Mitra Tewes

Bahriye Aktas

Anja Welt

Siemke Mueller

Siegfried Hauch

Rainer Kimmig

Sabine Kasimir-Bauer
email: sabine.kasimir-bauer@uk-essen.de

A E Bahriye

Aktas Ae

A E Siemke

Mueller Ae

A E Rainer

Kimmig Ae

Á A Welt

Á R Kimmig

Á S Kasimir-Bauer

Molecular profiling and predictive value of circulating tumor cells in patients with metastatic breast cancer: an option for monitoring response to breast cancer related therapies

Keywords: Circulating tumor cells, Molecular profiling, Therapy monitoring, Metastatic breast cancer

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The current therapies in early breast cancer are based on the primary tumor size, the presence or absence of axillary lymph node metastasis, grading of tumor differentiation, the receptor and HER2 status, age as well as the hormonal status of the patient. Most patients with stage I and II breast cancer will be cured of their disease by surgery, adjuvant chemotherapy and hormone therapy. However, a substantial number of them will develop recurrent carcinoma [START_REF] Goldhirsch | Meeting highlights: international expert consensus conference on the primary therapy of early breast cancer[END_REF]. This recurrence rate is explained by tumor cell dissemination into distant organs, preferentially bone marrow (BM), which often occurs prior to surgery. It has been demonstrated that these cells show a considerable heterogeneity in the expression of carcinoma-associated cell-surface molecules, including HER2, EpCAM, MUC1 and Lewis Y [START_REF] Braun | Tumor-antigen heterogeneity of disseminated breast cancer cells: implications for immunotherapy of minimal residual disease[END_REF]. Furthermore, they have a time-limited proliferation potential and, thus, may be dormant [START_REF] Pantel | Differential expression of proliferation-associated molecules in individual micrometastatic carcinoma cells[END_REF]. This phenomenon was also observed in blood samples of breast cancer patients even up to 22 years after diagnosis [START_REF] Meng | Circulating tumor cells in patients with breast cancer dormancy[END_REF]. Since existing therapies are aimed at proliferation pathways only, this might explain why adjuvant chemotherapy fails to prevent relapse in some patients with early breast cancer. In this context, it has been demonstrated that tumor cells frequently survive high-dose chemotherapy [START_REF] Kasimir-Bauer | Survival of tumor cells in stem cell preparations and bone marrow of patients with high-risk or metastatic breast cancer after receiving dose-intensive or high-dose chemotherapy[END_REF] and that tumor cells, still present after chemotherapy, represent an independent poor prognostic factor for reduced overall survival [START_REF] Braun | Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high-risk breast cancer patients[END_REF][START_REF] Wiedswang | Comparison of the clinical significance of occult tumor cells in blood and bone marrow in breast cancer[END_REF].

Thus, tumor cell detection in the BM is being regarded increasingly as a clinically relevant prognostic factor for breast cancer and a pooled analysis of BM findings in more than 4,700 patients documented the prognostic value of these cells with regard to reduced disease free and overall survival [START_REF] Braun | A pooled analysis of bone marrow micrometastasis in breast cancer[END_REF]. The above observations indicate that the detection and characterization of disseminated occult tumor cells may be an important marker to determine in patients with breast cancer. On the one hand, it may identify a subgroup of patients with early breast cancer who are at high risk of relapse. On the other hand, in patients with incurable metastatic breast cancer, there is an urgent need to identify a surrogate marker of tumor response to overcome current limitations of radiography and surgical biopsy in the serial monitoring of cancer therapy, especially in trials of costly new and targeted agents.

Since multiple BM aspirations from the same patient during follow-up are not easily tolerated, the peripheral blood would be a more convenient source of sampling for such longitudinal follow-up studies. In primary breast cancer, the predictive value of circulating tumor cells (CTC) still has to be proven. Whereas some groups showed that the presence of CTC in blood before and/or after adjuvant therapy is associated with poor prognosis and the over-expression of the estrogen-receptor [START_REF] Stathopoulou | Molecular detection of cytokeratin-19-positive cells in the peripheral blood of patients with operable breast cancer: evaluation of their prognostic significance[END_REF][START_REF] Giatromanolaki | Assessment of highly angiogenic and disseminated in the peripheral blood disease in breast cancer patients predicts for resistance to adjuvant chemotherapy and early relapse[END_REF][START_REF] Gaforio | Detection of breast cancer cells in the peripheral blood is positively correlated with estrogen-receptor status and predicts for poor prognosis[END_REF][START_REF] Jotsuka | Persistent evidence of circulating tumor cells detected by means of RT-PCR for CEA mRNA predicts early relapse: a prospective study in node-negative breast cancer[END_REF], other groups did not support these data [START_REF] Wiedswang | Comparison of the clinical significance of occult tumor cells in blood and bone marrow in breast cancer[END_REF][START_REF] Pierga | Clinical significance of immunocytochemical detection of tumor cells using digital microscopy in peripheral blood and bone marrow of breast cancer patients[END_REF][START_REF] Benoy | Real-time RT-PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer[END_REF]. Furthermore, apoptotic cells significantly contribute to CTC fractions in blood of breast cancer patients [START_REF] Mehes | Circulating breast cancer cells are frequently apoptotic[END_REF].

In contrast, the presence of [5 CTC in 7.5 ml of blood from metastatic breast cancer patients has been shown to predict shorter progression-free and overall survival and has superior and independent prognostic value of tumor burden and disease phenotype [START_REF] Cristofanilli | Circulating tumor cells, disease progression, and survival in metastatic breast cancer[END_REF][START_REF] Cristofanilli | Circulating tumor cells in metastatic breast cancer: biologic staging beyond tumor burden[END_REF]. However, besides isolating these cells, the need to determine the profiles of expression of genes and/or proteins of tumors is becoming increasingly important since changes in the phenotype of the tumor cells can occur after the original diagnosis (e.g. HER2, and receptors for estrogen and progesterone) and resistance to a treatment can only be inferred after the treatment has failed [START_REF] Hayes | Monitoring expression of HER-2 on circulating epithelial cells in patients with advanced breast cancer[END_REF]. PCR methods are generally more sensitive than microscopic methods and have the additional advantage of a high-throughput technology. Furthermore, using a multi-marker panel of cancer associated genes, RT-PCR was found to be the most sensitive technique for the detection of CTC in blood of breast cancer patients [START_REF] Bosma | Detection of circulating breast tumor cells by differential expression of marker genes[END_REF][START_REF] Ring | Detection of circulating epithelial cells in the blood of patients with breast cancer: comparison of three techniques[END_REF]. One approach involves the analysis of CTC-enriched samples by gene expression profiling [START_REF] Baker | Molecular detection of breast cancer cells in the peripheral blood of advanced-stage breast cancer patients using multimarker real-time reverse transcriptionpolymerase chain reaction and a novel porous barrier density gradient centrifugation technology[END_REF][START_REF] Hauch | The clinical significance of circulating tumour cells in breast cancer and colorectal cancer patients[END_REF].

In this study, we used the technology developed by Adn-aGen which combines immunomagnetic tumor cell selection targeting EpCAM and MUC1 (AdnaTest BreastCancerSelect), followed by multiplex RT-PCR for the transcripts EpCAM, MUC1 and HER2 (AdnaTest BreastCancerDetect). In addition, the transcripts of the estrogen (ER) and progesterone (PR) receptor were analyzed by an additional RT-PCR.

It was the purpose of the study to (a) detect and analyze CTC in the blood of metastatic breast cancer patients by molecular profiling during a follow-up of palliative chemo-, antibody-or hormonal therapy and (b) determine the ability of this method to predict the response to breast cancer related therapies.

Patients, materials and methods

Study design

We conducted a prospective single institution trial to purpose the molecular analysis of circulating tumor cells (CTC) in blood of breast cancer patients during palliative therapy and to determine the ability of this method to predict the response to breast cancer related therapies.

Patient population

The study was conducted at the Department of Obstetrics and Gynecology in collaboration with the Department of Internal Medicine (Cancer Research) at the University Hospital in Essen. In total, 42 patients have been studied since August 2006. The median time of follow up and sample collection was 9 months (range [START_REF] Kasimir-Bauer | Survival of tumor cells in stem cell preparations and bone marrow of patients with high-risk or metastatic breast cancer after receiving dose-intensive or high-dose chemotherapy[END_REF][START_REF] Braun | Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high-risk breast cancer patients[END_REF][START_REF] Wiedswang | Comparison of the clinical significance of occult tumor cells in blood and bone marrow in breast cancer[END_REF][START_REF] Braun | A pooled analysis of bone marrow micrometastasis in breast cancer[END_REF][START_REF] Stathopoulou | Molecular detection of cytokeratin-19-positive cells in the peripheral blood of patients with operable breast cancer: evaluation of their prognostic significance[END_REF][START_REF] Giatromanolaki | Assessment of highly angiogenic and disseminated in the peripheral blood disease in breast cancer patients predicts for resistance to adjuvant chemotherapy and early relapse[END_REF][START_REF] Gaforio | Detection of breast cancer cells in the peripheral blood is positively correlated with estrogen-receptor status and predicts for poor prognosis[END_REF][START_REF] Jotsuka | Persistent evidence of circulating tumor cells detected by means of RT-PCR for CEA mRNA predicts early relapse: a prospective study in node-negative breast cancer[END_REF][START_REF] Pierga | Clinical significance of immunocytochemical detection of tumor cells using digital microscopy in peripheral blood and bone marrow of breast cancer patients[END_REF][START_REF] Benoy | Real-time RT-PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer[END_REF].

Blood samples of 32/42 patients were analyzed during follow-up of their individual therapies. Ten patients could not be monitored for the following reasons: four patients died within two months after first blood sampling and six patients were lost for follow-up because palliative treatment was applied in other hospitals nearby their hometowns.

Eligibility criteria

The eligibility criteria were as follows: age C 18 years; patients with measurable or evaluable metastatic breast cancer; predicted life expectancy C2 months; Eastern Cooperative Oncology Group (ECOG) scores for performance status of 0-2; no severe uncontrolled co-morbidities or medical conditions; no second malignancies.

