Bianca Mostert
email: b.mostert@erasmusmc.nl

Jaco Kraan

Joan Bolt-De Vries

Petra Spoel

Anieta M Sieuwerts

Mieke Schutte

Annemieke M Timmermans

Renée Foekens

John W M Martens

Jan-Willem Gratama

Petra Van Der Spoel

John A Foekens

Stefan Sleijfer

Erasmus Mc

Detection of circulating tumor cells in breast cancer may improve through enrichment with anti-CD146

Keywords: Circulating tumor cells, breast cancer, CellSearch, CD146, minimal residual disease, EpCAM

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Introduction

The enumeration of circulating tumor cells (CTCs) in the peripheral blood of cancer patients is a promising tool to detect and monitor cancer earlier and less invasively than conventional methods [START_REF] Sleijfer | Circulating tumour cell detection on its way to routine diagnostic implementation?[END_REF][START_REF] Mostert | Circulating tumor cells (CTCs): detection methods and their clinical relevance in breast cancer[END_REF]. In recent years, several assays to detect CTCs have been introduced [START_REF] Mostert | Circulating tumor cells (CTCs): detection methods and their clinical relevance in breast cancer[END_REF]. CTCs measured with the FDA-approved CellSearch technique (Veridex™, Raritan, NJ) have prognostic value in metastatic breast cancer [START_REF] Cristofanilli | Circulating tumor cells, disease progression, and survival in metastatic breast cancer[END_REF][START_REF] Cristofanilli | Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer[END_REF] and changes in CTC counts during systemic therapy can serve as early marker for response [START_REF] Cristofanilli | Circulating tumor cells, disease progression, and survival in metastatic breast cancer[END_REF][START_REF] Cristofanilli | Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer[END_REF][START_REF] Smerage | The prognostic implications of circulating tumor cells in patients with breast cancer[END_REF][START_REF] Rack | Prognostic relevance of circulating tumor cells (CTCs) in peripheral blood of breast cancer patients before and after adjuvant chemotherapy: The German SUCCESS-Trial[END_REF].

Like most CTC detection assays in breast cancer, the CellSearch technique relies on EpCAM (epithelial cell adhesion molecule, CD326) expression on tumor cells. However, despite its robustness and proven clinical relevance, CellSearch identifies CTCs in only 60% of metastatic breast cancer patients [START_REF] Cristofanilli | Circulating tumor cells, disease progression, and survival in metastatic breast cancer[END_REF]. There are two possible explanations for this; CTCs are simply not present, or they cannot be detected by this assay.

In breast cancer, five different molecular subtypes have been identified [START_REF] Perou | Molecular portraits of human breast tumours[END_REF]. Recently, our group reported that in contrast to the other molecular subtypes, most normal-like breast cancer cell lines lack EpCAM expression and are missed using CellSearch technology [START_REF] Sieuwerts | Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells[END_REF]. Normal-like breast cancer accounted for 7.8% of all breast cancers in 344 breast cancer samples [START_REF] Smid | Subtypes of breast cancer show preferential site of relapse[END_REF] thus forming a substantial breast cancer subgroup. In accordance with our preclinical findings [START_REF] Sieuwerts | Response: Re: Anti-Epithelial Cell Adhesion Molecule Antibodies and the Detection of Circulating Normal-Like Breast Tumor Cells[END_REF], Spizzo et al., showed that 10.3% of 1715 invasive breast cancer patient samples lacked EpCAM expression [START_REF] Spizzo | High Ep-CAM expression is associated with poor prognosis in node-positive breast cancer[END_REF].

Using the lack of EpCAM expression on a subset of breast cancer cells as a model for EpCAMnegative CTCs, we embarked on a search for additional markers, ultimately aiming to improve CTC detection in all breast cancer patients, irrespective of the molecular subtype of their primary tumor. As a first attempt, CD146 was our marker of choice, based on our gene expression data [START_REF] Sieuwerts | Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells[END_REF], availability of anti-CD146 ferrofluids, and a recent study by Zabouo et al [START_REF] Zabouo | CD146 expression is associated with a poor prognosis in human breast tumors and with enhanced motility in breast cancer cell lines[END_REF], who showed that CD146 is expressed in a subset of primary breast cancers and is correlated with poor prognosis.

Methods

Breast cancer cell lines

In order to identify an additional marker expressed on EpCAM-negative breast cancer cells, we used our well-defined panel of 41 human breast cancer cell lines [START_REF] Elstrodt | BRCA1 mutation analysis of 41 human breast cancer cell lines reveals three new deleterious mutants[END_REF][START_REF] Hollestelle | Distinct gene mutation profiles among luminal-type and basal-type breast cancer cell lines[END_REF]. The intrinsic subtype of these cell lines has been determined by gene expression profiling previously [START_REF] Sieuwerts | Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells[END_REF].

CD146 mRNA expression levels

The transcript levels of the 41 cell lines were determined with Affymetrix GeneChip Exon 1.0 ST Arrays (Affymetrix UK Ltd., Wooburn Green, UK) as described previously [START_REF] Hollestelle | Distinct gene mutation profiles among luminal-type and basal-type breast cancer cell lines[END_REF].

Additionally, expression of CD146 was determined by real-time reverse transcriptase PCR. RNA was isolated from breast cancer cell lines with the RNeasy (Micro) kit (Qiagen, Valencia, CA). cDNA was prepared with the Superscript II RNase H-kit (Invitrogen, Carlsbad, CA). The resulting cDNA preparations were analyzed by real-time PCR in a 20 μL reaction volume in a MX3000P TM Real-Time PCR System (Agilent/Stratagene, Amsterdam, The Netherlands), using TaqMan™ Gene Expression Assays in combination with TaqMan Universal PCR Master Mix No AmpErase UNG (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Levels of hydroxymethylbilane (HMBS), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and glucuronidase, beta (GUSB) were used to control sample loading and RNA quality.

CD146 protein expression levels

Cells from cultured human breast cancer cell lines were incubated with fluorochromeconjugated monoclonal antibodies and presence of antigens was assessed as described before [START_REF] Sieuwerts | Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells[END_REF]. In brief, cells were incubated with CD34 conjugated with FITC (clone 8G12; BD Biosciences, San Jose, CA), CD146 conjugated with PE (clone P1H12; BD Biosciences) and anti-EpCAM conjugated with FITC (clone EBA-1; BD Biosciences). Cells were then analyzed on a FACSCanto flow cytometer (BD Biosciences). Unstained cells were used as a negative control. Tissue microarrays (TMAs) were prepared in duplicate from blocks of 37 formalin-fixed, paraffin-embedded (FFPE) breast cancer cell line cells that were cultured to near confluence in complete growth medium, and from blocks of 206 FFPE primary breast cancer specimens. These blocks and 23 FFPE normal-like primary breast cancer specimens were sectioned at 4 µm, mounted on StarFrost slides (Waldemar Knittel-GmbH, Braunschweig, Germany), dried, deparaffinized in xylene and rehydrated in graded solutions of ethanol and distilled water. Specimens were pretreated with retrieval buffer (DAKO, Glostrup, Denmark) at pH 6.0 for CD146 and pH 9.0 for CD34 for 40 min at 95-99°C in a water bath, cooled to room temperature for 20 min and rinsed in phosphate-buffered NaCL solution (PBS). This was followed by a 10-min blocking step with a 0.3% peroxide PBS solution and a 30-min blocking step with a 5% bovine serum albumin PBS solution. Slides were stained with monoclonal antibodies against CD146 (Abcam, Cambridge, UK, N1238) and CD34 (Neomarkers, Fremont, CA, clone QBE) and counterstained with the peroxidase-conjugated Envision technique (DAKO EnVision™+ System, HRP). Each core was scored by a well-trained technician and positive staining was recorded as 100%, 50-99%, 25-50%, 10-25%, 0-10% or 0% number of positive cells.

Enumeration of epithelial cells spiked in whole blood

Blood samples containing EDTA (7.5 mL aliquots of blood) from a single healthy male donor were obtained from CellSave Preservative Tubes (Veridex LLC). To each sample, a predefined amount (50-500) of cultured human breast cancer cells was added. To determine the actual viable cell number, a 100-μL aliquot of the cultured cells was incubated with 10 μL of 7AAD (Sigma-Aldrich, St. Louis, MO) and 100 μL of fluorescent beads (Beckman-Coulter, Miami, FL).

After 15 min incubation at room temperature, 2 mL PBS was added, and samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences). At least 10,000 beads were acquired to estimate the number of 7AAD-negative (viable) cells. The efficiency of retrieving the tumor cells was controlled by counting the number of viable cells that were drawn in triplicate by light microscopy after serial dilution.

To establish the number of recovered CTCs following spiking into blood from a healthy donor, samples were processed on the CellTracks AutoPrep analyzer (Veridex LLC) with the CellSearch Epithelial Cell kit (Veridex LLC). Briefly, ferrofluids coated with antibodies directed towards EpCAM were added to a blood sample, and CTCs bound to anti-EpCAM coupled ferrofluid were isolated from whole blood by magnets. Unbound cells and the remaining plasma were aspirated, followed by staining of the isolated cells with the nuclear dye 4',6-diamidino-2phenylindole (DAPI), anti-cytokeratin 8, 18 and 19 antibodies labeled with PE and CD45 antibodies labeled with APC. After incubation, CTCs were separated magnetically once more and unbound staining reagents removed. Finally, the cells were resuspended in a MagNest Cell Presentation Device (Veridex LLC). In this device, labeled cells are oriented by two magnets for analysis in the CellSpotter Analyzer (Veridex LLC) after which the number of CTCs are enumerated according to the manufacturer's instructions. as provided in the CellSearch Epithelial Cell kit. As CD146 enriches for circulating endothelial cells (CECs) [START_REF] Strijbos | Circulating endothelial cells in oncology: pitfalls and promises[END_REF], and CECs can express cytokeratin 18 [START_REF] Brakenhoff | Sensitive detection of squamous cells in bone marrow and blood of head and neck cancer patients by E48 reverse transcriptase-polymerase chain reaction[END_REF], a marker to exclude the cytokeratin 18-expressing subset of CECs was needed. As CD34 is a pan-endothelial marker [START_REF] Fina | Expression of the CD34 gene in vascular endothelial cells[END_REF], CD34 conjugated with FITC (clone 8G12; BD Biosciences) was added to the CellSearch Epithelial Cell kit, in a volume of 150 µL per sample plus an excess volume of 300 µL. The number of CTCs was determined on the CellSpotter Analyzer (Veridex LLC) according to the manufacturer's instructions. CD146+ CTCs were defined as DAPI+/CK+/CD45-/CD34-.

For combined anti-EpCAM and anti-CD146 enrichment, anti-EpCAM ferrofluids from the CellSearch Circulating Epithelial Cell kit and anti-CD146 ferrofluids from the CellSearch Circulating Endothelial Cell kit were combined in even parts to a total volume of 150 µL per sample plus an excess volume of 300 µL. The number of CTCs in samples enriched for CD146expressing and EpCAM-expressing events was determined and defined as mentioned above.

