Characterization of study patients and healthy volunteers
The present study was conducted at the Department of Obstetrics and Gynecology at the University Hospital in Essen. In total, 81 patients with primary breast cancer were studied from April 1998 until January 2003. Additionally, 10 healthy female controls aged between 30 and 50 years and with no history of cancer were recruited. Overall survival data of these patients were obtained from the local municipal registry; the median follow-up time was 6.2 years (range 0.2 to 9.8 years). Informed written consent was obtained from all patients, and the study was approved by the Local Essen Research Ethics Committee (05/2856). The clinical data of the patients are summarized in Table .
Patient characteristics at the time of primary diagnosis of breast cancer
The median age of the patients was 56 years (range 33 to 81 years). All four initial tumor stages were included, with a predominance of stage I and II. Most patients had ductal breast cancer and 49 women were node negative. High and moderately differentiated tumors were predominant. Two-thirds of the tumors were estrogen receptor (ER) negative and progesterone receptor (PR) positive, respectively. All patients undergoing breast-conserving therapy received an adjuvant radiation. Patients with hormone receptor-positive tumors received an adjuvant hormonal treatment with tamoxifen or an aromatase inhibitor. The chemotherapeutic adjuvant treatment mostly contained anthracyclines and taxanes.
For each of the 81 patients, the tumor type, TNM-staging and grading were assessed according to the World Health Organization-classification of tumors of the breast [16
] and the sixth edition of the TNM Classification System [17
]. The ER and PR receptor status were determined by immunohistochemistry.
Determination of serum tumor markers
For the quantitative determination of carcino embryonal antigen (CEA)/CA15-3 in human serum and plasma, 10 ml serum were collected and assessed using the Elecsys CEA/CA15-3 immunoassays (Roche, Mannheim, Germany). The serial measurement of CEA/CA15-3 was intended to aid in the management of cancer patients. These assays were performed on a Cobas® immunoassay analyzer in the central laboratory of University Hospital in Essen according to the manufacturer's instruction. The central laboratory has a valid certification for the performance of these assays following international guidelines.
Preparation of bone marrow
BM cells were isolated from heparinized BM (5000 U/ml BM) by Ficoll-Hypaque density gradient centrifugation (density 1.077 g/mol; Pharmacia, Freiburg, Germany) at 400 g for 30 minutes. Interface cells were washed (400 g for 15 minutes) and resuspended in PBS. For the detection of cytokeratin-positive (CK+) cells, 3 × 106 cells (1 × 106 per slide and area of 240 mm2) from each aspiration side were directly spun (400 g for 5 minutes) onto glass slides coated with poly-L-lysine (Sigma, Deisenhofen, Germany) using a Hettich cytocentrifuge (Tuttlingen, Germany).
After overnight air drying, staining of CK+ cells was performed using the Epimet® kit (Micromet, Munich, Germany). The identification of epithelial cells using this kit is based on the reactivity of the murine monoclonal antibody (Mab) A45-B/B3, directed against a common epitope of CK polypeptides. The kit uses Fab fragments of the pan-Mab conjugated with alkaline phosphatase molecules. The method includes: permeabilization of the cells by a detergent (5 minutes); fixation by a formaldehyde-based solution (10 minutes); binding of the conjugate Mab A45-B/B3-alkaline phosphatase to cytoskeletal CKs (45 minutes); and formation of an insoluble red reaction product at the binding site of the specific conjugate (15 minutes). Subsequently, the cells were counterstained with Mayer's hematoxylin for one minute and finally mounted with Kaiser's glyzerine/gelatine (Merck, Darmstadt, Germany) in Tris-EDTA buffer (Sigma, Deisenhofen, Germany). A conjugate of Fab-fragment served as a negative control. For each test a positive control slide with the breast carcinoma cell line MCF-7 (ATTC, Rockville, MD, USA) was treated under the same conditions. The microscopic evaluation was carried out independently by two investigators. Patients were evaluated as tumor cell-positive if at least one CK-positive cell was detected as analyzed by immunocytochemistry.