Patients had either a relapse of breast cancer diagnosed years before and were to start chemotherapy or a documented progressive breast cancer before receiving a new endocrine, chemo-or experimental therapy. Prior adjuvant treatment, radiation or any other treatment of metastatic disease were permitted.

Response criteria

Before starting a new treatment, patients underwent an evaluation of metastatic sites by ultrasound, X-ray or computer tomography. Blood samples were collected for laboratory evaluations, including CEA and Ca 15-3 as well as for the isolation and characterization of CTC. Re evaluations of disease status were done by the same techniques every 8-12 weeks, depending on the treatment schedule, until the loss or death of a patient (Table 1).

Response to therapy was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST). Complete Response (CR): disappearance of all target lesions; Partial Response (PR): at least 30% decrease in the sum of the LD (longest diameter) of target lesions, taking as reference the baseline sum LD; Progressive Disease (PD): at least 20% increase in the sum of the LD target lesions, taking as reference the smallest sum LD recorded since the treatment started or the appearance of one or more new lesions; Stable Disease (SD): neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum LD since the treatment started.

Immunohistochemical analysis

For each of the 42 patients, the tumor type, TNM-staging and grading were assessed according to the WHO-classification of tumors of the breast [START_REF] Ellis | Invasive breast carcinoma[END_REF] and the sixth edition of the TNM Classification System [START_REF] Sobin | International union against cancer. TNM classification of malignant tumours[END_REF]. The ER and PR receptor status were determined by immunohistochemistry. The DAKO-score for the expression of HER2 was determined with the HercepTest Ò , and the results were interpreted as follows: 0 = no membrane staining; 1+ = faint, partial membrane staining; 2+ = weak complete membrane staining in greater than 10% of invasive cancer; 3+ = intense complete membrane staining in greater than 10% of invasive cancer cells. FISH analysis in cases of 2+ staining as determined with the HercepTest was performed as described elsewhere [START_REF] Lal | HER-2 testing in breast cancer using immunohistochemical analysis and fluorescence in situ hybridization: a single-institution experience of 2, 279 cases and comparison of dual-color and single-color scoring[END_REF].

Sampling of biological material 2 9 5 ml EDTA blood was collected for isolation of CTC before the application of therapeutic substances with an S-Monovette Ò (Sarstedt AG & Co.) and stored at 4°C until further examination. The samples were processed immediately or not later than 4 h after blood withdrawal.

An additional serum sample was collected to determine tumor markers.

Healthy controls

5 ml blood was collected from 88 healthy donors aged 23-73 years for the determination of specificity and sensitivity of the AdnaTest and from 48 healthy donors for assaying the hormonal receptors.

Determination of serum tumor markers

CEA and CA 15-3 were determined using the Elecsys CEA/ CA 15-3 immunoassays (Roche, Mannheim, Germany) for the quantitative determination of CEA/CA 15-3 in human serum and plasma. The serial measurement of CEA/CA 15-3 was intended to aid in the management of cancer patients. These assays were performed in the central laboratory of our University Hospital on Cobas Ò immunoassay analyzers according to the manufacturer's instruction. The central laboratory has a valid certification for the performance of these assays following international guidelines.

Tumor cell enrichment/selection

Since August 2006 blood samples were taken from 42 patients with metastatic breast cancer. The AdnaTest BreastCancer (AdnaGen AG, Langenhagen, Germany) enables the immunomagnetic enrichment of tumor cells via epithelial and tumor associated antigens. Two antibodies against the epithelial antigen MUC1 and one antibody against the epithelial glycoprotein GA733-2 (EpCAM) are conjugated to magnetic beads (Dynabeads) for the labelling of tumor cells in peripheral blood [START_REF] Demel | Detection of tumour cells in the peripheral blood of patients with breast cancer. Development of a new sensitive and specific immunomolecular assay[END_REF][START_REF] Zieglschmid | Combination of immunomagnetic enrichment with multiplex RT-PCR analysis for the detection of disseminated tumor cells[END_REF]. In brief, the blood samples were incubated with a ready-to-use antibody mixture commercialized as AdnaTest BreastCancerSelect according to the manufacturer's instructions. The labelled cells were extracted by a magnetic particle concentrator (MPC). Subsequently, mRNA isolation from lysed, enriched cells was performed according to the manufacturer's instructions with the Dynabeads mRNA DIRECT TM Micro Kit (Dynal Biotech GmbH, Hamburg, Germany) that is included in the AdnaTest BreastCancerDetect. Reverse transcription resulted in cDNA, which was the template for tumor cell detection and characterization by multiplex RT-PCR. Sensiscript Ò Reverse Transcriptase (QIAGEN GmbH, Hilden, Germany) was used for the reverse transcription because of its high sensitivity (recommended for amounts of \50 ng RNA) in combination with oligo(dT) coupled Dynabeads of the mRNA DIRECT TM Micro Kit (Dynal Biotech GmbH) according to the manufacturer's instructions [START_REF] Lankiewicz | Quantitative real-time RT-PCR of disseminated tumor cells in combination with immunomagnetic cell enrichment[END_REF]. cDNA was synthesized in a thermocycler under the following conditions: Reverse transcription was performed at 37°C for 60 min followed by 3 min at 93°C for inactivation of the reaction. The resulting cDNA was stored at -20°C until further use.

Tumor cell detection

The Adnatest BreastCancerDetect was used for the detection of breast cancer-associated gene expression in immunomagnetically enriched tumor cells by reverse transcription and PCR. The analysis of tumor-associated mRNA isolated from circulating tumor cells was performed in a multiplex PCR for three tumor-associated transcripts: HER2, MUC1 and GA733-2. The thermal profile used for multiplex RT-PCR was as follows: After a 15 min denaturation at 95°C, 35 PCR cycles were carried out by denaturation at 94°C for 1 min, annealing/extension at 60°C for 1 min of and elongation for 1 min at 72°C. Subsequently, termination of the reaction was carried out at 72°C for 10 min followed by storage of the samples at 4°C. The primer sets for the ER and PR receptor were a gift from AdnaGen Ò AG (Langenhagen, Germany). ER and PR were detected on CTC performed with these reagents after the preparation of the cDNA and according to the instructions of the AdnaTest BreastCancerDetect. PCR was performed with the HotStarTaq Master Mix (QIAGEN GmbH). Actin was used as internal PCR positive control. The thermal profile used for the nested RT-PCR was as follows: After a 15min denaturation at 95°C, 37 cycles of PCR were carried out by denaturation at 94°C for 30 s, annealing/extension at 60°C for 30 s and elongation for 30 s at 72°C. Subsequently, termination of the reaction was carried out at 72°C for 5 min followed by storage of the samples at 4°C.

The primers generate fragments of the following sizes: GA733-2: 395 base pairs (bp), MUC1: 293 bp, HER2: 270 bp, PR: 270 bp, ER: 305 bp and actin: 114 bp. Visualization of the PCR fragments was carried out with a 2100 Bioanalyzer using the DNA 1000 LabChips (Agilent Technologies) and the Expert Software Package (version B.02.03.SI307).

Evaluation of data

The test is considered positive if a PCR fragment of at least one tumor associated transcript is clearly detected. Using the software package for evaluation of the data on the Agilent 2100 Bioanalyzer, peaks with a concentration of [0.15 ng/ll are positive for the transcripts GA733-2, MUC1 and HER2. Peaks that are not detected at the above setting are negative (concentration of \0.15 ng/ll). Peaks with a concentration of [0.60 ng/ll are positive for the ER transcript. The PR expression is considered positive when the transcript is detected without applying any cut-off.

Statistical analysis

Survival analysis was performed by analyzing Kaplan-Meier-life tables, whereby death is used as endpoint. Overall survival has been described as the overall survival during the time of monitoring. Kaplan-Meier survival curves separated for CTC(+) and CTC(-) are presented and compared via log rank test. Null-hypothesis of non different survival times is rejected, when P-values \ 0.05. The results were verified by Dr. Keller (ACOMED Statistics, Leipzig, Germany) using Software NCSS 2007 (Hintze J. (2007) NCSS, PASS and GESS. NCSS. Kaysville, Utah, US. www.ncss.com).

Results

Patient characteristics

A total of 42 patients was enrolled since August 2006. As shown in Table 2, the patients ranged in age from 29 to 74 years. All four initial tumor stages were included, with a predominance of stage II. Only eight patients had a primary metastatic breast cancer. Most patients had ductal breast cancer. Moderately and poorly differentiated tumors were predominant. The median number of sites of metastasis was two with a range from 1 to 4 sites. Most patients had visceral metastasis. 27 and 23 primary tumors were ER-and PRpositive, respectively; and 12 primary tumors had an overexpression of HER2 (DAKO-Score 3+). All patients with BET (breast conserving surgery) received an adjuvant radiation. Patients with hormone receptor-positive tumors (stage I-III) received an adjuvant hormonal treatment with tamoxifen or an aromatase inhibitor. The chemotherapeutic adjuvant treatment mostly contained anthracyclines and taxanes. Patients with metastatic tumors of the breast received different chemotherapeutic treatments including anthrayclines, taxane, vinorelbine and 5-FU. Most patients were extensively pretreated before starting the collection of the blood samples. 27 patients received an anthracycline-or taxane-based chemotherapy in an adjuvant or metastatic stetting before study start. Nearly all patients with HER2 3 + tumors received trastuzumab. One patient with HER2 overexpression of breast cancer did not receive trastuzumab because of a coronary artery disease and reduced general condition.

After starting measurement of CTC, patients underwent individual chemotherapeutical or hormonal treatment depending on the pre-treatment. A sub-group of 32/42 patients was monitored for CTC during chemotherapy over a median time period of 9 months (5-14 months). 10 patients could not be monitored for the following reasons: four patients died within two months after first blood sampling and six patients were lost for follow-up because palliative treatment was applied in other hospitals nearby their hometowns.