The pivotal differences between the classic CellSearch CTC enumeration assay and the new combined anti-EpCAM/anti-CD146 assay are highlighted in figure 1.

Blood samples

Blood samples were obtained from a laboratory volunteer and patients with metastatic breast cancer seen at the outpatient clinic. For each patient, 22.5 mL of blood was drawn into CellSave tubes and CTCs were enumerated after enrichment using anti-EpCAM, anti-CD146 and mixed anti-EpCAM/anti-CD146 ferrofluids, using 7.5 mL of blood for each enrichment method. Additionally, 10 healthy female donors (age 23-56) were tested for CD146+ events by enumerating CTCs in 7.5 mL blood after enrichment with anti-CD146 ferrofluids.

This study was approved by the Erasmus MC Institutional Review Board (METC protocol 2006-248), and all donors and patients gave their written informed consent.

Results

Breast cancer cell lines

As previously reported, our cell line panel consists of 10 normal-like, 5 basal-like, 5 erbb2 and 21 luminal breast cancers as determined by gene expression profiling according to the intrinsic subtypes of Perou and Sorlie [START_REF] Sieuwerts | Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells[END_REF][START_REF] Hollestelle | Distinct gene mutation profiles among luminal-type and basal-type breast cancer cell lines[END_REF]. As the MDA-MB-435 normal-like cell line has been the subject of debate in literature recently with doubts about its origin [START_REF] Ross | Systematic variation in gene expression patterns in human cancer cell lines[END_REF][START_REF] Rae | MDA-MB-435 cells are derived from M14 melanoma cells--a loss for breast cancer, but a boon for melanoma research[END_REF][START_REF] Chambers | MDA-MB-435 and M14 cell lines: identical but not M14 melanoma?[END_REF], we excluded this cell line from our experiments.

Candidate markers

We have previously shown that 8 out of these 9 cell lines lack EpCAM-expression [START_REF] Sieuwerts | Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells[END_REF].

Using these normal-like cell lines as a model for non-EpCAM expressing breast cancer, several candidate markers for the assessment of EpCAM-negative CTCs were identified by comparing gene expression data between molecular subtypes. Candidate markers required membrane expression of the proteins on the EpCAM-negative breast cancer cells and absence on hematopoietic cells, or, if present, hematopoietic cells expressing the marker should be easily excluded using additional markers. Several markers fulfilled these criteria, amongst which CAV1 [START_REF] Sieuwerts | Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells[END_REF], MUC1 [START_REF] Sieuwerts | Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells[END_REF] and MCAM (CD146). Given the availability of ferrofluids coated with CD146 antibodies [START_REF] Rowand | Endothelial cells in peripheral blood of healthy subjects and patients with metastatic carcinomas[END_REF], and the possibility to exclude interference of expression by hematopoietic cells (by CD45-counterstaining) and CECs (by CD34-counterstaining), we chose to further explore this marker.

CD146 and EpCAM expression

Based on Affymetrix micro-array and qRT-PCR data, 7 out of the 9 normal-like cell lines expressed high levels of CD146 mRNA. In addition, one basal-like cell line, SUM149PT, expressed CD146, resulting in a total of 8 cell lines expressing CD146 mRNA (Table 1).

As previously reported [START_REF] Sieuwerts | Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells[END_REF], we confirmed that cells from only 1 of the 9 normal-like cell lines (MDA-MB-157) expressed EpCAM (Table 1 and Figure 2a). Evaluation of CD146 expression by flow cytometry showed that 8 of the 9 normal-like cell lines had CD146 membrane expression at a level likely to be detected using CellSearch technology (i.e., a signal to noise ratio >5) (Table 1 and Figure 2a). Remarkably, cells from an early passage of the MDA-MB-157 cell line were CD146-positive, while cells from a later passage showed a CD146-positive and a CD146-negative population. In addition to these normal-like cell lines, two of 5 basal-like cell lines (SUM149 and SUM229) expressed both CD146 and EpCAM (Table 1 and Figure 2b).

To obtain an overview of CD146 expression across all different breast cancer subtypes, CD146 expression was determined by immunohistochemistry on a TMA of 37 breast cancer cell lines. A total of 17 cell lines stained positive for CD146, among which 8 were normal-likes, 2 basal-like, 4 luminal and 3 erbb2 cell lines. Most cell lines staining positive for CD146 showed a heterogeneous expression pattern (Figure 3a).

CD146 staining was also performed in a set of 23 normal-like primary breast tumors, 7 of which stained positive for CD146 on epithelial cells (Figure 3b).

CD34 to distinguish CTCs from CECs

The expression of CD146 on activated T-lymphocytes [START_REF] Pickl | MUC18/MCAM (CD146), an activation antigen of human T lymphocytes[END_REF] and CECs [START_REF] Smirnov | Global gene expression profiling of circulating endothelial cells in patients with metastatic carcinomas[END_REF] might interfere with the detection of CD146-positive CTCs. While leukocytes can be excluded with CD45, which is already incorporated in the standard CellSearch Circulating Epithelial Cell kit, we assessed whether CECs could be excluded with CD34, a pan-endothelial marker [START_REF] Strijbos | Circulating endothelial cells in oncology: pitfalls and promises[END_REF][START_REF] Fina | Expression of the CD34 gene in vascular endothelial cells[END_REF]. CD34 proved to be a suitable marker to distinguish CTCs from CECs, as none of the normal-like cell lines expressed CD34 as determined by flow cytometry (Table 1). In addition, CD34 staining was completely negative on a TMA containing 37 breast cancer cell lines (data not shown). CD34 staining on 206 primary breast tumors showed less than 10% CD34-positivity in only one tumor specimen, all other tumors were entirely CD34 negative.

EpCAM-negative breast cancer cells detected with anti-CD146 ferrofluids

To test whether EpCAM-negative breast cancer cells could be detected in healthy donor blood using CellSearch with anti-CD146 ferrofluids, a fixed number of cells from normal-like and basal-like cell lines was spiked into 7.5 mL healthy donor blood of the same donor (Figure 4b). As a control, a fixed number of luminal cells (CAMA-1) was spiked into 7.5 mL healthy donor blood (Figure 4b, bottom bar).

As previously reported [START_REF] Sieuwerts | Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells[END_REF], cells from 7 out of 9 normal-like cell lines could not be recovered with anti-EpCAM ferrofluids while cells from 1 of the other 2 normal-like cell lines (MDA-MB-231, Figure 4b) were partially detected (11%). Cells from the remaining cell line, MDA-MB-157, could be adequately recovered with anti-EpCAM ferrofluids, consistent with its EpCAMexpression. Of the 8 cell lines insufficiently recovered with anti-EpCAM, 5 could be detected with anti-CD146 (45-88% recovery). Additionally, the EpCAM-expressing cell line MDA-MB-157 was partially detected by anti-CD146 (11% recovery). After enrichment with mixed anti-EpCAM/anti-CD146 ferrofluids, cells from 6/9 normal-like cell lines were recovered at rates of 46 to 100%. Despite CD146 expression, cells from 3 normal-like cell lines could not be recovered (Figure 4b). Cells of the luminal cell line CAMA-1 were detected with a recovery approaching 100% using anti-EpCAM ferrofluids. Enrichment with anti-CD146 ferrofluids alone did not result in recovery of CAMA-1 cells. The combined use of anti-CD146 and anti-EpCAM ferrofluids still enabled detection of all spiked CAMA-1 cells (Figure 4b), suggesting that mixing both ferrofluids does not greatly compromise enrichment. In accordance, 2 basal-like cell lines expressing EpCAM and CD146 could be detected with both anti-EpCAM and anti-CD146 ferrofluids as well as with a mixture of both (Figure 4b).

CD146+ cells in healthy donors

In all blood samples from 10 healthy female donors and a healthy male donor enriched with anti-CD146 ferrofluids, either in mixture or alone, a small number of CD146+/CK+/DAPI+/CD45-/CD34+ cells was identified (median 1, range 0-7), most likely accounting for a subset of CECs from the healthy donors [START_REF]SAGE anatomic viewer: KRT18 expression in vascular tissue[END_REF]. In none of the healthy donors, CD146-expressing tumor cells, i.e., CD146+/CK+/DAPI+/CD34-cells, could be identified (data not shown).

CD146+ CTCs in breast cancer patients

To assess whether CD146-expressing CTCs do occur in breast cancer, CTCs were enumerated in 20 advanced breast cancer patients using anti-EpCAM, anti-CD146 and mixed ferrofluids. Of these 20 patients, 9 had CD146+ CTCs (Table 2). As expected, given the heterogeneity of breast cancer and the fact that some breast cancer cell lines express both EpCAM and CD146, 8 of these 9 CD146+ patients also had EpCAM+ CTCs. Enrichment with mixed ferrofluids yielded a higher CTC number compared to EpCAM enrichment alone in 8 patients (Table 2, patients 1, 2, 7, 9, 12. 13, 14 and 20). In six patients (Table 2, patients 4, 5, 15, 17, 18 and 19), the combined enrichment for CD146 and EpCAM resulted in a lower recovery of CTCs. In this small series of patients, CD146+ CTCs were present in patients with both hormone receptor positive and hormone receptor negative primary tumors.

Discussion

CTC detection with the CellSearch assay has proven its value as a prognostic marker in metastatic breast cancer [START_REF] Cristofanilli | Circulating tumor cells, disease progression, and survival in metastatic breast cancer[END_REF][START_REF] Cristofanilli | Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer[END_REF]. Additionally, a change in CTC numbers at first follow-up of therapy is associated with outcome [START_REF] Rack | Prognostic relevance of circulating tumor cells (CTCs) in peripheral blood of breast cancer patients before and after adjuvant chemotherapy: The German SUCCESS-Trial[END_REF][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][START_REF] Budd | Circulating tumor cells versus imaging--predicting overall survival in metastatic breast cancer[END_REF][START_REF] Pierga | Circulating tumor cell detection predicts early metastatic relapse after neoadjuvant chemotherapy in large operable and locally advanced breast cancer in a phase II randomized trial[END_REF]. Nevertheless, CTCs can be detected in only 60% of the metastatic breast cancer patients, suggesting that there is room for improvement in the CellSearch assay. As enrichment for EpCAM is the limiting first step in this and many other CTC enumeration assays, EpCAM expression on tumor cells is pivotal. However, evidence is accumulating that EpCAM is not a perfect marker for breast cancer CTCs [START_REF] Sieuwerts | Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells[END_REF][START_REF] Smid | Subtypes of breast cancer show preferential site of relapse[END_REF][START_REF] Sieuwerts | Response: Re: Anti-Epithelial Cell Adhesion Molecule Antibodies and the Detection of Circulating Normal-Like Breast Tumor Cells[END_REF].

