Maria Joa

Cardoso Ae

Jaime S Cardoso Ae

Thomas Wild

A E Wilfried

Krois Ae

Florian Fitzal

M J Cardoso

J S Cardoso

Á W Krois

Á F Fitzal

Comparing two objective methods for the aesthetic evaluation of breast cancer conservative treatment

Keywords: Breast cancer conservative treatment, Aesthetic evaluation, Objective methods, Software, BCCT.core, BAT

Abstract Two programs were recently developed for the aesthetic evaluation of results in breast cancer conservative treatment: the Breast Cancer Conservative Treatment cosmetic results (BCCT.core) and the Breast Analyzing Tool (BAT). Both make use of a face-only photographic view of the patient and were developed to overcome the lack of reproducibility observed with subjective visual evaluation. The BCCT.core analyses several parameters related to asymmetry, color differences and scar appearance, while the BAT considers only asymmetry measurements. The purpose of this study was to compare the performance of these two methods. Material and methods Digital pictures of 59 patients from Porto and 60 from Vienna were evaluated subjectively by two panels using the four-class Harris scale. The Porto photographs had a similar backlight and better quality, and were evaluated by an international panel of 23 experts. The Vienna photographs had different backlight and lower quality, and were evaluated by four students and two breast cancer specialists. All 119 cases were submitted to analysis using the BCCT.core and BAT. Agreement between the software programs and the subjective evaluation was calculated using kappa (k), weighted kappa statistics (wk) and error rate (er). Results In overall analysis, BCCT.core program obtained a better agreement with the subjective evaluation (k = 0.56; wk = 0,64; er = 0.20) than the BAT software (k = 0.39; wk = 0.46; er = 0.42) (P \ 0.0007). Results were again better for the BCCT.core program, when analysing the photographs obtained in Porto (k = 0.71; wk = 0.78; er = 0.14) than for the BAT (k = 0.35; wk = 0.41; er = 0.51) (P \ 0.0003) while no significant differences in agreement were obtained regarding the Vienna images (P [ 0.1). Conclusions The results suggest that the inclusion of multiple parameters in image analyses of aesthetic results has the potential to improve results. However, picture quality is probably important for analysis of other features besides asymmetry.

Introduction

Oncological outcome of breast cancer conservative treatment (BCCT) is considered to be equivalent to mastectomy in terms of disease free survival and overall survival [START_REF] Fisher | Twenty-year followup of a randomized trial comparing total mastectomy, lumpectomy, and lumpectomy plus irradiation for the treatment of invasive breast cancer[END_REF][START_REF] Veronesi | Twenty-year follow-up of a randomized study comparing breast-conserving surgery with radical mastectomy for early breast cancer[END_REF]. The absence of standardized tools for the aesthetic evaluation of this kind of treatment, however, limits the applicability of any comparative analysis of cosmetic outcome, one of its main objectives. Methods for evaluating aesthetic results of BCCT are traditionally considered as subjective or objective [START_REF] Sk | Cosmetic assessment of breast-conserving surgery for primary breast cancer[END_REF][START_REF] Christie | A comparison of methods of cosmetic assessment in breast conservation treatment[END_REF][START_REF] Sacchini | Quantitative and qualitative cosmetic evaluation after conservative treatment for breast cancer[END_REF][START_REF] Vrieling | Validation of the methods of cosmetic assessment after breast-conserving therapy in the EORTC ''boost versus no boost'' trial. EORTC Radiotherapy and Breast Cancer Cooperative Groups. European Organization for Research and Treatment of Cancer[END_REF]. Subjective methods have been largely used, either through direct patient evaluation or through a photograph, performed by one or several observers [START_REF] Sk | Cosmetic assessment of breast-conserving surgery for primary breast cancer[END_REF]. However, results of subjective evaluation show only a modest interobserver agreement, even when performed by experts [START_REF] Cardoso | Interobserver agreement and consensus over the esthetic evaluation of conservative treatment for breast cancer[END_REF][START_REF] Pezner | Limited usefulness of observer-based cosmesis scales employed to evaluate patients treated conservatively for breast cancer[END_REF]. Objective methods use measurements taken directly from the patient or from photographs, and are based essentially on asymmetries between treated and non-treated breasts [START_REF] Pezner | Breast retraction assessment: an objective evaluation of cosmetic results of patients treated conservatively for breast cancer[END_REF][START_REF] Van Limbergen | Cosmetic evaluation of breast conserving treatment for mammary cancer. 1. Proposal of a quantitative scoring system[END_REF]. Until now they have not been widely used in clinical practice, probably due to the absence of photographic documentation in most treated patients [START_REF] Van Limbergen | Cosmetic evaluation of breast conserving treatment for mammary cancer. 2. A quantitative analysis of the influence of radiation dose, fractionation schedules and surgical treatment techniques on cosmetic results[END_REF]. They are associated with increased reproducibility of assessment but it has been argued that they do not take into account the global appearance of aesthetic results [START_REF] Christie | A comparison of methods of cosmetic assessment in breast conservation treatment[END_REF][START_REF] Sacchini | Quantitative and qualitative cosmetic evaluation after conservative treatment for breast cancer[END_REF][START_REF] Vrieling | Validation of the methods of cosmetic assessment after breast-conserving therapy in the EORTC ''boost versus no boost'' trial. EORTC Radiotherapy and Breast Cancer Cooperative Groups. European Organization for Research and Treatment of Cancer[END_REF][START_REF] Krishnan | Form or function? Part 2. Objective cosmetic and functional correlates of quality of life in women treated with breast-conserving surgical procedures and radiotherapy[END_REF].

Two objective methods were recently developed for the objective evaluation of aesthetic results in BCCT, the breast cancer conservative treatment cosmetic result (BCCT.core) [START_REF] Cardoso | Turning subjective into objective: the BCCT.core software for evaluation of cosmetic results in breast cancer conservative treatment[END_REF] and the breast analyzing tool (BAT) [START_REF] Fitzal | The use of a breast symmetry index for objective evaluation of breast cosmesis[END_REF]. Both methods make use of a face-only photographic view of the patient. The BCCT.core analyses several parameters related to asymmetry, color differences and scar appearance while the BAT takes in consideration only measurements of asymmetry. In order to obtain an evaluation of software performance, we analyzed photographs from patients submitted to BCCT with both tools and compared the results with those obtained in a subjective evaluation performed by experts.

Material and methods

A total of 120 patients submitted to BCCT were photographed and evaluated. Of these 60 were photographed in Porto with identical backlight and background. Another 60 patients were photographed in Vienna using heterogeneous backlight and background.

Subjective evaluation

The 60 cases photographed in Porto were evaluated by a panel of 23 international experts, who were asked to classify the aesthetic result of each case in one of four Harris scale categories: excellent-treated breast nearly identical to untreated breast; good-treated breast slightly different from untreated; fair-treated breast clearly different from untreated but not seriously distorted; poortreated breast seriously distorted. A Delphi procedure was used to obtain a consensus between observers, which was possible in 59 of the 60 cases [START_REF] Cardoso | Interobserver agreement and consensus over the esthetic evaluation of conservative treatment for breast cancer[END_REF].

The 60 cases photographed in Vienna were evaluated by two experts (breast cancer surgeons from a cancer centre with more than 300 cases per year) and four non-experts (medical students) using a three-point scale (good, fair or poor). The mean values of evaluations were used as the final subjective score [START_REF] Fitzal | The use of a breast symmetry index for objective evaluation of breast cosmesis[END_REF].

To obtain a single scale for comparison with the software programs, excellent and good results from the Harris scale in the Porto series were merged together to form a three-point scale.

Objective evaluation

The 119 cases where a final subjective classification was obtained were evaluated by the BAT software. This software uses well-defined landmarks (jugulomamillary distance and distances from the nipples to the edge of the breast) and calculates the difference between left and right breasts. This difference in length is multiplied by the surface area difference and is noted as percent difference and as difference factor. The values obtained can be converted to a simplified 3-point Harris scale (good, fair, poor) [START_REF] Fitzal | The use of a breast symmetry index for objective evaluation of breast cosmesis[END_REF].

The BCCT.core software was also applied to evaluate the 119 digital photographs. This program automatically evaluates several indices used for the aesthetic evaluation of BCCT (asymmetry, skin colour change and scar visibility). The BCCT.core then uses artificial intelligence techniques to translate these measures into an overall objective classification of aesthetical results reported to the user as excellent, good, fair or poor [START_REF] Cardoso | Towards an intelligent medical system for the aesthetic evaluation of breast cancer conservative treatment[END_REF].

To obtain a homogeneous final classification, the BCCT.core evaluations were translated into a 3-point scale by merging good and excellent cases together.