Determination of the specificity for tumor cell selection and detection

A ROC analysis was performed using the highest fragment concentration obtained among the three markers (GA733-2, MUC1, and HER2) from 41 patients with metastatic breast cancer and 88 healthy donors (Fig. 1). The fragment concentrations (ng/ll) obtained from the 2100 Expert Software Package (version B.02.03.SI307) were used instead of the peak heights for the calculations in order to minimize device to device variations. The resulting ROC curve is shown in Fig. 1. The AdnaTest BreastCancer ROC curve yields an AUC (area under the curve) of 0.89, which suggests that the AdnaTest BreastCancer has reasonably small false positive and false negative rates across a reasonable range of cut off values. At a specificity level of 90% ([0.15 ng/ll) a clinical sensitivity of roughly 65.9% can be shown and even at 99% specificity level ([0.54 ng/ ll) a sensitivity of 43.9% would be reached. The statistical maximum for the diagnostic value (highest distance to Fig. 1 ROC curve for AdnaTest Breast Cancer. The ROC curve is shown as a grey line. The dashed lines describe the 95% confidence intervals. Data obtained from 41 women with metastatic breast cancer and 88 healthy donors were used for the calculation random guess line) is reached at a sensitivity of 80.5% and a corresponding specificity of 80%, respectively. For the determination of the specificity of the ER and PR assay 48 healthy donor samples were analyzed yielding to negative results for ER in 44/48 cases (92% specificity) if a cut-off value of 0.6 ng/ll was applied. For PR all healthy donor samples were found to be negative (data not shown).

Expression profiling of CTC of metastatic breast cancer patients

The results for CTC detection in 42 patients with metastatic breast cancer at the time of tumor progression or therapy switch are shown in Fig. 2. The overall detection rate for CTC was 52% (22/42 patients) with the expression rates of 86% for EpCAM (19/22 patients), 86% for MUC1 (19/22 patients) and 32% for HER2 (7/22 patients), respectively. Results for ER and PR could be obtained in a subset of 37 patients of which 46% were positive for CTC (17/37 patients) with a detection rate of 35% (6/17 patients) for ER and 12% (2/17 patients) for PR, respectively.

Comparison of antigen expression on the primary tumor with the expression on CTC

Table 3 shows the results for the expression of ER, PR and HER2 on the primary tumor as compared to the expression on CTC. Comparisons with the primary tumor were only performed in CTC+ patients (n = 22). In 5/11 (45%) patients with ER+ tumors, CTC were ER-and 7/9 (78%) patients with PR+ tumors did not express PR on CTC. Similar results were found for HER2 where 3/5 (60%) patients with HER2-positive tumors were HER2-negative on CTC. Interestingly, 5/17 (29%) patients with HER2negative primary tumors had HER2-positive CTC.

Palliative therapy monitoring of metastatic breast cancer patients

Figure 3 illustrates that the efficacy of therapy can be assessed through the detection of tumor cells. Patient 1 suffered from multiple liver (HEP), lung (PUL) and bone (OSS) metastases and was positive for ER and PR. Prior to therapy with paclitaxel, patient 1 showed an expression of GA733-2 and MUC1. After the onset of therapy, CTC immediately disappeared and in the course of four taxane therapy sessions, patient 1 was only positive again for CTC on one occasion.

Example 2 describes the therapy follow up of a patient that initially responded to a vinorelbine based therapy which was paralleled by a decreasing CA 15-3 level. However, after two months and seven therapy sessions no effect on the metastases was observed. The CTC findings confirm an initial response of that patient to the therapy, which eventually led after the third vinorelbine session to the disappearance of CTC. However, a reappearance of CTC after the fourth therapy session indicated an onset of therapy resistance.

Patient 3 had liver (HEP), lung (PUL) and bone (OSS) metastases and was positive for ER, PR and HER2. Prior to therapy with an anthracycline as well as during treatment, patient 3 showed an expression of three tumor-associated transcripts. Whereas CTC disappearance was related to clinical response in patient 1, progressive disease under therapy was related with positive findings of CTC in patient 3. Figure 1 shows the expression rate for the different transcripts analyzed on CTC. Multiplex-PCR for EpCAM, MUC1 and HER2 could be analyzed in all 42 patients. The black column shows the total positive rate for CTC found prior to application of chemotherapy. The dark grey column demonstrates the expression rate for EpCAM on CTC, the light grey column for MUC1, the white column for HER2, the cross-striped column for ER and the diagonally-striped column for PR, respectively Patient 4 had bone (OSS) and lymph node metastases, was positive for ER and PR but negative for HER2 on the primary tumor. Before therapy with capecitabine, this patient showed the expression for all markers analyzed. The upper part of the gel shows the results for the expression of GA733-2, MUC1 and HER2 and the lower part for the expression of the hormone receptors during the course of treatment. CTC completely disappeared after five chemotherapy sessions, which correlated with a decrease in CA 15-3 and CEA levels and a good clinical response. Therapy was then switched to hormonal therapy with an aromatase inhibitor for maintenance of the disease.

Correlation between CTC detection and therapy response

As shown in Fig. 4, CTC were not detected or disappeared shortly after onset of the therapy (CTC-) in 12/16 (75%) in the responder group. In 4/16 (25%) nonresponding patients no CTC were detected at any time point studied. In contrast, CTC were found in 13/16 (81%) patients with clinically confirmed progression during or after therapy (CTC+) whereas in 3/16 (19%) patients of this responder group CTC were still present after clinically confirmed partial remission.

In summary, breast cancer patients, CTC positive either before or during therapy, showed a correlation between presence, persistence or early disappearance of CTC with therapy response in 12 cases in the CTC negative group and in 13 cases in the CTC positive group, corresponding to a prediction rate of 78%. The concordance between clinical response and serum tumor marker levels of CA 15-3 and CEA measured during follow up was 67% in these patients (data not shown).

CTC-CTC+ Responder

75% 19%

Non-Responder

25% 81%

Fig. 4 Correlation between CTC detection and therapy response

Prognostic value of CTC detection

Overall survival was determined for the time frame of monitoring starting from the time point when patients had either a relapse of breast cancer or a documented progressive breast cancer before receiving a new therapy. The Kaplan-Meier analysis shown in Fig. 5 illustrates a shorter overall survival for CTC+ patients indicating an additional and highly significant prognostic value of CTC detection (P = 0.005). 16 of the 32 patients remained positive for CTC and 16 turned negative during therapy. During 14 months after therapy onset eight CTC+ patients have died in contrast to three patients of the CTC-group. Both patient groups did not differ in age and in the number of different therapies before entering the study.

Discussion

Our study shows that the presence or disappearance of CTC during the time course of individual treatment is a predictor of therapy response in metastatic breast cancer. Furthermore, molecular profiling of CTC with the Adna-Test may provide even more differentiated prognostic information and may help to monitor breast cancer related therapies, including chemotherapy, antibody-based chemotherapy and hormone therapy. With this test principle, we could demonstrate that CTC were detectable in 52% of the patients. Besides characterizing these cells for three different tumor cell associated transcripts by multiplex PCR, this test enables the additional analysis of transcripts, e.g. hormone receptors, which may be essential in controlling different kinds of breast cancer related therapies. In this regard, we here demonstrate for the first time that ER was expressed on 35% and PR on 12% of the CTC detected.

The AdnaTest BreastCancer was designed to detect rare epithelial cells in whole blood. To ensure the specificity required for the clinical application of mRNA expression assays for occult tumor cells, blood samples from 88 healthy controls were analyzed confirming a high specificity and sensitivity of this test. Thus, we can assume that the identified epithelial and cancer related transcripts in our patients derived from CTC.

Some recent studies showed the prognostic value of CTC in metastatic breast cancer. In this regard, the presence of[5 circulating tumor cells in 7.5 ml of blood from metastatic breast cancer patients has been shown to predict shorter progression-free and overall survival and has superior and independent prognostic value of tumor burden and disease phenotype, thus representing an important marker of tumor biology [START_REF] Cristofanilli | Circulating tumor cells, disease progression, and survival in metastatic breast cancer[END_REF][START_REF] Cristofanilli | Circulating tumor cells in metastatic breast cancer: biologic staging beyond tumor burden[END_REF]. The prognostic value of CTC could be confirmed in this study, demonstrating the validity of the RT-PCR method used in this study. However, besides showing the presence of CTC, there is an urgent need to characterize these cells to answer the efficacy of the applied therapy. Our results show that reliance on the immunophenotype of the primary tumor can be misleading since HER2 or the hormone receptors were often differently expressed on CTC as compared to the primary tumor. Similar data for the expression of HER2 on micrometastatic tumor cells from BM of patients with primary breast cancer have been recently published by different groups who independently indicated that HER2 expression on disseminated tumor cells differed from HER2 expression in the primary tumor and that the expression of HER2 in micrometastasis was correlated with poor prognosis [START_REF] Braun | ErbB2 overexpression on occult metastatic cells in bone marrow predicts poor clinical outcome of stage I-III breast cancer patients[END_REF][START_REF] Vincent-Salomon | HER2 gene status assessment in micrometastatic cells in bone marrow (BM) of breast cancer patients by fluorescence in situ hybridization[END_REF][START_REF] Becker | HER2 expression on disseminated tumor cells from bone marrow of breast cancer patients[END_REF][START_REF] Solomayer | Comparison of HER2 status between primary tumor and disseminated tumor cells in primary breast cancer patients[END_REF]. These observations might have clinical relevance for trastuzumab treatment. In this context, Meng et al. [START_REF] Meng | Circulating tumor cells in patients with breast cancer dormancy[END_REF] have recently shown that HER2-positive cells could be eliminated by trastuzumab. The expression of HER2 on CTC as compared to the primary tumor has rarely been investigated. Hayes et al. [START_REF] Hayes | Monitoring expression of HER-2 on circulating epithelial cells in patients with advanced breast cancer[END_REF] demonstrated in a monitoring study in patients with advanced breast cancer that it is possible to quantify HER2positive cells for rationally designed therapy using CTC. Interestingly, they found a phenotype conversion from HER2-negative to HER2-positive CTC in the time course of therapy in a subset of patients. These phenotypical differences can also be seen in three of our patients and have also been documented for EGFR in colorectal cancer [START_REF] Italiano | Epidermal growth factor receptor (EGFR) status in primary colorectal tumors correlates with EGFR expression in related metastatic sites: biological and clinical implications[END_REF].