In this study we demonstrated that CD146 is frequently present on EpCAM-negative breast cancer cell lines. CD146 is ubiquitously expressed on endothelial cells [START_REF] Bardin | S-Endo 1, a pan-endothelial monoclonal antibody recognizing a novel human endothelial antigen[END_REF] and melanoma cells [START_REF] Lehmann | MUC18, a marker of tumor progression in human melanoma, shows sequence similarity to the neural cell adhesion molecules of the immunoglobulin superfamily[END_REF]. A correlation with poor prognosis has recently been found in breast cancer; of 635 primary breast tumors, 7% were CD146-positive, and CD146 expression was associated with poor prognosis [START_REF] Zabouo | CD146 expression is associated with a poor prognosis in human breast tumors and with enhanced motility in breast cancer cell lines[END_REF]. Our finding that CD146 is present on EpCAM-negative breast cancer cell lines renders it a putative marker to detect EpCAM-negative CTCs. Expression of CD146 by other cells in blood such as CECs could hinder detection of CTCs. We found that the exclusion of CK+/DAPI+/CD45-/CD34+ events after anti-CD146 enrichment resulted in CTC-negative test results in healthy donors. Hematopoietic cells expressing CD146, such as activated T-lymphocytes [START_REF] Elshal | A unique population of effector memory lymphocytes identified by CD146 having a distinct immunophenotypic and genomic profile[END_REF] and NK-cells [START_REF] Despoix | Mouse CD146/MCAM is a marker of natural killer cell maturation[END_REF], can be identified according to their bright expression of CD45.

Using the definition of CD146+/CK+/DAPI+/CD45-/CD34-for CD146-positive CTCs we were able to detect cells from five out of eight normal-like cell lines when spiked in healthy blood.

For the remaining three cell lines, for which detection is limited despite CD146 expression, the assay may need further improvement, for instance by adding an additional enrichment or positive selection marker, such as different cytokeratins.

In the clinical setting, both EpCAM-positive and EpCAM-negative tumor cells are likely to be present in the blood of patients, given the heterogeneity in terms of EpCAM expression within primary tumors [START_REF] Sieuwerts | Response: Re: Anti-Epithelial Cell Adhesion Molecule Antibodies and the Detection of Circulating Normal-Like Breast Tumor Cells[END_REF]. As a consequence, one can imagine EpCAM-negative CTCs in patients with a primary tumor that is predominantly comprised of EpCAM-positive cells and vice versa.

Although we mostly observed CD146 expression in breast cancer cell lines with a normal-like subtype, the added value of CD146 is probably not limited to patients with normal-like primary tumors. CD146+ CTCs can be present in other patients too, as shown by the detection of CD146+ CTCs in patients with hormone receptor positive primary tumors, due to tumor heterogeneity or clonal selection during progression. Improved CTC detection in breast cancer using combined anti-CD146 and anti-EpCAM enrichment may be of value irrespective of the molecular subtype of the primary tumor.

To determine whether CD146-expressing CTCs are present in breast cancer patients, CTCs were captured with anti-EpCAM, anti-CD146 alone and a mixture of ferrofluids in twenty patients with advanced disease. Besides EpCAM-positive CTCs, CD146-positive CTCs were detected in 9 patients. In contrast, no CD146-expressing CTCs could be detected in healthy female controls. Although in a small series of patients, in line with the assumption that the combination of anti-EpCAM and anti-CD146 enrichment might improve CTC detection, a higher CTC count was found in 8 out of 20 patients when combining anti-EpCAM and anti-CD146 ferrofluids. However, a lower recovery was found in 6 patients using this approach. Whether this is due to technical reasons, such as suboptimal concentrations of the ferrofluids, should be evaluated before implementation into clinical studies. Importantly, the application of CD146 as an additional marker to detect CTCs is not restricted to the CellSearch assay alone, but may be useful for all assays depending on EpCAM-expression on the target cells.

Although based on a relatively small series of patients, the EpCAM-positive CTCs seem to outnumber the CD146-expressing CTCs. Nonetheless, even if CD146+ CTCs occur in only a small subset of patients, and may be of limited clinical relevance, minimizing false negative results is crucial for diagnostic purposes. Studies are being initiated to determine the exact incidence of CD146-positive CTCs in breast cancer patients and to reveal their clinical relevance.

In conclusion, we have shown that the majority of EpCAM-negative breast cancer cell lines can be detected using anti-CD146 ferrofluids. Furthermore, CD146-positive CTCs are present in breast cancer patients. It must be stressed that the clinical relevance of CD146positive CTCs remains to be established, and whether the prognostic value of CD146+ CTCs is equivalent to that of EpCAM+ CTCs will be determined in a large clinical study. Nevertheless, the detection of CTCs, although already well established in metastatic breast cancer, can become even more relevant when detecting EpCAM-positive as well as CD146-positive CTCs.

For

  the detection of cells expressing CD146, anti-CD146 ferrofluid from the CellSearch Circulating Endothelial Cell kit (Veridex LLC) was used in a volume of 150 µL per sample plus an excess volume of 300 µL. The sample was enriched for CD146-positive cells and further characterized by staining for the presence or absence of DAPI, cytokeratin 8, 18 or 19 and CD45

Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 1 7

 12341 Fig. 1 Setup of the CellSearch CTC enumeration assay (left column) versus the new anti-EpCAM/anti-CD146 assay (right column)Description of the makers used in each assay for enrichment, positive selection and negative selection, and the cell type associated with these selection criteria. CK; cytokeratin

Table 1

 1 CD146 mRNA and CD146, CD34 and EpCAM protein expression in normal-like and basal-like breast cancer cell lines.

		EpCAM		CD146			CD34
		membrane	mRNA	mRNA	membrane	membrane	membrane
		expression	expression	expression	expression	expression	expression
		FACS s/n a	Affymetrix b	qRT-PCR c	FACS s/n a	IHC	FACS s/n a
	Cell line						(%positivity)
	SUM102PT (n)	<5	569	0.6285	5-20		ND	<5
	SK-BR-7 (n)	<5	708	0.2736	20-200	100	<5
	MDA-MB-157 (n)	20-200	420	0.2300	5-20	d	100	<5
	Hs578T (n)	<5	235	0.1416	5-20		100	<5
	SUM159PT (n)	<5	281	0.0526	20-200	100	<5
	MDA-MB-231 (n)	<5	196	0.0238	20-200	100	<5
	SUM1315MO2 (n)	<5	44	0.0315	20-200	<10	<5
	MDA-MB-436 (n)	<5	65	0.0039	5-20		<10	<5
	BT549 (n)	<5	59	0.0027	5-20		25-50	<5
	SUM149PT (b)	5-20	301	0.0292	20-200	>50	<5
	MDA-MB-468 (b)	20-200	60	0.0005	<5		<10	<5
	BT20 (b)	20-200	53	0.0008	<5		0	<5
	SUM229PE (b)	20-200	49	0.0063	20-200	0	<5
	HCC1937 (b)	20-200	31	0.0010	<5		0	<5
	a Data are values of one representative experiment performed in duplicate, s/n; signal-to-noise
	ratio, s/n > 5 considered detectable expression,	b values >100 considered detectable expression;
	c qRT-PCR; quantitative real-time PCR, values >0.001, i.e., requiring ≥10 cycles to pass the
	threshold compared with the number of cycles required for the reference gene set, considered
	detectable expression, IHC; immunohistochemistry, d late passage of cell line, ND; not
	determined, (n); normal-like subtype, (b); basal-like subtype

Table 2

 2 CTC counts with anti-EpCAM, anti-CD146 and mixed anti-EpCAM/CD146 ferrofluids for 20 advanced breast cancer patients, and their primary tumor characteristics

	Patient number	Primary tumor characteristics			CTC count
		ER a	PR b	Her2	EpCAM+	CD146+	EpCAM/CD146+
	1	+	+	-	15	1	17
	2	+	-	+	19	1	25
	3	+	-	-	0	4	1
	4	+	+	-	38	5	20
	5	+	+	-	47	2	30
	6	+	+	NA	2	1	1
	7	-	+	-	28	5	40
	8	+	NA	+	0	0	0
	9	+	-	-	54	0	75
	10	+	+	-	0	0	0
	11	+	+	-	0	0	0
	12	+	+	-	0	0	1
	13	+	+	-	0	0	1
	14	+	+	-	1	0	2
	15	+	+	NA	2	0	1
	16	-	-	NA	0	0	0
	17	+	+	-	19	0	11
	18	+	+	-	155	24	148
	19	+	+	-	107	3	94
	20	+	+	-	0	0	1
	a ER; estrogen receptor,

b PR; progesterone receptor, NA; not available

Table 1

 1 CD146 mRNA and CD146, CD34 and EpCAM protein expression in normal-like and basal-like breast cancer cell lines.

		EpCAM		CD146		CD34
		membrane	mRNA	mRNA	membrane	membrane	membrane
		expression	expression	expression	expression	expression	expression
		FACS s/n a	Affymetrix b	qRT-PCR c	FACS s/n a	IHC	FACS s/n a
	Cell line					(%positivity)
	SUM102PT (n)	<5	569	0.6285	5-20	ND	<5
	SK-BR-7 (n)	<5	708	0.2736	20-200	100	<5
	MDA-MB-157 (n)	20-200	420	0.2300	5-20 d	100	<5
	Hs578T (n)	<5	235	0.1416	5-20	100	<5
	SUM159PT (n)	<5	281	0.0526	20-200	100	<5
	MDA-MB-231 (n)	<5	196	0.0238	20-200	100	<5

Acknowledgments

The authors wish to thank Antoinette Hollestelle and Anita Trapman for their precious help in providing the immunohistochemistry data.

This work was in part financially supported by the Netherlands Genomic Initiative (NGI)/Netherlands Organization for Scientific Research (NWO).


F Germán Rodríguez-González

Anieta M Sieuwerts

Marcel Smid

Maxime P Look

Marion E Meijer-Van Gelder

Vanja Weerd

Stefan Sleijfer

John W M Martens

John A Foekens

Vanja De Weerd

Fco Germán

PhD. Rodríguez González
email: f.rodriguezgonzalez@erasmusmc.nl

MicroRNA-30c expression level is an independent predictor of clinical benefit of endocrine therapy in advanced estrogen receptor positive breast cancer

Keywords:

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

Introduction

Current effective endocrine therapies include treatment with the ER-α antagonist tamoxifen or, more recently, aromatase inhibitors (AIs), that compromise the synthesis of estrogens. Despite their benefit in patients in ER-α positive breast cancer [START_REF] Jaiyesimi | Use of tamoxifen for breast cancer: twenty-eight years later[END_REF] a considerable number of patients with ER-positive breast cancers does not benefit from endocrine therapy due to de novo or acquired resistance [START_REF] Lewis | Selective estrogen receptor modulators (SERMs): mechanisms of anticarcinogenesis and drug resistance[END_REF]. Altered existing [START_REF] Girault | Role of estrogen receptor alpha transcriptional coregulators in tamoxifen resistance in breast cancer[END_REF][START_REF] Murphy | Relationship of coregulator and oestrogen receptor isoform expression to de novo tamoxifen resistance in human breast cancer[END_REF][START_REF] Murphy | Potential role of estrogen receptor alpha (ERalpha) phosphorylated at Serine118 in human breast cancer in vivo[END_REF][START_REF] Weis | Constitutively active human estrogen receptors containing amino acid substitutions for tyrosine 537 in the receptor protein[END_REF][START_REF] Yudt | Function of estrogen receptor tyrosine 537 in hormone binding, DNA binding, and transactivation[END_REF] or activation of novel signaling pathways [START_REF] Campbell | Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance[END_REF][START_REF] Gee | Epidermal growth factor receptor/HER2/insulin-like growth factor receptor signalling and oestrogen receptor activity in clinical breast cancer[END_REF] are considered to underlie resistant tumors and are thus major obstacles for endocrine therapy to be fully effective [START_REF] Chen | What do we know about the mechanisms of aromatase inhibitor resistance?[END_REF].