Statistical analysis

Agreement between the subjective evaluation and the BCCT.core and the BAT classifications was calculated using the kappa (k) and weighted kappa (wk) statistics, the latter allowing for some deviation from perfect agreement. A kappa score equal to 0 was considered to indicate poor agreement; 0.01-0.20 slight agreement; 0.21-0.40 fair agreement; 0.41-0.60 moderate agreement; 0.61-0.80 substantial agreement; 0.81-0.99 almost perfect; and 1.00 perfect agreement. The error rate of each method was also calculated, defined as the percentage of cases in which the software evaluation did not agree with the consensus. P values of 0.05 or less were considered statistically significant.

Results

The results of agreement between software and subjective evaluation are displayed in Table 1.

For the Porto photographs, agreement was substantially higher for the BCCT.core program. In the Vienna photographs, however the agreement with the subjective classification was only moderate, for both methods. Overall results revealed a significantly higher agreement with the BCCT.core program.

Discussion

With the warranty of oncological results identical to mastectomy [START_REF] Fisher | Twenty-year followup of a randomized trial comparing total mastectomy, lumpectomy, and lumpectomy plus irradiation for the treatment of invasive breast cancer[END_REF][START_REF] Veronesi | Twenty-year follow-up of a randomized study comparing breast-conserving surgery with radical mastectomy for early breast cancer[END_REF] cosmetic results are now also a major endpoint of BCCT. Their evaluation is fraught with difficulty, subjectivity and limited reproducibility. The BCCT.core and BAT softwares, were developed in different institutions but share the goals of providing a simple tool for reproducible evaluation of aesthetic results in BCCT [START_REF] Fitzal | The use of a breast symmetry index for objective evaluation of breast cosmesis[END_REF][START_REF] Cardoso | Towards an intelligent medical system for the aesthetic evaluation of breast cancer conservative treatment[END_REF] Different methodologies were used to arrive at a consensus analysis in the two datasets of photographs [START_REF] Cardoso | Interobserver agreement and consensus over the esthetic evaluation of conservative treatment for breast cancer[END_REF][START_REF] Fitzal | The use of a breast symmetry index for objective evaluation of breast cosmesis[END_REF]. The difficult and time consuming process of obtaining a consensus analysis in a large number of cases compelled us to use previous results obtained with different observers and scales.

This subset analysis quite unexpectedly leads to the most curious results obtained in this study. The BCCT.core program agreed more with the consensus in the Porto photographs and overall, but performed similarly to the BAT software in the Vienna patients.

Given the higher definition of the Porto photographs it can be hypothesised that the BAT software using only asymmetry measurements, maintains its performance independently of picture quality, while the BCCT.core that uses other parameters such as colour differences and scar appearance, performs less well in lower quality pictures [START_REF] Fitzal | The use of a breast symmetry index for objective evaluation of breast cosmesis[END_REF][START_REF] Cardoso | Towards an intelligent medical system for the aesthetic evaluation of breast cancer conservative treatment[END_REF] .

A good method of evaluation should be consistent and simple to use with all kinds of photographs but, on the other hand, some standards for picture quality (definition, backlight, background) are needed, if one is to expect discriminative power in evaluation of aesthetic results.

Breast Cancer Res Treat (2009) 116:149-152

Acknowledgements We wish to thank Prof. Ayres-de-Campos for his help in preparation of the manuscript. This work was partially funded by Fundac ¸a ˜o para a Cie ˆncia e a Tecnologia (FCT)-Portugal through project PTDC/EIA/64914/2006.


Sven Becker
email: sven.becker@med.uni-tuebingen.de

Graziella Becker-Pergola

Malgorzata Banys

Natalia Krawczyk

Diethelm Wallwiener

Erich Solomayer

Christina Schuetz

Tanja Fehm

A E Graziella Becker-Pergola

A E Malgorzata

Banys Ae

A E Diethelm

Wallwiener Ae

A E Christina

Schuetz Ae

Á M Banys

Á N Krawczyk

Á D Wallwiener

Á E Solomayer

Á C Schuetz

Á T Fehm

Evaluation of a RT-PCR based routine screening tool for the detection of disseminated epithelial cells in the bone marrow of breast cancer patients

Keywords: Disseminated tumor cells, Immunocytochemistry, Minimal residual disease, Bone marrow, Breast cancer, RT-PCR

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

Introduction

Treatment decisions for breast cancer patients are mostly based on TNM-status, tumor grade, hormone receptor status and HER2-status [START_REF] Goldhirsch | International expert consensus on the primary therapy of early breast cancer[END_REF]. Despite complete surgical resection and optimal systemic therapy a subset of patients still suffers from relapse. Patients who are at high-risk for relapse can be identified by the presence of clinically occult disseminated tumour cells (DTCs). Numerous studies, including a large pooled analysis, provide level I evidence that the presence of such cells in the bone marrow (BM) is associated with an unfavourable clinical outcome [START_REF] Braun | A pooled analysis of bone marrow micrometastasis in breast cancer[END_REF][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] Solomayer | Time independence of the prognostic impact of tumor cell detection in the bone marrow of primary breast cancer patients[END_REF]. Currently, immuncytochemistry using pancytokeratin antibodies is the gold standard for the detection of DTCs [START_REF] Borgen | Standardization of the immunocytochemical detection of cancer cells in BM and Blood: I. Establishment of objective criteria for the evaluation of immunostained cells[END_REF]. A consensus has been published for the standardised detection of DTC's as well as the clinical implications of their detection [START_REF] Fehm | A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation[END_REF]. Recently, RT-PCR based methods have been established as an alternative to ICC. Various markers were evaluated including mammaglobin and cytokeratin 19 (CK19) [START_REF] Benoy | Real-time RT-PCR correlates with immunocytochemistry for the detection of disseminated epithelial cells in bone marrow aspirates of patients with breast cancer[END_REF][START_REF] Datta | Sensitive detection of occult breast cancer by the reverse-transcriptase polymerase chain reaction[END_REF][START_REF] Reinholz | Evaluation of a panel of tumor markers for molecular detection of circulating tumor cells in women with suspected breast cancer[END_REF][START_REF] Bosma | Detection of circulating breast tumor cells by differential expression of marker genes[END_REF][START_REF] Gradilone | Detection of CK19, CK20 and EGFR mRNAs in peripheral blood of carcinoma patients: correlation with clinical stage of disease[END_REF][START_REF] Eaton | Immunobead RT-PCR: a sensitive method for detection of circulating tumor cells[END_REF]. CK19 is an intermediate filament cytokeratin and stably expressed in epithelial tissues but not in mesenchymal haemopoietic cells. We have developed an assay for the detection of DTCs targeting the CK19 transcript with a real-time RT-PCR using the Light Cycler system. Our focus was the possibility of routine clinical application. 385 patients were screened over a 12 month period using both methods. Endpoint of our study was to compare ICC and RT-PCR-positivity rates.

Materials and methods

This study was approved by the University of Tu ¨bingen Ethics Committee in compliance with the Declaration of Helsinki ethical principles for medical research involving human subjects. After written informed consent, BM samples were obtained from 385 breast cancer patients who were treated at the Tuebingen University Breast Center (Tuebingen University Hospital, Tuebingen, Germany) over a 12 month period. 15-20 ml of BM were aspirated from the anterior iliac crest into syringes containing heparin under local or general anesthesia using the Jamshidi technique [START_REF] Jamshidi | Bone marrow biopsy with unaltered architecture: a new biopsy device[END_REF]. Time of aspiration was noted on the test tube and samples arrived in our in-house laboratory within 30 min. Samples were divided into two parts: (a) 7-10 ml were transferred into a new tube and immediately processed for RT-PCR detection and (b) 7-10 ml were set aside for subsequent ICC processing. RT-PCR-Samples were processed immediately and mixed with 50 ml RNA/DNA Stabilization Reagent for Blood and BM (Roche Diagnostics), causing cell lysis and stabilization of nucleic acids (guanidinium thiocyanate effect). Samples were then immediately frozen at minus 20°C until further processing. Subsequent analysis was performed in batches of 5-10 samples.

RT-PCR assay

MRNA was extracted using the mRNA Isolation Kit for Blood and BM (Roche Diagnostics). The system uses a magnetic glass particle technology in a two step procedure and yields purified mRNA. MRNA isolation was performed according to the manufacturer's instructions and eluted in 10 ll DEPC treated H 2 O. Light Cycler One-Step RT-PCR for CK19 using the RNAMaster Hybridization Probes Kit (Roche Diagnostics) was performed and yielded a product of 309 bp. 5 ll of mRNA were subjected to RT-PCR with a Mn[OAc] 2 concentration of 3 mM, 0.5 lM of each primer (primer A: AGATCCTGAGTGACATGCGAA; primer B: CCCAGCTGGGCTTCAATAC) and 0.125 lM of each probe (probe A: CCCTTCAGGGGTCTTGAGATGAGCx, probe B: LCRedGCAGTCACAGCTGAGCATGAAAGCp) were used. Design and synthesis of primers and probes are specific for the CK19 gene (Tibmolbio) and avoid amplification of the CK19 pseudogene [START_REF] Ruud | Identification of a novel cytokeratin 19 pseudogene that may interfere with reverse transcriptase polymerase chain reaction assays used to detect micrometastatic tumor cells[END_REF]. Reverse Transcription was performed at 60°C for 20 min immediately preceding the PCR (Hotstart at 95°C for 30 s and 50 cycles: 95°C for 1 s, 55°C for 15 s, 72°C for 13 s). PCR products were then analysed using the Fit Points Method of the Light Cycler software. Specificity was ensured by a negative control of 5 ml blood from a healthy volunteer that was included in the experiment. In addition one H 2 O sample was run in parallel to check for possible contamination of RT-PCR reagents and equipment. One positive control, consisting of preparations of MCF-7 cells spiked with 5 ml blood from a healthy volunteer was included with each batch. Data analysis was performed with the LC software version 3.5 Fit Points method and results were compared to a previously established standard curve of positive control samples with a detection limit of 5 MCF-7 cells mixed with 5 ml blood containing 3 9 10 6 PBMCs. The housekeeping gene GAP-DH was amplified with specific primers (Tibmolbio) to check for RNA integrity.

Preliminary testing RNA stability assays were performed before patient samples were analysed using direct RNA stabilisation after various time intervals. These tests showed highly time sensitive RNA degradation starting immediately after BM sampling [START_REF] Becker | Time is an important factor when processing samples for the detection of disseminated tumor cells in blood/bone marrow by reverse transcription-PCR[END_REF]. Preceding the analysis of clinical specimens, a dilution series of tissue cultured breast cancer cells (MCF-7) cells helped to define the sensitivity of the assay of 5 tumor cell in 3 9 10 6 PBMCs and is shown in Fig. 1. One positive control consisting of RNA from 5 isolated tumor cells mixed with 3 9 10 6 PBMCs from a healthy individual was included with each patient batch in order to verify specific amplification of the CK19 gene and ensure adequate sensitivity.

Detection of CK positive cells by ICC

Tumor cell isolation and ICC detection was performed according to recommendations for the standardized detection of DTC's by the Collaborative BM Micrometastases Group [START_REF] Fehm | A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation[END_REF]. BM was diluted with Phosphate Buffered Saline (PBS) and mononucleated cells were obtained by density gradient centrifugation over Biocoll (density 1077 g/ml, Biochrom). Cells were washed with PBS and-if necessary-red blood cells were lysed with Lysis buffer. Cytospins were prepared with 10 6 cells using a cytocentrifuge (Hettich). Fixation of cells occurred in a 0.5% neutral buffered formalin solution for 10 min. Automated staining was performed on the Autostainer (DAKO Cytomation) using the A45B-B3 monoclonal antibody for pancytokeratin (Mikromet) at a dilution of 1:400 and AP-AAP detection (Dako Cytomation). The A45B-B3 antibody is directed against common cytokeratin epitopes including the CK heterodimers 8/18 and 8/19. A positive control consisting of preparations of MCF-7 cells mixed with PBMC's was included with each batch of samples, as well as a negative control, consisting of PBMC's from healthy volunteers. In order to check for unspecific staining, isotype matched myeloma protein was included as a staining control (Sigma). For each patient 2 9 10 6 cells were analyzed on two slides. Analysis was performed on the Automated Cell Imaging System ACIS Ò (Chromavision). Criteria for DTC-detection were based on recommendations of the European ISHAGE Working group for standardization of tumor cell detection [START_REF] Borgen | Standardization of the immunocytochemical detection of cancer cells in BM and Blood: I. Establishment of objective criteria for the evaluation of immunostained cells[END_REF].

Statistics

Chi-squared test was used to examine the relationship between categorical variables. Statistical analysis was performed using SPSS (version 11.5). P-values less than 0.05 were considered statistically significant.

Results

Patient characteristics

BM samples from 385 breast cancer patients were analyzed. 305 samples were obtained at the time of surgery for primary breast cancer. Sixty-three patients had a history of breast cancer and underwent secondary BM aspiration during follow-up at our outpatient clinic. The remaining seventeen patients were diagnosed with recurrent breast cancer. Clinical data of patients are shown in Table 1.

BM analysis by ICC

135 of 385 patients (35%) had DTC's in their BM. The number of cells ranged from 1 to 8 cells per 10 6 MNC's. Figure 2 demonstrates examples of CK positive cells in the BM of a primary breast cancer patient. The highest positivity rates were seen in primary breast cancer patients who underwent primary systemic chemotherapy. 15 of 63 (24%) follow up samples of breast cancer patients with no evidence of disease were DTC positive.

BM analysis by RT-PCR

The CK19 detection rate by RT-PCR was 35%. Figure 3 demonstrates a typical result of CK19 positive samples obtained with the Fit Points Method of the Light Cycler version 3.5 (Roche Diagnostics). In primary breast cancer, 117 of 305 patients (38%) were DTC positive. After The positivity rates of both methods were 35%. 49% of the BM samples (187 of 385) were positive by at least one method. 14% of the samples were positive only by RT-PCR and another 14% only by ICC. Both methods showed a statically significant correlation (P \ 0.01).

In 73% of the patients (280 of 385 cases) identical results were obtained. 198 (51%) patients were BM negative by both methods. Positive results by both methods were obtained in 82 (21%) patients (Table 2a). In order to rule out that discordant results obtained by the two methods was the result of inferior sensitivity of the RT-PCR, we compared the number of CK? cells detected by ICC with PCR positivity. Table 2b demonstrates that the number of detected tumor cells on ICC did not correlate with RT-PCR results (P = 0.365). Thus, RT-PCR correctly evaluates the BM-status for presence or absence of CK-positive cells, irrespective of their number, which would correspond to the clinical significance of the ''positive'' of ''negative'' BM-status as established by ICC.

Discussion

Over the past 15 years, the standard method of detection of disseminated tumor cells (DTCs) in the BM of breast cancer patients has been immunocytochemistry (ICC). This method has shown that DTCs are correlated independently with early disease relapse and decreased survival [START_REF] Wiedswang | Detection of isolated tumor cells in bone marrow is an independent prognostic factor in breast cancer[END_REF][START_REF] Janni | Clinical relevance of occult metastatic cells in the bone marrow of patients with different stages of breast cancer[END_REF]. DTC's have the potential to become targets for new therapeutic modalities, which would make routine evaluation of the BM-status an important aspect of breast cancer care. Recent efforts to standardize the ICC-approach underline its growing clinical relevance and the need for an accurate and generally accepted assessment of BM-status [START_REF] Fehm | A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation[END_REF]. While ICC has emerged as the method of choice for DTCdetection, it has not been without criticism. Differences of detection rates between research groups using ICC have been attributed to the use of different cell preparation techniques, different antibodies as well as different cytomorphologic diagnostic criteria. The most consistent results have been obtained with the A45B-B3 antibody. Recent consensus meetings have recommended using this antibody and introduced standardized technical and diagnostic criteria [START_REF] Fehm | A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation[END_REF]. Another problem has been the subjective aspect of cytologic evaluation. Furthermore the implementation of BM-ICC into routine clinical practice is labor intensive and costly. Efforts continue to provide an alternative, both from a methodological as well as from a logistic point of view. The focus of interest has been RT-PCR. Different research groups have established RT-PCR-protocols for the detection of DTCs [START_REF] Xenidis | Predictive and prognostic value of peripheral blood cytokeratin-19 mRNA-positive cells detected by real-time polymerase chain reaction in node-negative breast cancer patients[END_REF][START_REF] Gilbey | The detection of circulating breast cancer cells[END_REF][START_REF] Ring | Circulating tumour cells in breast cancer[END_REF][START_REF] Silva | Detection of epithelial messenger RNA in the plasma of breast cancer patients is associated with poor prognosis tumor characteristics[END_REF][START_REF] Kahn | RT-PCR amplification of CK19 mRNA in the blood of breast cancer patients: correlation with established prognostic parameters[END_REF][START_REF] Aerts | A real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect breast carcinoma cells in peripheral blood[END_REF][START_REF] Berois | Molecular detection of cancer cells in bone marrow and peripheral blood of patients with operable breast cancer: comparison of CK19, MUC1 and CEA using RT-PCR[END_REF]. Only few have compared their results to ICC as shown in Table 3 [START_REF] Benoy | Real-time RT-PCR correlates with immunocytochemistry for the detection of disseminated epithelial cells in bone marrow aspirates of patients with breast cancer[END_REF][START_REF] Smith | Response of circulating tumor cells to systemic therapy in patients with metastatic breast cancer: comparison of quantitative polymerase chain reaction and immunocytochemical techniques[END_REF][START_REF] Schroder | Detection of micrometastatic breast cancer by means of real time quantitative RT-PCR and immunostaining in perioperative blood samples and sentinel nodes[END_REF][START_REF] Schoenfeld | The detection of micrometastases in the peripheral blood and bone marrow of patients with breast cancer using immunohistochemistry and reverse transcriptase polymerase chain reaction for keratin 19[END_REF]. RT-PCR based assays have the advantage of being highly sensitive and reproducible. They offer an objective, observer-independent way of assessing the presence of cytokeratin-bearing cells in the BM. However, due to key methodological difficulties such as the inherent instability of the CK-related RNA, its implementation remains a challenge. Our group has established a one-step real-time RT-PCR based protocol using the Light Cycler system. Our focus was the establishment of a protocol suited for clinical routine and comparable to ICC with regard to sensitivity, specificity and reproducibility. Based on our findings and those of other research groups regarding RNA-degradation we did not use any preenrichment strategy [START_REF] Becker | Time is an important factor when processing samples for the detection of disseminated tumor cells in blood/bone marrow by reverse transcription-PCR[END_REF][START_REF] Naume | Increased sensitivity for deteion of micrometastases in bone marrow/peripheral blood stem-cell products from bresast cancer patients by negative immunomagnetic separation[END_REF]30]. RNA degradation starts immediately after BM is obtained and pre-enrichment uses up additional time. Because RNA-levels of CK19-positive cells within a background of hematopoietic cells are at the threshold of detection, even small levels of degradation can lead to false negative results. Without pre-enrichment, rapid RNA stabilization (\30 min after the sample was obtained) was essential. After the addition of the RNAstabilizer, samples were stored at minus 20°C until further analysis, allowing sample batching, which is important for routine clinical use. The intermediate filament CK19 has emerged as the PCR-marker of choice for microdisseminated breast cancer cells [START_REF] Benoy | Real-time RT-PCR correlates with immunocytochemistry for the detection of disseminated epithelial cells in bone marrow aspirates of patients with breast cancer[END_REF][31][START_REF] Ring | Detection of circulating epithelial cells in the blood of patients with breast cancer: comparison of three techniques[END_REF][START_REF] Slade | Quantitative polymerase chain reaction for the detection of micrometastases in patients with breast cancer[END_REF][START_REF] Kruger | Reverse transcriptase/polymerase chain reaction detection of cytokeratin-19 mRNA in bone marrow and blood of breast cancer patients[END_REF][START_REF] Gradilone | Detection of CK19, CK20 and EGFR mRNAs in peripheral blood of carcinoma patients: correlation with clinical stage of disease[END_REF]. CK19 is stably expressed in epithelial tissues and tumors but not usually in mesenchymal and/or haematopoietic cells [START_REF] Bosma | Detection of circulating breast tumor cells by differential expression of marker genes[END_REF][START_REF] Gradilone | Detection of CK19, CK20 and EGFR mRNAs in peripheral blood of carcinoma patients: correlation with clinical stage of disease[END_REF]. Alternative markers tested by different groups have been Muc-1, urokinase type plasminogen activator receptor (uPAR), EpCAM and mammaglobin each originally considered breast cancer specific [START_REF] Pierga | Real-time quantitative PCR determination of urokinase-type plasminogen activator receptor (uPAR) expression of isolated micrometastatic cells from bone marrow of breast cancer patients[END_REF][START_REF] De Cremoux | Detection of MUC1-expressing mammary carcinoma cells in the peripheral blood of breast cancer patients by real-time polymerase chain reaction[END_REF][START_REF] Ferrucci | New comprehensive gene expression panel to study tumor micrometastasis in patients with high-risk breast cancer[END_REF]. Some of these markers, however, have been detected in peripheral blood of a control group of healthy patients [START_REF] Xenidis | Predictive and prognostic value of peripheral blood cytokeratin-19 mRNA-positive cells detected by real-time polymerase chain reaction in node-negative breast cancer patients[END_REF][START_REF] Silva | Detection of epithelial messenger RNA in the plasma of breast cancer patients is associated with poor prognosis tumor characteristics[END_REF] and data regarding their clinical significance remains scarce. Based on the available evidence regarding the significance of CKpositive cells in the BM, it is clear that RT-PCR would have to look at established CK-markers, too. Table 4 compares the test-specific characteristics of RT-PCR and The expression patterns of these epithelial cytokines are very similar to that of CK19 and they are not expected to provide more specificity than detection of the single marker CK19. In fact, the A45B-B3 antibody was initially used for the detection of non-breast cancer epithelial tumor cells. CK 18, for example, has been widely used as a marker for DTC-detection in colon cancer, but has been found to be less relevant in breast cancer. Significant problems remain. It has been shown that haematopoietic cells potentially (though rarely) express CK19 at very low levels [START_REF] Ko | High percentage of false-positive results of cytokeratin 19 RT-PCR in blood: a model for the analysis of illegitimate gene expression[END_REF][START_REF] Bostick | Limitations of specific reverse-transcriptase polymerase chain reaction markers in the detection of metastases in the lymph nodes and blood of breast cancer patients[END_REF]. If sensitivity is too high, ''illegitimately'' expressed mRNA transcripts would be detected and specificity suffers. One way to solve this problem is to use quantitative RT-PCR, which requires a defined cut-off, above which expression of a gene is considered tumor cell specific. However, due to the heterogeneity of tumor cells, they too express CK19 at very different levels, so it has been difficult to define an exact cut off value that does not arbitrarily exclude tumor cells, reducing the value of the test overall.

So far, only one study has looked at the follow-up of patients with RT-PCR positive BM: the presence of CK19-mRNA in BM was correlated with a significantly reduced overall survival [START_REF] Benoy | Real-time RT-PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer[END_REF]. Our data provides good evidence that RT-PCR might play an important part in the detection of DTC's. The test correlates well with the established standard and could serve to accumulate clinical data which then needs to be compared to clinical follow up as well. Survival data of patients recruited for this study will be available within the next 3-5 years. In summary, looking at the largest patient group to date, we report on a pragmatic and easy to use RT-PCR based assay for DTC-detection in the BM of breast cancer patients. Our results show good correlation with the existing standardized ICC. If the BMstatus of breast cancer patients becomes more important for their management, new test-options need to be explored. The RT-PCR approach is fast, can be performed in batches and requires no cytologist-evaluation. Being largely automated it can easily be implemented in established laboratory routine.

Fig. 1

 1 Fig. 1 Determination of sensitivity of the RT-PCR assay by Fit Points method of the Cycler Software package. A 10-fold dilution series of MCF-7 breast cancer cells (5 9 10 3 , 5 9 10 2 , 5 9 10 1 , 5 9 10 0 ; squares) mixed with 3 9 10 6 PBMCs determined the

Fig. 2

 2 Fig. 2 Criteria for evaluation of positive DTC's in bone marrow and examples from primary breast cancer patients

Fig. 3

 3 Fig. 3 Amplification curves obtained by the Light Cycler version 3.5 software package. The x-axis shows the cycle number of the PCR where amplification started. The y-axis shows fluorescence intensity. Five of 11 samples amplified the CK19 gene

Table 1

 1 Patient characteristics

	Patients enrolled	n = 385	ICC positive	RT-PCR positive
	Primary breast cancer	305	111 (36%)	117 (38%)
	Prior surgery/no neoadjuvant chemotherapy	277	95 (34%)	103 (37%)
	Stage I	164	54 (33%)	64 (39%)
	Stage II	73	25 (34%)	24 (33%)
	Stage III	30	13 (43%)	12 (40%)
	Stage IV	10	3 (30%)	3 (30%)
	After primary systemic therapy	28	16 (57%)	14 (50%)
	Recurrencies	17	9 (53%)	5 (29%)
	Secondary bone marrow aspiration	63	15 (24%)	12 (19%)
	Total	385	135 (35%)	134 (35%)

Table 2

 2

	Positivity rates obtained by ICC and RT-PCR	RT-PCR negative	RT-PCR positive	Total
	(a)
	ICC Negative	198 (51%)	55 (14%)	253 (65%)
	ICC Positive	53 (14%)	82 (21%)	135 (35%)
	Total	251 (65%)	134 (35%)	385 (100%)
	(b)
	1 CK? Cell ICC	24 (46%)	28 (54%)	52 (100%)
	2 CK? Cells ICC	12 (32%)	26 (68%)	38 (100%)
	C3 CK? Cells ICC	17 (38%)	28 (62%)	45 (100%)
	Total positive ICC	53 (39%)	82 (61%)	135 (100%)	P = 0.365