Studies comparing the expression of hormone receptors on the primary tumor with those on CTC rarely have been published up to now. Using the CellSearch SM system followed by PCR and FISH analysis, Reuben et al. [START_REF] Reuben | Genomic of circulating tumor cells in metastatic breast cancer[END_REF] demonstrated that they were able to find ER in 15%, PR in 0% and HER2 in 10% of CTC in 15 patients with metastatic sites. Whereas a significant association between primary and metastatic tumors could be shown with respect to the presence of ER, PR and HER2, it seemed that CTC differed significantly from those tumor cells with regards to the expression of hormone-receptor and HER2 status. Analyzing a comparable number of patients, our study shows a higher percentage of hormone receptor positive CTC, which might be explained by the different methods used. Nevertheless, both studies clearly show that HER2, ER and PR were differently expressed on the tumor and on CTC. In contrast to the documented switch in antigen expression, it has been reported that chromosomal abnormalities in CTC obtained from patients with metastatic epithelial cancers matched those in primary lesions [START_REF] Fehm | Patterns of aneusomy for three chromosomes in individual cells from breast cancer tumors[END_REF]. Thus, these data suggest that CTC may represent a unique and heterogeneous cell population which should be investigated further.

Little is known about the prognostic significance of CTC in primary breast cancer. A very recently published study by Pachmann et al. [START_REF] Pachmann | Monitoring the response of circulating epithelial tumor cells to adjuvant chemotherapy in breast cancer allows detection of patients at risk of early relapse[END_REF] analyzed CTC in 91 non-metastatic primary breast cancer patients by laser scanning cytometry of anti-epithelial cell adhesion molecule-stained epithelial cells from whole unseparated blood before and during adjuvant chemotherapy. They concluded that an increase of CTC of 10-fold or more at the end of therapy was a strong predictor of relapse and a surrogate marker for the aggressiveness of the tumor cells. Although a different method has been applied, this study clearly underlines the importance of CTC as a new predictive factor even in primary disease. Furthermore, this study confirms the conclusions drawn in CTC studies for metastatic breast cancer patients.

Generally, serum tumor markers and radiological determination of sites and number of metastatic lesions are used for the assessment of tumor burden by clinical and laboratory methods. Whereas cost effective radiological assessments can only be performed to a certain degree, the clinical utility of serum tumor markers in predicting prognosis and monitoring treatment still remains controversial [START_REF] Bast | Update of recommendations for the use of tumor markers in breast and colorectal cancer: clinical practice guidelines of the American Society of Clinical Oncology[END_REF]. The concordance between clinical response and serum tumor marker levels of CA 15-3 and CEA measured during follow up was 67% in our patients as compared to the CTC detection which predicted 78% of the therapy outcome. Although serum markers are helpful and easily measurable from the clinical point of view, the detection and especially the characterization of CTC may be more helpful in individual therapy decisions. Furthermore, the detection and molecular characterization of CTC allows an access to expression profiles of many potential therapy targets (like ER, PR and HER2). CTC might be useful as a surrogate marker for patient stratification and for stratifying patients into well defined clinical studies. In this context, it recently has been demonstrated that assessment of CTC is an earlier, more reproducible indication of disease status than current imaging methods [START_REF] Budd | Circulating tumor cells versus imaging-predicting overall survival in metastatic breast cancer[END_REF]. Furthermore, Hayes et al. [START_REF] Hayes | Circulating tumor cells at each follow-up time point during therapy of metastatic breast cancer patients predict progression-free and overall survival[END_REF] clearly demonstrated that the detection of elevated CTC at any time during therapy is an accurate indication of subsequent rapid disease progression and mortality for metastatic breast cancer patients. These results are similar to our findings showing a significant shorter overall survival for CTC positive patients over the time course of therapy as compared with patients showing a disappearance of or no CTC. In addition, our test system also offers a ''semiquantification'' of circulating tumor load during the time course of therapy by the calculation of marker concentration so that even in a patient with the persistence of CTC a partial response could be documented.

Of course, it should be emphasized that our study mainly consisted of patients having experienced different kinds of first up to fourth line therapies and are now undergoing palliative treatment. Thus, there is a limitation of the study when patients have progressive disease and do not respond to any kind of treatment anymore. At that time point, the assessment of CTC will not be useful anymore. Nevertheless, including CTC detection at first diagnosis of metastasis might help to better treat a patient individually in addition to standard regimens.

In general, it is difficult to draw any conclusions concerning the prognostic value of CTC at the moment since there is no ''gold standard'' for a comparison making interpretations of the published data difficult. Nevertheless, following the recommendations of the REMARK criteria for tumor marker studies [START_REF] Mcshane | REporting recommendations for tumor MARKer prognostic studies (REMARK)[END_REF] we have shown that the detection and analysis of CTC in the time course of breast cancer related therapies is not only a useful prognostic tool but also predictive for response to therapy in metastatic breast cancer. Furthermore, the molecular profiles of CTC can be helpful for the identification of drug targets present during the adjuvant and palliative situation. In addition, phenotype changes as described for HER2 and EGFR in the course of primary disease versus metastatic disease may have a crucial influence on the efficacy of drugs. Since this study is an ongoing trial, the evaluation of more patients will show whether the significant differences already documented for CTC+ and CTC-patients will be further confirmed and strengthened by more cases.

Fig. 2

 2 Fig. 2 Expression of breast cancer related transcripts on CTC.Figure1shows the expression rate for the different transcripts analyzed on CTC. Multiplex-PCR for EpCAM, MUC1 and HER2 could be analyzed in all 42 patients. The black column shows the total positive rate for CTC found prior to application of chemotherapy. The dark grey column demonstrates the expression rate for EpCAM on CTC, the light grey column for MUC1, the white column for HER2, the cross-striped column for ER and the diagonally-striped column for PR, respectively

Patient 4 M1Fig. 3

 43 Fig. 3 Monitoring of palliative therapy of breast cancer patients. Before each therapy unit (TU) blood samples were analyzed in duplicates. The time course of treatment including the analysis of ER and PR (lower part) is shown for Patient 4

Fig. 5

 5 Fig.5Kaplan-Meier analysis. Overall survival is describing the overall survival during the time of monitoring. 8/16 CTC+ patients died within 12 months in contrast to 3/16 patients in the CTCnegative group. A Kaplan-Meier survival plot was calculated to estimate the significance of this observation. The presence of CTC was a significant prognostic factor with respect to poor overall survival (P = 0.005) (censored patients: still alive and under observation)

Table 1

 1 Flow chart for the evaluation and re-evaluation of patients

	Time	Visit no. 1 before	Follow	Follow
		start of a new	up every	up every
		treatment	8-12 weeks	2-4 weeks a
		regimen for
		metastatic
		breast cancer
	Imaging	X	X
	Clinical investigation X	X
	Laboratory (CEA,	X	X
	Ca 13-5 e.c.)
	CTC detection in	X		X
	peripheral blood

a Dependent on treatment regimen

Table 2

 2 Patient characteristics at baseline

			Table 2 continued
		n (Patients)							n (Patients)
	Total numbers of pts	42	Trastuzumab					10
	Median age (years)	50 (range 29-74)	Hormone therapy				15
	Initial staging		Not known					6
	I	4
	IIA	14
	IIB	7
	IIIA	4
	IIIB	4
	IV	8
	Not known	1
	Primary tumor
	Histology
	Ductal	28
	Lobula ¨r	6
	Medulla ¨r	1
	Not known	7
	Grading
	1	2
	2	2 0
	3	1 8
	Not known	2
	Estrogen receptor
	Pos	27
	Progesteron receptor
	Pos	23
	HER2 overexpression
	Pos (3+)	1 2
	Sites of metastasis
	Median number	2 (range 1-4)
	Visceral	34
	Nonvisceral	9
	Cerebral	2
	Elevated markers
	CEA	13				CTC
	CA 15-3	20		100%					N	N
	Not known Adjuvant treatment	10		80%					(healthy donor) 88	(metastatic disease) 41
	Chemotherapy Radiation Hormone	23 14 17	Sensitivity	40% 60%					ROC CURVE AUC SE low. CI (0.95) 0.8234 P1 0,8907 0.0352
	Trastuzumab	2							upp. CI (0.95) 0.9389
	Not known	6		20%					Specificity Sensitivity
	Metastatic treatment Chemotherapy line 1st	9		0% 100%	80%	40% Specificity 60%	20%	0%	95% 90% 80%	61.0% 65.9% 80.5%
	2nd	7
	3rd	4
	4th and more	16

Table 3

 3 Comparison of antigen expression on the primary tumor with the expression on CTC

	PCR-results	Primary tumor	Primary tumor
		IHC-positive	IHC-negative
	ER-positive (PCR)	6	0
	ER-negative (PCR)	5	6
	PR-positive (PCR)	2	0
	PR-negative (PCR)	7	7
	Her2-positive (PCR)	2	5
	Her2-negative (PCR)	3	12
	CTC, Circulating tumor cells; ER, estrogen receptor; IHC, immuno-
	histochemistry; PCR, polymerase chain reaction; PR, progesterone
	receptor

Breast Cancer Res Treat (2009) 115:581-590


George Pentheroudakis

Konstantine T Kalogeras

Ralph M Wirtz

Irene Grimani

George Zografos

Helen Gogas

Udo Stropp

Dimitrios Pectasides

Dimosthenis Skarlos

Guido Hennig

A E Konstantine

T Kalogeras

A E Ralph

M Wirtz Ae

A E George

Zografos Ae

A E Udo

Stropp Ae

A E Dimosthenis

Skarlos Ae

A E Epaminondas

Samantas Ae

Dimitrios Bafaloukos

A E Pavlos

Papakostas Ae

Haralabos P Kalofonos

A E Nicholas

Pavlidis Ae

George Fountzilas

Á N Pavlidis

Á G Fountzilas

Á I Grimani

Á U Stropp

Á G Hennig

P Papakostas

Gene expression of estrogen receptor, progesterone receptor and microtubule-associated protein Tau in high-risk early breast cancer: a quest for molecular predictors of treatment benefit in the context of a Hellenic Cooperative Oncology Group trial

Keywords: Estrogen receptor, Progesterone receptor, Microtubule, MAP-Tau, Predictive value, Breast cancer