MiRNAs are non-coding, single-stranded RNAs, which in their mature form constitute a novel class of gene regulators [START_REF] Bartel | MicroRNAs: genomics, biogenesis, mechanism, and function[END_REF]. Due to partial sequence homology to the 3´ untranslated region (3´ UTR) of target mRNAs, single-stranded mature miRNAs bind to their targets thereby blocking translation or causing mRNA degradation [START_REF] Carthew | Origins and Mechanisms of miRNAs and siRNAs[END_REF].

Altered miRNA expression levels have been described in breast cancer [START_REF] Cummins | The colorectal microRNAome[END_REF][START_REF] Iorio | MicroRNA gene expression deregulation in human breast cancer[END_REF][START_REF] Scott | Coordinate suppression of ERBB2 and ERBB3 by enforced expression of micro-RNA miR-125a or miR-125b[END_REF], and specific miRNAs have been associated with the occurrence of metastasis and poor prognosis [START_REF] Adams | Involvement of microRNAs in breast cancer[END_REF][START_REF] Foekens | Four miRNAs associated with aggressiveness of lymph node-negative, estrogen receptor-positive human breast cancer[END_REF][START_REF] Huang | Expression of microRNA-21 in invasive ductal carcinoma of the breast and its association with phosphatase and tensin homolog deleted from chromosome expression and clinicopathologic features[END_REF][START_REF] Ma | Tumour invasion and metastasis initiated by microRNA-10b in breast cancer[END_REF][START_REF] Tavazoie | Endogenous human microRNAs that suppress breast cancer metastasis[END_REF]. In cultured breast cancer cell lines hsa-miRNA-221 and -222 expression can induce tamoxifen resistance due to negative regulation of ER-α or direct targeting of p27Kip1 [START_REF] Miller | MicroRNA-221/222 confers tamoxifen resistance in breast cancer by targeting p27Kip1[END_REF][START_REF] Zhao | MicroRNA-221/222 negatively regulates estrogen receptor alpha and is associated with tamoxifen resistance in breast cancer[END_REF]. These latter studies suggest a role for miRNAs in endocrine therapy resistance in breast cancer. In this work, we have studied in ERpositive beast cancer the association of selected miRNAs with clinical benefit of tamoxifen therapy for advanced disease.

Materials and Methods

Patients

The Medical Ethical Committee of the Erasmus Medical Center Rotterdam, the Netherlands, approved our study design (MEC 02.953). This retrospective study used 246 coded, ER protein-positive (≥ 10 fmol/mg protein) primary tumor tissues from patients diagnosed between 1981 and 1995. This study was conducted in accordance with the Code of Conduct of the Federation of Medical Scientific Societies in the Netherlands (http:/www.fmvv.nl) and where possible adheres to the REMARK criteria for reporting on prognostic/predictive biomarkers [START_REF] Mcshane | REporting recommendations for tumor MARKer prognostic studies (REMARK)[END_REF]. Patients were treated either with breast-conserving surgery (34%) or with modified mastectomy (66%). An axillary dissection was performed in 93% of the patients (n=228). None of the patients received neoadjuvant therapy or hormonal adjuvant treatment (hormonenaïve). Twenty-eight patients received adjuvant CMF (cyclophosphamide, methotrexate, 5-fluorouracil) and 18 patients anthracycline-containing chemotherapy. Relevant clinicopathologic characteristics of the patients and their primary tumor are given in Table 1. Thirty patients presented with distant metastasis at diagnosis or developed distant metastasis within 1 month after primary surgery (M1-patients). These 30 patients and the 216 M0-patients who developed a recurrence during follow-up (32 patients with localregional relapse; 184 patients with distant metastasis) were treated with tamoxifen (40 mg daily) as first line therapy for advanced disease. Of the 216 M0-patients, the median time between primary surgery and start of therapy was 29 months (range, 2-134 months). At the time of surgical removal of the primary tumor the median age of the patients was 60 years (range, 26-89 years) and at start of tamoxifen therapy for advanced disease 62 years (range, 29-90 years). Response to tamoxifen therapy was defined by standard Union International Contre le Cancer (UICC) criteria [START_REF] Hayward | Assessment of response to therapy in advanced breast cancer: a project of the Programme on Clinical Oncology of the International Union Against Cancer[END_REF]. Objective response was observed in 45 patients (12 complete remissions, CR; and 33 partial remissions, PR). Seventy-eight patients experienced an increase in tumor size of 25% or more, or showed new tumor lesions within 3 months (progressive disease, PD). The remaining 123 patients with no objective response or PD were considered as patients with no change (NC). These patients with NC were categorized into 112 patients who had NC > 6 months (defined as stable disease, SD) and 11 patients with NC ≤ 6 months. The time that 50% of the patients survived without progression of the disease was for CR: 41 months, PR: 14 months, SD: 14 months, NC ≤ 6 months: 6 months, and for PD: 3 months. Hundred and fifty-seven patients (64%) showed clinical benefit (CR+PR+SD) of tamoxifen therapy. The median follow-up time of patients alive after surgery was 86 months (range, 10-165 months), and 25 months (range, 1-95 months) after start of tamoxifen therapy. At the end of the follow-up period, 229 patients had developed tumor progression and 181 patients had died.

Tissue processing, RNA isolation, multiplex cDNA synthesis and miRNA quantification

Tissue inclusion, processing, sectioning and total RNA extraction was done as before [START_REF] Sieuwerts | Which cyclin E prevails as prognostic marker for breast cancer? Results from a retrospective study involving 635 lymph node-negative breast cancer patients[END_REF][START_REF] Sieuwerts | How ADAM-9 and ADAM-11 differentially from estrogen receptor predict response to tamoxifen treatment in patients with recurrent breast cancer: a retrospective study[END_REF].

Only ER-positive cases (≥10 fmol/mg cytosolic protein or more) determined as described [START_REF]Revision of the standards for the assessment of hormone receptors in human breast cancer[END_REF][START_REF] Foekens | Prognostic value of estrogen and progesterone receptors measured by enzyme immunoassays in human breast tumor cytosols[END_REF] were included. As described in detail [START_REF] Foekens | Four miRNAs associated with aggressiveness of lymph node-negative, estrogen receptor-positive human breast cancer[END_REF], 50 ng of total RNA was reverse-transcribed using up to 30 different reverse transcription (RT) primers (10 nM each) in 12 μl using the TaqMan MicroRNA Reverse Transcription Kit (ABI) according to the manufacturer"s instructions [START_REF] Foekens | Four miRNAs associated with aggressiveness of lymph node-negative, estrogen receptor-positive human breast cancer[END_REF]. Using cDNA synthesized from 0.5 ng of total RNA, individual miRNAs were separately quantified in duplicate essentially as described [START_REF] Foekens | Four miRNAs associated with aggressiveness of lymph node-negative, estrogen receptor-positive human breast cancer[END_REF] by real-time PCR using individual TaqMan human MicroRNA Assay kits (Table 2). A standard curve prepared from a pool of RNA obtained from a randomly selected set of 33 human breast tissues included in this study was added in each cDNA synthesis and PCR run, and the resulting data were used to normalize for in-between experimental variations. In all cDNA synthesis runs a minus RT reaction was always incorporated. PCR efficiency, linearity, and the upper and lower detection limits of each individual miRNA assay was validated [START_REF] Foekens | Four miRNAs associated with aggressiveness of lymph node-negative, estrogen receptor-positive human breast cancer[END_REF]. To control for RNA input and integrity, two miRNAs, hsa-miR-132 and hsa-miR-374, were used as reference to normalize and quantify miRNA expression levels as previously reported [START_REF] Foekens | Four miRNAs associated with aggressiveness of lymph node-negative, estrogen receptor-positive human breast cancer[END_REF].

Statistics

For statistical computations, STATA statistical package 10.1 (STATA Corp., College Station, TX) was used. Differences in miRNA levels in subgroups of patients were assessed with the Mann-Whitney U test or Kruskal-Wallis test, including a Wilcoxon-type test for trend. In these tests, patient and tumor characteristics were used as grouping variables. The strengths of the associations between continuous variables were tested with the Spearman rank correlation (r s ). To reduce the skewness most variables were log or Box-Cox transformed. Logistic regression analysis was used to examine the relation of miRNA levels with clinical benefit of tamoxifen therapy and for the calculation of the odds ratio (OR) and its 95% confidence interval (CI). The Cox proportional hazard model was used to calculate the hazard ratio (HR) and its 95% CI in the analyses of PFS. The starting point of PFS being defined as the start of treatment with tamoxifen for recurrence and the endpoint as the first detection of progression of the disease or death. Survival curves were generated using the method of Kaplan and Meier [START_REF] Kaplan | Non-parametric estimation from incomplete observations[END_REF] and the log rank test was used to test for differences. A two-sided P-value < 0.05 was considered statistically significant.

Pathways and global testing

Our Affymetrix microarray gene expression data (entries GSE2034 and GSE5327) were combined with gene expression data from HG-U133-plus2 chips. Data were preprocessed and normalization was performed as previously described [START_REF] Wang | Geneexpression profiles to predict distant metastasis of lymph-node-negative primary breast cancer[END_REF]. Next, separately for each chip-type, data were normalized before combining the data-sets. Absence of a bias based on the chip-type (the 133A-data and plus 2 data) was checked as described [START_REF] Yu | Pathway analysis of gene signatures predicting metastasis of node-negative primary breast cancer[END_REF]. For 71 of the 246 patients included in this study, we have both miRNA and mRNA expression data available for pathway analysis. The Global Test [START_REF] Goeman | A global test for groups of genes: testing association with a clinical outcome[END_REF] was used (version 4.4.0) to associate Biocarta pathways (http://www.biocarta.com/) to samples expressing high or low amounts of a particular miRNA. For "high" or "low" assignment of samples, the 20 samples with the highest and the 20 samples with the lowest expression of a particular miRNA were selected. A P-value was considered significant when the false discovery rate (FDR)-adjusted P value of the test [START_REF] Foekens | Four miRNAs associated with aggressiveness of lymph node-negative, estrogen receptor-positive human breast cancer[END_REF] as well as P-value calculated by 1,000 re-samplings were both < .05. The contribution of individual genes in a pathway was evaluated using calculated z-scores [START_REF] Goeman | A global test for groups of genes: testing association with a clinical outcome[END_REF]; genes with z-scores >1.96 were considered significant contributors to the pathway. R version 2.4.1 (http://www.cran.r-project.org) was used to run the Global Test package.

