Stella Apostolaki

Maria Perraki

Galatea Kallergi

Maria Kafousi

Savvas Papadopoulos

Athanasios Kotsakis

Athanasios Pallis

Nikolaos Xenidis

Lyda Kalmanti

Kostas Kalbakis

Stella Apostolaki Ae

A E Galatea

Kallergi Ae

A E Savvas

Papadopoulos Ae

A E Athanasios

Pallis Ae

A E Lyda

Kalmanti Ae

A E Sofia

Agelaki Ae

Antonia Kalykaki

A E Christos

Stournaras Ae

Efstathios Stathopoulos

A E Vassilis

Georgoulias Ae

Dimitris Mavroudis

Á M Perraki

Á V Georgoulias

Á D Mavroudis

Á C Stournaras

Á E Stathopoulos

Á A Pallis

Á N Xenidis

Á L Kalmanti

Á K Kalbakis

Á S Agelaki

Á A Kalykaki

Detection of occult HER2 mRNA-positive tumor cells in the peripheral blood of patients with operable breast cancer: evaluation of their prognostic relevance

Keywords: HER2 mRNA, Breast cancer, Circulating tumor cells, Prognosis

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

Introduction

Breast cancer is considered a systemic disease because early tumor cell dissemination may occur even in patients with small tumors; using immunocytochemistry or molecular techniques, several investigators have shown that epithelial cells can be identified in the bone marrow (disseminated tumor cells; DTCs) aspirates or the peripheral blood (circulating tumor cells; CTCs) of otherwise metastases-free patients with breast cancer [START_REF] Pantel | Detection and clinical importance of micrometastatic disease[END_REF]. The detection of occult tumor cells either in the bone marrow or the peripheral blood has been demonstrated to represent an independent prognostic factor for disease relapse and reduced survival [START_REF] Braun | Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II or III breast cancer[END_REF][START_REF] Diel | Micrometastatic breast cancer cells in bone marrow at primary surgery: Prognostic value in comparison with nodal status[END_REF][START_REF] Mansi | Bone marrow micrometastases in primary breast cancer: prognostic significance after 6 years' follow-up[END_REF][START_REF] Stathopoulou | Molecular detection of cytokeratin-19-positive cells in the peripheral blood of patients with operable breast cancer: Evaluation of their prognostic significance[END_REF][START_REF] Xenidis | Predictive and prognostic value of peripheral blood cytokeratic-19 mRNA positive cells detected by real time PCR in node-negative breast cancer patients[END_REF]. Indeed, a meta-analysis involving 4,703 patients with stage I-III breast cancer confirmed the independent prognostic value of DTCs [START_REF] Braun | A pooled analysis of bone marrow micrometastasis in breast cancer[END_REF].

Several markers, corresponding to molecules encoded by genes that are thought to be tissue specific and expressed on epithelial but not on hematopoietic cells, have been used to detect occult tumor cells in the bone marrow or the peripheral blood of patients with breast cancer. Among these markers are cytokeratin-19 (CK19), mammaglobin, maspin, and carcinoembryonic antigen (CEA) [START_REF] Braun | Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II or III breast cancer[END_REF][START_REF] Diel | Micrometastatic breast cancer cells in bone marrow at primary surgery: Prognostic value in comparison with nodal status[END_REF][START_REF] Mansi | Bone marrow micrometastases in primary breast cancer: prognostic significance after 6 years' follow-up[END_REF][START_REF] Stathopoulou | Molecular detection of cytokeratin-19-positive cells in the peripheral blood of patients with operable breast cancer: Evaluation of their prognostic significance[END_REF][START_REF] Zach | Detection of circulating mammary carcinoma cells in the peripheral blood of breast cancer patients via a nested reverse transcriptase polymerase chain reaction assay for mammaglobin mRNA[END_REF][START_REF] Luppi | Sensitive detection of circulating breast cancer cells by reverse-transcriptase polymerase chain reaction of maspin gene[END_REF][START_REF] Gerhard | Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction[END_REF][START_REF] Stathopoulou | Molecular detection of cancer cells in the peripheral blood of patients with breast cancer: Comparison of CK-19, CEA and maspin as detection markers[END_REF].

Immunophenotyping of DTCs and CTCs revealed that they often express the HER2/neu (p185-erbB2) oncoprotein [START_REF] Brandt | Isolation of bloodbored epithelium-derived c-erb-B2 oncoprotein-positive clustered cells from the peripheral blood of breast cancer patients[END_REF][START_REF] Solomayer | Comparison of HER2 status between primary tumor and disseminated tumor cells in primary breast cancer patients[END_REF][START_REF] Braun | erbB2 overexpression on occult tumor metastatic cells in bone marrow predicts poor clinical outcome of stage I-II breast cancer patients[END_REF]. The HER2/neu oncoprotein is a transmembrane glycoprotein receptor sharing sequence homology with the epidermal growth factor receptor [START_REF] Schechter | The neu oncogene: an erb-B-related gene encoding a 185000-Mr tumour antigen[END_REF][START_REF] Slamon | Human breast cancer: correlation of relapse and survival with amplification of the HER2/neu oncogene[END_REF]. Amplification of HER2/neu, occurring in approximately 15-30% of human primary breast cancers, is associated with a higher probability of disease relapse [START_REF] Slamon | Studies of the HER-2/neu proto oncogene in human breast cancer and ovarian cancer[END_REF][START_REF] Toikkanen | Prognostic significance of HER-2 oncoprotein expression in breast cancer: a 30-year follow-up[END_REF]. Braun et al. [START_REF] Braun | erbB2 overexpression on occult tumor metastatic cells in bone marrow predicts poor clinical outcome of stage I-II breast cancer patients[END_REF] using a double labeling assay showed that 60% of the studied patients had DTCs which co-expressed the cytokeratin-18 (CK-18) and HER2/neu molecules; the detection of these CK-18?/HER2? cells was an independent prognostic factor for overall survival. A more recent study in 35 patients with stage I-III breast cancer showed the immunocytochemical detection of cytokeratin?/HER2?CTCs in 48.6% of the patients; the detection of these double-positive CTCs was also correlated with a significantly decreased relapse-free and overall survival [START_REF] Wulfing | HER 2 positive circulating tumor cells indicate poor clinical outcome in stage I to III breast cancer patients[END_REF].

There are only a few data in the literature concerning the value of HER2/neu oncoprotein as a marker of occult tumor cells in patients with breast cancer. A qualitative nested reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the detection of HER2 mRNA has already been described [START_REF] Wasserman | A seminested RT-PCR assay for HER2/neu: initial validation of a new method for the detection of disseminated breast cancer cells[END_REF][START_REF] Thomas | Absence of Her2/neu gene expression in osteosarcoma and skeletal Ewing's sarcoma[END_REF]. In the present study we evaluated a nested RT-PCR assay to detect HER2 mRNApositive occult tumor cells in the peripheral blood (CTCs) of patients with operable (stage I and II) breast cancer; our findings demonstrate that the detection of HER2 mRNApositive CTCs represents an independent prognostic factor associated with decreased disease-free interval and overall survival.

Methods

Cell lines

The human mammary carcinoma cell lines MCF-7, T47D, MDA-415 and SKBR3, as well as the COLO-205 colon adenocarcinoma cell line and ARH-77 myeloma cell line (all obtained from the American Type Culture Collection, Rockville, MD) were used; the MDA-415 cells do not express HER2/neu, the SKBR3 cells highly express the HER2/neu molecule while the MCF-7 and T47D cells present an intermediate expression of HER2/neu. Cells were cultured in Dulbecco's modified Eagle's medium (Gibco Life Sciences, BRL, Grand Island, NY, USA) supplemented with 10% bovine fetal serum (Gibco), L-glutamine 2 mmol (Sigma Chemical Company, St. Louis, MI, USA) and pyruvate 1 mmol/l (Sigma). Cell cultures were maintained in 5% CO2 air, and cells grown in monolayer were harvested by washing the dishes once with phosphate-buffered saline (PBS), pH 7.3, and then incubating the cells with PBS containing EDTA 0.53 mmol/l and 0.05% trypsin (Gibco) for 10-15 min at 37°C. The cells were washed with PBS and then passed through 25-gauge 5/8 needles to dissociate them. Cells were counted and viability was assessed by trypan blue dye exclusion.

Patients and clinical samples

Peripheral blood (20 ml in EDTA) was obtained from: (i) 61 patients with stage III-IV breast cancer; (ii) 20 patients with operable (Astler-Coller stages B 2 and C) colorectal cancer; (iii) 31 healthy female blood donors, (iv) 12 women operated for a benign breast fibroadenoma and (v) 216 patients with operable (stages I and II) breast cancer approximately 3-5 weeks after the surgical procedure and before the initiation of any systemic (adjuvant chemotherapy or hormonotherapy) or loco-regional (adjuvant radiation) treatment. The stage of disease was determined according to the tumornode-metastasis classification of the Union Internationale contre le Cancer and independent of the results of the RT-PCR assay. All patients had the primary breast tumor completely resected and dissection of level I and II axillary lymph nodes. In all patients immunohistochemical analysis of tumor tissue for the expression of estrogen (ER) and progesterone (PR) receptors as well as for HER2 molecule was performed according to standard techniques. Immunohistochemical detection of HER2 was performed using the CB11 monoclonal antibody (NCL-CB11, Novocastra Lab., London, UK) and scoring was based on the criteria recommended by DACO A/S (0-3 ? scale). Only membrane staining intensity was taken into account. Patients with breast-conserving surgery received radiation therapy and patients with ER/PR-positive tumors received tamoxifen 20 mg/day for 5 years. All patients received adjuvant chemotherapy with 5-fluouracil/epirubicin/cyclophosphamide (FEC; n = 111), sequential docetaxel and epirubicin/ cyclophoshamide (T/EC; n = 61), cyclophosphamide/ methotrexate/5-fluorauracil (CMF; n = 33) and other regimens (n = 11). No patient received adjuvant Herceptin since all patients were enrolled before the results of the adjuvant Herceptin trials were reported. Before the initiation of adjuvant treatment, the baseline diagnostic evaluation to exclude the presence of distant metastases included plain chest radiography, mammography, ultrasonography of the abdomen, computed tomography of the thorax and abdomen if indicated and whole body bone scan. No patient showed evidence of distant metastases at the time of enrollment onto the study. All patients were regularly followed every three months for the first 2 years, every 6 months for the three subsequent years and yearly thereafter. All patients gave their written informed consent to participate in the study, which was approved by the Ethics and Scientific Committees of our Institution.