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Breast adenocarcinoma is the most common malignant tumor in females with 60-70% of affected patients presenting with localised disease. These women become disease-free following surgical resection of the tumor and may be candidates for adjuvant systemic therapy aiming at eradication of micrometastatic deposits [START_REF] Ebctc Group | Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials[END_REF]. Estrogen receptor (ER) protein expression, studied by means of immunohistochemical (IHC) staining, is the gold standard for the selection of patients who will be managed with hormonal therapy, though its predictive value for benefit from such treatment is modest (30-60%) [START_REF] Bonneterre | Anastrozole versus tamoxifen as first-line therapy for advanced breast cancer in 668 post-menopausal women: results of the Tamoxifen or Arimidex Randomised Group Efficacy and Tolerability study[END_REF][START_REF] Mouridsen | Superior efficacy of letrozole versus tamoxifen as first-line therapy for postmenopausal women with advanced breast cancer: results of a phase III study of the International Letrozole Breast Cancer Group[END_REF]. Several clinicopathological parameters, including tumor size, grade, ER status, axillary nodal status, patient age, and Human Epidermal growth factor Receptor-type 2 (HER2) overexpression are taken into consideration in order to identify which patients are most likely to benefit from administration of adjuvant cytotoxic chemotherapy and which drugs should be used [START_REF] Henderson | The relationship between prognostic and predictive factors in the management of breast cancer[END_REF][START_REF] Trudeau | Selection of adjuvant chemotherapy for treatment of node-positive breast cancer[END_REF]. Still, the power of these complex predictive models is at best moderate: patients may be undertreated or overtreated. Ultimately, we are unable to reliably predict the true binary outcome of recurrence versus no recurrence at the level of the individual, as these clinicopathological guidelines do not capture the rich molecular heterogeneity of the malignancy and are by no means able to pinpoint the exact clinical behaviour of breast tumors [START_REF] Pusztai | Molecular classification of breast cancer: limitations and potential[END_REF].

Even when conventional IHC studies were used, evidence emerged that incremental ER protein expression was associated with higher probability of benefit from endocrine 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]. However, (IHC) studies show considerable intra-and inter-laboratory variability even when reporting binary results, due to variation in fixation time, antigen retrieval and staining methods, observer subjectivity or image acquisition threshold differences [START_REF] Layfield | Interlaboratory variation in results from immunohistochemical assessment of estrogen receptor status[END_REF][START_REF] Rhodes | Reliability of immunohistochemical demonstration of estrogen receptors in routine practice: interlaboratory variance in the sensitivity of detection and evaluation of scoring systems[END_REF][START_REF] Rudiger | Quality assurance in immunohistochemistry: results of an interlaboratory trial involving 172 pathologists[END_REF]. Accordingly, efforts at quantification of ER and progesterone receptor (PgR) protein expression face problems of technical feasibility and reproducibility. The advent of robust, sensitive and reproducible molecular techniques that reliably quantify expression of one or several genes offers promise for more accurate quantification of ER and PgR. Reverse-transcriptase polymerase chain reaction (RT-PCR) techniques analysing messenger RNA (mRNA) extracted from formalin-fixed paraffin-embedded tumors have recently been developed, able to provide normalised gene expression data over a broad dynamic range. Preliminary reports suggest satisfactory correlation of quantitative mRNA results with categories of IHC protein expression [START_REF] Bianchini | Use of estrogen receptor expression by quantitative RT-PCR to identify an ERnegative subgroup by IHC who might benefit from hormonal therapy[END_REF][START_REF] Badve | ER and PR assessment in ECOG 2197: comparison of locally determined IHC with centrally determined IHC and quantitative RT-PCR[END_REF][START_REF] Gong | Determination of estrogen-receptor status and ERBB2 status of breast carcinoma: a gene-expression profiling study[END_REF]. Still, the prognostic and predictive value of the continuous range of tumoral ER and PgR gene transcription data for patient survival and benefit from therapy has not been examined to date.

Tau is a microtubule-associated protein (MAP-Tau) that stabilises tubulin polymerisation. Early preclinical and clinical reports suggest that the MAP-Tau gene is induced by ER and that expression of Tau protein modulates sensitivity of malignant cells to taxanes [START_REF] Bhat | Microtubule-associated proteins as targets in cancer chemotherapy[END_REF]. Low levels of Tau protein expression render mitotic and cytoskeletal microtubules more sensitive to disruption by paclitaxel, affecting mitosis and cell signalling. We retrospectively examined gene expression of ER, PgR and MAP-Tau in high-risk localised breast carcinomas of women enrolled in a prospective, randomised Hellenic Cooperative Oncology Group (HeCOG) trial of adjuvant chemotherapy in order to study their correlation and concordance with IHC expression of the relevant proteins as well as their utility for predicting clinical benefit from adjuvant hormonal therapy and taxane-containing chemotherapy.

Materials and methods

Clinical trial

The HeCOG prospective trial HE10/97 randomised a total of 595 high-risk (T1-3N1M0 or T3N0M0) breast cancer patients in the period 1997-2000, to receive either four cycles of epirubicin followed by four cycles of intensified cyclophosphamide, methotrexate and 5-fluorouracil combination chemotherapy (E-CMF) or three cycles of epirubicin followed by three cycles of paclitaxel and three cycles of intensified CMF (E-T-CMF) [START_REF] Fountzilas | Postoperative dose-dense sequential chemotherapy with epirubicin, followed by CMF with or without paclitaxel, in patients with high-risk operable breast cancer: a randomised phase III study conducted by the Hellenic Cooperative Oncology Group[END_REF]. Chemotherapy cycles were administered every 2 weeks and patients received granulocyte-colony stimulating factor (GCSF) support. The clinical protocol and translational research studies were approved by the HeCOG Protocol Review Committee, by the Institutional Review Board of Kyanous Stavros Hospital and the AHEPA University Hospital. All patients provided written informed consent prior to enrolment.

Molecular and immunohistochemical studies

This was a retrospective translational research study among patients who had been enrolled in a prospective clinical trial. Accordingly, collection of formalin-fixed paraffinembedded (FFPE) tumor tissue samples was possible in 279 patients only, due to logistical/organisational barriers. An experimental method based on proprietary magnetic beads from Siemens Healthcare Diagnostics (Cologne, Germany) resulted in mRNA isolation from 274 FFPE blocks. For all tumor samples included in the analysis the number of malignant cells represented at least 75% of all nucleated cells per section as verified by hematoxylin-eosin staining. Kinetic reverse transcription polymerase chain reaction (kRT-PCR) was applied for assessment of the expression of estrogen receptor (ESR1), progesterone receptor and MAP-Tau genes using gene specific Taq-Man TM based Primer/Probe sets. Forty cycles of nucleic acid amplification were applied and the cycle threshold (CT) values of the target genes were identified. CT values were normalized by subtracting the CT value of the housekeeping gene RPL37A from the CT value of the target gene (DCT). RNA results were then reported as 40-DCT values, which would correlate proportionally to the mRNA expression level of the target gene.

In short, each FFPE slide (5 lm thick) was deparaffinized in xylol and ethanol, the pellet was washed with ethanol and dried at 55°C for 10 min. The pellet was then lysed and proteinised overnight at 55°C with shaking. After adding a binding buffer and the magnetic particles (Siemens Healthcare Diagnostics, Cologne, Germany) nucleic acids were bound to the particles within 15 min at room temperature. On a magnetic stand, the supernatant was aspirated and the beads were washed several times with a washing buffer. After adding elution buffer and incubating for 10 min at 70°C the supernatant was aspirated on a magnetic stand without touching the beads. The quality and quantity of RNA was checked by measuring absorbance at 260 and 280 nm. Pure RNA has an A260/A280 ratio of 1.9-2.0. Human reference total RNA pooled from ten human cell lines (Stratagene, La Jolla, California, USA) was used as a positive control. RNA-free DNA extracted from tumor tissues was used as a negative control.

ER and PgR protein expression was evaluated immunohistochemically in the pathology department of the different centers involved in the study, by means of streptavidin-biotin complex immunoperoxidase assay on 5 lm sections from FFPE tumor tissue. Tumor samples were categorised as ER or PgR-positive if at least 1% of malignant cells stained for ER or PgR protein, respectively.

Statistical methodology

In the absence of a reliable gold standard and following distributional studies, we used the 25th percentile of observed hormonal receptor mRNA expression levels and the median of observed MAP-Tau mRNA levels as thresholds for categorization of tumors to positive or negative cases. The chosen cut-offs were found to be close to the natural cut-offs in distributional studies (Fig. 1a-c). Moreover, cut-offs on the quartiles offer easily interpretable, reproducible and objective results. Fisher's exact test was applied to compare standard clinical and pathological factors between ER, PgR and MAP-Tau mRNA levels. Spearman's rank correlation was used as a measure of association between the three biomarkers. The Mann-Whitney test was used to compare MAP-Tau levels with ER and PgR expression levels.

Overall percentages of agreement, uncorrected for agreement by chance, were calculated and given along with the 95% confidence intervals. The degree of agreement corrected by chance was estimated using the Kappa coefficient. Kappa estimations were interpreted following the guidelines described by Landis and Koch [START_REF] Landis | The measurement of observer agreement for categorical data[END_REF]: j B 0, poor agreement; j[0 but B0.20, slight agreement; j[0.20 but B0.40, fair agreement; j [ 0.40 but B0.60, moderate agreement; j [ 0.60 but B0.80, substantial agreement; and j [ 0.80-1.00, almost perfect agreement.

Disease-free survival (DFS) was defined as the interval from study entry to first locoregional recurrence, first distant metastasis, contralateral breast cancer, secondary neoplasm, death from the disease, or death from any cause non-related to breast cancer, whichever occurred first [START_REF] Hudis | Proposal for standardized definitions for efficacy end points in adjuvant breast cancer trials: the STEEP system[END_REF]. Overall survival (OS) was measured from study entry until death from any cause. Surviving patients were censored at the date of last contact. Estimation of diseasefree and overall survival was performed using the Kaplan-Meier product-limit method and differences between survival curves were assessed with the log-rank test. The Cox proportional hazards model was used to assess the strength of the association of OS and DFS with various clinical and histological variables in the presence of treatment group, ER, PgR, and MAP-Tau mRNA expression levels. A backward selection procedure with removal criterion P [ 0.10, identified a subclass of significant variables among the following: menopausal status (pre versus post), tumor grade (I-II versus III-Undifferentiated), tumor size (B2 cm vs. 2-5 cm vs. [5 cm), and number of positive lymph nodes (0-3 vs. C4). Multivariate P values were based on Wald statistics. Results of this study were presented according to reporting recommendations for tumor marker prognostic studies [START_REF] Mcshane | Reporting recommendations for tumor marker prognostic studies[END_REF].