Results

Identification of candidate miRNAs predictive of clinical benefit

To identify miRNAs potentially related to the type of response to tamoxifen therapy, we exploited our previously generated data set [START_REF] Foekens | Four miRNAs associated with aggressiveness of lymph node-negative, estrogen receptor-positive human breast cancer[END_REF]. This dataset comprised miRNA expression levels of 249 miRNAs of 38 ER-positive breast cancer patients with lymph-node negative disease with welldocumented clinical follow-up. Fifteen of these 38 breast cancer patients, who were all hormone-naïve, experienced a relapse and received tamoxifen monotherapy as first-line treatment for recurrent disease.

Ten patients had clinical benefit of this therapy while five did not and progressed within six months. A two-tailed Mann-Whitney test ranked the following five miRNAs, hsa-miR-422a (P = 0.004), hsa-miR-30a-3p (P = 0.004), hsa-miR-187 (P = 0.005), hsa-miR-30c (P = 0.014) and hsa-miR-182 (P = 0.027), as the most significantly differentially expressed between those patients benefiting from therapy and those who failed therapy.

To independently validate the predictive value of these five putative predictors of benefit of tamoxifen therapy, we measured their expression levels by quantitative real time RT-PCR in 246 ER positive primary tumors of hormone-naïve breast cancer patients who received tamoxifen monotherapy as first-line treatment for advanced disease.

Association of miRNA expression levels with patient and tumor characteristics

The association of the levels of the five candidate predictive miRNAs with patient and tumor characteristics are summarized in Table 1. The patient"s age was positively correlated with hsa-miR-30c (P = 0.001) and hsa-miR-182 (P = 0.009) levels. Similarly, these two miRNAs were associated with menopausal status (P = 0.036; P = 0.030, respectively). The levels of none of the miRNAs analysed were significantly associated other clinical and pathological parameters such as nodal status, tumor size and grade, and disease-free interval.

ER mRNA, PgR mRNA and miRNA levels were analysed as continuous variables. Spearman rank correlation showed significant positive association (all P < 0.001) between the levels of ER mRNA, and those of hsa-miR-30a-3p (r s = 0.298), hsa-miR-30c (r s = 0.309), and hsa-miR-182 (r s = 0.276).

Furthermore, PgR mRNA levels were also positively correlated (all P < 0.001) with hsa-miR-30a-3p (r s = 0.444), hsa-miR-30c (r s = 0.336) and hsa-miR-182, (r s = 0.177). In contrast, hsa-miR-422a levels were negatively correlated with those of ER (r s = -0.134, P = 0.036) and PgR (r s = -0.153, P = 0.016). In addition, hsa-miR-30a-3p expression levels were positively correlated (P < 0.001) with those of hsa-miR-30c (r s = 0.744). Hsa-miR-182 (r s = 0.310), and hsa-miR-30c levels were correlated with those of hsa-miR-182 (r s = 0.410).

Univariate and multivariate analysis for clinical benefit of tamoxifen therapy

In univariate logistic regression analysis using continuous variables, increasing levels of hsa-miR-30a-3p (OR 1.51, 95% CI 1.16-1.96, P = 0.002), hsa-miR-30c (OR 3.87, 95% CI 2.16-6.94 P < 0.001), and hsa-miR-182 (OR 1.54, 95% CI 1.09-2.16, P = 0.013), were associated with clinical benefit of tamoxifen treatment. No significant associations were observed for hsa-miR-422a or hsa-miR-187 in any of the analyses (not shown). Therefore, these miRNAs are not discussed any further as they were probably false discoveries. In multivariate analysis, when adding the miRNAs separately to the base model comprising the traditional predictive factors (age, menopausal status, dominant site of relapse, disease-free interval and tumor ER and PgR levels), only hsa-miR-30c was significantly associated with clinical benefit (OR 3.15, 95% CI 1.62-6.12, P = 0.001). To express the effect size of the clinical benefit of patients based on the different predictive miRNAs, the patients were categorized into four groups based on their quartile miRNA expression levels. Patients in the fourth quarter with the 25% highest expression had clinical benefit than those in the first quarter, i.e., 77% (47 out of 61) versus 48% (31 out of 64) for hsa-miR-30a-3p, 80% (49 out of 61) versus 47% (30 out of 64) for hsa-miR-30c, and 79% (48 out of 61) versus 55% (34 out of 62) for hsa-miR-182. Similar to the analysis of the miRNAs as a continuous variable, only hsa-miR-30c remained as an independent predictive variable when analyzed as a categorical variable (Table 3).

Association of miRNA levels with PFS

The relationship of miRNA levels with PFS was determined with the Cox proportional hazards model and visualized by Kaplan-Meier survival curves using the quarters as mentioned above. Patients in the fourth quarter, with the highest expression levels had the longest PFS for all three miRNAs.

Compared with patients with tumor miRNA levels in the first quarter, the HRs for patients in the fourth quarter ranged from 0.47 for hsa-miR-30c, via 0.51 for hsa-miR-30a-3p, to 0.57 for hsa-miR-182 (Figure 1).

Pathways and global testing

For 71 of the 246 patients Affymetrix (U133A or plus2) transcriptome information was available as well. Using the Global Test [START_REF] Goeman | A global test for groups of genes: testing association with a clinical outcome[END_REF], we associated biological pathways with the significant miRNAs (Supplementary Table 1). Hsa-miR-30c was found to be the most significantly coupled to ""HER, signal transduction and oncology pathway"" (comparative P = 0.014, FDR-adjusted P = 0.007), in which higher ER-α and HER4 expression levels and lower EGFR expression levels were seen in tumors having high hsa-miR-30c expression levels compared with those expressing low levels (Figure 2A). Additionally, hsa-miR-30c and hsa-miR-30a-3p were both negatively associated to the "RAC1 cell motility signaling pathway" (comparative P = 0.025, FDR-adjusted P = 0.015, and comparative P = 0.016, FDR-adjusted P = 0.016, respectively). MRNA expression levels of platelet-derived growth factor receptor α (PDGFRα), Wiskott-aldrich syndrome protein 1 (WASF1), cdk5 regulatory subunit 1, (CDK5K1), and chimerin 1 (CHN1) genes were all inversely correlated with the expression levels of these two miRNAs. (Figure 2 B,C).

Hsa-miR-30a-3p was also significantly associated with "Ceramide signaling pathway" (comparative P = 0.019, FDR-adjusted P = 0.012). (Figure 2 D). In which anti-apoptotic BCL2, NF-κB1 and MAP2K4 were over-expressed in tumors having high versus those having low hsa-miR-30a-3p expression (Supplementary Table 1).

Our analysis did not reveal a significant association of hsa-miR-182 with any of the pathways included in the analysis.

Discussion

Given the importance to get better insight into the mechanisms accounting for outcome to hormonal therapy in breast cancer, we explored in this study the association between five putative candidate miRNAs and outcome in ER-α positive breast cancer patients who received tamoxifen therapy for advanced disease. We found an association of high expression levels of hsa-miR-30a-3p, hsa-miR-30c and hsa-miR-182 with clinical benefit and longer PFS. According to published predicted target databases, none of the miRNAs included in this study have the ER mRNA as a target. This suggests that, although indirect effects are possible, the effects of the studied miRNAs on tamoxifen response are not directly related to modulation of ER levels.

To unravel the mechanisms by which these miRNAs affects outcome to tamoxifen, global testing together with gene expression data were used to reveal associations between these miRNAs and biological pathways. In these analyses, the most significant and independent predictor of clinical benefit, hsa-miR-30c, was positively linked to ER-α and negatively to EGFR. Cross-talk between EGFR and ERα has earlier been implicated in tumor cell response to tamoxifen [START_REF] Klijn | The clinical significance of epidermal growth factor receptor (EGF-R) in human breast cancer: a review on 5232 patients[END_REF][START_REF] Osborne | Growth factor receptor cross-talk with estrogen receptor as a mechanism for tamoxifen resistance in breast cancer[END_REF][START_REF] Shou | Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer[END_REF]. Furthermore, hsa-miR-30c and the correlated and possibly co-regulated hsa-miR-30a-3p were both negatively correlated to "RAC1 cell motility signaling pathway". The driving growth factor receptor of this pathway, PDGFRα, is causally linked to tamoxifen resistance, since we have shown previously [START_REF] Meijer | Functional screen for genes responsible for tamoxifen resistance in human breast cancer cells[END_REF] that increased expression of PDGFRα allows the human breast cancer cell line ZR-75-1 to grow in the presence of tamoxifen. Downstream Rho/Rac/Cdc42-like GTPases and their activating (RhoGAP) proteins such CHN1 (Chimerin1, also called ARHGAP2) have multiple functions including the modulation of the plasticity of tumor cells [START_REF] Katoh | Identification and characterization of ARHGAP27 gene in silico[END_REF][START_REF] Sanz-Moreno | Rac activation and inactivation control plasticity of tumor cell movement[END_REF] but there is no link of this with endocrine resistance. Not apparent from the pathway analysis is that another GTPase, RHOBTB3, as well as the GTPase modulator VAV3 are also implicated or associated within tamoxifen resistance [START_REF] Meijer | Functional screen for genes responsible for tamoxifen resistance in human breast cancer cells[END_REF]. Thus, there is mounting evidence of specific GTPase signaling pathways in modulation of endocrine therapy response. In addition, pathway analysis connects hsa-miR-30a-3p positively to BCL2 mediated cellular survival/ceramide induced apoptosis, which is in line with a favorable role of increased BCL2 expression in breast cancer treated with endocrine therapy [START_REF] Dos Santos | Bcl-2 oncogene expression in estrogen receptor-positive and negative breast carcinoma[END_REF][START_REF] Gasparini | Expression of bcl-2 protein predicts efficacy of adjuvant treatments in operable node-positive breast cancer[END_REF][START_REF] Linke | A multimarker model to predict outcome in tamoxifen-treated breast cancer patients[END_REF][START_REF] Silvestrini | p53 and bcl-2 expression correlates with clinical outcome in a series of node-positive breast cancer patients[END_REF][START_REF] Yang | Prognostic value of Bcl-2 in invasive breast cancer receiving chemotherapy and endocrine therapy[END_REF]. As BCL2 is an ER-α regulated gene, high BCL2 is indicative of an intact ER-α pathway driving tumor growth thereby potentially explaining sensitivity to endocrine therapy [START_REF] Perillo | 17beta-estradiol inhibits apoptosis in MCF-7 cells, inducing bcl-2 expression via two estrogen-responsive elements present in the coding sequence[END_REF].