All blood samples were obtained at the middle of vein puncture after the first 5 ml of blood were discarded. This precaution was undertaken in order to avoid contamination of blood with epithelial cells during sample collection. Peripheral blood samples were diluted with PBS (vol/vol) and cells were dissociated by passing them through 25gauge 5/8 needles. Peripheral blood mononuclear cells (PBMCs) were obtained by gradient density centrifugation using Ficoll-Hypaque 1,077 (Sigma) at 1,200 g for 30 min at 4°C. The interface cells were carefully collected, washed twice with 50 ml of sterile PBS and pelleted. Cell pellets were kept at -80°C until RNA extraction. Total RNA isolation was performed by using Trizol LS reagent (Gibco) according to the manufacturer's instructions. All RNA preparations and handling steps took place in a laminar flow hood, under RNAse-free conditions. The isolated RNA was dissolved in diethylpyrocarbonate-treated water and stored at -80°C until used. RNA concentration was determined by absorbance readings at 260 nm with the Hitachi UV-VIS (U-2000) spectrophotometer (Tokyo, Japan). RNA integrity was tested by PCR amplification of the b-actin housekeeping gene. RNA extracted from breast cancer cell lines (MCF-7 and SKBR3) and COLO 205 cell line was used as positive and negative controls, respectively.

Nested reverse transcription-polymerase chain reaction (RT-PCR)

A previously described nested RT-PCR assay for the detection of HER2 mRNA [START_REF] Thomas | Absence of Her2/neu gene expression in osteosarcoma and skeletal Ewing's sarcoma[END_REF] with minor modifications was used [START_REF] Bozionellou | Trastuzumab administration can effectively target chemotherapyresistant cytokeratin-19 messenger RNA-positive tumor cells in the peripheral blood and bone marrow of patients with breast cancer[END_REF]. Reverse transcription of RNA was carried out with the Thermoscript RT-PCR system (Invitrogen, Paisley, UK). cDNA was synthesized according to the manufacturer's instructions. Two different PCR reactions, with the respective negative controls, were performed with each sample in order to amplify fragments of HER2 and bactin. The sequences of primers used for HER2 (synthesized by Gencet, Paris, France) were as follows: 5 0 TCCTCC TCGCCCTCTTGC3 0 (sense Her-2-A); 5 0 GCGGGTCTCC ATTGTCTA3 0 (antisense Her-2-B); 5 0 AGCCGCGAGCA CCCAAGT3 0 (sense Her-2-C); 5 0 TTGGTGGGCAGGTA GGTGAGTT3 0 (antisense Her-2-D). To amplify cDNA, 2 ll were subjected to first PCR in 20 ll buffer [10 mM of HCL, buffer (pH 8.3), 50 mM KCL and 2.5 mM MgCl 2 ] containing 1 mM deoxynucleotide triphosphate, 3 mM of primers (Her-2-A and Her-2-B) and 2.5 units platinum Taq DNA polymerase (Invitrogen). For the second round of amplification (nested RT-PCR) a 2 ll aliquot of first PCR product was added to the same PCR buffer with 1 mM deoxynucleotide triphosphate, 3 mM of primers (Her-2-C and Her-2-D) and 2.5 units platinum Taq DNA polymerase. The nested RT-PCR was performed using a modified touchdown program as previously described [START_REF] Bozionellou | Trastuzumab administration can effectively target chemotherapyresistant cytokeratin-19 messenger RNA-positive tumor cells in the peripheral blood and bone marrow of patients with breast cancer[END_REF]. All PCR products were electrophoresed in agarose 2% gel, stained with ethidium bromide and photographed under UV conditions. In order to determine the sensitivity of the assay, MCF-7, T47D and SKBR3 cells were mixed with normal PBMCs in a cell ratio ranging from 1:10 to 1:10 6 , and total RNA was extracted from these cell dilutions and tested for HER2 mRNA by nested RT-PCR.

Double staining for cytokeratin and HER2 expression and confocal laser scanning microscopy

Alexa Fluor anti-rabbit IgG and FITC anti-mouse IgG were obtained from Molecular Probes, Inc. (Eugene, Oregon, USA). Polyclonal anti-pan-cytokeratin antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and anti-HER2 monoclonal antibody from Oncogene (Calbiochem, Germany). For morphological studies by confocal laser microscopy, cytospins of mononuclear cells from patients with breast cancer were fixed with cold aceton:methanol 9:1 (vol/vol) for 20 min and stained for cytokeratins with polyclonal anti-rabbit antibody and for HER2 with monoclonal anti-mouse antibody for 45 min each. Subsequently, cells were incubated with secondary antibodies, Alexa Fluor anti-rabbit IgG and FITC-anti-mouse IgG for 45 min each. Slides were analyzed using a confocal laser scanning microscopy module (Leica, Lasertechnik, Heidelberg, Germany).

Fluorescence in situ hybridization for HER2

Cytospins with PBMCs (5 9 10 5 cells/slide) were prepared and stored at -70°C until used. Then, frozen slides were left at room temperature for 15 min, immersed in 70% acetic acid for 30 s, washed and dehydrated in ETOH, immersed in cold acetone for 2 min and air dried. Thereafter, they were placed in 29 SCC solution (pH 5.3) for 2 min at 73°C. Proteolysis with pepsin was performed for 5 min at 37°C and, the slides were placed in 29 SSC solution for 1 min at room temperature with post fixation in formalin buffer solution for 5 min and in 29 SSC for 1 min and dehydrated in ETOH. For hybridization the PathVysion HER2 DNA probe (Vysis, Downers, IL, USA) was used at 85°C for 2 min and overnight at 37°C. The next morning, slides were placed in the post-hybridization wash buffer for 2 min at 72°C, 10 ll of DAPI were added and the covered slides were stored at -20°C. The microscopic examination was performed using an Axioplane 2 Nikon microscope with Vysis filters and the images were processed with the Metasystem's ISIS FISH imaging system (Alltussheim, Germany). For HER2/neu probe we used single-band filters for orange, green and DAPI. Cells with more than four orange signals or a ratio of HER2/neu orange signals to CEP 17 green signals [2 in cases of aneuploidy were considered as FISH HER2-positive.

Statistical analysis

The main tools of analysis were logistic regression and the Cox proportional hazards model [START_REF] Armitage | Statistical methods in medical research[END_REF] for outcomes related to point events and time variables, respectively. To select those factors with an independent significant influence on outcomes, both analyses were carried out in a stepwise (unconditional backward) fashion [START_REF] Armitage | Statistical methods in medical research[END_REF]. Before the application of these methods, univariate analyses were performed for a preliminary exploration of marked associations. Univariate analyses included contingency tables, t or Mann-Whitney U tests, log-rank tests, and simple Cox regression analyses [START_REF] Armitage | Statistical methods in medical research[END_REF].

Results

Sensitivity and specificity of nested PCR for HER2 mRNA? cells in the peripheral blood

In preliminary experiments, RNA extracted from the MCF-7, T47D, SKBR3, MDA-415, ARH-77 and COLO-205 cells was amplified by nested PCR using the set of HER2 primers as described in Materials and Methods. The MCF-7, T47D and SKBR3 cell lines were consistently positive (amplified product of 147 bp; Fig. 1, lanes 1-3), whereas the MDA-415, ARH-77 and COLO-205 were negative (Fig. 1, lanes 4-6); in addition, no HER2 amplified transcripts could be detected by nested RT-PCR using mRNA from SKBR3 amplified in the absence of Taq polymerase (Fig. 1, lane 7) as well as from genomic DNA (Fig. 1, lane 8), indicating that any contaminating DNA would not amplify using the above mentioned pair of primers.

The sensitivity of the detection of HER2 mRNA-positive cells was evaluated by mixing MCF-7 or SKBR3 cells with PBMCs from healthy blood donors in a cell ratio ranging from 0: 10 6 to 10 6 : 0 cells, which mimics the clinical setting for the detection of occult tumor cells in patients' peripheral blood. A representative experiment using the SKBR3 cell line showing a dose-dependent 147 bp signal corresponding to the HER2 mRNA, is presented in Fig. 2; conversely, the 154 bp signal, which corresponds to the b-actin gene, gave the same intensity in all dilutions (Fig. 2). In 3 of 3 identical experiments, this assay could detect 1 MCF-7 cell among 10 5 and 1 SKBR3 cell among 10 6 normal PBMCs.