Results

Patient and tumor characteristics

Patients included in the present study were comparable in terms of basic characteristics (age, menopausal status, ER status, PgR status, proportion of patients receiving hormonal treatment, number of positive nodes and tumor size) with patients from the HE10/97 trial that were not included. Two-hundred and seventy-four patients (median age 51) harboured breast carcinomas, most commonly hormonereceptor positive by IHC (77% of patients), T2-3 (70%) of moderate to poor differentiation (96%), from which neoplastic FFPE material underwent molecular analysis for gene expression of ER, PgR and MAP-Tau. After modified radical mastectomy or breast conserving surgery and nodal dissection, malignant metastases in axillary lymph nodes were present in all but four cases, with presence of four or more involved nodes in 77% of patients. Slightly more patients were managed with adjuvant E-CMF chemotherapy than E-T-CMF. With the exception of grade, basic clinicopathological characteristics were well balanced when the 274 analysed patients were stratified by adjuvant chemotherapy arm (Table 1). Following the completion of adjuvant chemotherapy, 92% of the 274 women received some form of hormonal therapy, mostly tamoxifen. Among women with IHC ER-negative tumors who took tamoxifen, a proportion had PgR-unknown or PgR-positive tumors, while some received hormonal therapy despite having both ER and PgR-negative tumors, a practice not followed anymore in HeCOG participating centres.

Correlation of molecular and clinicopathological parameters

Although mRNA isolation was successful in 274 out of 279 tumor blocks, the study of several other genes in companion projects resulted in availability of high-quality mRNA in only 271 (MAP-Tau) and 263 (ER, PgR) blocks respectively. The correlations of ER, PgR and MAP-Tau mRNA status (positive or negative) with standard clinical and pathologic factors are shown in Table 2. The associations of ER mRNA and MAP-Tau mRNA status with administration of adjuvant hormonal therapy and histological grade were statistically significant. Histological grade was also significantly associated to PgR mRNA status. Moreover, MAP-Tau mRNA status was significantly associated with patient menopausal status. Specifically, patients with negative ER, PgR or MAP-Tau mRNA levels had significantly higher grade (ER negative: 78% vs. positive: 41%, P \ 0.001; PgR negative: 67% vs. positive: 45%, P = 0.003; MAP-Tau negative: 64% vs. positive: 36%, P \0.001). Significantly more patients with positive ER and MAP-Tau mRNA levels received adjuvant hormonal therapy (ER negative: 85% vs. positive: 95%, P = 0.012; MAP-Tau negative: 85% vs. positive: 98%, P \ 0.001). Premenopausal patients were significantly more numerous in patients with positive MAP-Tau mRNA levels (MAP-Tau negative: 43% vs. positive: 58.5%, P = 0.015). On the contrary, mRNA status of any of ER, PgR and MAP-Tau did not exhibit significant correlations with patient age, adjuvant chemotherapy arm (E-CMF versus E-T-CMF), administration of adjuvant radiotherapy, tumor size, and number of involved axillary lymph nodes.

Tumoral MAP-Tau mRNA status was significantly associated with both ER (Spearmann rank correlation r = 0.52, P \ 0.001) and PgR mRNA status (Spearmann rank correlation r = 0.64, P \ 0.001). In fact, positive MAP-Tau mRNA expression was seen in 67% of cases with positive ER mRNA expression versus only 3% of cases with negative ER mRNA expression (Fisher exact test, P \ 0.001). Similarly, positive MAP-Tau mRNA expression was observed in 61% of cases with positive PgR mRNA expression versus 20% of cases with negative PgR mRNA status (Fisher exact test, P \ 0.001). The distribution of tumoral MAP-Tau mRNA levels in accordance to ER mRNA status is shown in Fig. 2.

Concordance between immunohistochemical and molecular hormonal receptor studies

Two hundred and ten patients (77%) were ER and/or PgRpositive by IHC. The observed overall agreement between the determination of hormonal receptor status by kRT-PCR (mRNA) and IHC was 77% (95% confidence interval (CI): 71.5%-82%) for ER and 71% (95% CI: 65%-79%) for PgR. Among ER-mRNA positive tumors, 82.5% stained positively for the ER protein whereas 17.5% did not. Among ER-mRNA negative tumors, 61% were IHC negative but 39% stained positively for the ER protein. These rates of concordance and disagreement between the RT-PCR and IHC methodologies were quite similar for the PgR parameter as well: among PgR-mRNA positive tumors, 26% exhibited negative protein staining, while among PgR-mRNA negative tumors 37.5% stained positively for the PgR protein.

Despite the good observed agreement between the two methods, the Kappa statistic (chance-corrected measure of agreement) between kRT-PCR and IHC was 0.41 (95% CI: 0.29-0.54) for ER, a marker of moderate agreement, and 0.33 (95% CI: 0.20-0.45) for PgR, a marker of fair agreement between methodologies. Using the 25th percentile of the ER and PgR mRNA values as the threshold and considering the more widely used IHC protein expression as the benchmark, the relatively new methodology of kRT-PCR had satisfactory sensitivity (85.5-86.5%) and positive versus 23% relapses and 16% deaths among those with MAP-Tau positive carcinomas. In contrast, 33% of PgR-mRNA negative cases suffered a relapse and 21% died versus 33.5% relapsed and 24% dead patients with PgR-mRNA positive tumors. Univariate Cox regression analysis, adjusted for treatment, showed a non-significant trend for decreased risk of death in patients with ER-mRNA positive tumors (hazard ratio (HR) = 0.62, 95% CI: 0.36-1.05, Wald-P = 0.077), as well as a trend towards a decreased risk of malignant relapse (HR = 0.65, 95% CI: 0.41-1.01, Wald-P = 0.06), in comparison to those affected by ER-mRNA negative breast carcinomas. In contrast, the status of tumoral PgR mRNA did not carry any predictive significance for either risk of death or malignant relapse. Patients with MAP-Tau mRNA positive tumors had a significantly decreased risk of death compared with those with MAP-Tau mRNA negative tumors (HR = 0.49, 95% CI: 0.29-0.83, Wald-P = 0.008) and a significantly decreased risk of disease relapse (HR = 0.50, 95% CI: 0.32-0.78, Wald-P = 0.002). Kaplan-Meier survival curves representing the probability of survival and disease-free survival as a function of MAP-Tau mRNA status are presented in Fig. 3 (log-rank test for DFS P = 0.002, for OS P = 0.006). However, neither MAP-Tau mRNA nor ER mRNA expression status were predictive for benefit from adjuvant treatment with paclitaxel (E-T-CMF arm) for either disease-free survival or overall survival (DFS and OS tests for interaction Wald P = 0.65 and P = 0.73 for MAP-Tau, P = 0.32 and P = 0.99 for ER, respectively). Restricting analysis to ER positive patients, MAP-Tau mRNA expression status remained non-predictive for benefit from adjuvant treatment with paclitaxel (DFS and OS tests for interaction Wald P = 0.50 and P = 0.88 for MAP-Tau among ER positive patients).

The impact of paclitaxel on OS and DFS in MAP-Tau negative and MAP-Tau positive patients are shown in Fig. 4a andb, respectively. Same analysis among ER positive patients is presented in Figs. 5a and5b, respectively.

Multivariate Cox regression analysis (Table 4), including menopausal status, tumor grade, size, number of positive lymph nodes, treatment group, ER, PgR, and MAP-Tau mRNA expression levels, revealed that only MAP-Tau and number of involved axillary nodes remained significant independent predictors of OS, while MAP-Tau, number of positive nodes and tumor grade independently predicted DFS. More specifically, positive MAP-Tau was associated with a decreased risk of death (HR = 0.46, 95% CI: 0.25-0.85, Wald-P = 0.01). As expected, four or more positive nodes were associated with an increased risk of death (HR = 2.33, 95% CI: 1.05-5.16, Wald-P = 0.04). Regarding DFS, positive MAP-Tau mRNA status was associated with a decreased risk of relapse (HR = 0.53, 95% CI: 0.32-0.89, Wald-P = 0.02), while four or more positive nodes (HR = 2.30, 95% CI: 1.21-4.38, Wald-P = 0.01) and higher tumor grade (HR = 1.56, 95% CI: 0.98-2.50, Wald-P = 0.06) significantly increased the risk of malignant recurrence. In both multivariate models described above, MAP-Tau mRNA status was not predictive for the benefit of adjuvant treatment with paclitaxel either for OS or for DFS (test for interaction: Wald-P = 0.99 and Wald-P = 0.92, respectively).

As one of the advantages of quantitative measurement of hormonal receptor mRNA expression is a continuous dynamic range of output, we sought to determine whether increasing levels of hormone receptor mRNA correlated with increasing benefit from adjuvant hormonal therapy in a ''dose-dependent'' manner. We tested this hypothesis in 203 patients harbouring breast carcinomas immunohistochemically positive for ER and/or PgR, who received adjuvant hormonal manipulations. Data were split into four equal groups, using the quartiles as cut off points.

Using Cox regression analysis, we examined the association of very low (ER, PgR mRNA expression B first quartile), low (first quartile \ ER, PgR mRNA expression B second quartile), moderate (second quartile \ ER, PgR mRNA expression B third quartile), and high (ER, PgR mRNA expression [ third quartile) mRNA levels of ER and PgR with disease-free survival. There was no evidence of association between increasing ER or PgR mRNA expression and benefit from hormonal therapy (Wald P values adjusted for treatment group: P = 0.54 for ER mRNA and P = 0.505 for PgR mRNA). In comparison to tumors with very low ER mRNA expression, the hazard ratios for malignant recurrence in tumors with low, moderate and high expression were 1.08 (95% CI: 0.51-2.31, Wald-P = 0.835), 1.61 (95% CI: 0.79-3.29, Wald-P = 0.19) and 1.12 (95% CI: 0.52-2.39, Wald-P = 0.77), respectively. Similarly, in comparison to tumors with very low PgR mRNA expression, the hazard ratios for relapse in patients with low, moderate and high mRNA-expressing tumors were 1.06 (95% CI: 0.54-2.10, Wald-P = 0.86), 0.61 (95% CI: 0.28-1.32, Wald-P = 0.21) and 0.97 (95% CI: 0.48-1.97, Wald-P = 0.93), respectively.