Searching publicly available data bases, it appeared that the highly correlated and highly homologous hsa-miR-30c and -30a share many predicted targets such as PPARGC1B, Makorin-3, UBAC1, PTPDC1 (http://microrna.sanger.ac.uk). However, for hsa-miR30-a we quantified the star strand, hsa-miR-30a-3p, which can be also released from the hsa-miR-30a miRNA stemloop. This miRNA targets an entirely different set of genes i.e., POU4F1, ZIC4, FOXJ3, BRCA1 (http://pictar.mdcberlin.de). The putative targets of these miRNAs, however, do not obviously pinpoint to a specific mechanism nor are they connected to the above-mentioned pathways revealed by data mining. As a consequence, thus far the putative targets are not informative and further research is needed to elucidate the exact mechanisms underlying the association of the found miRNAs and outcome to tamoxifen.

In conclusion, this study showed a connection between hsa-miR-30c, hsa-miR-30a-3p and hsa-miR-182 expression levels and benefit of conventional endocrine therapy. Only hsa-miR-30c was independent of traditional predictive markers suggesting that measuring this miRNA may have added clinical value. At present these markers are not ready for clinical application. First of all the findings need to be firmly validated in additional independent cohorts, and secondly the current markers on their own are insufficiently strong for clinical decision making and the results should be interpreted as hypothesis generating. Apart from this, the miRNAs revealed to be predictive for outcome to tamoxifen, may be tested for their ability to predict response to aromatase inhibitors as well, the other main type of endocrine therapy currently prescribed. Finally, the pathways connected to these miRNAs as well as their shared predicted target genes may provide new clues for mechanism driving tamoxifen resistance. For PFS analysis, samples were divided in quarters (Q 1 -Q 4 ) based on the quartile levels of hsa-miR-30a-3p (A), hsa-miR-30c (B), and hsa-miR-182 (C). Patients at risk at 12 and 24 month time intervals are indicated at the bottom of each graph. The color of a bar represents positive (red) or negative (green) association.

Legends to the figures

The number of horizontal markers in a bar indicates the significance and the height of a bar the contribution of a gene to the pathway.

Figure 1 .

 1 Figure 1. Kaplan Meier survival curves.

Figure 2 .

 2 Figure 2. Pathways associated with the tumor subgroup having high versus low hsa-miR-30c (A, B) or hsa-miR-30a-3p (C, D).

Table 1 . Associations of biological factors with clinico pathological factors Charactristics Median miRs levels (interquartile range) after normalization to the housekeeper m Patients hsa-miR-30a-3p hsa-miR-30c hsa-miR-182 hsa-miR-187 hsa-m Age (years)

 1

	≤55			92	0.59 (0.81)	0.94 (0.77)	0.80 (0.75)	0.19 (0.70)	0.99
	56-70			90	0.68 (1.00)	1.01 (0.95)	1.03 (1.18)	0.19 (0.61)	0.95
	>70 Menopausal status	*	64	0.76 (1.80) P = 0.092 ‡	1.34 (1.23) P = 0.001 ‡	0.95 (1.12) P = 0.009 ‡	0.26 (1.31) P = 0.064 ‡	1.08 P =
	Premenopausal		59	0.60 (0.80)	0.87 (0.69)	0.78 (0.81)	0.15 (0.67)	0.96
	Postmenopausal	187	0.70 (1.17) P = 0.257 ¶	1.08 (1.07) P = 0.036 ¶	0.96 (1.08) P = 0.030 ¶	0.21 (0.79) P = 0.159 ¶	1.01 P =
	Dominant site of relapse
	Local regional relapse	32	0.48 (0.73)	0.88 (0.97)	0.81 (0.43)	0.22 (0.67)	1.13
	Bone			128	0.70 (1.27)	1.05 (0.81)	1.01 (0.99)	0.22 (0.81)	1.00
	Visceral			86	0.56 (0.85) P = 0.318 §	1.01 (1.10) P = 0.816 §	0.91 (1.34) P = 0.131 §	0.20 (0.63) P = 0.623 §	0.94 P =
	Disease-free interval (months)
	≤12			64	0.51 (0.68)	0.88 (0.89)	0.91 (0.85)	0.21 (1.37)	1.13
	13-36			108	0.74 (1.13)	1.05 (0.98)	0.94 (1.14)	0.18 (0.48)	0.99
	>36 Tumor Size (cm)	†		74	0.86 (1.35) P = 0.173 §	1.14 (0.99) P = 0.163 §	1.01 (0.91) P = 0.945 §	0.22 (0.84) P = 0.219 §	0.93 P =
	≤2			65	0.60 (1.41)	0.98 (0.93)	1.01 (0.78)	0.19 (0.83)	0.95
	2-5			146	0.66 (0.99)	1.02 (0.85)	0.90 (1.08)	0.21 (0.63)	1.04
	>5			35	0.66 (0.89)	1.28 (1.05)	0.91 (1.50)	0.09 (0.54)	0.93

*

Acknowledgements

This study was financially supported by the Association for International Cancer Research (Grant number 07-0609) and the Netherlands Genomics Initiative (NGI)/Netherlands Organisation for Scientific Research (NWO).


Bent Ejlertsen Kirsten Vang Nielsen
email: kirsten.vang@dako.com

Sven Müller

Susanne Møller

Birgitte B Rasmussen

Eva Balslev

Anne-Vibeke Laenkholm

Peer Christiansen

Henning T Mouridsen

Amplification of may predict resistance to adjuvant tamoxifen in postmenopausal patients with hormone receptor positive breast cancer

Keywords: ESR1, estrogen receptor, gene amplification, tamoxifen, endocrine therapy, gene aberrations, breast cancer

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Introduction

The estrogen receptor (ER) is the earliest established and most widely used biomarker in breast cancer and it is recommended that the tumor ER status be determined in all breast cancer patients [START_REF] Harris | American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer[END_REF][START_REF] Goldhirsch | Progress and promise: highlights of the international expert consensus on the primary therapy of early breast cancer 2007[END_REF]. ER has both a prognostic and predictive value. ER status is a prognostic factor, but shifts from a positive prognostic factor initially to a slightly negative prognostic indicator five to six years after diagnosis [START_REF] Bentzon | Prognostic effect of estrogen receptor status across age in primary breast cancer[END_REF]. ER is the target of tamoxifen. The efficacy of adjuvant endocrine therapies is associated with the presence of ER in the primary tumor [START_REF] Ebctcg | Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials[END_REF], and patients with ER negative breast cancer are unlikely to benefit from tamoxifen. Unfortunately, endocrine therapies do not benefit all patients with ER positive tumors and in a search for explanations for this, many features of ER have been studied in the past [START_REF] Schiff | Endocrinology and hormone therapy in breast cancer: new insight into estrogen receptor-alpha function and its implication for endocrine therapy resistance in breast cancer[END_REF], including ER and ERβ protein, ER mRNA, and numerous ER cofactors, e.g. AIB1 [START_REF] Osborne | Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer[END_REF][START_REF] Kirkegaard | Amplified in breast cancer 1 in human epidermal growth factor receptor -positive tumors of tamoxifen-treated breast cancer patients[END_REF]. Array techniques have provided information on ER target genes and response elements [START_REF] Lin | Discovery of estrogen receptor alpha target genes and response elements in breast tumor cells[END_REF]. Also studies investigating polymorphisms [START_REF] Hill | Estrogen receptor expression in human breast cancer associated with an estrogen receptor gene restriction fragment length polymorphism[END_REF] and mutations within the ESR1 gene that codes for ER have been reported.

Over a 17 years period, gene amplification of the ESR1 gene, encoding the estrogen receptor, has been the focus of very few studies [START_REF] Nembrot | Estrogen receptor gene amplification is found in some estrogen receptor-positive human breast tumors[END_REF][START_REF] Watts | Oestrogen receptor gene structure and function in breast cancer[END_REF][START_REF] Schuur | Monoallelic amplification of estrogen receptor-alpha expression in breast cancer[END_REF][START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF][START_REF] Tomita | Estrogen receptor alpha gene ESR1 amplification may predict endocrine therapy responsiveness in breast cancer patients[END_REF], although gene amplification is the major mechanism behind the cancer related changes of many oncogenes [START_REF] Albertson | Gene amplification in cancer[END_REF], including ERBB2 (HER2) [START_REF] Slamon | Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene[END_REF], TOP2A [START_REF] Järvinen | Characterization of Topoisomerase II Gene Amplification and Deletion in Breast Cancer[END_REF], CCND1 [START_REF] Kirkegaard | Genetic alterations of CCND1 and EMSY in breast cancers[END_REF] and MYC [START_REF] Corzo | The MYC oncogene in breast cancer progression: from benign epithelium to invasive carcinoma[END_REF].

The first ESR1 gene studies were based on Southern blotting technique and showed contradictory results [START_REF] Nembrot | Estrogen receptor gene amplification is found in some estrogen receptor-positive human breast tumors[END_REF][START_REF] Watts | Oestrogen receptor gene structure and function in breast cancer[END_REF]. With more sensitive techniques, ESR1 was included in the list of possible genes located in loci that frequently undergo amplification or deletions in breast cancer [START_REF] Hicks | Novel patterns of genome rearrangement and their association with survival in breast cancer[END_REF]. Recently, the frequency of ESR1 amplifications was established [START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF][START_REF] Tomita | Estrogen receptor alpha gene ESR1 amplification may predict endocrine therapy responsiveness in breast cancer patients[END_REF] to be approximately 20% among 2000 breast cancer specimens analyzed by FISH 05-06-2010 4 (fluorescence in situ hybridization) technique, and ESR1 amplification was reported to be associated with good response to adjuvant tamoxifen treatment. The initial ESR1 FISH study [START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF] has led to much debate about the frequency of ESR1 amplification in breast cancer and the implication of the methodological variation for the estimation of the frequency [START_REF] Albertson | Conflicting evidence on the frequency of ESR1 amplification in breast cancer[END_REF][START_REF] Brown | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Horlings | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Vincent-Salomon | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Reis-Filho | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Holst | Reply to "ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF], but direct comparison of ESR1 FISH with quantitative PCR [START_REF] Tomita | Estrogen receptor alpha gene ESR1 amplification may predict endocrine therapy responsiveness in breast cancer patients[END_REF] showed the superiority of the FISH method for detection of these low level amplifications.

The present study [START_REF] Ejlertsen | Amplification of ESR1 may predict resistance to adjuvant tamoxifen in postmenopausal patients with hormone receptor positive breast cancer[END_REF] was initiated before publication of the study by Holst et al. [START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF], and our hypothesis does not take their result into account. We hypothesized that copy number changes of the ESR1 gene confer resistance to tamoxifen because amplification is an abnormal status and normal ER protein expression (ER positive status) is requisite for response to tamoxifen. We designed a study to analyze the frequency of abnormal copy number of ESR1 according to outcome in breast cancer patients having adjuvant treatment with tamoxifen.

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Material and Methods

Patient samples

Tissue blocks were collected from the archives at the department of Pathology at Herlev and Roskilde Hospitals. From a nationwide, previously described cohort of 1115 Danish postmenopausal patients allocated to tamoxifen 20 mg daily for 5 years following radical surgery from 1995 to 2006 for early hormone receptor positive breast cancer [START_REF] Moller | The clinical database and the treatment guidelines of the Danish Breast Cancer Cooperative Group (DBCG); its 30-years experience and future promise[END_REF], we extracted a subgroup whose tumour blocks were located at one of the two participating pathology departments and who had a recurrence less than 4 years (61 patients) or were without recurrence at least 7 years after initiation of adjuvant tamoxifen (48 patients).