No HER2 mRNA-positive cells could be detected in the peripheral blood of 31 healthy female blood donors, 12 patients operated for benign breast fibroadenomas as well as 20 patients with colorectal cancer; conversely, HER2 mRNA-positive cells could be detected in the blood of 18 out of 61 (29.5%) patients with advanced breast cancer. All samples obtained from healthy female blood donors, patients with colorectal cancer and patients with benign fibroadenomas or malignant breast tumors were positive for b-actin mRNA when tested with RT-PCR, indicating the presence of intact RNA and successful first-strand cDNA preparation.

Detection of peripheral blood HER2-positive cells by immunofluoresence

The presence of HER2(?) cells was further investigated by double staining experiments of cytospin smears, prepared from PBMCs of HER2 mRNA-positive patients, using anticytokeratins and anti-HER2 antibodies and confocal laser microscopy analysis. Figure 3 demonstrates representative CK-and HER2-positive cells (panel a and b, respectively) as well as double-stained CK ? /HER2 -and CK ? /HER2 ? cells (panel C and D, respectively); as it was expected, the HER2 protein was localized in the cellular membrane while cytokeratin filaments were distributed all over the cytoplasm. HER2-positive cells could be detected by immunostaining in 15 (33%) of 45 patients; those included 14 of 28 cytokeratin-positive and one of 17 cytokeratinnegative patients. However, the expression of HER2 on CK-positive cells was heterogeneous; in some patients all cytokeratin-positive cells were also HER2-positive whereas in others only a proportion of cytokeratin-positive cells co-expressed the HER2 molecule. In order to further characterize the HER2-expressing cells, peripheral blood smears from 5 patients, who had detectable HER2 mRNApositive cells, were analyzed by FISH for HER2 gene amplification. A typical FISH HER2-positive case demonstrating gene amplification with a HER2 to CEP17 ratio [4 is shown in Fig. 4.

Detection of peripheral blood HER2 mRNA-positive CTCs in patients with operable breast cancer

Table 1 indicates the clinical characteristics of the 216 patients with stage I-II breast cancer according to the presence of HER2 mRNA-positive cells before the initiation of any systemic adjuvant treatment. The median age of the patients was 55 years and most of them (62.5%) were postmenopausal. One hundred and four (48.1%) patients had histological grade III tumors, measuring C2 cm (in 79.4% of them) while 141 (65.3%) presented lymph node involvement; in addition, ER-positive and HER2-positive (3?) tumors had 126 (60.3%) and 24 (11.1%) of the patients, respectively (Table 1). The median follow-up time for the entire group of patients was 78 months (range, 5-108).

Fig. 3 Confocal laser microscopy analysis of PBMCs cytospin smears prepared from breast cancer patients using anti-cytokeratins with Alexa Fluor (red colour) and anti-HER2 antibodies with FITC (green colour) a CK? cells stained with anti-cytokeratin antibody (9600), b HER2? cells stained with anti-HER2 antibody (9600), c Double stained CK?/HER2cells (9600), d Double stained CK?/HER2? cells (9600) Fig. 4 Two breast cancer cells with HER2/neu gene copy amplification. Red dots represent the HER2/neu signal and green the CEP 17. The HER2 to CEP17 ratio is [START_REF] Mansi | Bone marrow micrometastases in primary breast cancer: prognostic significance after 6 years' follow-up[END_REF] Circulating HER2 mRNA-positive cells could be detected in 53 (24.5%) out of 216 patients. In univariate analyses, there was no statistically significant association between the detection of HER2 mRNA-positive cells and the patients' menstrual status, stage of the disease, size and differentiation grade of the tumor, number of involved lymph nodes, estrogen or progesterone positivity and HER2 status of the primary tumor (Table 1).

Clinical relevance of the detection of peripheral blood HER2 mRNA-positive CTCs in patients with early breast cancer

Relapse

During the follow-up period, 51 (23.6%) patients developed distant metastases. The incidence of clinical relapses was significantly higher in patients with detectable HER2positive CTCs compared with those without before the initiation of any systemic treatment (49.1 vs. 15.3%; P = 0.001) (Table 2). The median disease-free interval (DFI) for patients with detectable HER2 mRNA-positive CTCs was 81 months (range, 7-104) whereas it has not been reached (range, 5-100 months) for patients without HER2 mRNA-positive CTCs (log-rank test; P \ 0.0001) (Fig. 5a). Univariate analysis including standard clinicopathologic parameters demonstrated that the presence of HER2 mRNA-positive CTCs [Hazard ratio (HR): 3.717, 95% CI 2.15-6.44; P \ 0.0001], the high histology grade (HR: 2.254, 95% CI 1.25-4.08; P = 0.007), C4 involved axillary lymph nodes (HR: 2.292, 95% CI 1.32-3.99; P = 0.003) were significantly associated with decreased DFI (Table 3). Multivariate analysis demonstrated that the presence of HER2 mRNA-positive CTCs, (HR: 4.045, 95% 4).

Survival

During the follow-up period, 30 (13.8%) patients died because of disease progression. The incidence of death was 26.4% (14 out of 53 patients) and 9.8% (16 out of 163 patients) in patients with and without detectable HER2 mRNA-positive CTCs, respectively (P = 0.005) (Table 2). The median overall survival times for patients with and without detectable HER2 mRNA-positive CTCs have not been reached (range, 10-106) and (range, 5-108), respectively (log-rank test, P = 0.004; Fig. 5b). As shown in Table 3, the univariate analysis revealed that the detection of HER2 mRNA-positive CTCs (HR: 2.740; 95% CI 1.34-5.61; P = 0.006) and the involvement of four or more axillary lymph nodes (HR: 2.926; 95% CI, 1.43-6.00; P = 0.003) were significantly associated with decreased overall survival. Multivariate analysis revealed that detection of peripheral blood HER2 mRNA-positive CTCs (HR: 2.761, 95% CI: 1.35-5.66; P = 0.003) and involvement of C4 axillary lymph nodes (HR: 2.947, 95% CI: 1.44-6.04; P = 0.006) were independent prognostic factors associated with a decreased overall survival (Table 4).

Discussion

The early tumor cell dissemination away from the primary tumor as well as the failure of adjuvant treatment to eradicate them may account for the high risk of relapse in patients with operable solid tumors [START_REF] Pantel | Detection and clinical importance of micrometastatic disease[END_REF]. Several studies have shown that the detection of bone marrow (DTCs) [START_REF] Braun | Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II or III breast cancer[END_REF][START_REF] Diel | Micrometastatic breast cancer cells in bone marrow at primary surgery: Prognostic value in comparison with nodal status[END_REF][START_REF] Mansi | Bone marrow micrometastases in primary breast cancer: prognostic significance after 6 years' follow-up[END_REF] or peripheral blood [CTCs; [START_REF] Stathopoulou | Molecular detection of cytokeratin-19-positive cells in the peripheral blood of patients with operable breast cancer: Evaluation of their prognostic significance[END_REF][START_REF] Xenidis | Predictive and prognostic value of peripheral blood cytokeratic-19 mRNA positive cells detected by real time PCR in node-negative breast cancer patients[END_REF] tumor cells is associated with an increased risk of relapse and reduced survival in patients with stage I/III breast cancer. In most of the studies, the detection of CTCs was performed using CK19 as a marker of epithelial cells. The development of a multimarker approach for the detection of CTCs may increase the specificity and the sensitivity of this procedure allowing for a more accurate identification of those patients with early breast cancer who are at increased risk for relapse and death.

The HER2 oncoprotein could be a useful marker for the detection of CTCs in patients with breast cancer. Indeed, previous studies have demonstrated that DTCs [START_REF] Solomayer | Comparison of HER2 status between primary tumor and disseminated tumor cells in primary breast cancer patients[END_REF][START_REF] Braun | erbB2 overexpression on occult tumor metastatic cells in bone marrow predicts poor clinical outcome of stage I-II breast cancer patients[END_REF] and CTCs [START_REF] Brandt | Isolation of bloodbored epithelium-derived c-erb-B2 oncoprotein-positive clustered cells from the peripheral blood of breast cancer patients[END_REF][START_REF] Wulfing | HER 2 positive circulating tumor cells indicate poor clinical outcome in stage I to III breast cancer patients[END_REF] frequently express the HER2 oncoprotein. Using a double immunocytochemical staining technique, Solomayer et al. detected HER2 expression on DTCs in 20 out of 137 (14%) breast cancer patients [START_REF] Solomayer | Comparison of HER2 status between primary tumor and disseminated tumor cells in primary breast cancer patients[END_REF] while Braun et al. found co-expression of HER2 in 31 out of 52 (60%) women with CK18-positive DTCs [START_REF] Braun | erbB2 overexpression on occult tumor metastatic cells in bone marrow predicts poor clinical outcome of stage I-II breast cancer patients[END_REF]. Moreover, Brandt et al. found cytokeratin/c-erbB-2 positive clustered CTCs in 19 out of 46 (41%) breast cancer patients [START_REF] Brandt | Isolation of bloodbored epithelium-derived c-erb-B2 oncoprotein-positive clustered cells from the peripheral blood of breast cancer patients[END_REF], and Wulfing et al. reported immunocytochemical detection of cytokeratin?/HER2?CTCs in 48.6% of patients [START_REF] Wulfing | HER 2 positive circulating tumor cells indicate poor clinical outcome in stage I to III breast cancer patients[END_REF]. In the present study we evaluated a nested RT-PCR assay to detect peripheral blood cells expressing HER2 mRNA. Specificity experiments demonstrated that the 147 bp HER2 mRNA transcripts could be identified in breast cancer cells expressing the HER2 molecule (MCF-7, T47D and SKBR3 cells) but not in breast cancer cells which do not express HER2 (MDA-415) or in tumor cells from a colorectal (Colo 205) and myeloma (ARH-77) cell line which, also, do not express HER2. The specificity of the used RT-PCR assay was further demonstrated by the observation that no HER2 mRNA-positive cells could be identified in female healthy blood donors, patients with benign fibroadenomas as well as in patients with colorectal cancer. In addition, the detection of circulating HER2 mRNA-positive cells was independent of the prior surgical excision of the primary tumor since they could be identified in 20% of patients who were enrolled in a neo-adjuvant chemotherapy program before the initiation of any systemic treatment (data not shown) suggesting that HER2 mRNA-positive CTCs were not shed into the circulation during the manipulations for the surgical excision of the primary tumor but rather their detection reflects their potential to migrate through the blood vasculature. It is noteworthy that the sensitivity of the used nested RT-PCR assay was dependent on the cell line (1/10 5 and 1/10 6 normal PBMC for the MCF-7 and SKBR3 cells, respectively) suggesting that the degree of HER2 expression may be an important limiting factor for the detection of HER2 mRNA-positive cells. These findings are in the same range of sensitivity as the RT-PCR assays detecting CK-19 mRNA-positive cells [START_REF] Stathopoulou | Molecular detection of cytokeratin-19-positive cells in the peripheral blood of patients with operable breast cancer: Evaluation of their prognostic significance[END_REF][START_REF] Stathopoulou | Real-time quantification of CK-19 mRNA positive cells in peripheral blood of breast cancer patients using the LightCycler system[END_REF]. Double staining of CTCs with anti-pancytokeratin and anti-HER2 antibodies demonstrated that 50% of the patients with CK-positive cells, also displayed HER2positive cells and, practically, in all but one patient, circulating HER2-positive cells co-expressed cytokeratins. However, in some patients distinct cell populations expressing cytokeratins (CKs) only or CKs and HER2 could be detected. This finding is in agreement with the already described heterogeneity of circulating occult tumor cells [START_REF] Brandt | Isolation of bloodbored epithelium-derived c-erb-B2 oncoprotein-positive clustered cells from the peripheral blood of breast cancer patients[END_REF][START_REF] Wulfing | HER 2 positive circulating tumor cells indicate poor clinical outcome in stage I to III breast cancer patients[END_REF]. In addition, FISH analysis showed that CTCs present HER2/neu gene amplification, indicating the malignant nature of cytokeratin-positive cells as already has been reported [START_REF] Bozionellou | Trastuzumab administration can effectively target chemotherapyresistant cytokeratin-19 messenger RNA-positive tumor cells in the peripheral blood and bone marrow of patients with breast cancer[END_REF][START_REF] Muller | Detection of genetic alterations in micrometastatic cells in bone marrow of cancer patients with fluorescence in situ hybridization[END_REF]. Previous studies have demonstrated that expression of HER2 molecule on DTCs and CTCs was independent of its expression on the primary tumor cells [START_REF] Brandt | Isolation of bloodbored epithelium-derived c-erb-B2 oncoprotein-positive clustered cells from the peripheral blood of breast cancer patients[END_REF][START_REF] Solomayer | Comparison of HER2 status between primary tumor and disseminated tumor cells in primary breast cancer patients[END_REF][START_REF] Braun | erbB2 overexpression on occult tumor metastatic cells in bone marrow predicts poor clinical outcome of stage I-II breast cancer patients[END_REF][START_REF] Wulfing | HER 2 positive circulating tumor cells indicate poor clinical outcome in stage I to III breast cancer patients[END_REF]; the absence of any statistical correlation between the expression of HER2 molecule on the primary tumor cells and the detection of circulating HER2 mRNA-positive cells in our study further confirms the above observations. The reasons for the discrepancy of HER2 status between occult tumor cells and primary tumor cells are not obvious. It has been proposed that these cells may represent an early disseminated subclone of HER2-positive cells of the primary tumor, which is microscopically undetectable because of their very low frequency. Previous studies have demonstrated that cytokeratin/HER2 double-positive clustered peripheral blood cells have a high potential for locomotion suggesting that they might be precursor cells responsible for the development of DTCs [START_REF] Brandt | Isolation of bloodbored epithelium-derived c-erb-B2 oncoprotein-positive clustered cells from the peripheral blood of breast cancer patients[END_REF]; furthermore, HER2positive CTCs have a growth and survival advantage [START_REF] Brandt | Isolation of bloodbored epithelium-derived c-erb-B2 oncoprotein-positive clustered cells from the peripheral blood of breast cancer patients[END_REF]. In addition, it has been shown that HER2 gene amplification can be acquired during breast cancer progression [START_REF] Meng | HER2 gene amplification can be aquired as breast cancer progresses[END_REF]. This hypothesis is further supported by the recently reported 14% discordance of HER2 overexpression between the primary tumor and the corresponding metastases in patients with breast cancer [START_REF] Zidan | Comparison of HER-2 overexpression in primary breast cancer and metastatic sites and its effect on biological targeting therapy of metastatic disease[END_REF]. Despite all the above evidence, our data should be interpreted with caution since different methods have been used to detect HER2 expression in primary tumor cells and circulating HER2 mRNA-positive CTCs.

Based on these data we evaluated the detection of HER2 mRNA as a marker of CTCs in patients with operable breast cancer. Our findings demonstrated that 24.5% of patients with stage I-II breast cancer had detectable HER2 mRNA-positive CTCs before the initiation of any systemic adjuvant treatment. Moreover, the detection of HER2 mRNA-positive CTCs was not associated with other important clinical or pathological tumor parameters (Table 3). This positivity rate for HER2-expressing CTCs is in accordance with previous reports using immunocytochemical detection methods [START_REF] Brandt | Isolation of bloodbored epithelium-derived c-erb-B2 oncoprotein-positive clustered cells from the peripheral blood of breast cancer patients[END_REF][START_REF] Wulfing | HER 2 positive circulating tumor cells indicate poor clinical outcome in stage I to III breast cancer patients[END_REF]. Moreover, the pooled analysis of 4,703 early breast cancer patients reported detection of DTCs in 30.6% of patients at the time of primary surgery [START_REF] Braun | A pooled analysis of bone marrow micrometastasis in breast cancer[END_REF] and in another study co-expression of HER2 was found in 60% of patients presenting CKpositive DTCs [START_REF] Braun | erbB2 overexpression on occult tumor metastatic cells in bone marrow predicts poor clinical outcome of stage I-II breast cancer patients[END_REF]. Therefore, taken together and despite the different detection method used in our study, the HER2 positivity rate is in general agreement with the literature. Interestingly, the detection of HER2 mRNA-positive CTCs revealed important prognostic information since it was associated with reduced DFS and overall survival; moreover, multivariate analysis revealed that the detection of HER2 mRNA-positive CTCs was an independent prognostic factor for reduced DFS and overall survival (Table 4). These findings are in agreement with the prognostic value of the detection of HER2 mRNA-positive CTCs after the completion of adjuvant chemotherapy in the same patient cohort which has already been published [START_REF] Apostolaki | Circulating HER2 mRNA-positive cells in the peripheral blood of patients with stage I and II breast cancer after the administration of adjuvant chemotherapy: evaluation of their clinical relevance[END_REF].

At the present time, the probability of cure for patients with operable breast cancer may be increased by reducing the incidence of distant metastasis. Since chemotherapy cannot in many cases eliminate the disseminated [START_REF] Braun | Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in the bone marrow of high-risk breast cancer patients[END_REF] or/ and the circulating [30] tumor cells, it is important to target these occult tumor cells by using novel therapeutic approaches, such as monoclonal antibodies or other drugs acting on specific molecular targets. In a previous study, the administration of transtuzumab, a humanized anti-HER2 monoclonal antibody, resulted in a significant decrease of the HER2-positive CTCs [START_REF] Bozionellou | Trastuzumab administration can effectively target chemotherapyresistant cytokeratin-19 messenger RNA-positive tumor cells in the peripheral blood and bone marrow of patients with breast cancer[END_REF]. Therefore, prospective trials targeting the residual HER2 mRNApositive occult tumor cells in patients with early stage breast cancer could demonstrate the importance of a ''secondary'' adjuvant therapeutic approach.

Fig. 1

 1 Fig. 1 Nested RT-PCR from total RNA of MCF-7 (lane 1), T47D (lane 2), SKBR3 (lane 3), MDA-415 (lane 4), ARH-77 (lane 5), COLO-205 (lane 6) cells lines, from SCBR3 cells amplified in the absence of Taq polymerase (lane 7) and from SKBR3 genomic DNA (lane 8). A standard b-actin RT-PCR assay was also used to demonstrate the presence of intact RNA

Fig. 5

 5 Fig. 5 Disease-free survival (a) and overall survival (b) of patients with early stage breast cancer according to the detection of HER 2 mRNA-positive CTCs before adjuvant chemotherapy

Table 1

 1 Patient characteristics according to the detection of HER mRNA-positive CTCs before the initiation of adjuvant therapy

	Clinicopathologic parameter	All patients	Pre-chemotherapy		P-value
		(n = 216)	HER2 mRNA ?	HER2 mRNA -
			(n = 53)	(n = 163)
		n (%)	n (%)	n (%)
	Age (median, range)	55 (28-79)	55 (28-74)	55 (30-79)	0.866
	Menopausal status
	Premenopausal	81 (37.5)	21 (39.6)	60 (36.8)	0.745
	Postmenopausal	135 (62.5)	32 (60.4)	103 (63.2)
	Disease stage
	I	68 (31.5)	14 (26.4)	54 (33.1)	0.399
	II	148 (68.5)	39 (73.6)	109 (66.8)
	Tumor size
	\2.0 cm	44 (20.6)	7 (13.2)	37 (22.7)	0.170
	C2.0 cm	172 (79.4)	46 (86.8)	126 (77.3)
	Histology grade
	I/II	112 (51.8)	26 (49.1)	86 (52.8)	0.747
	III	104 (48.1)	27 (50.9)	77 (47.2)
	Axillary lymph nodes
	0-3	153 (70.8)	38 (71.7)	115 (70.6)	0.999
	C4	63 (29.2)	15 (28.3)	48 (29.4)
	Estrogen receptor
	Negative	83 (38.4)	23 (43.4)	60 (36.8)	0.514
	Positive	126 (60.3)	29 (54.7)	97 (59.5)
	Unknown	7 (3.2)	1 (1.9)	6 (3.7)
	Progesteron receptor
	Negative	132 (61.1)	30 (56.6)	102 (62.6)	0.504
	Positive	76 (35.2)	21 (39.6)	55 (33.7)
	Unknown	8 (3.7)	2 (3.8)	6 (3.7)
	HER2/neu expression on primary tumor
	Negative (0-2 ? )	188 (87.0)	47 (88.7)	141 (86.5)	0.803
	Positive (3 ? )	24 (11.1)	5 (9.4)	19 (11.7)
	Unknown	4 (1.9)	1 (1.9)	3 (1.8)

Table 2

 2 Incidence of clinical relapses and disease-related deaths according to the HER2 mRNA-positive cells before the initiation of adjuvant chemotherapy

	HER2 mRNA status	Relapse n (%)	Non-relapse n (%)	P-value	Dead n (%)	Alive n (%)	P-value
	Negative (n = 163)	25 (15.3)	138 (84.7)	0.001	16 (9.8)	147 (90.2)	0.005
	Positive (n = 53)	26 (49.1)	27 (50.9)		14 (26.4)	39 (73.6)

Table 3

 3 Univariate analysis for DFS and overall survival of patients with early breast cancer

		Parameter	Hazard ratio	95% CI	P-value
		DFS
		Menopausal status (post vs. pre)	1.399	0.774-2.527	0.266
		Disease stage (II vs. I)	1.666	0.872-3.183	0.122
		Tumor size ([2 vs. B2 cm)	2.191	0.934-5.136	0.071
		Histology grade (III vs. I/II)	2.254	1.247-4.076	0.007
		Axillary lymph nodes (C4 vs. \4)	2.292	1.318-3.986	0.003
		Estrogen receptors (negative vs. positive)	1.713	0.989-2.967	0.055
		Progesteron receptors (negative vs. positive)	1.090	0.607-1.958	0.773
		HER2 mRNA (positive vs. negative)	3.717	2.145-6.442	\0.0001
		Overall survival
		Menopausal status (post vs. pre)	1.094	0.520-2.299	0.813
		Disease stage (II vs. I)	2.534	0.970-6.623	0.058
		Tumor size ([2 vs. B2 cm)	7.954	1.083-58.395	0.041
		Histology grade (III vs. I/II)	1.559	0.737-3.297	0.246
		Axillary lymph nodes (C4 vs. \4)	2.926	1.427-6.001	0.003
		Estrogen receptors (negative vs. positive)	1.965	0.959-4.028	0.065
		Progesteron receptors (negative vs. positive)	1.488	0.659-3.362	0.339
		HER2 mRNA (positive vs. negative)	2.740	1.337-5.614	0.006
	Table 4 Multivariate analysis for DFS and overall survival of	Parameter	Hazard ratio	95% CI	P-value
	patients with early breast cancer	DFS
		HER2 mRNA pre-chemotherapy (positive)	4.045	2.285-7.161	0.0001
		Axillary lymph nodes (C4 positive)	5.743	2.171-15.195	0.0004
		Histology grade (III)	4.246	1.777-10.143	0.001
		Overall survival
		HER2 mRNA pre-chemotherapy (positive)	2.761	1.347-5.658	0.003
		Axillary lymph nodes (C4 positive)	2.947	1.437-6.045	0.006

Breast Cancer Res Treat (2009) 117:525-534

Acknowledgements Supported in part by grants from the Cretan Association for Biomedical Research (CABR), Pfizer Hellas and cofunded by the European Social Fund and National Resources.


Josefa Salgado
email: jsalgadog@unav.es

Carmen Gil

A E Maitane

Robles Ae

Jesu ´s Garcı ´a-Foncillas

Á C Gil

Á M Robles

Á J Garcı

A novel BRCA1 mutation in a Spanish patient with ovarian cancer

Keywords: BRCA1, Direct automatic sequencing, Germline mutation, Ovarian cancer

We report a novel germline 3536delT mutation in BRCA1 gene, detected in a 43-year-old woman with bilateral ovarian adenocarcinoma. The mutation originates a premature stop codon at position 1154 and has not been documented in any published report to the best of our knowledge.

Introduction

Breast cancer is the most commonly occurring cancer among women. It accounts for 22% of all female cancers and the estimated annual incidence of breast cancer worldwide is about one million cases. Many risk factors have been identified but a positive family history remains among the most important ones established, with firstdegree relatives of the patient having approximately twofold elevated risk. It is currently estimated that approximately 20-25% of this risk is explained by known breast cancer susceptibility genes 1 and 2 (BRCA1 and BRCA2). BRCA1 is a tumor suppressor gene which appears to be implicated, among others, in DNA repair, cell proliferation regulation and apoptosis. All these issues have been discussed in detail in several reviews [START_REF] Venkitaraman | Cancer susceptibility and the functions of BRCA1 and BRCA2[END_REF][START_REF] Narod | BRCA1 and BRCA2: 1994 and beyond[END_REF][START_REF] Oldenburg | Genetic susceptibility for breast cancer: how many more genes to be found?[END_REF]. Firstdegree relatives of patients, that are BRCA1 mutations carriers, have 50% probability of inheriting mutations. Women will develop breast carcinoma in 50-80% of cases and an ovarian tumor in 20-40%, while in males the risk of develop a breast cancer is less than 6%. Moreover, women who are carriers of a known mutation show a 40-60% risk of developing a second breast tumor, while men with BRCA1 mutations are at higher risk, not only for tumors of the prostate or colon, but also of the pancreas. According to the BIC (Breast Cancer Information Core) database, since the mutations are distributed widely all over the coding regions of the BRCA1 gene, in order to identify its mutations, the whole gene must be sequenced. This report concerns a Spanish woman with a family history of BRCA associated breast cancer, in whom a novel germline mutation was identified.

Case report

The patient, who is indicated by an arrow in pedigree (Fig. 1a), was a multiparous, non-Ashkenazi woman, 43 years old, who, 3 years previously, had been diagnosed and treated for a bilateral ovarian adenocarcinoma in other center. During genetic counseling in our center, the genealogical tree of the patient was reconstructed going back for three generations, and revealed a history of grade I breast cancer, together with several other types of tumors including breast and ovarian cancer in other family members of grade II. The clinical history of this patient leads us to consider that the ovarian adenocarcinoma diagnosed could be BRCA1 and/or BRCA2-associated. Genomic DNA was obtained from a peripheral blood sample and automatically extracted (MagNaPure, Roche). Direct sequencing of the complete BRCA1 and BRCA2 genes was done by standard methods on an automated sequencer ABI PRISM Ò 3130XL Genetic Analyzer (Applied Biosystems) and compared to a consensus wild-type sequence constructed for each exon and partial intronic regions of the genes (Genbank No U14680, BRCA1 and Genbank NM_000059, BRCA2). A 3536delT frameshift mutation in exon [START_REF] Saal | Recurrent gross mutations of the PTEN tumor suppressor gene in breast cancers with deficient DSB repair[END_REF], not yet reported in the BIC database, was detected in BRCA1 (Fig. 1b). The deletion was confirmed by repeated analysis including reverse-primer sequencing. The analysis of exon 11 of the gene BRCA1 was offered to other members of the family being the patient's sister, diagnosed with breast cancer at the age of 41, participative in this study. The genetic analysis showed exactly the same 3536delT frameshift mutation in the sister. Furthermore, a screening of the 3636delT mutation was done in a group of 134 patients with hereditary and familiar ovarian and/or breast cancer in our center. We did not find the mutation in the other families studied.

Discussion

BRCA1 gene encodes an 1863 amino acid protein with two main protein-interaction domains: an N-terminal RING finger and two tandem BRCT domains at the Cterminal end [START_REF] Koonin | BRCA1 protein products ... Functional motifs[END_REF]. The Stop codon at position 1154 detected in our patient would, in principle, generate a truncated protein that lack both BRCT domains. We have previously detected novel BRCA1 variants [START_REF] Salgado | Structure-based assessment of BRCA1 and BRCA2 mutations in a small Spanish population[END_REF], but this is the first case we find a mutation that clearly originates a truncated BRCA1 protein and, moreover, we have the complete pedigree information of the family. In vivo, BRCA1 forms a stable heterodimer with the structurally related protein BARD1. The resulting heterodimer functions as a potent E3 ubiquitin ligase [START_REF] Brzovic | Binding and recognition in the assembly of an active BRCA1/BARD1 ubiquitin-ligase complex[END_REF][START_REF] Baer | The BRCA1/BARD1 heterodimer, a tumor suppressor complex with ubiquitin E3 ligase activity[END_REF]. Different studies have shown that two tandem BRCT motifs, such as those arrayed at the C-termini of BRCA1 and BARD1, can form a structural domain that specifically binds phosphorylated peptide sequences [START_REF] Manke | BRCT repeats as phosphopeptide-binding modules involved in protein targeting[END_REF][START_REF] Yu | The BRCT domain is a phospho-protein binding domain[END_REF]. Therefore, the BRCT motifs of BRCA1 can control distinct cell cycle checkpoints through phosphor-dependent interactions with different cellular proteins. In this way, the transient G2/M checkpoint, induced by ionizing radiation, is dependent on recognition of S327-phosphorylated isoforms of the CtIP protein by the BRCT motifs of BRCA1 [START_REF] Yu | DNA damage-induced cell cycle checkpoint control requires CtIP, a phosphorylation-dependent binding partner of BRCA1 C-terminal domains[END_REF]. The interaction between these same motifs and the S990phosphorylated BACH1/BRIP1/FancJ protein is required for activation of the G2 accumulation checkpoint [START_REF] Yu | The BRCT domain is a phospho-protein binding domain[END_REF]. On the other hand, it is known that BRCA1 is involved in double-strand DNA break (DSB) repair. Recently it has been shown that loss of PTEN is highly associated with basal-like breast cancer subtype, in both human sporadic and BRCA1-associated hereditary breast cancers. Frequent ''gross'' PTEN mutations (intragenic chromosome breaks, inversions, deletions and micro copy number aberrations) were specifically identified in BRCA1-deficient tumors [START_REF] Saal | Recurrent gross mutations of the PTEN tumor suppressor gene in breast cancers with deficient DSB repair[END_REF]. BRCA1 hereditary dysfunction is thought to be tumorigenic primarily owing to defective BRCA1-dependent repair, which precipitates and accumulation of secondary mutations in genes as PTEN. Certain BRCA1 mutations are found with greater frequency in specific ethnic groups, where a restricted number of BRCA1 ''founder mutations'' have been identified [START_REF] Hodgson | A survey of the current clinical facilities for the management of familial cancer in Europe. European Union BIOMED II demonstration project: familial breast cancer: audit of a new development in medical practice in European centres[END_REF]. Further extension of the analysis of the mutation we have found to other families in the same geographical area, will make it possible for us to describe the phenotype features associated. At the same time, a study conducted on a population with the same mutation might be of help in determining disease penetrance.

Fig. 1

 1 Fig. 1 (a) Pedigree with the case reported indicated by an arrow. Black circle indicates women affected by cancer. Diagonal slash indicates deceased. White square and circle indicates man and woman not affected by disease. Br = breast cancer, Ov = ovarian cancer, Colon = colon cancer. (b) Sequence analysis of the exon11 of BRCA1 gene in the case reported. The arrow indicates the T deleted at position 3536

Breast Cancer Res Treat (2009) 113:71-73

Acknowledgments We are grateful to the patients, and their families, for their participation in our research project. We thank other members of our Clinical Genetics Unit at the University Clinic of Navarra for their support and encouragement.


Ben Calster

Isabelle Vanden Bempt

Maria Drijkoningen

Nathalie Pochet

Jiqiu Cheng

Sabine Huffel

Wouter Hendrickx

Julie Decock

Huei-Jean Huang

Karin Leunen

Ben Van

Calster Ae

Isabelle Vanden

Bempt Ae

Maria Drijkoningen Ae

A E Jiqiu

Cheng Ae

Sabine Van Huffel

A E Wouter

Hendrickx Ae

A E Huei

Jean Huang

A E Karin

Leunen Ae

Frederic Amant

A E Patrick

Berteloot Ae

Robert Paridaens

A E Hans

Wildiers Ae

Erik Van Limbergen

A E Caroline

Weltens Ae

Dirk Timmerman

A E Toon

Van Gorp

A E Ann

Smeets Ae

Walter Van Den Bogaert

A E Ignace

Vergote Ae

Marie-Rose Christiaens

Ae Patrick Neven

B Van Calster

Á I Vanden Bempt

Á M Drijkoningen

Á N Pochet

Á J Cheng

Á S Van Huffel

Á W Hendrickx

Á J Decock

H.-J Huang

Á K Leunen

Á F Amant

Á P Berteloot

Á R Paridaens

Á H Wildiers

Á E Van Limbergen

Á C Weltens

Á D Timmerman

Á T Van Gorp

Á A Smeets

Á W Van Den Bogaert

Á I Vergote

M.-R Christiaens

Á P Neven

Axillary lymph node status of operable breast cancers by combined steroid receptor and HER-2 status: triple positive tumours are more likely lymph node positive

Keywords: HER-2, Steroid receptors, Lymph node, Breast cancer

Introduction

In breast cancer, the three predictive markers ER, PR and HER-2 have an independent prognostic value [START_REF] Esteva | Prognostic molecular markers in early breast cancer[END_REF]. HER-2 is over-expressed in about 15-20% of cases [START_REF] Akiyama | The product of the human c-erbB-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity[END_REF][START_REF] Slamon | Human breast cancer: correlation of relapse and survival with amplification of the HER-2 oncogene[END_REF][START_REF] Slamon | Studies of the HER-2 proto-oncogene in human breast and ovarian cancer[END_REF]. Its prognostic significance has been obscured by an association with other poor prognostic markers like tumour grade, S phase fraction and a negative steroid receptor status [START_REF] Slamon | Human breast cancer: correlation of relapse and survival with amplification of the HER-2 oncogene[END_REF][START_REF] Slamon | Studies of the HER-2 proto-oncogene in human breast and ovarian cancer[END_REF][START_REF] Paik | Pathologic findings from the National Surgical Adjuvant Breast and Bowel Project: prognostic significance of erbB-2 protein overexpression in primary breast cancer[END_REF][START_REF] Ross | The HER-2 gene and protein in breast cancer 2003: biomarker and target of therapy[END_REF]. Furthermore, differences in HER-2 detection methods and cutoff values, the small numbers of patients in the HER-2 + cohorts, confounding effects of treatment and short length of follow-up made the interpretation of HER-2 as a prognostic marker difficult. However, with short follow-up it became clear that HER-2 is prognostic for disease free and overall survival in node positive and with longer follow-up also in node negative breast cancers [START_REF] Ross | The HER-2 gene and protein in breast cancer 2003: biomarker and target of therapy[END_REF]. ER is expressed in 80-90% of all breast cancers [START_REF] Elledge | Clinical aspects of estrogen and progesterone receptors[END_REF]. The ER is prognostic but annual recurrence of breast cancer by ER status varies over time [START_REF] Esteva | Prognostic molecular markers in early breast cancer[END_REF][START_REF] Elledge | Clinical aspects of estrogen and progesterone receptors[END_REF][START_REF] Saphner | Annual hazard rates of recurrence for breast cancer after primary therapy[END_REF]. The PR is expressed in 70-80% of all breast cancers [START_REF] Elledge | Clinical aspects of estrogen and progesterone receptors[END_REF] and also PR is considered as a time-dependent prognostic factor in ER + breast cancers [START_REF] Esteva | Prognostic molecular markers in early breast cancer[END_REF][START_REF] Grann | Hormone receptor status and survival in a population-based cohort of patients with breast carcinoma[END_REF]. Steroid and HER-2 receptors are strongly associated [START_REF] Zeillinger | HER-2 amplification, steroid receptors and epidermal growth factor receptor in primary breast cancer[END_REF][START_REF] Cui | Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase[END_REF][START_REF] Lee | Cross-talk among estrogen receptor, epidermal growth factor, and insulin-like growth factor signaling in breast cancer[END_REF][START_REF] Konecny | Quantitative association between HER-2/neu and steroid hormone receptors in hormone receptor-positive primary breast cancer[END_REF][START_REF] Huang | Association between tumour characteristics and HER-2 by immunohistochemistry in 1362 women with primary operable breast cancer[END_REF][START_REF] Cui | Biology of progesterone receptor loss in breast cancer and its implications for endocrine therapy[END_REF]. The joint expression of steroid receptors has a greater predictive value than the expression of each receptor on its own. Furthermore, the joint IHC expression of ER, PR and HER-2 is prognostic as it reflects the prognostic value of breast cancer phenotypes defined by micro-array studies [START_REF] Sorlie | Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications[END_REF][START_REF] Sorlie | Repeated observation of breast tumor subtypes in independent gene expression data sets[END_REF][START_REF] Fan | Concordance among geneexpression-based predictors for breast cancer[END_REF][START_REF] Carey | Race, breast cancer subtype and survival in the Carolina breast cancer study[END_REF]. Breast cancers are therefore better presented by their combined receptor expression than by each receptor status alone.

The ALN status is one of the best independent prognostic factors for disease free and overall survival of breast cancer but some tumours are already systemic even if ALN are not involved [START_REF] Esteva | Prognostic molecular markers in early breast cancer[END_REF][START_REF] Fisher | Biological and clinical considerations regarding the use of surgery and chemotherapy in the treatment of primary breast cancer[END_REF]. Women with an ER -, ER + PR -or HER-2 + breast cancer experience a shorter disease free period than women with an ER + PR + HER-2 -breast cancer despite the fact that these tumours are not necessarily more likely ALN positive [START_REF] Dunnwald | Hormone receptor status, tumor characteristics, and prognosis: a prospective cohort of breast cancer patients[END_REF]. Rates of ALN involvement in the different prognostic breast cancer subgroups based on their combined IHC expression of steroid receptors and HER-2 status have not been systematically described. We examined the frequency of ALN involvement for these different prognostic breast cancer subtypes. Such differences may not necessarily reflect differences in breast cancer outcome as variables like treatment are important but may differentiate breast cancers on the base of their steroid receptor and HER status for lymphatic spread and local aggressiveness.

Materials and methods

Charts from 2227 cases with an operable breast cancer, treated since January 2000 at Leuven University Hospital, Belgium, were evaluated. All women had a classical ALN dissection, mostly level I-II. After June 2003, patients with a cT1 tumour had the sentinel lymph node procedure. A classical ALN dissection was only performed if the sentinel node was involved. Tumour grading was performed according to the Ellis and Elston grading system [START_REF] Elston | Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up[END_REF]. Lymph nodes were examined by H&E staining using 3 sections per node; sentinel lymph nodes and those from lobular breast cancers classified as negative on H&E were additionally stained with epithelial markers. Cases were examined by IHC for the expression of ER, PR and HER2 (NLC-ER-6F11, NCL-PR-312 and CB11 respectively, Novocastra Laboratories, Newcastle-on-Tyne, UK). Since 2005, highly sensitive rabbit monoclonal antibodies are used for the assessment of ER and PR expression (SP1 and SP2 respectively, Labvision Corporation, Fremont, CA, USA). IHC staining was performed according to standard procedures for clinical purposes. Briefly, 4 lm thick paraffin sections were cut. Heat-induced epitope retrieval was carried out in a calibrated water bath (95-99°C) and antibody complexes were visualized by EnVision+ (DakoCytomation, Glostrup, Denmark) and diaminobenzidine. For ER and PR, any nuclear staining of invasive tumor cells was considered as positive. HER-2 immunostaining was scored according to the guidelines for HercepTestÒ and an equivocal HER-2 status was further investigated by Fluorescence in situ Hybridisation or FISH (PathVision, Vysis, Downers Grove, IL, USA). All clinico-pathological data of the patient were entered in a breast cancer database.

The following factors predicting the axillary lymph node involvement were examined: patient's age at diagnosis (B versus [50 years), maximal microscopic tumour size (B versus [20 mm), tumour grade (grade 1 to 3), ER, PR and HER-2 status (negative versus positive). The receptor status variables were combined with NNN, NNP, PNN, PNP, PPN and PPP as possible values where PPP stands for triple positive or ER + PR + HER-2 + breast cancers.

Likelihood ratio v 2 -tests were used for univariate analysis and logistic regression for multivariate analysis. We considered the following variables to predict lymph node involvement in all tumours: patient's age at diagnosis, largest microscopic tumour size, tumour grade, ER-status, PR-status, HER-2 status and the combined receptor status with 6 predefined possible values NNN, NNP, PNN, PNP, PPN and PPP. For the multivariate logistic regression model, we considered patient's age, tumour size, tumour grade, and the combined steroid and HER-2 receptor status as possible independent predictors. The presence of multicollinearity was investigated and the assumption of linearity in the logit was checked [START_REF] Hosmer | Applied logistic regression[END_REF]. It was also checked whether any strong interaction would affect the possible independent effect of group. Finally, regression diagnostics were investigated as explained by Hosmer and Lemeshow [START_REF] Hosmer | Applied logistic regression[END_REF]. One should always be careful with P-values, therefore more attention was paid to odds ratios and their confidence intervals [START_REF] Sterne | Sifting the evidence-what's wrong with significance tests[END_REF]. In particular, in large datasets p-values can become very small while the effect is actually small and not highly significant in a clinical sense.

Results

Table 1 showed the clinicopathological features of all 2227 cases as well as univariate results. In univariate analysis a large tumour size (OR per cm increase in size = 1.580) and a high tumour grade (compared with grade 1; OR for grade 2 = 1.896 and for grade 3 = 2.900) were strongly related to positive ALN status. A positive HER-2 status was also related to a positive ALN status [35.8% HER-2 - vs 45.7% HER-2 + ; OR = 1.508] but this effect was mainly due to the relationship between triple positive tumours and positive ALN status [35.7% non-PPP vs 56.2% PPP; OR = 2.309]. ER and PR expression did not predict the ALN status. Finally, there appeared to be only a small negative effect of age on ALN status (OR per 10 year increase in age = 0.914). In multivariate logistic regression analysis, high tumour size (OR per cm increase in size = 1.540), high tumour grade (OR per grade increase = 1.384) and being PPP were predictive for a positive ALN status. Table 2 presents the final logistic regression analysis: PPP tumours were more strongly related to positive ALN status than any other combined phenotype. The OR ranged from 1.883 (PPP versus PNN) to 3.471 (PPP versus PNP). Age was also related to ALN status, albeit not in a straightforward way: age was related to negative ALN status for women up to 70 years while it was related to positive ALN for women older than 70 years. The Hosmer-Lemeshow test for goodness-of-fit gave a P-value of 0.2136. No interactions affected the effect of being PPP on ALN status.

Table 3 illustrated the proportion of grade 3 tumours, median tumour size and patient's age at diagnosis in different ER/PR/HER-2 phenotypes. Women with a triple positive breast cancer were more likely to be diagnosed at a younger age than tumours of any other phenotype. Tumours in this group were often high grade and larger. The difference between triple positive tumours on the one hand, and triple negative tumours and tumours overexpressing HER-2 with a negative PR-status (ER + PR -HER-2 + and ER -PR - HER-2 + ) on the other hand is less clear.

Finally, Table 4 represented the ALN status comparing PPP cases with non-PPP cases adjusting for tumour grade. When only considering grade 3 lesions, the sample proportion of ALN positive tumours was 43% higher for PPP cases than for non-PPP cases (relative risk = 61.4/42.8 = 1.43).

Discussion

We found that triple positive breast cancers-ER + PR +- HER-2 + -had an odds of ALN involvement of 130.9% higher than breast cancers of any other ERPRHER-2 phenotype (OR = 2.309). Triple positive breast tumours were diagnosed at a younger age and tumour characteristics like tumour size and grade could have been the main reason for this higher probability of ALN involvement [START_REF] Ravdin | Prediction of axillary lymph node status in breast cancers by prognostic indicators[END_REF][START_REF] Gajdos | Lymphatic invasion, tumor size, and age are independent predictors of axillary lymph node metastases in women with T1 breast cancers[END_REF][START_REF] Gann | Factors associated with axillary lymph node metastasis from breast carcinoma[END_REF][START_REF] Silverstein | Predicting axillary nodal positivity in 2282 patients with breast carcinoma[END_REF][START_REF] Chua | Frequency and predictors of axillary lymph node metastases in invasive breast cancer[END_REF][START_REF] Viale | Predicting the status of axillary sentinel lymph nodes in 4351 patients with invasive breast carcinoma treated in a single institution[END_REF][START_REF] Patani | Predictors of axillary lymph node metastasis in breast cancer: a systemic review[END_REF]. However, the multivariate logistic regression analysis identified ''being triple positive'' as an independent predictor for lymph node involvement. Our observation is another proof of the heterogeneity of the natural history of breast carcinomas acquiring new insights in breast cancer biology and tumour cells dissemination.

HER-2 over-expression in operable breast cancer has only in 8 out of 23 studies been associated with a positive ALN-status [START_REF] Huang | Association between tumour characteristics and HER-2 by immunohistochemistry in 1362 women with primary operable breast cancer[END_REF][START_REF] Revillion | ERBB2 oncogene in human breast cancer and its clinical significance[END_REF]. Reasons why a predictive role of HER-2 for ALN involvement has not been reported are that subgroup analysis by combined steroid receptor expression was not done, ER + PR + HER-2 + tumours are rare (in this series only 6.2% of all cases) and HER-2 as a predictor for the ALN status is overlapping with other poor prognostic factors (high tumour grade, a large tumour size, younger age at diagnosis) already predicting ALN involvement [START_REF] Patani | Predictors of axillary lymph node metastasis in breast cancer: a systemic review[END_REF]. Therefore, our results need being cross checked with other large series. Bartlett et al. recently also identified significant interactions between HER-2, ER-expression and lymph node involvement [START_REF] Bartlett | Human epidermal growth factor receptor 2 status correlates with lymph node involvement in patients with estrogen receptor (ER)-negative, but with grade in those with ER-positive early-stage breast cancer suitable for cytotoxic chemotherapy[END_REF]. They found a different probability of HER-2 over-expression with lymph node involvement by ER-status in a very high risk group of early-stage breast cancers selected for adjuvant chemotherapy. Our confirmatory findings of this interaction in a non-selected patient group of consecutive cases is therefore interesting. Controversy also remains regarding the value of steroid receptor expression as a reliable predictor for the ALN status [START_REF] Patani | Predictors of axillary lymph node metastasis in breast cancer: a systemic review[END_REF]. Some studies reported no value for both ER and PR [START_REF] Gajdos | Lymphatic invasion, tumor size, and age are independent predictors of axillary lymph node metastases in women with T1 breast cancers[END_REF][START_REF] Chua | Frequency and predictors of axillary lymph node metastases in invasive breast cancer[END_REF] whereas others pointed to a lower risk of ALN metastases for tumours negative for either receptor SE, standard error; OR, odds ratio; CI, confidence interval; LR, likelihood ratio; Df, degrees of freedom; P, P-value a Odds ratio is computed for each 10-year increase in age b Odds ratio is computed for each centimeter increase in size c Odds ratio is computed for each increase in grade [START_REF] Gann | Factors associated with axillary lymph node metastasis from breast carcinoma[END_REF] or for PR only [START_REF] Ravdin | Prediction of axillary lymph node status in breast cancers by prognostic indicators[END_REF][START_REF] Silverstein | Predicting axillary nodal positivity in 2282 patients with breast carcinoma[END_REF][START_REF] Viale | Predicting the status of axillary sentinel lymph nodes in 4351 patients with invasive breast carcinoma treated in a single institution[END_REF]. Our findings are in line with these last reports. ALN invasion was least likely in PNP (25.8%) and most likely in PPP (56.2%) breast cancers referring to an interaction between PR and HER-2 for tumour cell migration and lymph node invasion. It remains unclear whether the small subgroup of triple positive breast carcinomas might deserve a specific treatment if our observation of increased cell migration or motility is confirmed. Nonetheless, it is worth reporting that triple positive breast cancers are more likely lymph node positive. Our findings are probably also yet another reason to test HER-2 in a breast cancer population which by definition has a low risk to be HER-2 + [START_REF] Taucher | Do we need HER-2/ neu testing for all patients with primary breast carcinoma?[END_REF][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]. The predictive role of HER-2 for endocrine agents, chemotherapeutic regimen and trastuzumab (HerceptinÒ) in ER + breast cancers has already been proven [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][START_REF] Shou | Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer[END_REF][START_REF] Pritchard | HER2 and responsiveness of breast cancer to adjuvant chemotherapy[END_REF].