Discussion

In our patient cohort, the concordance between immunohistochemical and kRT-PCR determination of ER and PgR status was good (77% for ER, 71% for PgR) but clearly inferior to rates of agreement reported by Badve et [START_REF] Bianchini | Use of estrogen receptor expression by quantitative RT-PCR to identify an ERnegative subgroup by IHC who might benefit from hormonal therapy[END_REF][START_REF] Badve | ER and PR assessment in ECOG 2197: comparison of locally determined IHC with centrally determined IHC and quantitative RT-PCR[END_REF][START_REF] Gong | Determination of estrogen-receptor status and ERBB2 status of breast carcinoma: a gene-expression profiling study[END_REF]. After excluding chance agreement, as estimated by the Kappa coefficient, only fair to moderate agreement was found between IHC and RT-PCR determination of ER and PgR. However, in most studies the investigators actually intended to identify those kRT-PCR mRNA cutoff values that would provide the highest concordance with the IHC status of the target protein. On the contrary, we regarded IHC ER and PgR protein expression determination as the most widely used analytical assay, but not the gold standard, in view of its moderate predictive value for benefit from endocrine therapies, semiquantitative nature of results and inter-or intra-laboratory reporting variability [START_REF] Bonneterre | Anastrozole versus tamoxifen as first-line therapy for advanced breast cancer in 668 post-menopausal women: results of the Tamoxifen or Arimidex Randomised Group Efficacy and Tolerability study[END_REF][START_REF] Mouridsen | Superior efficacy of letrozole versus tamoxifen as first-line therapy for postmenopausal women with advanced breast cancer: results of a phase III study of the International Letrozole Breast Cancer Group[END_REF][START_REF] Layfield | Interlaboratory variation in results from immunohistochemical assessment of estrogen receptor status[END_REF][START_REF] Rhodes | Reliability of immunohistochemical demonstration of estrogen receptors in routine practice: interlaboratory variance in the sensitivity of detection and evaluation of scoring systems[END_REF][START_REF] Rudiger | Quality assurance in immunohistochemistry: results of an interlaboratory trial involving 172 pathologists[END_REF]. Using distributional studies of mRNA values and taking into account hormonal receptor protein staining as a gross template, we sought to study the predictive value and correlation of a more conservative threshold, the 25th percentile of mRNA values, in order not to miss an effect of low, but potentially biologically significant level of messenger RNA.

With the use of this threshold, among patients with hormone receptor mRNA-positive tumors, 17-25% lacked immunohistochemical protein staining. This observation could be the result of mRNA failing to translate to protein (alternate splicing, aberrant polyadenylation, microRNAmediated or other form of translational silencing) or rapid catabolism of the hormone receptor protein. Protein degradation in the tumor block by inadequate fixation, problematic antigen retrieval, protein staining underestimation in old archival material, or finally, RNA amplification of normal tissue along with the tumor could also be the culprits for absence of protein staining [START_REF] Rhodes | Reliability of immunohistochemical demonstration of estrogen receptors in routine practice: interlaboratory variance in the sensitivity of detection and evaluation of scoring systems[END_REF][START_REF] Goldstein | Minimum formalin fixation time for consistent estrogen receptor immunocytochemical staining of invasive breast carcinoma[END_REF][20][START_REF] Morris | RNA-mediated transcriptional gene silencing in human cells[END_REF]. Conversely, when tumoral hormone receptor mRNA was categorised as negative, immunohistochemical protein staining was evident in at least 1% of tumor cells in 37-39% of cases. This discrepancy could reflect the difficulty capturing short-lived, sensitive to degradation mRNA from FFPE tumor blocks, as well as the different fixation and turnover times of ribonucleic acids in relation to proteins [START_REF] Ravo | Quantitative expression profiling of highly degraded RNA from formalin-fixed paraffin-embedded breast tumour biopsies by oligonucleotide microarrays[END_REF][START_REF] Van Maldegem | Effects of processing delay, formalin fixation and immunohistochemistry on RNA recovery from formalin-fixed paraffin-embedded tissue sections[END_REF]. Accumulation of the protein due to aberrant catabolism may also be a factor.

Despite the moderate at best agreement between IHC and kRT-PCR, the ER mRNA status, according to the objective threshold values we set, did show a trend for predictive significance. Estrogen receptor protein expression was shown to be a weak prognostic factor for survival and a moderate predictive factor for benefit from hormonal manipulations in patients with early breast cancer [START_REF] Ebctc Group | Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials[END_REF]. Its prognostic information may be carried by a multi-gene signature, for which the ER gene itself is either a key regulator or simply the best-known surrogate marker. profile mRNA transcripts from snap-frozen breast carcinomas of 38 patients [START_REF] Pusztai | Gene expression profiles obtained from fine-needle aspirations of breast cancer reliably identify routine prognostic markers and reveal large-scale molecular differences between estrogen-negative and estrogen-positive tumours[END_REF]. They reported that ER positive tumors, as assessed by IHC were strongly associated with transcriptional activity of 105 genes involved in regulation of transcription, cellular proliferation, differentiation, intracellular trafficking of proteins, and signal transduction, in contrast to ER negative tumors. Most of these genes are not known to be regulated by ER, suggesting that differences between IHC ER positive and negative tumors are not attributable to the presence or absence of ER function, but rather reflect different molecular phenotypes. These findings were supported by identification of other gene signatures associated with the presence of ER (Paik et al.,22 genes and Symmans et al.,200 genes) [START_REF] Paik | A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer[END_REF][START_REF] Symmans | Measurements of estrogen receptor and reporter genes from microarrays determine receptor status and time to recurrence following adjuvant tamoxifen therapy[END_REF]. However, the validity of the above findings was challenged by the low number of tumor samples, the high number of assessed genes and technical variances implicating statistical problems (i.e. over-fitting).

Pusztai et al. recently used DNA microarrays in order to

In our study of patients with high-risk, predominantly node-positive early breast carcinomas, ER mRNA positive status had a favourable prognostic significance for decreased risk of relapse and death, which was of borderline statistical significance. Ninety two percent of the patients received some form of adjuvant hormonal therapy, including as many as 69% of ER mRNA negative patients. Moreover, all did receive chemotherapy. Accordingly, it is unclear whether the transcriptional activity of ER carries prognostic significance for outcome irrespectively of the therapy administered, in which case it would reflect inherent biologic characteristics of the tumor, or whether it possesses predictive utility for benefit from antineoplastic or hormonal therapy.

We sought to exploit the availability of a dynamic range of continuous mRNA data to look for an association of increasing hormone receptor gene transcription with enhanced benefit from anti-estrogen therapy but found none. It is unlikely that sample size precluded identification of a modest ''dose-response'' effect, in view of the absence of any trend for association. Presence of hormone receptor mRNA levels above a certain biological threshold may well be irrelevant, if low levels ensure presence of an amount of protein adequate for biologic effects [START_REF] Webb | Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high-and lowaffinity EGFR in A431 cells[END_REF]. Alternatively, increasing concentrations of mRNA may not linearly translate to protein synthesis, since it is modulated by several other regulatory mechanisms [START_REF] Yasuda | The RNA continent[END_REF]. Ultimately, ER and PgR may not be the sole or determining genes that define response or resistance to hormonal therapy.

We found that ER, PgR and MAP-Tau mRNA positive status correlated to low histological grade. In view of recent evidence suggesting that high-grade tumors are driven by a distinct genetic programme, rather than simply lacking some markers of differentiation, tumors with active ER, PgR or MAP-Tau gene function may represent a group with indolent characteristics in terms of proliferation, anaplasia, invasion, and cell cycle control [START_REF] Ramaswamy | Multiclass cancer diagnosis using tumour gene expression signatures[END_REF]. The association of tumoral MAP-Tau gene transcription with premenopausal patient status, a marker of more aggressive tumor biology, may be a random one in a relatively small sample or may suggest that a subgroup of premenopausal women harbour breast carcinomas of more benign biology. Alternatively, this could be a result of high plasma estrogen levels in premenopausal patients, with increased expression of MAP-Tau in ER positive tumors of comparatively benign nature, compared to the more aggressive ER negative, potentially BRCA1 or BRCA2-positive, premenopausal breast cancer tumors. In line with this, MAP-Tau gene transcription was strongly associated with ER and PgR gene transcription, an observation that confirms prior evidence that MAP-Tau is an ER-inducible gene. Tau contains an imperfect estrogen response element upstream of its promoter and is an estrogen-inducible protein in cultured neurons and in the MCF7, ZR75 and T47D breast cancer cell lines [START_REF] West | Predicting the clinical status of human breast cancer by using gene expression profiles[END_REF][START_REF] Matsuno | Modulation of protein kinases and microtubuleassociated proteins and changes in ultrastructure in female rat pituitary cells: effects of estrogen and bromocriptine[END_REF]. Interestingly, MAP-Tau is the target of multiple signalling pathways and thereby may represent an important checkpoint for microtubule and cellular functions beyond its interplay with hormonal activities. Microtubules are intrinsically dynamic polymers composed of ab-tubulin heterodimers and constitute essential components of the mitotic spindle and cytoskeleton. Microtubule dynamics control mitosis and cellular motility. Moreover, microtubules serve as scaffolds for signalling molecules, gene transcription and cell cycle activity [START_REF] Bhat | Microtubule-associated proteins as targets in cancer chemotherapy[END_REF][START_REF] Jordan | Microtubules as a target for anticancer drugs[END_REF]. Several proteins bind to microtubules and either stabilize or destabilize their polymerisation. Microtubule-associated protein Tau, one of the most extensively studied, is found in neurons, breast epithelium and breast cancer. Preclinical experiments have shown that the phosphorylated form of MAP-Tau binds to tubulin, resulting in promotion and stabilisation of microtubules. MAP-Tau can compete with paclitaxel for binding to the inner surface pocket of microtubules, a finding that suggests that microtubules assembled in the presence of Tau may be less susceptible to taxane binding and pharmacological hyperpolymerisation [START_REF] Kar | Repeat motifs of Tau bind to the insides of microtubules in the absence of taxol[END_REF]. Indeed, Rouzier et al. reported that MAP-Tau gene expression was inversely associated with pathologic complete response (pCR) of breast carcinomas to neoadjuvant paclitaxel-based chemotherapy. Among 82 patients treated with T-FAC induction regimen, pCR occurred in 44% of MAP-Tau mRNA-negative tumors, but only in 17% of MAP-Tau mRNA-positive ones (P = 0.04) [START_REF] Rouzier | Microtubule-associated protein tau: a marker of paclitaxel sensitivity in breast cancer[END_REF].