Table 3

 3 Comparison of RT-PCR and ICC

		Smith et al. [26]	Benoy et al. [8]	Schroder et al. [27]	Schoenfeld et al. [28]	Becker et al. [16]
	Total n both tests performed	133	25	31	65	385
	Blood/bone marrow	Blood	Bone marrow	Blood	Bone marrow	Bone marrow
	M status patients	M?	M?	M0	M0	M0
	Positivity rate RT-PCR CK19	50%	80%	50%	35%	35%
	Positivity rate ICC pCK	42%	63% a	0% b	22%	35%
	Same result both methods	71%	75%	67%	74%	73%
	Correlation ICC/PCR	kappa 0.43	P \ 0.0001	n.a.	n.a.	P \ 0.001
	Sensitivity RT-PCR assay	1 in 10 7 cells	5 in 10 7 cells	1 in 10 6 cells	1 in 10 6 cells	5 in 3 9 10 6 cells
	Sensitivity ICC assay	n.a.	1 in 10 6 cells	1 in 2 9 10 6 cells	[1 in 10 5 cells	1 in 10 6 cells
	a Total (n = 24) samples analysed
	b Total (n = 149) samples analysed

Table 4

 4 Comparison of ICC and RT-PCR protocols considering aspects for routine application of protocols Having established an easy to use CK19-RT-PCR assay, we prospectively compared the results of our RT-PCR assay with routine ICC assessment of the BM of 385 breast cancer patients. The results show a significant correlation between the RT-PCR based method and ICC,

		One-step RT-PCR	ICC
	Marker/Antigen	CK19	CK8, CK18 and CK19
	Sensitivity	5 MCF-7 cells/3 9 10 6 PBMCs	1 MCF-7 cell/10 6 PBMC's
	Enrichment strategy	None a	Density gradient
	Specificity b	Primers CK19	Monoclonal PanCK antibody
	Time needed for assay	200 min	300 min
	Time for entire procedure	1 day	2 days c
	Cost (material only)	€ 35,-/assay	€ 13,-/assay
	Subjective element	No	Yes
	Stability of target	RNA, instable molecule	Protein, stable
	Morphological evaluation	No	Yes

a Advantage: (a) RNA stabilization feasible within minutes, (b) saves time b Evaluation of-negative controls (blood) were obtained from healthy blood donors-positive controls (cells from breast cancer cell line spiked with PBMCs of healthy donors)-Isotype control (MOPC21 antibody to check for unspecific staining) c Cytospins dry overnight ICC.

Breast Cancer Res Treat (2009) 117:227-233

Acknowledgements This work was supported by the University of Tu ¨bingen AKF Foundation (Angewandte Klinische Forschung).


S H Teertstra

J Muller

J L Wesseling

H Peterse

E J Bartelink

K G A Rutgers

Gilhuijs
email: k.gilhuijs@nki.nl

K E Pengel

Ae C E Loo

Ae H J Teertstra

Ae S H Muller

Ae J Wesseling

Ae J L Peterse

Ae H Bartelink

Ae E J Rutgers

Ae K G A Gilhuijs

J L K E Peterse

Á C E Pengel

Á H J Loo

Teertstra

S H Muller

Á K G A Gilhuijs

H Bartelink

The impact of preoperative MRI on breast-conserving surgery of invasive cancer: a comparative cohort study

Keywords: MRI, Breast cancer, Breast-conserving therapy, Incomplete excision

The impact of preoperative MRI on breast-conserving surgery of invasive cancer: a comparative cohort study

Introduction

Breast-conserving therapy (BCT), consisting of wide local excision (WLE) of the tumor combined with postoperative radiotherapy, is the standard of care in loco-regional treatment of early (\3 cm) breast cancer. Large prospective randomized trials have demonstrated that the survival rates after BCT are equivalent to those obtained after radical mastectomy [START_REF] Van Dongen | Long-term results of a randomized trial comparing breast-conserving therapy with mastectomy: European Organization for Research and Treatment of Cancer 10801 trial[END_REF][START_REF] Clarke | Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: an overview of the randomised trials[END_REF][START_REF] Bartelink | Recurrence rates after treatment of breast cancer with standard radiotherapy with or without additional radiation[END_REF]. The 5-year local-recurrence rate after BCT is about 5% with survival rates of approximately 92% [START_REF] Clarke | Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: an overview of the randomised trials[END_REF][START_REF] Bartelink | Recurrence rates after treatment of breast cancer with standard radiotherapy with or without additional radiation[END_REF][START_REF] Vrieling | Can patient-, treatment-and pathologyrelated characteristics explain the high local recurrence rate following breast-conserving therapy in young patients?[END_REF][START_REF] Kreike | Continuing risk of ipsilateral breast relapse after breast-conserving therapy at long-term follow-up[END_REF]. The local-recurrence rate is, however, approximately twice as large in patients with incompletely excised tumors and in patients 40 years of age or younger [START_REF] Clarke | Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: an overview of the randomised trials[END_REF][START_REF] Bartelink | Recurrence rates after treatment of breast cancer with standard radiotherapy with or without additional radiation[END_REF][START_REF] Vrieling | Can patient-, treatment-and pathologyrelated characteristics explain the high local recurrence rate following breast-conserving therapy in young patients?[END_REF][START_REF] Kreike | Continuing risk of ipsilateral breast relapse after breast-conserving therapy at long-term follow-up[END_REF]. Results from major trials with long-term follow-up demonstrated that improvement of local control results in better survival, and complete gross excision of the primary tumor has been shown to maximize local control [START_REF] Clarke | Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: an overview of the randomised trials[END_REF][START_REF] Bartelink | Recurrence rates after treatment of breast cancer with standard radiotherapy with or without additional radiation[END_REF][START_REF] Vrieling | Can patient-, treatment-and pathologyrelated characteristics explain the high local recurrence rate following breast-conserving therapy in young patients?[END_REF][START_REF] Kreike | Continuing risk of ipsilateral breast relapse after breast-conserving therapy at long-term follow-up[END_REF][START_REF] Smitt | The importance of the lumpectomy surgical margin status in long-term results of breast conservation[END_REF].

To select the optimal therapeutic strategy, the size, growth pattern and location of the lesion must be accurately visualized. Conventional imaging by mammography and ultrasonography fails to accurately assess tumor extent in approximately one-third of patients eligible for BCT [START_REF] Faverly | Breast carcinomas of limited extent: frequency, radiologic-pathologic characteristics, and surgical margin requirements[END_REF]. Several studies reported a higher sensitivity of contrastenhanced magnetic resonance imaging (CE MRI) to detect invasive breast cancer and to visualize disease extent compared to the standard workup using conventional imaging [START_REF] Boetes | Breast tumors: comparative accuracy of MR imaging relative to mammography and US for demonstrating extent[END_REF][START_REF] Hata | Magnetic resonance imaging for preoperative evaluation of breast cancer: a comparative study with mammography and ultrasonography[END_REF][START_REF] Berg | Diagnostic accuracy of mammography, clinical examination, US, and MR imaging in preoperative assessment of breast cancer[END_REF][START_REF] Deurloo | Contrast-enhanced MRI in breast cancer patients eligible for breast-conserving therapy: complementary value for subgroups of patients[END_REF]. These observations have led to changes in the surgical management of patients [START_REF] Deurloo | Additional breast lesions in patients eligible for breast-conserving therapy by MRI: impact on preoperative management and potential benefit of computerised analysis[END_REF][START_REF] Braun | Influence of preoperative MRI on the surgical management of patients with operable breast cancer[END_REF][START_REF] Bilimoria | Evaluating the impact of preoperative breast magnetic resonance imaging on the surgical management of newly diagnosed breast cancers[END_REF][START_REF] Grobmyer | Is there a role for routine use of MRI in selection of patients for breast-conserving cancer therapy?[END_REF][START_REF] Bedrosian | Changes in the surgical management of patients with breast carcinoma based on preoperative magnetic resonance imaging[END_REF][START_REF] Houssami | Accuracy and surgical impact of magnetic resonance imaging in breast cancer staging: systematic review and meta-analysis in detection of multifocal and multicentric cancer[END_REF]. Preoperative breast MRI for women with known breast cancer is, however, controversial due to the increase in cost, mastectomy rate and larger excisions, without knowing the benefit in local control [START_REF] Plevritis | Ethical issues in contrastenhanced magnetic resonance imaging screening for breast cancer[END_REF][START_REF] Morrow | Magnetic resonance imaging in the preoperative evaluation of breast cancer: primum non nocere[END_REF][START_REF] Morrow | Magnetic resonance imaging in the breast cancer patient: curb your enthusiasm[END_REF][START_REF] Sh | Contrast-enhanced MRI of the breast: accuracy, value, controversies, solutions[END_REF][START_REF] Bleicher | MRI and breast cancer: role in detection, diagnosis, and staging[END_REF][START_REF] Orel | Who should have breast magnetic resonance imaging evaluation?[END_REF]. The therapy of most patients is not converted after preoperative MRI, but little is known whether the high sensitivity of CE MRI improves the precision of BCT in this group of patients.