Why HER-2 over-expression favours axillary metastases more in ER + PR + cases than in breast cancers with another joint ERPR status is also puzzling. Lange et al. suggested already in 1998 that progestins sensitize breast cancer cells for growth factor and cytokine signalling [START_REF] Mackey | Trastuzumab prolongs progressive-free survival in hormone-dependent and HER-2 positive metastatic breast cancer[END_REF]. Furthermore, the role of plasminogen activator inhibitor (PAI-1), another predictor for ALN metastases, is limited to tumours expressing PR [START_REF] Lange | Convergence of progesterone and epidermal growth factor signaling in breast cancer. Potentiation of mitogen-activated protein kinase pathways[END_REF]. Also, a recent study performed on endometrial stromal cells has shown that epithelial growth factors and PR are needed for maximal PAI-I overexpression [START_REF] Schneider | Accumulation of uPA-PAI-I complexes inside the tumour cells is associated with axillary nodal invasion in progesterone-receptorpositive early breast cancer[END_REF].

Furthermore, cross talk between ER and the growthfactor-signalling pathways suggests a growth advantage if HER-2 is expressed in ER + breast cancer cells [START_REF] Paik | Pathologic findings from the National Surgical Adjuvant Breast and Bowel Project: prognostic significance of erbB-2 protein overexpression in primary breast cancer[END_REF][START_REF] Lee | Cross-talk among estrogen receptor, epidermal growth factor, and insulin-like growth factor signaling 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][START_REF] Lockwood | Regulation of plasminogeen activator inhibitor I expression by interaction of epidermal growth factor with progestin during decidualization of human endometrial stromal cells[END_REF][START_REF] Russell | Transcriptional repression of the neu protooncogene by estrogen stimulated estrogen receptor[END_REF][START_REF] Tandon | HER-2/neu oncogene protein and prognosis in breast cancer[END_REF][START_REF] Borg | ERBB2 amplification in breast cancer with a high rate of proliferation[END_REF][START_REF] Mccann | Prognostic significance of c-erbB-2 and estrogen receptor status in human breast cancer[END_REF][START_REF] Allred | HER-2 in nodenegative breast cancer: prognostic significance of overexpression influenced by the presence of in situ carcinoma[END_REF][START_REF] Love | HER-2 overexpression and response to oophorectomy plus tamoxifen adjuvant therapy in estrogen receptor-positive premenopausal women with operable breast cancer[END_REF][START_REF] Peiro | Prognostic implication of HER-2 status in steroid receptor positive, lymph node negative breast carcinoma[END_REF]. Saal et al. recently described PI3K/AKT signalling through PIK3CA mutations that correlate with hormone receptors, HER-2 and lymph node metastases. The mutation was present in 58% among ER + HER-2 + node positive cases but in less than 7% of ER -HER-2 -tumours [START_REF] Saal | PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma[END_REF]. PIK3CA mutations may explain enhanced invasion of breast cancer cells to lymph nodes. ER -tumours and especially the basal like phenotype might posses a distinct mechanism of metastatic spread [START_REF] Foulkes | The prognostic implication of the basal-like (cyclin E high/p27 low/p53+/ glomeruloid-microvascular-proliferation+) phenotype of BRCA1related breast cancer[END_REF].

Whether triple positive breast cancers have a worse outcome than other ER + breast cancers in the luminal B group may be suggested by our findings but absence of information about the different subgroup's outcome is a major weakness of this report. However, such data are currently not mature enough to report and will also reflect the predictive value of different treatment modalities in different receptor subgroups. Another potential weakness of our report may be that we based the ALN status on results from a mixture of complete axillary node dissection and sentinel lymph node procedure. However, it has previously been shown that predictors for ALN status are independent of how the lymph node resection was performed although the metastatic detection rate in lymph nodes may be higher using the sentinel node procedure as more thorough histologic examination of the sentinel lymph node results in a higher number of metastases detected [START_REF] Viale | Predicting the status of axillary sentinel lymph nodes in 4351 patients with invasive breast carcinoma treated in a single institution[END_REF]. Another weakness may be that we did not consider other predictors for ALN status as tumour localisation within the breast, tumour vascularisation, lymphangiogenesis, protein and genetic markers for ALN involvement [START_REF] Patani | Predictors of axillary lymph node metastasis in breast cancer: a systemic review[END_REF]. Another important information which is missing is whether triple positive breast cancers, apart from being more likely lymph node positive, also involved a higher number of lymph nodes as compared to breast cancers with a non-triple positive phenotype. Such information may strengthen our findings. A strength of our study is that cases were unselected and consecutively treated in one centre. Also, analyses for ER, PR and HER-2 were confirmed by one pathologist. Successful quality control and quality assurance programs were guaranteed in our laboratory according to requested guidelines. Another strength of our study is that we defined HER-2 + on the base of membrane staining when they either had a DAKO score 3 + or 2 + with a positive FISH test.

Although our study does contrast substantially with gene micro-array studies showing that gene expression profiles of node positive and node negative tumours do not differ, the present study highlights the association between the combined steroid receptor expression, the HER-2 status and qualitative ALN involvement. HER-2 defined by a DAKO score 3 + or by FISH for those with a DAKO score 2 + is a predictor for ALN involvement in consecutive women with an ER + PR + operable breast cancer. Whether other pathways are involved in breast cancer cell migration in conjunction with the steroid and HER-2 receptors through PIK3CA mutations or the PAI-I system is a working hypothesis.

Table 1

 1 Descriptive statistics

	Lymph node status
	Statistic
	Variable

Table 2

 2 Results from the final logistic regression model

	Variable	Level	Parameter estimate (SE)	OR (95% CI)	LR chi-square	Df	P
	Age (B70 years)		-0.0147 (0.0051)	0.864 a (0.782-0.954)	8.29	1	0.0040
	Age ([70 years)		0.0303 (0.0166)	1.353 a (0.977-1.875)	3.28	1	0.0701
	Size (mm)		0.0432 (0.0030)	1.540 b (1.451-1.634)	281.52	1	\0.0001
	Grade [1-3]		0.3247 (0.0802)	1.384 c (1.182-1.619)	16.62	1	\0.0001
	Group	PPP vs PPN	0.6429 (0.1977)	1.902 (1.290-2.801)	21.32	5	0.0007
		PPP vs PNP	1.2445 (0.4694)	3.471 (1.383-8.696)
		PPP vs PNN	0.6328 (0.2465)	1.883 (1.161-3.049)
		PPP vs NNP	0.9577 (0.3039)	2.606 (1.437-4.717)
		PPP vs NNN	1.0108 (0.2447)	2.748 (1.701-4.444)

Table 3

 3 Proportion being grade 3, median tumour size and median age by ER, PR and HER-2 status NNN: ER -PR -HER-2 - e PNP and NNP: ER + PR -HER-2 + and ER -PR -HER-2 +

	Prognostic factor	a PPP 56.2% ALN +	b PPN 36.0% ALN +	c PNN 35.2% ALN +	d NNN 34.7% ALN +	e PNP and NNP 33.6%
		N = 137	N = 1579	N = 199	N = 193	ALN + N = 119
	Grade 3, %	73.7%	27.4%	40.2%	90.2%	81.5%
	Size, median (range)	25 (4-130)	20 (1-160)	20 (2-80)	24 (1-160)	23 (1-100)
	Age, median (range)	51 (27-86)	57 (26-95)	62 (27-91)	55 (26-90)	57 (32-88)
	ALN: Axillary lymph node status
	( + ): Positive
	( -): Negative
	a PPP: ER + PR + HER-2 +
	b PPN: ER + PR + HER-2 -
	c PNN: ER + PR -HER-2 -

d

Table 4

 4 ALN-status by tumour grade comparing PPP with non-PPP lesions

	Tumor grade	a PPP N = 137			b Non-PPP N = 2090
		ALN +	ALN -	All	ALN +	ALN -	All
	1	0 (0.0%)	3 (100%)	3	70 (22.2%)	246 (87.8%)	316
	2	15 (45.5%)	18 (54.5%)	33	341 (34.4%)	650 (65.6%)	991
	3	62 (61.4%)	39 (38.6%)	101	335 (42.8%)	448 (57.2%)	783
	ALN: Axillary lymph node status
	( + ): Positive
	( -): Negative
	a PPP: ER + PR + HER-2 +

b Non-PPP: ER + PR + HER-2 -, ER + PR -HER-2 -, ER -PR -HER-2 -, ER + PR -HER-2 + , ER -PR -HER-2 +

Breast Cancer Res Treat (2009) 113:181-187