We report here for the first time in the literature that positive MAP-Tau mRNA expression strongly predicts decreased risk of relapse and death in patients with highrisk early breast cancer, suggesting that it carries either prognostic information for patient outcome or predictive information for benefit from antineoplastic and/or hormonal therapy. In fact, the observed weak predictive utility of ER mRNA expression was lost in multivariate analysis, suggesting that it may be exerted via induction of MAP-Tau gene transcription. The critical regulatory properties of MAP-Tau for microtubule dynamics make it a potential key protein in controlling mitosis, migration and cell cycle activity, possibly resulting in its strong predictive significance for benefit from antineoplastic and/or hormonal therapy.

In contrast to the findings reported by Rouzier et al., MAP-Tau mRNA status did not predict clinical benefit from taxanes in our cohort, as it had the same predictive utility in both E-CMF treated and E-T-CMF treated patients. Selection of a different cut-off was unlikely to change this finding and our exploratory analyses using different threshold values confirmed this. Andre et al. found high MAP-Tau gene transcriptional activity to be a bifunctional predictor of endocrine sensitivity and chemotherapy resistance [START_REF] Andre | Microtubule-associated protein tau is a bifunctional predictor of endocrine sensitivity and chemotherapy resistance in estrogenreceptor positive breast cancer[END_REF]. As the majority of the E-T-CMF patients in our cohort received endocrine therapy after chemotherapy, it is possible that any predictive significance of low MAP-Tau mRNA expression for taxane benefit was cancelled out by resistance to hormonal therapy, resulting in no detectable differences in outcome. Moreover, in the entire HE10/97 trial, the addition of taxanes reduced the hazard of death only in ER-negative patients [START_REF] Fountzilas | Postoperative dose-dense sequential chemotherapy with epirubicin, followed by CMF with or without paclitaxel, in patients with high-risk operable breast cancer: a randomised phase III study conducted by the Hellenic Cooperative Oncology Group[END_REF]. Given the correlation of ER and MAP-Tau gene activity, it is probable that ER negative patients who benefit from taxanes are mostly MAP-Tau negative. However, this effect may have been lost in our cohort, as the number of patients evaluated for MAP-Tau mRNA expression in the present study was reduced to less than half, due to limitations in tissue availability. Finally, it is always possible that MAP-Tau has no true predictive value for response to taxanes, or that other molecular characteristics abrogate it: tubulin mutations, variable expression of tubulin isoforms, expression of multidrug resistance proteins, or BCL2 may contribute to taxane resistance in tumors with low MAP-Tau expression. Indeed, Rody et al. performed gene expression profiling in breast carcinomas from 50 patients treated with taxane-based induction chemotherapy and failed to find any predictive significance of MAP-Tau gene expression for pCR [START_REF] Rody | Gene expression profiling of breast cancer patients treated with docetaxel, doxorubicin, and cyclophosphamide within the GEPARTRIO trial: HER2, but not topoisomerase II alpha and microtubule-associated protein tau, is highly predictive of tumour response[END_REF].

To conclude, we showed the feasibility of obtaining quantitative mRNA expression data of ER, PgR and MAP-Tau from widely available FFPE breast tumor tissue, though the choice of biologically relevant threshold values needs insights from the fields of molecular and systems biology. The use of a dynamic range of continuous hormone receptor mRNA data has yet no established value in quantifying the benefit from endocrine manipulations, as mRNA levels are modulated by several other parameters before exerting biological effects. MAP-Tau is an ERassociated gene that carries a strong predictive significance for patient outcome, though its effects on sensitivity to chemotherapy and hormonal therapy are still unclear. Study of larger and more homogeneous populations of breast cancer patients is warranted in order to unravel these issues and exploit their potential predictive utility for devising tailored therapeutic strategies.

Fig. 1

 1 Fig. 1 Distribution of ER (a), PgR (b) and MAP-Tau (c) mRNA levels in the studied population. Solid vertical lines on the graphs correspond to the chosen cut-offs (25th percentile for ER and PgR and the median for MAP-Tau) and dashed lines to the natural cut-offs as determined by the corresponding distributions

Fig. 2

 2 Fig. 2 MAP-Tau mRNA levels in ER mRNA positive and negative cases (Mann-Whitney test, P \ 0.001)

Fig. 3

 3 Fig. 3 Overall survival (OS) (a) (log rank P = 0.006) and DFS (b) (log rank P = 0.002) by MAP-Tau mRNA expression

Fig. 4

 4 Fig. 4 OS (left) and DFS (right) for E-T-CMF (red line) and E-CMF (blue line) in MAP-Tau negative and MAP-Tau positive patients

Fig. 5

 5 Fig. 5 OS (left) and DFS (right) for E-T-CMF (red line) and E-CMF (blue line) in MAP-Tau negative and MAP-Tau positive patients restricted to ER positive patients

Table 1

 1 Patient and tumor characteristics

	a Distribution of grade differs
	significantly (P \ 0.001). This
	result is identical to the
	corresponding result in the
	clinical paper

predictive value (74-82.5%) but modest specificity (45-53%) and negative predictive value (61-62.5%). Metrics of concordance between the two methods for ER and PgR determination are summarised in Table

3

.

Clinicopathological/molecular variables and patient outcome

Survival status of the 274 patients was updated in October 2007. At the time of analysis (February 2008), the median follow up time was 8 years. During this time, 90 patients (33%) had developed a relapse and 62 patients (23%) had died. Malignant relapses occurred in 43% and deaths in 31% of the ER-mRNA negative cases versus 30% relapsed and 21% dead patients with ER-mRNA positive tumors. Similarly, malignant relapses were seen in 41% and deaths in 29% of patients harbouring MAP-Tau negative tumors

Table 2

 2 Relationship between mRNA ER, PgR and MAP-Tau levels and standard clinical and pathological factors

		ER mRNA level (N = 263)	PgR mRNA level (N = 263)	MAP-Tau mRNA level (N = 271)
		Negative	Positive	P-value	Negative	Positive	P-value	Negative	Positive	P-value
	Age
	Median	50	52	0.31	51	51	0.90	52	50	0.27
	Range	27.5-74	22.5-76		22.5-74	27-76		22.5-74.5	27-76
		N (%)	N (%)	P-value	N (%)	N (%)	P-value	N (%)	N (%)	P-value
	Menopausal status
	Premenopausal	34 (52)	101 (51)	0.89	31 (47)	104 (53)	0.48	59 (43)	79 (58.5)	0.015
	Postmenopausal	31 (48)	97 (49)		35 (53)	93 (47)		77 (57)	56 (41.5)
	Treatment group
	E-T-CMF	32 (49)	89 (45)	0.57	26 (39)	95 (48)	0.25	59 (43)	59 (44)	1.0
	E-CMF	33 (51)	109 (55)		40 (61)	102 (52)		77 (57)	76 (56)
	Adjuvant HT
	No	10 (15)	10 (5)	0.012	8 (12)	12 (6)	0.11	20 (15)	3 (2)	\0.001
	Yes	55 (85)	188 (95)		58 (88)	185 (94)		116 (85)	132 (98)
	Adjuvant RT
	No	17 (27)	37 (19)	0.21	17 (27)	37 (19)	0.21	33 (25)	24 (18)	0.18
	Yes	46 (73)	160 (81)		47 (73)	159 (81)		100 (75)	111 (82)

Italic values denote relationships statistically significant at P \ 0.05

Table 3

 3

	ER	PgR	ER/PgR
	(%)	(%)	(%)

Agreement between kRT-PCR and IHC status of ER, PgR and ER/PgR a Proportion of agreement observed between IHC and kRT-PCR methods b Kappa coefficients are expressed as percentages NPV, negative predictive value; PPV, positive predictive value The third column ER/PgR represents hormone receptor status (any of the two receptors ER, PgR positive versus both ER and PgR negative)

Table 4

 4 Estimated hazard ratios (HRs) and 95% confidence intervals (CIs) for OS and DFS-multivariate analysis

		HR	95% CI	Wald P-value
	Overall survival
	Treatment group
	E-T-CMF	1
	E-CMF	1.09	0.65-1.82	0.74
	Positive nodes
	0-3	1
	C4	2.33	1.05-5.16	0.04
	MAP-Tau mRNA levels
	Negative	1
	Positive	0.46	0.25-0.85	0.01
	ER mRNA levels
	Negative	1
	Positive	0.67	0.33-1.36	0.26
	PgR mRNA levels
	Negative	1
	Positive	1.78	0.86-3.67	0.12
	Disease-free survival
	Treatment group
	E-T-CMF	1
	E-CMF	1.15	0.74-1.78	0.53
	Positive nodes
	0-3	1
	C4	2.30	1.21-4.38	0.01
	Tumor grade
	I-II	1
	III-Undifferentiated	1.56	0.98-2.50	0.06
	MAP-Tau mRNA levels
	Negative	1
	Positive	0.53	0.32-0.89	0.02
	ER mRNA levels
	Negative	1
	Positive	0.87	0.46-1.63	0.66
	PgR mRNA levels
	Negative	1
	Positive	1.33	0.72-2.46	0.36

Breast Cancer Res Treat (2009) 116:131-143

Breast Cancer Res Treat (2009) 116:131-143

Acknowledgements

The authors wish to thank all HeCOG study coordinators for data collection, Ms. E. Fragou and Ms. D. Katsala for study monitoring, Ms. Th. Spinari for tissue sample collection and Ms. M. Moschoni for data management. Supported by HeCOG research grant HE R 10/97.