The aim of this study was to assess whether preoperative MRI reduces the risk of an incomplete tumor excision, and to assess consequences in patient management.

Materials and methods

Patient cohort

All women with invasive breast cancer and eligible for BCT on the basis of conventional imaging and palpation presenting at our hospital between March 2002 and July 2004 were consecutively included. Women who underwent chemo-or hormone-therapy prior to surgery and women with only primary ductal carcinoma in situ (DCIS) were not included. Detection and staging of breast cancer was performed by mammography, ultrasonography, and physical examination. Ultrasonography was also used to detect pathological loco-regional lymph nodes [START_REF] Van Rijk | Ultrasonography and fine-needle aspiration cytology can spare breast cancer patients unnecessary sentinel lymph node biopsy[END_REF]. Proof of breast cancer was obtained using fine-needle aspiration (FNA) or core biopsy. Treatment plans were established in consensus by a multi-disciplinary team of breast cancer specialists (radiologists, surgeons, medical oncologists, radiation oncologists, pathologists, and nurse practitioners).

In 2000, the MARGINS (Multi-modality Analysis and Radiogical Guidance IN breast conServing therapy) study started at our hospital. The aim of this single-institutional study was to investigate the use of conventional imaging in combination with MRI to improve the definition of the extent and localization of the tumor. In the MARGINS study, women with pathology-proven breast cancer and eligible for BCT were recruited for an additional preoperative breast MRI (the MRI group). The MRI results were discussed in a subsequent multi-disciplinary meeting at which time the initial treatment plan was confirmed or adjusted. In the current study, patients not recruited (the non-MRI group), either refused to participate, could not be imaged by MRI prior to the scheduled surgery date, or had contraindications for MRI, such as claustrophobia.

All data were obtained after written informed patient consent and approval of the institutional review board (the MRI group) or from established clinical guidelines and waiver of consent (the non-MRI group).

Prospective guidelines for management of additional findings at MRI Guidelines were established to handle additional findings detected at CE MRI, aiming to minimize treatment changes on benign findings [START_REF] Deurloo | Additional breast lesions in patients eligible for breast-conserving therapy by MRI: impact on preoperative management and potential benefit of computerised analysis[END_REF]. In short, if additional findings were sufficiently close to the index lesion, BCT was pursued with larger wide-local excision margins to include the additional findings. Depending on the size of the breast, the total diameter of disease in patients eligible for BCT typically does not exceed 3 cm. If additional MRI findings were further away from the index lesion, attempts were made to obtain proof of malignancy by second-look targeted ultrasonography and FNA or biopsy. If pathology confirmed malignant disease over a region too large to allow cosmetically acceptable BCT, a conversion to mastectomy was advised. If pathology proof could not be obtained, BCT was advised and follow-up by MRI. The final treatment plan was implemented after consultation with the patient.

Magnetic resonance imaging

Magnetic resonance imaging was performed with a 1.5-tesla scanner (Magnetom, Siemens Medical Systems, Erlangen, Germany) using a coronal FLASH-3D technique. Both breasts were imaged in prone orientation, using a dedicated double-breast array coil. One series was acquired before the injection of contrast agent, and four series were acquired after intravenous injection of contrast agent (Prohance, Bracco-Byk Gulden, Konstanz, Germany); 0.1 mmol/kg body weight, at a rate of 2-4 ml/s. The series were acquired at intervals of approximately 120 s. The following MRI parameters were used: 3D coronal T1-weighted sequence, repetition time 8.1 ms, echo time 4.0 ms, isotropic voxels of 1.35 9 1.35 9 1.35 mm 3 and no fat suppression. Subtraction images were available to examine initial and late enhancement. Radiologists with experience in breast MRI read the examinations. If multiple lesions were visible, the total area including normal breast tissue between lesions, was indicated as the total disease extent.

Surgery

The surgical procedures were performed according to accepted surgical standards, with the aim to achieve tumorfree margins with the best possible cosmetic outcome [START_REF] Rutgers | Quality control in the locoregional treatment of breast cancer[END_REF][START_REF] Rutgers | Sentinel node procedure in breast carcinoma: a valid tool to omit unnecessary axillary treatment or even more?[END_REF]. The procedures were carried out or directly supervised by fully trained surgeons specialized in breast surgery.

Results of preoperative diagnostic imaging were available in the operating theater. For non-palpable lesions, surgeons were typically guided by a gamma-ray detection probe, after preoperative intra-lesional injection of 99m Tc-nanocolloid (Nanocoll; GE-Healthcare, Eindhoven, The Netherlands) administered under ultrasound guidance [START_REF] Van Rijk | Sentinel node biopsy and concomitant probe-guided tumor excision of nonpalpable breast cancer[END_REF]. Wide local excisions in our hospital are performed according to the technique described by Aspegren et al. [START_REF] Aspegren | Standardization of the surgical technique in breast-conserving treatment of mammary cancer[END_REF]: the incision is typically radial, skin is removed only for cosmetic reasons, tumor resection is performed with an intended macroscopic margin of at least 1 cm, generally down to the fascia and reconstruction of the breast parenchyma is done by mobilization of breast tissue in order to minimize the cavity.

Pathology

At pathology, the excision specimens were handled according to a fixed protocol based on radiographic and pathological assessment, described by Egan [START_REF] Egan | Multicentric breast carcinomas: clinical-radiographic-pathologic whole organ studies and 10-year survival[END_REF]. Specimens were inked to indicate their orientation in the breast, sectioned into 5 mm parallel slices and radiographs were obtained. Cross sections of the tumor, surrounding breast tissue and nearest margins, as well as additional grossly or radiologically suspicious areas, were sampled for microscopic examination. Pathologists, experienced in breast pathology, assessed the specimens to report the largest diameter of invasive cancer, in situ components, growth pattern, margins, histological grade and type.

Grade was determined according to the Bloom and Richardson method resulting in well (grade 1), moderately (grade 2), or poorly (grade 3) differentiated carcinoma [START_REF] Bloom | Histological grading and prognosis in breast cancer; a study of 1409 cases of which 359 have been followed for 15 years[END_REF][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]. An incomplete excision was defined as an excision that resulted in extension of the tumor into the edge of specimen. Incomplete excisions were rated as focally incomplete (tumor present in the resection margin in less than two low power fields) or extensively positive (tumor present in the resection margin in two or more low power fields).

Statistical analysis SPSS Version 15.0; SPSS Chicago, Ill, was used for all analyses. To explore the difference in patients and tumor characteristics between the MRI and the non-MRI group, Students t-tests were performed for normally distributed continuous variables, and Mann-Whitney-U and Fisher's exact test for the non-normally distributed variables.

Multivariate analysis using logistic regression (LR) was performed to determine which factors were significant explanatory variables for incomplete surgery. Backward LR based on step-wise feature selection (f-to-entry: 0.05, fto-remove: 0.10) was applied.

The following patient and tumor characteristics were fitted into the model to explore the association with incompletely excised tumor: age, palpable (yes/no), lymph node metastases (present/not present), tumor size, tumor histological grade [START_REF] Van Dongen | Long-term results of a randomized trial comparing breast-conserving therapy with mastectomy: European Organization for Research and Treatment of Cancer 10801 trial[END_REF][START_REF] Clarke | Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: an overview of the randomised trials[END_REF][START_REF] Bartelink | Recurrence rates after treatment of breast cancer with standard radiotherapy with or without additional radiation[END_REF] and MRI (done/not done). Subset analysis, using similar procedures, was performed for presence of various tumor types on the excision boundary (IDC, ILC and DCIS).

Results

Patient characteristics

At baseline the MRI group consisted of 173 women with 175 tumors (2 contra-lateral tumors prior to MRI). The non-MRI group consisted of 176 women with 180 breast tumors (4 contra-lateral tumors). The mean age was 56.1 years, standard deviation (SD) 9.9 years (range: 28-82) and 59.6 years, SD 11.1 years (range: 31-89) in the MRI group and non-MRI group, respectively (P = 0.02).

No significant differences were found between the tumor characteristics in the two groups: lymph node stage, palpability, tumor size, histological tumor type, and grade. The tumor type was invasive ductal carcinoma (IDC) in 274 (77.2%) of the cases. Invasive lobular carcinoma (ILC) was found in 53 (14.9%) of all cases. Patient characteristics are listed in Table 1 and Fig. 1. The rate of patient inclusion in the MRI group and non-MRI group was comparable throughout the investigated study period.

Additional findings and patient management

In the MRI group, therapy was converted to mastectomy in 16/173 (9.2%) women after detection of additional pathology-proven disease (Table 1; Fig. 1). In 19 women (11.0%), tumor with a larger extent was found than anticipated using conventional imaging. Fifteen patients (8.7%) were advised a mastectomy because of larger tumor size (n = 3) and multi-focal disease spread, up to 9 cm (n = 12) (Table 2). In all cases, the spread of disease was confirmed by extensive histopathological examination of the specimens. In four cases (2.3%) a wider excision was performed than initially planned. Three women (1.7%) were found to have additional contra-lateral breast cancer, (not visible at conventional imaging). These MRI-detected contra-lateral tumors were not included in the current study. In one woman with a bilateral tumor the treatment was changed to bilateral mastectomy, according to the wish of the patient. The other two women underwent bilateral BCT. A total of 157 women with 159 tumors continued with wide-local excision.

In addition to malignant findings, 21 benign lesions in 175 breasts (12.0%) were detected at preoperative MRI. Three lesions were considered benign on the basis of ultrasound characteristics, four were proved to be benign after target ultrasound with fine-needle aspiration (FNA). Thirteen lesions were occult at target ultrasonography and considered benign by follow-up. The median follow-up time was 54.1 months (range: 37.0-70.1 months). The only therapy change for a benign lesion was a wider local excision than initially planned which included a fibroadenoma close to the index tumor.

Incomplete surgical excision

Surgical excision of the tumor was incomplete in 22 of the 159 procedures (13.8%) in the MRI group, and in 35 of the 180 procedures (19.4%) in the non-MRI group (P = 0.17). The types of disease at the margins of the incomplete

Management after incomplete surgery

In the MRI group, 8/159 (5.0%) additional surgical procedures were performed after WLE. A re-excision was done in four patients and in another four a mastectomy. In the non-MRI group, 19/180 (10.6%) additional surgical procedures were performed after WLE (P = 0.06). Ten reexcisions were done and nine mastectomies. In 11 of the 22 women in the MRI group and in 13 of the 35 in the non-MRI group incomplete surgery was followed by a higher boost dose at radiotherapy to treat the incompletely excised component rather than additional surgery (Table 4).

Multivariate results

At multivariate analysis, no factors were associated with incomplete tumor excision in the overall patient cohort. At subset analysis, significant association was found only between absence of MRI and incompletely excised IDC (P = 0.02) (Table 3). The hazard ratio was 0.18 (CI: 0.04-0.81), suggesting that these patients were at a relatively lower risk of incomplete excision of IDC when a preoperative MRI was performed. None of the other co-variates (age, palpability, lymph node status, tumor size, and grade) were significantly associated with incomplete surgical excision, and no factors were found to be associated with presence of ILC or a DCIS component on the excision boundary.

Discussion

We reported our experiences with a clinical guideline established to manage additional findings at preoperative contrast-enhanced MRI of the breast. The impact of this guideline was assessed on the precision of BCT in a retrospective cohort of 349 women treated in the same time period. A total of 173 women underwent preoperative MRI.

In the entire cohort no significant difference in incomplete excision between the MRI and the non-MRI group was found (P = 0.17). Further stratification by subset analysis showed significant association between preoperative MRI and reduced rates of incompletely excised infiltrating ductal carcinoma (IDC) (P = 0.02). These findings may provide input to future studies investigating the potential benefit of preoperative MRI in subgroups of patients.

MRI led to treatment change in 11% of patients after pathology proof of malignancy The rate of therapy changes due to benign findings were small (one change to larger wide local excision for a benign finding); no changes to mastectomy were caused by benign findings. MRI also detected contra-lateral cancer occult at conventional imaging in 1.7% of the patients.

The impact of preoperative MRI appeared twofold. Firstly women with more extensive pathology-proven disease were filtered away from the standard BCT eligibility and thus received a more extensive surgical treatment (mastectomy or a wider excision). The increased mastectomy rate in the MRI group raises the concern that MRI leads to over-treatment. Indeed, increased mastectomy rates were also reported in other studies [START_REF] Deurloo | Additional breast lesions in patients eligible for breast-conserving therapy by MRI: impact on preoperative management and potential benefit of computerised analysis[END_REF][START_REF] Braun | Influence of preoperative MRI on the surgical management of patients with operable breast cancer[END_REF][START_REF] Bedrosian | Changes in the surgical management of patients with breast carcinoma based on preoperative magnetic resonance imaging[END_REF]. It is likely that additional malignant disease also occurred in the non-MRI group. This assumption is supported by studies that reported more extensive disease in diverse groups of breast tumors [START_REF] Faverly | Breast carcinomas of limited extent: frequency, radiologic-pathologic characteristics, and surgical margin requirements[END_REF][START_REF] Holland | Histologic multifocality of Tis, T1-2 breast carcinomas. Implications for clinical trials of breast-conserving surgery[END_REF][START_REF] Rosen | Residual'' mammary carcinoma following simulated partial mastectomy[END_REF][START_REF] Anastassiades | Multicentricity in breast cancer. A study of 366 cases[END_REF][START_REF] Coombs | Multifocal and multicentric breast cancer: does each focus matter?[END_REF]. Consequently, a considerable part of undetected multi-focal tumor may be controlled by adjuvant treatment such as radiotherapy. Nonetheless, it is also known that radiotherapy is ineffective to eradicate large residual tumor burden [START_REF] Clarke | Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: an overview of the randomised trials[END_REF][START_REF] Bartelink | Recurrence rates after treatment of breast cancer with standard radiotherapy with or without additional radiation[END_REF][START_REF] Vrieling | Can patient-, treatment-and pathologyrelated characteristics explain the high local recurrence rate following breast-conserving therapy in young patients?[END_REF]. It remains to be proven whether conversion of therapy to mastectomy on the basis of additional MRI-detected disease has impact on the 15-20-year local recurrence rates after BCT [18-21, 22, 36-38]. This may be of particular concern in younger patients where the local recurrence rate is approximately twice as large as in older patients. In a retrospective analysis, Fisher and colleagues reported that preoperative MRI reduced the incidence of local recurrence [START_REF] Fischer | The influence of preoperative MRI of the breasts on recurrence rate in patients with breast cancer[END_REF]. Impact of preoperative MRI on surgical precision was not addressed. Solin and colleagues reported that the use of MRI in the staging of the patients was not associated with an improvement in local control after BCT [START_REF] Solin | Relationship of breast magnetic resonance imaging to outcome after breast-conservation treatment with radiation for women with early-stage invasive breast carcinoma or ductal carcinoma in situ[END_REF]. One of the study limitations, indicated by the authors, was a significantly lower age of women in the MRI group, which makes differences in local recurrence difficult to interpret.

In contrast to Fisher et al., Solin and colleagues took differences in age and adjuvant treatment into account in their analysis. Local recurrence was not an end-point of our current study.

A second impact of MRI appears to be the high accuracy at which the extent and location of IDC is visualized. This provided the surgeons with a better preoperative assessment resulting in reduced rates of incompletely excised IDC. Despite observations from other studies demonstrating superior sensitivity of MRI for ILC compared to that of conventional breast imaging [START_REF] Mann | The value of MRI compared to mammography in the assessment of tumour extent in invasive lobular carcinoma of the breast[END_REF][START_REF] Boetes | The role of MRI in invasive lobular carcinoma[END_REF][START_REF] Quan | Magnetic resonance imaging detects unsuspected disease in patients with invasive lobular cancer[END_REF][START_REF] Rodenko | MR imaging in the management before surgery of lobular carcinoma of the breast: correlation with pathology[END_REF][START_REF] Weinstein | MR imaging of the breast in patients with invasive lobular carcinoma[END_REF][START_REF] Mann | MRI compared to conventional diagnostic work-up in the detection and evaluation of invasive lobular carcinoma of the breast: a review of existing literature[END_REF], we were unable to translate these findings to reduced rates of incompletely excised ILC. Several explanations may exist. First, the number of patients with ILC was relatively small in our cohort. Secondly, all patients in our study were eligible for BCT on the basis of conventional imaging thus excluding tumors larger than 3 cm that were also included in other studies. Thirdly, evidence of multi-focal disease elsewhere in the breast may be difficult to extract from the cut margins of the WLE specimens at histopathology due to the finite tissue sampling and the typically diffuse multi-nodular pattern of growth of ILC [START_REF] Krecke | Invasive lobular carcinoma of the breast: mammographic findings and extent of disease at diagnosis in 184 patients[END_REF][START_REF] Arpino | Infiltrating lobular carcinoma of the breast: tumor characteristics and clinical outcome[END_REF]. Some of these considerations also hold for DCIS. Moreover, variable sensitivity of MRI for the detection of pure DCIS has been observed in other studies [START_REF] Berg | Diagnostic accuracy of mammography, clinical examination, US, and MR imaging in preoperative assessment of breast cancer[END_REF][START_REF] Bluemke | Magnetic resonance imaging of the breast prior to biopsy[END_REF][START_REF] Kuhl | MRI for diagnosis of pure ductal carcinoma in situ: a prospective observational study[END_REF][START_REF] Santamaria | Preoperative MRI of pure intraductal breast carcinoma-a valuable adjunct to mammography in assessing cancer extent[END_REF][START_REF] Rosen | BI-RADS MRI enhancement characteristics of ductal carcinoma in situ[END_REF].

