatment. Nuclear expression of p-AKT was significantly reduced with treatment. Tumors that had a reduction in Ki67 with treatment exhibited a significant reduction in cytoplasmic p-AKT. p-S6 staining was significantly reduced independently of Ki67 (p<0.001 for two sites of phosphorylation).

Conclusions: RAD001 5mg/ daily is safe and tolerable in postmenopausal early breast cancer patients and inhibits the mTOR pathway and its downstream effectors, significantly reducing tumor cell proliferation. Tumors with high Ki67, high p-AKT and HER-2 positivity may be more responsive to * Sabine VS, Faratian D and Bartlett JMS. Validation of cleaved caspase-3 antibody staining as a marker of apoptosis in breast cancer.

mTOR inhibition with RAD001. This is the first study to report results of RAD001 5mg as a single agent in early breast cancer.

INTRODUCTION

The mammalian target of rapamycin (mTOR) is a 289kDa serine/threonine protein kinase that belongs to the phosphatidylinositol-3OH kinase related kinase (PIKK) family [START_REF] Kunz | Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression[END_REF][START_REF] Brown | A mammalian protein targeted by G1-arresting rapamycin-receptor complex[END_REF][START_REF] Chiu | RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/ rapamycin complex[END_REF][START_REF] Sabatini | RAFT1: A mammalian protein that binds to FKBP12 in a rapamycindependent fashion and is homologous to yeast TORs[END_REF][START_REF] Sabers | Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells[END_REF]. mTOR is deregulated during oncogenesis resulting in disruption of a cascade of signals that regulate basic cellular functions including proliferation, apoptosis and angiogenesis [6]. Multiple mechanisms have been proposed for mTOR deregulation, including mutation of phosphatidylinositol 3-kinase (PI3K), gene amplification of AKT/PKB (protein kinase B) [7] and mutation or silencing of the phosphatase and tensin homolog (PTEN) tumor suppressor gene which, in the normal cell, acts to inhibit the activation of the PI3K-AKT cascade [8-10]. These alterations within the PI3K/PTEN axis result in hyperactivation of the AKT/ PKB family proteins, which are associated with resistance to apoptosis, increased cell growth, cell proliferation and altered cellular energy metabolism mediated via activation of mTOR [11]. mTOR exists in two distinct complexes within the mammalian cell, one consisting of mTOR, G-protein beta-subunit-like (GβL) and raptor, (mTORC1) and the other includes mTOR, GβL and rictor (mTORC2) [12][13][14][START_REF] Wullschleger | Molecular organization of target of rapamycin complex 2[END_REF]. These two mTOR complexes have distinct functions with mTORC1 having downstream effects including a negative feedback loop to regulate AKT through ribosomal S6 protein and insulin receptor substrate 1 (IRS1) [START_REF] Sarbassov | Phosphorylation and regulation of Akt/ PKB by the rictor-mTOR complex[END_REF]. mTORC2 (mTOR rictor complex) functions as a phosphoinositol-dependent kinase (PDK), activating AKT, and inhibition of mTORC2 reverses this effect. These distinct functions of mTORC1 and mTORC2 with respect to AKT have been established using in vitro model systems [12][13][14][START_REF] Sarbassov | Phosphorylation and regulation of Akt/ PKB by the rictor-mTOR complex[END_REF]. What remains to be determined is whether therapeutic inhibitors of mTOR act on mTORC1 and thus will upregulate AKT activity, or upon mTORC2 with possible inhibition of AKT.

Phospho-70S6 kinase (S6K1) and eIF4E binding proteins (4E-BPs, also known as translational repressor protein PHAS-1) are two well characterized downstream effectors of mTOR activity that are activated through the mTORC1 complex [START_REF] Burnett | RAFT1 phosphorylation of the translational regulators p70S6kinase and 4E-BP1[END_REF]. Activated S6K1 phosphorylates the 40S ribosomal protein S6 to promote mRNA translation with terminal oligopyrimidine (TOP), including ribosomal proteins, elongation factors and insulin growth factor -II [10,[START_REF] Wullschleger | TOR signaling in growth and metabolism[END_REF][START_REF] Sarbassov | Growing roles for the mTOR pathway[END_REF]. RAD001 (everolimus, Novartis) is a novel macrolide that bears a hydroxylethyl chain giving it increased polarity for oral formulation. RAD001 is licensed for use in multi-drug immunosuppression after transplantation, and has high affinity for the 12-kDa FK506-binding protein (FKBP-12); when bound to this protein RAD001 interacts with and inhibits mTOR. It is thought that the main interaction is with mTORC1 but there is evidence to suggest that with prolonged rapamycin treatment mTORC2 is also inhibited [START_REF] Sarbassov | Prolonged Rapamycin Treatment Inhibits mTORC2 Assembly and Akt/PKB[END_REF]. RAD001 is currently being evaluated in renal, neuroendocrine, colorectal, lung, hematological and lung tumors, as a single agent and in combination with antiangiogenic or endocrine agents following promising results in Phase I and II studies [START_REF] Motzer | Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebocontrolled phase III trial[END_REF][START_REF] Yee | Phase I/II study of the mammalian target of rapamycin inhibitor everolimus (RAD001) in patients with relapsed or refractory hematologic malignancies[END_REF][START_REF] Baselga | Phase II randomized study of neoadjuvant everolimus plus letrozole compared with plac ebo plus letrozole in patients with estrogen receptor-positive breast cancer[END_REF][START_REF] Kris | Combined inhibition of mTOR and EGFR with everolimus (RAD001) and gefitinib in patients with non-small cell lung cancer who have smoked cigarettes: A phase II trial[END_REF].