Later revision of the patient data led to exclusion of one patient who had recurrence after 5 years from the early recurrence group and two patients where the tissue block did not contain tumor tissue; one from each recurrence group. All tumors were hormone receptor positive with more than 10% stained cells. All samples except two were analyzed on tissue microarrays (TMAs). Two samples were analyzed on whole sections. The TMAs were constructed by a technologist supervised by a pathologist (EB). A HE staining was available to identify areas of invasive breast tumor. The biomarker study was conducted according to the REMARK recommendations [START_REF] Mcshane | Reporting recommendations for tumor marker prognostic studies (REMARK)[END_REF].

Collection of tissue samples from normal breasts

For assay validation we analyzed ESR1 gene copy numbers in 120 samples collected consecutively and retrospectively at the department of Pathology at Herlev University Hospital from breast reduction operations with no identified malignancy. Areas of normal breast glands suitable for tissue microarray (TMA) construction were identified by a 05-06-2010 6 pathologist (EB). TMAs were constructed using Advanced Tissue Arrayer, ATA-100 (Chemicon International, Temecula, CA, USA) from formalin-fixed, paraffin-embedded normal breast tissue. Duplicate 2.0mm tissue cores from each donor block were reembedded into recipient paraffin block containing 40 tissue cores.

Collection of tissue samples from ER negative tumors

To validate the observation of ESR1 deletions we identified 9 ER negative breast tumors from the tissue archives at Dako A/S which contains anonymous tissue blocks from various types of cancer. Information on some tumor characteristics was available, but patient characteristics including outcome were unknown.

FISH analysis

Samples from tumor tissue and normal tissue were analyzed for ESR1 copy number changes using FISH with a probe covering the ESR1 gene at 6q25 and a reference probe covering the centromere of chromosome 6. The RP11-450E24 and RP11-54K4 BAC clones (Fig. 1) that make up the ESR1 probe applied in this study covers all coding sequence of the ESR1 gene. Nearly 30 % of the ESR1 probe covers up-and downstream flanking sequences including approximately 4 % of the neighboring SYNE1 gene.

However, as the SYNE1 overlapping part make up less than 6 % of the ESR1 probe, separate amplification or deletion of SYNE1 would not be detectable by use of the ESR1 probe. The ESR1 targeted BAC clones were labeled with Texas Red fluorochrome by Nick translation. The centromere 6 targeted references probe was based on a mixture of PNA (peptide nucleic acid) oligoes labeled with fluorescein (FITC). A mixture of alu PNA 05-06-2010 7 oligoes were used as blocking agent. The FISH protocol described below is optimized for a DNA/PNA mixture. Further details are described elsewhere [START_REF] Nielsen | Combined Use of PNA and DNA for Fluorescence In Situ Hybridization (FISH)[END_REF]. Prior to FISH analysis, the slides were deparaffinized twice in Xylene for 5 minutes followed by rinse in 96% and 70% ethanol (each twice for 2 minutes). FISH was performed with Dako Histology FISH accessory kit (K5599, Dako A/S, Glostrup, Denmark) according to the manufacturer's instructions. Briefly the method consist of pretreatment for 10 minutes at 95-99 0 C in pretreatment solution followed by pepsin treatment for 10 minutes at room temperature (K5599, vial 2, RTU, pH 2.0). After denaturation at 82 0 C for 5 minutes and overnight hybridization at 45 0 C, excess of probe is removed by stringent wash at 65 0 C for 10 minutes. After dehydration in ethanol, the slides are mounted in antifade with DAPI (K5599, vial 5). Figure 2 illustrates the appearance of ESR1 amplification using a Lieca DM microscope equipped with a Lieca Image System and filters for Texas Red, FITC and DAPI. The ratio was calculated as the number of signals for the gene probe divided by the number of signals for the centromere 6 reference probe. Cases were scored as ESR1 FISH amplified when the ratio was 2.0 and deleted when the ratio was < 0.8.

Slide evaluation

The slides were evaluated according to the TOP2A FISH scoring guidelines (from Dako K5333 USA package insert, 1 st edition 2008.01.18, approved by the American Food and Drug Administration (FDA)). These guidelines specifically allow detection of both amplifications and deletions compared to the HER2 guidelines [START_REF] Wolff | American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer[END_REF] that focus on amplifications. For a nucleus to be included in the scoring, the HER2 guidelines require that it contains signals of both colors. The TOP2A guidelines only require the presence of a green reference signal for a nucleus to be included in the scoring. The reason for this 05-06-2010 8 difference is that the truncated nuclei in cut sections of a deleted, diploid tissue sample will either contain one or zero red gene signals and omission of all nuclei with zero red signals will prevent detection of most deleted cases. A previously conducted pilot study [START_REF] Olsen | Amplification of HER2 and TOP2A and deletion of TOP2A genes in breast cancer investigated by new FISH probes[END_REF] showed that two different counting methods gave identical results: Either the signals were counted in 60 nuclei or sufficient nuclei were included until a total of 60 red gene probe signals were counted along with the green reference probe signals in the same nuclei. An average of 35 nuclei was counted. The latter method has the advantage that the highest number of cells will be counted in the deleted and normal cases, while the lowest number of cells will be counted in highly amplified cases. Highly amplified cases are often obvious to identify just by looking in the microscope, but are more time demanding to evaluate if 60 nuclei have to be scored.

Statistical Analysis

The DBCG Data Center collected and analyzed the data. Associations between ESR1 status and other clinicopathological characteristics were analyzed by chi-square or Fischer's exact test. P-values are two-tailed. Disease free survival (DFS) was defined as the duration of survival without invasive loco-regional recurrence, distant metastasis, contralateral breast cancer, second primary nonbreast invasive cancer or death irrespective of cause. DFS was analyzed unadjusted using the log-rank test. For

Results

Archival tissue from primary tumor was retrieved from 97 of the 109 patients (89%). FISH analysis for ESR1 was successful in 91 of the 97 patients (94%). Patient characteristics are seen in Table 1. Patients in the early recurrence group had more positive lymph nodes (P<0.001) and more tumors with higher grade (P=0.04) compared to patients in the nonrecurrence group. The distribution of ESR1/CEN-6 ratios in patients without recurrence for 7 or more years compared to patients experiencing recurrence within the first 4 years is seen in Figure 3a. Ratios were found in the range of 0.50 to 3.13. Overall 14% of the patients had amplification of the ESR1 gene. All amplifications were in the low level range with a ratio from 2.00-3.13. Nine amplifications were found in diploid tumors (< 2 average references signals per nucleus) and 4 in triploid tumors (< 3 average reference signals per nucleus). If no reference probe had been included most of the amplifications would have been missed as only 4 of the samples had an average of 5 or more ESR1 gene signals/nucleus. In addition, we observed ESR1 deletion in four tumors, two in each recurrence group. The deletions were in the range of 0.50-0.76. Three deletions were in diploid tumors and one in a triploid tumor. If no reference probe had been included only 3 deletions would have been detected. Amplification was observed in 11 of 50 (22%) patients with recurrence within the first four years of tamoxifen treatment, compared to two of 41 (5%) patients who were free of recurrence at least 7 years after initiation of tamoxifen treatment (Table 2). The difference is statistically significant (P=0.033; Fishers exact test). Compared to patients with ESR1 normal tumors, patients with ESR1 amplifications had a significant shorter disease-free (P=0.0054) and overall survival (P=0.0004) as illustrated in the Kaplan-Meyer plots of For assay validation we analyzed ESR1 gene copy numbers in 120 normal breast samples (Fig. 3b and Fig. 5) and found neither amplifications nor deletions. In each sample 60 nuclei were evaluated. An average of 1.78 red signals representing the ESR1 genes was counted and 1.69 green signals representing the centromeric reference sequence. The ESR1/CEN-6 ratio varied from 0.96 to 1.29 with a mean of 1.06. The ratios were distributed normally (Fig. 5) with a standard deviation of 0.04.

To validate the observation of deletions, we performed ESR1 FISH on nine ER negative, but otherwise anonymous tissue samples. All 9 samples had ratios below 1.00 in the range from 0.61-0.96 (data not shown), and 6 of the samples had ESR1 deletion, indicating that the frequency of ESR1 deletion may be higher in an ER negative patient series.

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Discussion

The estrogen receptor (ER) is encoded by the ESR1 gene localized on chromosome 6q25.1 and copy number changes of ESR1 have only been the subject of a very limited number of studies in contrast to the intensive study of the ER protein and its cofactors [START_REF] Schiff | Endocrinology and hormone therapy in breast cancer: new insight into estrogen receptor-alpha function and its implication for endocrine therapy resistance in breast cancer[END_REF].

The present study reports on the existence of both ESR1 amplifications and deletions. The frequency of amplification varies from 5% in a group of patients without recurrence to 22% in a group of patients with recurrence within the first four years after initiation of treatment.

We found amplification in ESR1 to be a strong independent prognostic marker of poor survival in postmenopausal patients allocated to five years of tamoxifen following radical surgery for early hormone receptor positive breast cancer. The amplifications were in the low level range compared to ERBB2 (HER2) amplifications and this may be the explanation for the infrequent report of ESR1 amplifications. However, all amplifications were well outside the normal range established in 120 normal breast samples.

Genetic alterations including copy number changes are the major cancer causing mechanism [START_REF] Albertson | Gene amplification in cancer[END_REF], but amplification of ESR1 has been the focus in few studies [START_REF] Nembrot | Estrogen receptor gene amplification is found in some estrogen receptor-positive human breast tumors[END_REF][START_REF] Watts | Oestrogen receptor gene structure and function in breast cancer[END_REF][START_REF] Schuur | Monoallelic amplification of estrogen receptor-alpha expression in breast cancer[END_REF][START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF]. Regulation of the estrogen receptor has primarily been studied at the protein and mRNA level and many studies have been devoted to ER as a transcription factor, e.g. ER regulation of other genes [START_REF] Lin | Discovery of estrogen receptor alpha target genes and response elements in breast tumor cells[END_REF]. However, if the ESR1 gene itself is abnormal, in structure or copy number, it may not be capable of regulating other genes normally. In a study using Southern hybridization, no major structural rearrangements of ESR1 were identified in a series of 188 primary breast cancers [START_REF] Hill | Estrogen receptor expression in human breast cancer associated with an estrogen receptor gene restriction fragment length polymorphism[END_REF] and subsequent studies using related techniques for the study of polymorphisms, e.g. restriction fragment length polymorphism (RFLP) and loss of heterozygosity (LOH), have confirmed that structural changes within the ESR1 05-06-2010 gene are uncommon in breast cancers and do not explain differences in protein expression and ER status [START_REF] Watts | Oestrogen receptor gene structure and function in breast cancer[END_REF][START_REF] Andersen | Oestrogen receptor (ESR) polymorphisms and breast cancer susceptibility[END_REF][START_REF] Yaich | Analysis of the PvuII restriction fragment-length polymorphism and exon structure of the estrogen receptor gene in breast cancer and peripheral blood[END_REF][START_REF] Otis | Loss of heterozygosity of p53, BRCA1, VHL, and estrogen receptor genes in breast carcinoma: correlation with related protein products and morphologic features[END_REF].