Study design

A randomized clinical trial (RCT) is the standard study design to provide level-I evidence for medical intervention studies. Level-II evidence is available showing that breast MRI visualizes disease extent more accurately than conventional breast imaging [START_REF] Boetes | Breast tumors: comparative accuracy of MR imaging relative to mammography and US for demonstrating extent[END_REF][START_REF] Hata | Magnetic resonance imaging for preoperative evaluation of breast cancer: a comparative study with mammography and ultrasonography[END_REF][START_REF] Berg | Diagnostic accuracy of mammography, clinical examination, US, and MR imaging in preoperative assessment of breast cancer[END_REF][START_REF] Deurloo | Contrast-enhanced MRI in breast cancer patients eligible for breast-conserving therapy: complementary value for subgroups of patients[END_REF]. This evidence has created much confidence in favor of MRI in patients as well as in practitioners, making it difficult to implement prospective comparative level-I studies. To our knowledge, there are currently only two RCTs that address aspects discussed in the present study. The first one is the COMICE (comparative effectiveness of magnetic resonance imaging in breast cancer) trial [START_REF] Kneeshaw | Current applications and future direction of MR mammography[END_REF], investigating the role of the MRI in the preoperative staging of breast cancer. The second one is the MONET (MR mammography Of Nonpalpable BrEast Tumors) trial [START_REF] Peters | Breast MRI in nonpalpable breast lesions: a randomized trial with diagnostic and therapeutic outcome-MO[END_REF], evaluating whether MRI will improve breast cancer management for non-palpable tumors. Neither trials have been designed specifically to answer the question whether MRI will reduce local recurrence after long-term follow-up. Moreover, potential differences in how MRI findings are incorporated into clinical decision-making may lead to differences in outcome and cost-benefit between hospitals.

The design and results of the present study may be useful in follow-up studies to examine differences in local recurrence between ''MRI-proven unifocal'' breast tumors and tumors where only conventional imaging was performed. Studies concerning the cost effectiveness of MRI and the selection of patients for partial breast irradiation (PBI) may also benefit from these findings.

Study limitations

An obvious drawback of non-randomized studies is that they are prone to diverse bias and confounders. The women in the current study were not randomized, but consecutively recruited for a preoperative MRI scan. They were treated in the same time period as the patients in the non-MRI group, were included at similar rates during the investigated study period, under the same circumstances and had the same tumor characteristics. It allowed us to explore the potential impact of our preoperative guidelines on the management of additional MRI findings.

Although there was no selection on age in the current study we did observe a lower mean age in the MRI group, comparable to the observations of Solin et al. [START_REF] Solin | Relationship of breast magnetic resonance imaging to outcome after breast-conservation treatment with radiation for women with early-stage invasive breast carcinoma or ductal carcinoma in situ[END_REF]. We analyzed the allocation procedure but could not find an explanation for this finding. It is likely that older women refused more often to undergo preoperative MRI. The multivariate analysis was performed taking the differences in age into account. Premenopausal women are at increased risk of local recurrence because of dense breast tissue (resulting in difficulty to visualize tumor extent) and increased tumor aggressiveness [START_REF] Bartelink | Recurrence rates after treatment of breast cancer with standard radiotherapy with or without additional radiation[END_REF][START_REF] Vrieling | Can patient-, treatment-and pathologyrelated characteristics explain the high local recurrence rate following breast-conserving therapy in young patients?[END_REF][START_REF] Kreike | Continuing risk of ipsilateral breast relapse after breast-conserving therapy at long-term follow-up[END_REF]. Nonetheless, despite the younger age of the women in the MRI group, this group still showed more complete excisions of IDC than the non-MRI group. This effect was not caused by larger excision volumes in the MRI group compared to the non-MRI group.

Conclusions

Preoperative MRI did not significantly affect the overall rate of incomplete tumor excision, but it yielded significantly lower rate of incompletely excised IDC. Preoperative MRI detected disease with larger extent in 11% of all patients eligible for BCT on the basis of conventional imaging and palpation, thus leading to a-prior changes in treatment.

Table 1

 1 Patient and tumor characteristics

	a Mastectomies due to disease
	with larger extent (n = 15) and
	contra-lateral disease (n = 1)
	detected at MRI (see also
	Table 2). Values between
	parentheses are percentages

excisions are shown in Table

3

. Incomplete excision of invasive disease was 8/159 (5%) and 19/180 (10.6%) in the MRI and in the non-MRI group, respectively, (P = 0.06).

Table 2

 2 Primary mastectomy due to disease with larger extent in the MRI group

	Breast Cancer Res Treat (2009) 116:161-169
	Fig. 1 Overview surgery in the MRI and the non-MRI group;	Initially eligible for BCT:
	main results	349 women
		355 tumors
		173women
		175 tumors
		MRI
		16 women
		16 tumors
		Mastectomy
		157 women	176 women
		159 tumors	180 tumors
		WLE
		-complete: 137/159 (86.2%)	-complete: 145/180 (80.5%)
		-incomplete for IDC 2/126 (1.6%)	-incomplete for IDC 11/136 (8.1%)
	Variable	15 Women
		15 tumors
	Age (years)	Mean: 47.7 years; SD 9.3 years
	B40	3 (20.0)
	41-50	8 (53.3)
	51-60	2 (13.3)
	[60	2 (13.3)
	Laterality
	Left	7 (46.7)
	Right	8 (53.3)
	Histological grade
	1	4 (26.7)
	2	7 (46.7)
	3	4 (26.7)
	Nodal status
	N0	9 (60.0)
	N1	5 (33.3)
	N2	1 (6.7)
	N3	0
	Histological type
	IDC	11 (73.3)
	ILC	1 (6.7)
	Others	3 (20.0)
	Values between parentheses are percentages

Table 3

 3 Incomplete surgery in the MRI versus the non-MRI group

	Histological type	Incompletely	MRI				Non-MRI				P-value
	primary tumor	excised disease	M	%	F	E	M	%	F	E
	IDC	IDC		2	1.6	1	1	11	8.1	2	9	0.02
	N = 126 vs. 136	in situ	12	9.8	6	6	12	8.6	4	8	n.s
	MRI vs. non-MRI
	ILC	ILC		6	23.1	4	2	5	19.2	2	3	n.s.
	N = 26 vs. 26	in situ	1	3.8	0	1	3	11.5	1	2	n.s.
	MRI vs. non-MRI
	Others	Others	0	-	-	-	1	5.6	0	1	n.s.
	N = 7 vs. 18	IDC		0	-	-	-	1	5.6	1	0	n.s.
	MRI vs. non-MRI	ILC		0	-	-	-	1	5.6	1	0	n.s.
		in situ	1	14.3	1	0	1	5.6	0	1	n.s.
		Total		22	13.8	12	10	35	19.4	11	24	0.17
	M = Number of incompletely excised disease
	F = Focally incomplete
	E = Extensively incomplete
	% = Percentage of incompletely excised disease
	n.s. = Not significant
	Table 4 Management after incomplete surgery
			MRI	Non-MRI
			(N = 159)	(N = 180)
	Re-excision		4 (2.5)	10 (5.6)
	Mastectomy		4 (2.5)	9 (5.0)
	Higher radiotherapy boost	11 (6.9)	13 (7.2)
	dose
	No change in treatment		1 (0.6)	1 (0.6)
	Unknown		2 (1.3)	1 (0.6)
	Others			1 (0.6) a
	Total		22 (13.8)	35 (19.4)
	a One woman with extensive incompletely excised IDC refused
	further treatment Values between parentheses are percentages

Breast Cancer Res Treat (2009) 116:161-169

Acknowledgments The authors thank Angelique Schlief, Anita Paape, Wil van Waardenberg, Eline Deurloo, Wilma Oughlane-Heemsbergen, and Robert Lindeboom for their contribution. This work was financially supported by The Dutch Cancer Foundation, grant number NKB 2004-3082