The aims of this clinical and biological study were to assess the safety and tolerability of RAD001 in postmenopausal women with early breast cancer, to assess the effects of 5mg RAD001 on tumor proliferation and apoptosis, and to investigate the effect of RAD001 as an mTOR inhibitor on the mTOR pathway.

METHODS

Ethical approval for this study was obtained from Northern and Yorkshire MREC through the UK national application system for the clinical and biological study and use of tissue (MREC reference 04/MRE03/89). All patients gave written informed consent for all aspects of the study.

Clinical Study

The clinical study was a pre-operative 2 week study of patients with operable early breast cancer. All patients were postmenopausal females, defined as >12 months since last menses or in patients whose last menses was <12 months before starting treatment, FSH >35IU/L and LH >40 IU/L. Patients with operable early breast cancer (T1-3, N0-1, M0) proceeding to primary surgery were eligible. Patients were not selected on the basis of tumor positivity for estrogen receptor (ER), progesterone receptor (PR) or epidermal growth factor (HER-2). Patients were dispensed 14 days of RAD001 5mg once daily so they took the last dose on the day of surgery. Patients were advised to contact the research team in the event of any side effects. A daily patient diary was completed by the patient and examined on Day 13 (preoperative admission day) to assess compliance with medication and monitoring of side effects experienced by patients, if these had not already been notified to the research team during the study period. Pre-treatment blood samples were taken on the day of enrollment in addition to the routine blood samples that had been taken within 10 days of study entry as part of preoperative assessment and screening for study suitability.

All patients entering this study had diagnostic core biopsies taken at time of presentation with a symptomatic breast lump. The tissue was processed in accordance with local pathology department policy. Additional core biopsies were snap frozen in liquid nitrogen for expression analysis from both diagnostic core biopsies and post-treatment surgical excision specimens. The surgical excision specimen was processed by the pathology laboratory and once all diagnostic procedures completed, the tumor blocks were retrieved and sectioned for study purposes.

Immunohistochemistry

Staining was performed on all samples collected including those from patients who did not complete the full 14 days of treatment. Pre-and post-treatment sections were stained within the same staining run and anonymized prior to scoring.

Immunohistochemistry for Ki67 (MIB-1, diluted 1:50; Dako, UK), estrogen receptor (ER, NCL-L-ER-6F11/2, diluted 1:25, Novocastra Laboratories, UK) and progesterone receptor (PR, PgR636 diluted 1:100, Dako) were performed as follows: epitope retrieval was performed in citrate buffer pH6.0 for 6 minutes in a microwave, slides were treated with 3% H 2 O 2 for 5 minutes before washing and incubating with appropriate primary antibody at room temperature (RT) for 1 hour for ER and 30 minutes for PR and Ki67 before incubation with secondary antibody (Dako EnVision+, K5007) for 30 minutes at RT, visualized with DAB chromogen for 10 minutes at RT and counterstained with haematoxylin.

Immunohistochemistry for phospho-mTOR (serine 2448, 49F9, diluted 1:50) and phospho-S6 (serine 235/236 (clone 2211, diluted 1:400) and 240/244 (clone 2215, diluted 1:100) (all Cell Signaling Technology) were performed as follows: epitope retrieval was performed in citrate buffer pH6.0 at 96 C for 10 (p-S6) and 20 minutes (p-mTOR), slides were treated with 3% H 2 0 2 for 10 minutes (p-mTOR) and 30 minutes (p-S6), and with casein (p-mTOR) or 10% normal goat serum (p-S6) before washing and incubating over night at 4 C with the relevant antibody before continuing with secondary antibody incubation as described above.

Immunohistochemistry for phospho-AKT (serine 473; clone 14-5 diluted 1:6, Dako, Denmark) was performed as follows: epitope retrieval was performed in TRIS/ EDTA buffer pH9.0 at 97ºC for 20 minutes, slides were then treated with 3% H 2 0 2 for 5 minutes before washing and incubating with primary antibody for 30 minutes at RT followed by incubation with secondary antibody as before.

Immunohistochemistry for apoptosis by active caspase-3 (Abcam ab 2302, UK) was performed as described previously [START_REF] Kirkegaard | Expression of tumor necrosis factor alpha converting enzyme in endocrine cancers[END_REF].

Immunohistochemical testing for HER-2 was performed routinely as per national guidelines. All samples scored as 2+ for HER-2 status by immunohistochemistry were tested by fluorescent in situ hybridisation (FISH).

Expression of HER-2 was defined as tumors staining 3+, or 2+ and FISH positive, while all patients with tumors scored as 0 or 1+ were considered not to express HER-2.

All samples except Ki67 and apoptosis were scored blind using the histoscore method [START_REF] Mccarty | Use of a monoclonal antiestrogen receptor antibody in the immunohistochemical evaluation of human tumors[END_REF] performed by two scorers. Ki67 was counted using a previously described and validated method of scoring the percentage of stained cells as a proportion of the total cancer cells [27]. A semi-automated method of assessing apoptosis using the Ariol SL-50 image analysis system (Genetix) was employed: sections were scanned at high resolution (20x) and tumor areas manually selected. Tumor cells were scored using a previously validated algorithm. * Sections were checked by a pathologist to ensure that only tumor cells were scored.

Statistical analysis

The number of patients required for this study was 30 evaluable patients to have 80% power to detect a reduction in Ki67 in 50% of patients. Analysis of variance (ANOVA) was performed to determine the significance of changes in expression of immunohistochemical results and blood results.

Results were analyzed on an intention to treat (ITT) basis, after initial ANOVA showed no difference between those completing 14 days and those who took RAD001 for less than 14 days for change in proliferation as measured by Ki67, this being the primary outcome; neither was there any difference between those completing the full course or stopping early for the other markers of the mTOR pathway assessed.

ANOVA was also employed to determine differences between those that had a marked reduction in Ki67, "responders" (R), and those who did not, "nonresponders" (NR). Estimations of "responders" and "non-responders" were based upon dual observer analysis of Ki67 staining, the 95% confidence interval for the mean difference in Ki67 counts between two observers was taken as the minimum difference which could be regarded as a change in Ki67 status between pre-and post-treatment samples [28]. Due to the nonnormal distribution of the data, Ki67 scores were log transformed prior to analysis. The change between pre and post treatment scores for each patient was calculated as the difference in the logged scores.

In order to examine the linear relationship between the immunohistochemical and blood results with the Ki67 scores, Pearson's Correlation (P) was used.

ANOVA was used to examine the change between pre and post treatment scores for all the measurements for each patient.

For the purpose of sub-group analysis data were split about the median value to differentiate between those with high and low expression for each variable.

RESULTS

Clinical study

31 patients were recruited to this study. Five patients did not complete 14 days of treatment with RAD001 5mg due to side effects. One patient was withdrawn due to a lack of study drug availability after 11 days.

The median patient age at diagnosis was 68 years (range 51-89), (Table 1) and all patients had a WHO performance scale of 0-2. Performance status scale did not change within the treatment period for any patient.

Twelve of 31 patients reported no symptoms while taking RAD001 5mg for the two week treatment period. All adverse events reported are shown in Table 2.

Five patients withdrew during the two week treatment period due to adverse events, of which two patients had more than one symptom that contributed to their withdrawal. Median duration of treatment in those that withdrew was 9.5 days (range 4-13 days). Four patients had mouth ulcers which were of sufficient impact with pain on swallowing or chewing to result in the patient withdrawing from the study, although one patient continued her medication until planned admission to hospital on Day 13. There was no correlation between tolerability to RAD001 and age or WHO performance scale.

Safety bloods were taken pre-and post-treatment with RAD001. As expected from previous data available for RAD001 treatment, there was a significant reduction in white cells, neutrophils and platelet count in the intention-to-treat population [START_REF] Motzer | Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebocontrolled phase III trial[END_REF][START_REF] Baselga | Phase II randomized study of neoadjuvant everolimus plus letrozole compared with plac ebo plus letrozole in patients with estrogen receptor-positive breast cancer[END_REF]29,30]. There was no link between reduction in the numbers of white cells or neutrophils with postoperative infections, nor with reduction in platelets and postoperative bleeding problems. There was a significant increase in random cholesterol with treatment with RAD001, which is in agreement with previous findings [START_REF] Motzer | Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebocontrolled phase III trial[END_REF][START_REF] Yee | Phase I/II study of the mammalian target of rapamycin inhibitor everolimus (RAD001) in patients with relapsed or refractory hematologic malignancies[END_REF][START_REF] Kris | Combined inhibition of mTOR and EGFR with everolimus (RAD001) and gefitinib in patients with non-small cell lung cancer who have smoked cigarettes: A phase II trial[END_REF]30]. There was no correlation between changes in blood parameters and reduction in Ki67 during treatment with RAD001.

Biological endpoints

All patients proceeded to surgery after the 14 day study period (Table 1). The Nottingham Prognostic Index was calculated for each patient [31]. Seven patients had an NPI in the good prognosis category, 16 were in the intermediate prognosis group and 8 were in the poor prognostic category.

There was a significant correlation (Spearmans rank correlation (r)) between pre-treatment proliferation (Ki67%) and tumor grade for both core biopsy (r=0.59; 95%CI 0.29, 0.78; p=0.0013) and excision specimens (r=0.61; 95%CI 0.32, 0.79; p=0.0009), see below). No correlations were found between pretreatment Ki67 and either tumor size, type, presence of LVI, node positivity or NPI.

Tumors that were of higher grade had a significantly greater reduction in proliferation after RAD001 treatment (r=0.41; 95%CI 0.07, 0.67; p=0.024).

Tumor size, type, presence of LVI, node positivity and NPI were not found to correlate with the change in proliferation with treatment.

Excellent intra-and inter-observer correlations were observed for histoscores (Pearson's correlation coefficient (P)=0.984, p<0.0001) and proliferation counts (P=0.8846, p<0.0001).

There was a significant reduction in proliferation following treatment as assessed by Ki67 with an absolute median decrease of 8.5% (p=0.019; Figure 1). Using log transformed data, the post-treatment levels of Ki67 were 26% of those before treatment (95% CI 5-56%). Those that had a high proliferation at diagnosis had the greatest absolute decrease in Ki67 (based on log transformed data P=0.36, p=0.046) with RAD001 treatment.

Tumor samples were split into two groups: "responders" (R) (n=18) and nonresponders (NR) (n=13) using the 95%CI of the absolute value for change in Ki67% for further analysis. Pre-treatment scores for ER, PR, p-S6, p-mTOR, p-AKT and apoptosis were not significantly different for tumors that responded to RAD001 with a reduction in Ki67 counts and those that did not respond.

There was no significant change in levels of apoptosis as assessed using activated caspase-3 with RAD001 treatment for the ITT population (median pre-treatment 0.6% (95%CI 0.46, 0.81), median post-treatment 0.22% (95%CI 0.15, 0.68); p=NS). There was evidence to suggest a positive linear relationship between initial Ki67 (log transformed) and the change in apoptosis (P=0.37, p=0.04), in that those tumors initially high in proliferation had the greatest increase in apoptosis with RAD001 treatment (Figure 2).

There was no significant difference in change in apoptosis with treatment between responders and non-responders.

While tumor ER positivity was not an entry requirement into the study, all tumors from patients entered into the study had some ER positive staining prior to treatment. There was a significant decrease in ER histoscore from pre-to post-treatment samples (mean decrease 40.5 histoscore units; 95% CI (16.9-64.2), p=0.001), with a significant difference between those with high (n=19) and low (n=12) ER pre-treatment (data split about the median 240) (p=0.009). Those with lower pre-treatment ER scores had a significant decrease in ER, (mean decrease 77.5, 95%CI 43.3-111.7) compared with those with high ER pre-treatment that had no significant change (mean decrease 17.21, 95% CI -10.00-44.42). The change in ER did not correlate with pre-treatment Ki67 nor the change in Ki67 after treatment. There was no significant change in expression of progesterone receptor with treatment.

Eight patients had tumors that were positive for HER-2 on IHC with a score of 3+. All those who had FISH testing for score 2+ were negative. Of those that were HER-2 positive, four were ER poor, and all were PR poor or negative.

Response by Ki67 was seen in both HER-2 positive and HER-2 negative tumors. Of the 8 HER-2 positive tumors, 7 responded with a reduction in Ki67 (87.5%), but of 23 HER-2 negative patients, only 11 had a response (47.8%) (Figure 3). This numerical difference in response between HER-2 positive and HER-2 negative tumors did not reach statistical significance (p=0.10).

Tumors that were HER-2 positive had a greater reduction in p-S6 (ser 240/244 p=0.03) than HER-2 negative tumors.

For both the phosphorylation sites assessed there was a significant reduction in phosphorylation of S6 kinase with RAD001 treatment in all samples. For p-S6 s235/236 the mean histoscore pre-treatment was 129.4 and this fell to 58.3 post treatment (mean decrease 71.2, 95%CI 41.9, 100.4, p<0.001). For p-S6 s240/244 the mean histoscore pre-treatment with RAD001 was 142.0 and 31.5 post treatment (mean decrease 110.5, 95%CI 82.9-138.2, p<0.001; Figure 4). There was no evidence of a relationship between pre-treatment p-S6 for either site of phosphorylation or change in p-S6 with treatment and levels of proliferation either at baseline or with treatment. There was a trend toward greater reduction in p-S6 in those tumors that responded to RAD001 but this did not reach statistical significance for either s235/236 (responders(R) mean decrease 89.3, 95%CI 51.7, 127.0; non-responders (NR) mean decrease 46.0 95%CI 1.7, 90.3; p=NS) or s240/244 (R mean decrease 124.5, 95%CI 88.5, 160.6; NR mean decrease 91.2, 95%CI 48.8, 133.6, p=NS) sites of phosphorylation. Tumors that had a greater reduction in p-S6 (ser 240/244) had a greater reduction in nuclear p-AKT after RAD001 treatment (P=0.37, p=0.04).

There was no significant change in phospho-mTOR overall in the ITT population following treatment with RAD001. There was no correlation between pretreatment scores of p-mTOR and pretreatment Ki67, but there was evidence of a positive linear relationship between Ki67 pretreatment and change in p-mTOR with treatment (P=0.43, p=0.0168), suggesting that highly proliferative tumors had an decrease in phosphorylation of mTOR after RAD001 treatment. There was no significant difference between responders and non-responders in pre-treatment or change in p-mTOR scores with RAD001.

Both nuclear and cytoplasmic staining for p-AKT was observed, and separate scores were calculated for both sites of staining. There was a significant decrease in nuclear p-AKT (mean decrease 20.9, 95%CI 5.02, 36.79, p=0.012) with RAD001 treatment. There was no evidence of a relationship between initial nuclear p-AKT and change in nuclear p-AKT with either initial Ki67 or change in Ki67 with treatment. There was no significant difference between responders and non-responders in initial expression or change in nuclear AKT scores with RAD001 treatment.

There was no significant change in levels of cytoplasmic p-AKT with RAD001 treatment. Patients with a pre-treatment score above the median (70) fell by an average of 58.6 (95% CI 25.0, 92.1) with treatment, while the p-AKT scores of patients with pre-treatment p-AKT below median 70 had no significant change with treatment (mean increase 12.87, 95% CI decrease of 21.7 to increase of 47.5, NS). Thus those with high pre-treatment p-AKT had a significant decrease in p-AKT expression with treatment, and those with low pre-treatment expression had no change. The change between the two groups was significant (p=0.0051). There was a correlation between high pretreatment levels of Ki67 and high pre-treatment cytoplasmic expression of p-AKT (P=0.45, p=0.0105), and a correlation between the change in Ki67 with treatment and change in cytoplasmic p-AKT with treatment (P=0.45, p=0.0106), where those with a significant reduction in Ki67 also had a reduction in p-AKT with treatment. There was a correlation between pretreatment Ki67 and the change in cytoplasmic p-AKT with treatment (P=0.35, p=0.0505), suggesting that those with high Ki67 pre-treatment had the greatest reduction in p-AKT after RAD001 treatment.

There was a significant reduction in scores for cytoplasmic p-AKT in those tumors that were classified as responding to RAD001 (mean decrease 52, 95%CI 19.7-84.4) compared with those who did not respond, in which there was no significant change (mean increase 14.8, 95%CI decrease 23.2increase 52.9). The difference between the groups was significant (p=0.0105,

Figure 5).

In multivariate analysis, no pre-treatment variables were found to predict for a response to RAD001 as seen by reduction in Ki67.

DISCUSSION

RAD001 at a dose of 5mg appears to be tolerable to the majority of patients.

The most commonly reported side effect was mouth ulcers/ mucositis. Four of the eight patients who experienced this side effect withdrew from the study with grade I or II (mild or moderate) symptoms; these patients had pain on swallowing but were able to maintain an adequate oral intake of liquids and solids. One of these patients had mouth ulcers at the time of entry into the study and withdrew on day 9 of treatment. Four other patients experienced mouth ulcers but remained in the study as they felt that their symptoms were not interfering with their quality of life. The mouth ulcers resolved within 2 days in all patients after stopping RAD001. The rate of mouth ulcers in this study (25%) is less than in other reported studies of RAD001 at 5mg or 10mg daily dose, which may be related to treatment duration. The selection of 5mg once daily as the dose for this study was on the basis of the available information at the time of recruitment beginning in 2005. Pharmacodynamic studies since published have suggested 5-10mg daily as an appropriate dosing schedule [29,30]. Reductions of S6 were the same with 5mg (n=12) or 10mg (n=12) daily in solid advanced tumors, but there was only partial inhibition of eIF-4G with 5mg compared to complete inhibition with 10mg, with inter-patient variability for 4E-BP1. Side effect profiles were similar for both dose schedules, with more fatigue in the 10mg group. The rate of mouth ulcers was higher in this advanced study (33%) than in our study of the same dose. In a combination study of letrozole (2.5mg) and RAD001 at a dose of 10mg daily for 16 weeks in postmenopausal women with early breast cancer more than half of patients required a dose reduction or dose interruption [START_REF] Baselga | Phase II randomized study of neoadjuvant everolimus plus letrozole compared with plac ebo plus letrozole in patients with estrogen receptor-positive breast cancer[END_REF].

The dose of 5mg RAD001 used in our study may be more tolerable than the 10mg dose and remains effective as a biochemical inhibitor of the mTOR pathway.

There were significant reductions in white cells, particularly neutrophils and platelets in this study. This is of concern in the pre-operative setting, although there was no obvious increase in infective complications in the patients participating in this study, and in particular among those who had reduced neutrophil counts. Those patients with reduced neutrophil counts had the expected response to surgery with a raise in neutrophil counts observed postoperatively. Similarly, there was no increase in bleeding complications in the overall group, nor in those who had a reduction in platelets compared with the population of patients operated upon in Edinburgh Breast Unit at the time of the study.

The primary outcome measured in this pre-operative study was the change in proliferation with RAD001 treatment as measured by percentage of Ki67 positive cells. A significant decrease in Ki67 with 14 days of RAD001 at 5mg daily was seen in this study, confirming the effectiveness of RAD001 at reducing proliferation in breast tumors, particularly those that are highly proliferative at baseline. In a few neoadjuvant studies a reduction in Ki67 to ≤1% has been used as defining "complete cell cycle response". In the current study, however, we used a significant reduction in Ki67 after treatment to define cell cycle response as the significance of reductions to ≤1% remains unclear and may not reflect clinical response. We therefore felt it more appropriate to consider the absolute reductions in Ki67% dichotomized based upon median count in line with previous studies [32,33]. The measurement of Ki67 has attracted interest recently as a possible predictor of response to endocrine therapy [32]. Previous pre-operative studies performed in Edinburgh have used Ki67 as an outcome measure to allow comparison between agents and the individual efficacy of agents as anti-proliferative agents [34,35], and this study using RAD001 has favorable results for reduction in proliferation. In the current study, tumors with the highest Ki67 pre-treatment, i.e. the most proliferative tumors, were those that had the greatest reductions in Ki67 with treatment, the greatest absolute increase in apoptosis with treatment, and reductions in p-mTOR and p-AKT. There was also evidence of a positive correlation between change in p-AKT and Ki67 after RAD001 treatment. This would support previous findings that more aggressive or resistant cancers tend to have higher proliferation rates, and greater expression of p-AKT [36][37][38][START_REF] Kirkegaard | Akt activation predicts outcome in breast cancer patients treated with tamoxifen[END_REF][START_REF] Zhou | Activation of the Akt/ Mammalian target of rapamycin/ 4E-BP1 pathway by ErbB2 overexpression predicts tumor progression in breast cancers[END_REF]. This study has found that tumors that respond to RAD001 (determined by Ki67 reduction) were those that highly expressed Ki67 and p-AKT, indicating a potential use for RAD001 in more proliferative tumors and in tumors that are resistant to other treatments, or as a combination therapy to prevent resistance or prolong sensitivity to other agents such as endocrine agents. A study of RAD001 in cell lines also found an increased sensitivity to RAD001 in cells that had high expression of p-AKT [START_REF] Beeram | Akt-induced endocrine therapy resistance is reversed by inhibition of mTOR signaling[END_REF]. In the previously mentioned combination study of letrozole 2.5mg and RAD001 at a dose of 10mg daily for 16 weeks in postmenopausal women with early breast cancer [START_REF] Baselga | Phase II randomized study of neoadjuvant everolimus plus letrozole compared with plac ebo plus letrozole in patients with estrogen receptor-positive breast cancer[END_REF], there was a greater reduction in Ki67 at 15 days in those receiving letrozole and RAD001 compared with those receiving letrozole alone. The extent of reduction in Ki67 correlated moderately with clinical evaluation of response.

In a single tumor with a marked increase in proliferation (Ki67 increased from 3.6%-47.2%) the biomarker evidence was consistent with a lack of mTOR inhibition and upregulation of AKT signaling. Further studies would be required to explore this observation as it raises the possibility that there are tumor types that are resistant to mTOR inhibition.

There was a significant reduction of downstream effects of mTOR with RAD001 treatment demonstrated by the significant reduction in phosphorylation of S6 at both sites of phosphorylation. The extent of reduction at either site did not correlate with either the reduction in Ki67 or p-mTOR, although there appeared to be a correlation with reductions in nuclear p-AKT in tumors that had a greater reduction in p-S6 with RAD001 treatment, and greater reductions in p-S6 in HER-2 positive tumors. Reductions in p-S6 have previously been shown to correlate to tumor response to mTOR inhibition in sarcoma, and may be a potential predictive marker [START_REF] Iwenofu | Phospho-S6 ribosomal protein: a potential new predictive marker for targeted mTOR therapy[END_REF].

While there was no significant change in phosphorylation of mTOR (s2448) overall, there was some evidence to suggest that those tumors that had a high Ki67 pre-treatment were more likely to have an decrease in p-mTOR after RAD001 treatment. There is debate from published data as to the mechanisms behind phosphorylation of mTOR at ser2448, but the most recent data would suggest that ser2448 is predominantly a site for mTORC1 and ser 2481 for TORC2, with p-S6 functioning as the kinase for ser2448 [START_REF] Rosner | mTOR phosphorylated at s2448 binds to raptor and rictor[END_REF][START_REF] Generali | Down-regulation of Phosphatidylinositol 3-Kinase/ AKT/ Molecular Target of Rapamycin metabolic pathway by primary letrozole-based therapy in human breast cancer[END_REF].

Current understanding of the mTOR pathway and the two distinct complexes formed by mTOR with raptor (mTORC1) and rictor (mTORC2) has been based upon work with rapamycin, hence the naming of raptor (rapamycin associated companion to mTOR) and rictor (rapamycin insensitive companion to mTOR). One of the aims of this study was to determine the action of RAD001 upon mTOR and whether rictor is indeed insensitive to rapamycinlike mTOR inhibitors in vivo. The result that would be anticipated based on the current literature is that if only mTOR raptor is inhibited then the downregulation of mTOR, p-S6 and upregulation of p-AKT would be expected to occur by negative feedback. The negative associations of upregulation of AKT include resistance to apoptosis, increased cell growth and proliferation, increased resistance to endocrine treatment and increased risk of metastasis and reduced survival. If however, an mTOR inhibitor inhibits both mTOR raptor and mTOR rictor then it would be expected that downregulation of mTOR and p-S6, and also downregulation of p-AKT would occur. In this study it was found that in all tumors there was a significant reduction in expression of nuclear p-AKT after treatment with RAD001, indicating that both raptor and rictor complexes of mTOR are inhibited. Similarly, tumors with high cytoplasmic expression of p-AKT pre-treatment had a reduction in p-AKT in the cytoplasm after treatment with RAD001, and in those tumors defined as responding to RAD001 by reduction in proliferation with treatment, there was a significant reduction in expression of cytoplasmic p-AKT. However, tumors with a low p-AKT cytoplasmic expression treated with RAD001 had no change or an increase in p-AKT cytoplasmic expression with RAD001 treatment, indicating that tumors with high p-AKT expression may respond better to RAD001 than those with low cytoplasmic p-AKT.

No association was observed between response and PI3K mutations (data not shown [START_REF] Sabine | Mutation of PI3KCA in post-menopausal with breast cancer and response to RAD001 treatment[END_REF]. Although 26% of samples (8/31) harbored mutations, the sample size of this study is too small to draw conclusions relating to PI3K mutations and RAD001 response. In the combination study of neoadjuvant RAD001 and letrozole tumors with mutations in the PIK3CA exon 9 helical domain had a relatively small antiproliferative response to letrozole alone but a good response to RAD001 plus letrozole [START_REF] Baselga | Phase II randomized study of neoadjuvant everolimus plus letrozole compared with plac ebo plus letrozole in patients with estrogen receptor-positive breast cancer[END_REF].

There was a significant reduction in ER scores with RAD001 treatment, particularly in those that had moderate to low ER levels pre-operatively. It has previously been shown that a reduction in ER can occur due to variation when comparing core biopsies and tissue sections, although no such changes have been shown for PR or Ki67 [46,[START_REF] Iqbal | MIB-1 assessments in breast cancers[END_REF] This may account for the loss of ER staining particularly in those with low staining on core biopsy. There is increasing evidence for the role of PI3K/ AKT pathway in modulation of the ER signaling pathway [START_REF] Bhat-Nakshatri | AKT alters genomeside estrogen receptor alpha binding and impacts estrogen signaling in breast cancer[END_REF], and this is of particular interest with the development of the concept of combining hormonal treatment with agents that block signal transduction. Tumors were not selected for this study on the basis of tumor type, ER, PR or HER-2 status, although all tumors had some level of ER positivity. There appeared to be a greater response in tumors that were HER-2 positive, with greater reductions in p-S6. This is in keeping with results from a study of another mTOR inhibitor CCI779 (Wyeth Pharmaceuticals) in metastatic breast cancer during which no responses were seen in HER-2 positive tumors [START_REF] Chan | Phase II study of Temsirolimus (CCI-779), a novel inhibitor of mTOR, in heavily pretreated patients with locally advanced or metastatic breast cancer[END_REF].

Combining a broad range of baseline tumor characteristics may have affected the results of this study, as these represent biologically different tumors with potentially different mechanisms and drivers for proliferation, and different clinical outcomes [50]. Further studies should focus on more defined populations to avoid this potential dilution of results, in particular in tumors with high proliferation, high p-AKT and HER-2 positivity.

The results of this study confirm that mTOR inhibition with RAD001 at 5mg daily dose has an effect upon ER signaling pathways as a single agent in addition to reducing proliferation. It remains to be determined whether this reduction in ER may lead to a change in responsiveness to anti-estrogens.

Further

  studies are warranted to clarify the mechanism for this to optimize the potential for combination therapy and to clarify the role of RAD001 as a single agent. 27 Going JJ (1994) Efficiently estimated histologic cell counts. Hum Pathol 25: 333-336 28 Kirkegaard T, Edwards J, Tovey S, McGlynn LM, Krishna SN, Mukherjee R et al (2006) Observer variation in immunohistochemical analysis of protein expression, time for a change? Histopathology 48(7):787-794 29 Tabernero J, Rojo F, Calvo E et al (2008) Dose-and Schedule-dependent inhibition of the mammalian target of rapamycin pathway with everolimus: a phase I tumor pharmacodynamic study in patients with advanced s olid tumors. J Clin Oncol 26: 1603-1610 30 O'Donnell A, Faivre S, Burris HA et al (2008) Phase I pharmacokinetic and pharmacodynamic study of the oral mammalian target of rapamycin inhibitor everolimus in patients with advanced solid tumors. J Clin Oncol 26: 1588-1595 31 Galea MH, Blamey RW, Elston CE, Ellis IO (1992) The Nottingham Prognostic Index in primary breast cancer. Breast Cancer Res Treat 22:207-219 32 Dowsett M, Smith I, Ebbs SR, on behalf of the IMPACT Trialists (2005) Short-term changes in Ki-67 during neoadjuvant treatment of primary breast cancer with anastrozole or tamoxifen alone or combined correlate with recurrence-free survival. Clin Cancer Res 11: 951s-958s 33 Viale G, Giobbie-Hurder A, Regan MM et al (2008) Prognostic and Predictive Value of Centrally Reviewed Ki-67 Labeling Index in Postmenopausal Women W ith Endocrine-Responsive Breast Cancer: Results From Breast International Group Trial 1-98 Comparing Adjuvant Tamoxifen With Letrozole. J Clin Oncol 28: 5569-5575 34 Murray J, Young OE, Renshaw L et al (2009) A randomised study of the effects of letrozole and anastrozole on oestrogen receptor positive breast cancers in postmenopausal women. Breast Cancer Res Treat 114(3): 495-501 35 Young O, Renshaw L. Macaskill EJ et al (2008) Effects of fulvestrant 750mg in premenopausal women with oestrogen-receptor-positive primary breast cancer. Eur J Cancer 44:391-399 36 Knuefermann C, Lu Y, Liu B et al (2003) HER2/ PI3K/ Akt activation leads to a multidrug resistance in human breast adenocarcinoma c ells. Oncogene 22: 3205-3212 37 Tokanuga E, Kataoka A, Kimura Y et al (2006) The association between Akt activation and resistance to hormone therapy in metastatic breast canc er. Eur J Cancer 42: 629-635 50 Sorlie T, Perou CM, Tibshirani R, et al (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 98: 10869-10874

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Table 1

 1

	Patient demographics	n (%)
		(unless otherwise specified)
	Mean age (range, yrs)	67.5 (51-89)
	Mean tumor size (range, mm)	23.9 (11-41)
	Clinical tumor stage: T1	8 (25.8)
	T2	23 (74.2)
	T3	0
	Clinical nodal stage: N0	28 (90.3)
	N1	3 (9.7)
	N2	0
	Surgery: Breast conservation	23 (74.2)
	Mastectomy	8 (25.8)
	Tumor type: IDC	27 (87.1)
	Lobular	1 (3.2)
	Mucinous	1 (3.2)
	Tubular	2 (6.5)
	Tumor grade: 1	8 (25.8)
	2	12 (38.7)
	3	11 (35.5)

Table 1 : Baseline patient and tumor demographics. All patients (n=31) were postmenopausal females, with a WHO performance scale of 0-2 with early breast cancer. IDC, invasive ductal carcinoma.

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