Preparation of serum and leukocytes
From each patient, 10 ml whole blood was collected in routine S-Monovette® tubes (Sarstedt AG&Co, Nümbrecht, Germany) and immediately stored at 4°C. Serum and leukocyte preparations were performed within four hours. The blood samples were centrifuged at 2500 g for 10 minutes. The upper phase contained the blood serum, from which 3 to 4 ml was removed for the extraction and analysis of the circulating DNA. The remaining 16 to 17 ml blood was supplemented up to 50 ml with lysis buffer containing 0.3 M sucrose, 10 mM Tris-HCl pH 7.5, 5 mM MgCl2 and 1% Triton X100 (Sigma, Taufkirchen, Germany). Following incubation for 15 minutes on ice, the isolation and purification of the leukocytes were carried out by two centrifugation steps at 2500 g, 4°C for 20 minutes.
Preparation of paraffin-embedded tumor tissue
Tumor tissue of 22 patients was available. Specimens were retrieved from the Institute of Pathology and Neuropathology of the University Hospital of Essen. Tumor pieces of 3 mm in size were processed from paraffin-embedded tumor blocks and embedded in paraffin again to perform six sections of 10 to 20 μm thickness. The sections were dewaxed in 1 ml xylene on a shaker incubator at 45°C for five minutes. After centrifugation at full speed and room temperature for five minutes, the supernatant was removed. Pellets were washed in 1 ml of ethanol and centrifuged at full speed for five minutes. The supernatant was removed, and the pellet was dried at 45°C for two to five minutes until the ethanol had evaporated.
DNA extraction and fluorescence-labeled PCR
For the PCR-based fluorescence microsatellite analyses we used our former microsatellite method without extended fractionation step [15
], because the fractionation technique which separates blood DNA in short and long DNA fragments was not yet established [18
] when blood samples were collected between 1998 and 2003.
Genomic DNA was extracted from tumor tissues, leukocytes and serum of peripheral blood using the QIAamp Blood DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Quantification and quality of the extracted DNA were determined spectrophotometrically using the BioPhotometer (Eppendorf, Hamburg, Germany) or the NanoDrop Spectrometer ND-1000 (Peqlab Biotechnologie, Erlangen, Germany). To determine the lowest portion of tumor-specific DNA which can be flawlessly detected, dilution experiments were performed. For this study we mixed and amplified known quantities and proportions of normal leukocyte and serum DNA, as described before [18
Serum, tumor and leukocyte (reference) DNA were amplified with a PCR using primer pairs binding to microsatellite markers as summarized in Table . PCR conditions were described before [15
]. To confirm the microsatellite alterations, each PCR was repeated at least twice.
Microsatellite markers used for loss of heterozygosity analysis
Evaluation of PCR products
The fluorescence-labeled PCR products were separated by capillary gel electrophoresis and detected on an automated Genetic Analyzer 310 (Applied Biosystems, Freiburg, Germany). Fragment length and fluorescence intensity were evaluated by the GeneScan software. The 500-ROX size marker (Applied Biosystems, Freiburg, Germany) served as an internal standard. The LOH incidence was determined by calculating the ratio of the intensities of the two alleles from a serum or tumor sample corrected by the ratio of the intensities of the two alleles from the corresponding leukocyte sample which served as reference DNA. LOH was interpreted if the final quotient was less than 0.6 or more than 1.67. Homozygous and non-analyzable peaks were designated as non-informative cases.
The statistical analyses were performed using the SPSS software package, version 13.0 (SPSS Inc. Chicago, IL, USA). The chi-square or two-tailed Fischer's exact test, and the univariate binary logistical regression were used to identify possible associations between the occurrence of DTC in BM, DNA concentrations and LOH patterns in blood and the following established risk factors of the patients with primary breast cancer: age, histology results, tumor stage (TNM), nuclear grade, ER and PR status, presence of tumor markers CEA and CA15-3, relapse time, menopause status, family history, and use of chemotherapy, radiotherapy and hormone therapy. In addition, the Mann and Whitney-U and the Wilcoxon-W test for the non-parametric comparison of two independent and dependent variables were used, respectively. Kaplan-Meier plots were drawn on to estimate overall survival and recurrence, and the log rank test was used for statistical analyses. A P value of less than 0.05 was considered as statistically significant.