The first ESR1 gene amplification studies were based on Southern blotting technique and showed contradictory results [START_REF] Nembrot | Estrogen receptor gene amplification is found in some estrogen receptor-positive human breast tumors[END_REF][START_REF] Watts | Oestrogen receptor gene structure and function in breast cancer[END_REF]. In accordance with our observations, a study from 1990 using Southern hybridization reported low level amplification ranging from 1.6 to 3fold in 6 of 14 ER-positive tumors, while no amplification could be detected among 8 ERnegative tumors included in the study [START_REF] Nembrot | Estrogen receptor gene amplification is found in some estrogen receptor-positive human breast tumors[END_REF]. The frequency of amplification could not be confirmed in a study from 1992 [START_REF] Watts | Oestrogen receptor gene structure and function in breast cancer[END_REF] as only a single low level amplification among 29 ERpositive tumors was reported and the existence of ESR1 amplifications has not historically been widely appreciated [START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF][START_REF] Albertson | Conflicting evidence on the frequency of ESR1 amplification in breast cancer[END_REF]. However, the sensitivity of the Southern hybridization method may not allow detection of low level amplifications and deletions. With more sensitive techniques, ESR1 was included in the list of possible genes located in loci that frequently undergo amplification or deletions in breast cancer [START_REF] Hicks | Novel patterns of genome rearrangement and their association with survival in breast cancer[END_REF].

Two recent FISH studies [START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF][START_REF] Tomita | Estrogen receptor alpha gene ESR1 amplification may predict endocrine therapy responsiveness in breast cancer patients[END_REF] report the frequency of ESR1 amplifications to be in the range of 20-23%. Our study supports the existence of ESR1 amplifications varying from 5% in the group of patients with relatively good prognosis to 22% in the poor prognosis group of patients, and amplifications were significantly associated with poor out-come following treatment with Tamoxifen. In contrast to the two previous studies, we exclusively analyzed the prognostic implications of ESR1 aberrations within an ER positive cohort, and the exclusion of ER negative patients partly explains the differences observed between studies.

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to an estimated frequency of about 11% in the total population [START_REF] Moller | The clinical database and the treatment guidelines of the Danish Breast Cancer Cooperative Group (DBCG); its 30-years experience and future promise[END_REF]. The discrepancies mimic early days of HER2 diagnostics, when different patient populations, methods, scoring guidelines and cut-off levels led to enormous debate until firm guidelines were established [START_REF] Wolff | American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer[END_REF]. ESR1 amplifications seem to be at a lower level than HER2 and will thus be more difficult to detect as demonstrated by Tomita and coworkers [START_REF] Tomita | Estrogen receptor alpha gene ESR1 amplification may predict endocrine therapy responsiveness in breast cancer patients[END_REF]. Therefore, conflicting results must be expected when different methods are used [START_REF] Nembrot | Estrogen receptor gene amplification is found in some estrogen receptor-positive human breast tumors[END_REF][START_REF] Watts | Oestrogen receptor gene structure and function in breast cancer[END_REF][START_REF] Schuur | Monoallelic amplification of estrogen receptor-alpha expression in breast cancer[END_REF][START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF][START_REF] Tomita | Estrogen receptor alpha gene ESR1 amplification may predict endocrine therapy responsiveness in breast cancer patients[END_REF][START_REF] Brown | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Horlings | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Vincent-Salomon | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Reis-Filho | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Holst | Reply to "ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF] and the majority of platforms may not detect the ESR1 amplifications [START_REF] Albertson | Conflicting evidence on the frequency of ESR1 amplification in breast cancer[END_REF].

The 13 amplified tumors of the present study had an average of 3-7 ESR1 signals per nucleus while Holst et al [START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF] reports a higher average number og signals. Using their definition of "ESR1 gain" (ESR1/CEN-6 ratio >1.0), the majority of our cases (68=73%) would have been classified as having gain. Our assay validation showed no copy number variation among 120 normal breast samples. It is worth noting that the majority of normal samples would have been scored as having "ESR1 gain" using the criteria of ESR1 ratio >1.0 from Holst et al. [START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF]. One possible explanation for this difference is the use of different signal counting guidelines, especially for two signals not separated by the diameter of a signal. In line with HER2 scoring guidelines [START_REF] Wolff | American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer[END_REF], we consider such a "duplet" signal as only one signal created by chromatin folding. Although Holst et al. [START_REF] Holst | Reply to "ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF] reports high intra-observer reproducibility, no intra-laboratory reproducibility studies have been performed, which could have revealed differences of signal counting instructions. Our assay included a reference probe to estimate the ploidy level of the samples. If only the red gene signals had been used in the assay, as in a single color FISH assay, most of the low level amplifications would have been missed and only 4 amplifications have an 05-06-2010 16 average of more than 5 gene signals/nucleus. Thus, using the cut-off criteria of Reis-Filho et al. [START_REF] Reis-Filho | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Drury | Gene copy number variability of oestrogen receptor alpha in breast cancer[END_REF] would have reduced our frequency of amplifications to 4%.

In this patient series, we also identified 4 tumors with ESR1 deletion, two in each outcome group, and confirmed the existence of deletions by analysis of nine ER negative breast tumor samples. This is the first identification of ESR1 deletions in patient samples by FISH technique, but an in vitro study [START_REF] Zhang | Feasibility of simultaneous fluorescence immunophenotyping and fluorescence in situ hybridization study for the detection of estrogen receptor expression and deletions of the estrogen receptor gene in breast carcinoma cell lines[END_REF] using simultaneous fluorescence immunophenotyping and FISH detected ESR1 deletions in four of six breast cancer cell lines. However, as no correlation to ER expression could be found, this solitary study concluded that ESR1 deletions are not the major cause of absent or reduced ER expression in breast cancer cell lines. Although the ESR1 deletions may not correlate with ER protein expression, the observation on patient samples deserves further analysis.

In summary, our data support that ESR1 amplification is a frequent feature of ER positive tumors [START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF][START_REF] Tomita | Estrogen receptor alpha gene ESR1 amplification may predict endocrine therapy responsiveness in breast cancer patients[END_REF]. We observe ESR1 gene amplification in 14% of the tumors, ranging from 5% in patients with superior outcome to 22% in patients with inferior outcome on adjuvant treatment with tamoxifen. The ESR1 amplifications are significantly associated with poor disease-free and overall survival, and in this respect our data disagree with recent ESR1 FISH observations [START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF][START_REF] Tomita | Estrogen receptor alpha gene ESR1 amplification may predict endocrine therapy responsiveness in breast cancer patients[END_REF]. The clinical utility of ESR1 copy number changes deserves more attention until the exact importance for patient diagnostics has been established. Table 1. Patient characteristics correlated with recurrence groups.

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Legends to figures

  multivariate analysis the Cox proportional hazards regression model was applied to assess the adjusted hazard ratio (HR) of DFS by ESR1 status, and to explore interactions. Factors included in the multivariate analyses were age, tumor size (0-2 cm, >2-5 cm, >5 cm), nodal status (0-3, 4-9, > 9 positive), histological type and grade (ductal grade I, ductal grade II, 05-06-2010 9 ductal grade III, unknowns, missing or other histological types). Statistical analyses were done with the SAS 8.2 program package. Ethics All data used and presented was collected and analyzed by the DBCG registry. The biological substudy was conducted according to the Helsinki declaration and approved by the Scientific Ethical Committee of the Capital Region (1H-D-2007-0034, August 17th 2007). 05-06-2010 10

Figure 4 .

 4 Figure 4. ESR1 status is shown according to patient characteristics in Table3 and Cox

Figure 1 .

 1 Figure 1. Localization and coverage of the ESR1/CEN-6 probe mixture (A). The ESR1

Figure 2 .

 2 Figure 2 . ESR1 amplification in a breast cancer sample. In the individual filters, the red ESR1 signals (A), the green centromere 6 signals (B) and the DAPI counterstaining (C) is

Figure 3 .

 3 Figure 3. Distribution of ESR1/CEN-6 ratios in (A) 50 patients with early recurrence

Figure 4 .

 4 Figure 4. Recurrence free survival (A) and overall survival (B) of patients with and without

Figure 5 .

 5 Figure 5. The distribution of ESR1/CEN-6 ratios in 120 samples from normal breast tissue.

Table 2 .

 2 Distribution of tumors with ESR1 amplification and deletion among patients with early recurrence and patients with late or no recurrence.

Table 3 .

 3 Patient characteristics correlated to ESR1 status.

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Acknowledgements

We thank Signe Lykke Nielsen for excellent technical assistance. Supported by the Danish Research Council (Grant No. 271060542) and Danish Ministry of Internal Affairs and Health (Grant No. 2006-12103-272). 05-06-2010

All patients in the present study were ER positive with more than 10% stained cells as demanded for receiving adjuvant endocrine treatment. We have deliberately focused on the additive role of ESR1 amplifications in a patient cohort selected for treatment by ER immunohistochemistry and have not tried to compare ER protein level with ESR1 gene status. Both the work of Holst et al. [START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF] and Tomita et al. [START_REF] Tomita | Estrogen receptor alpha gene ESR1 amplification may predict endocrine therapy responsiveness in breast cancer patients[END_REF] show that although all patients with ESR1 amplification have high protein expression (Allred score 7-8), only approximately half of the patients with high protein expression have amplifications. Thus, high protein expression apparently is not always the result of amplification, as in e.g. HER2 protein overexpression. This lack of consistence might be one of the reasons for our finding in the present work, that amplification is a predictor of resistance, with amplification reflecting gene disturbance with loss of normal function.

The initial ESR1 FISH study [START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF] has led to much debate about the frequency of ESR1 amplification in breast cancer and correspondences from four groups report on frequencies of only 0-10% using a variety of methods, including comparative genomic hybridization (GCH), FISH, CISH and quantitative PCR [START_REF] Albertson | Conflicting evidence on the frequency of ESR1 amplification in breast cancer[END_REF][START_REF] Brown | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Horlings | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Vincent-Salomon | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Reis-Filho | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Holst | Reply to "ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF]. Some of the methodological debate has been resolved by the most recent study [START_REF] Tomita | Estrogen receptor alpha gene ESR1 amplification may predict endocrine therapy responsiveness in breast cancer patients[END_REF] that confirms the data by Holst et al. [START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF], but also compares the ESR1 FISH data directly with quantitative PCR data from the same patient material. The study found 22.6% ESR1 amplifications using FISH compared to 1.4% ESR1 amplifications using q-PCR, thus providing an explanation for the reported discrepancy among the other studies [START_REF] Brown | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Horlings | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Vincent-Salomon | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Reis-Filho | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF]