Effect of Ibandronate on Disseminated Tumor Cells in the Bone Marrow of Patients with Primary Breast Cancer: A Pilot Study

Patients and Methods

Study design. We conducted a retro- and prospective single institution trial to determine the role of bisphosphonates on DTC in BM for patients with non metastatic-breast cancer. Patients with diagnosed breast cancer and DTCs at the time of primary diagnosis who presented at our clinic from 1998-2007 were invited to have their BM analyzed again for DTCs. In cases of persistence of DTCs in the BM, patients received a 6-month oral bisphosphonate therapy with ibandronate (50 mg per day). After 6 months, the BM was re-analyzed for DTCs.

Eligibility criteria. The eligibility criteria were as follows: histologically proven breast cancer, evidence of DTCs at time of primary diagnosis, completion of adjuvant treatment with operation and systemic chemotherapy, no local or distant metastasis, no severe uncontrolled co-morbidities or medical conditions, no second malignancies.

Prior adjuvant chemotherapy and radiation were permitted, as was therapy with clodronate. For those undergoing adjuvant endocrine therapy with tamoxifen or an aromatase inhibitor, continuing during the study was allowed. Before starting the study, all patients underwent an evaluation for local relapse and distant metastasis by clinical examination, ultrasound and mammography of the breast.

Patient population and patient characteristics. The study was conducted at the Department of Obstetrics and Gynecology in Essen. In total, 54 breast cancer patients with first diagnosis between 1998-2003 (n=21), or 2004-2007 (n=33), and who had DTCs in their BM aspirates at the time of primary diagnosis gave their written consent to participate in the study. Patients' characteristics at the time of diagnosis are shown in Table I. The patients ranged in age from 39 to 85 years. Most patients had a ductal carcinoma (n=40) of the breast; moderately (G2) differentiated tumors were predominant. Axillary lymph node dissection had been carried out in cases of clinically involved lymph nodes or after a positive sentinel lymph node biopsy (n=17 patients). Thirty-one patients had small tumors (pT1) and breast-conserving therapy was performed in 34 patients. Forty primary tumors were ER + and PR +, respectively, and 9 primary tumors had overexpression of HER2 (DAKO, Score 3+).

Therapy. Thirty-four patients received adjuvant chemotherapeutic treatment consisting of anthracyclines and taxanes. All patients with breast-conserving surgery received an adjuvant radiation. Patients with hormone receptor-positive tumors were treated with tamoxifen or aromatase inhibitors. Most of the patients (43/54) followed our recommendation and received oral clodronate therapy (2 × 520 mg per day) for at least 2 years. Only 11 patients did not receive bisphosphonate treatment at the time of primary diagnosis.

Collection and analysis of BM. Between 10 and 20 ml BM were aspirated under local anesthesia with mepivacain from the anterior iliac crests of 54 patients and processed within 24 hours. All specimens were obtained after written informed consent and collected using protocols approved by the Institutional Review Board (05/2856). Tumor cell isolation and detection was performed based on the recommendations for standardized tumor cell detection recently published by the German Consensus group of Senology (8). 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 min. Interface cells were washed (400×g for 15 min) and resuspended in phosphate-buffered saline (PBS). A total of 1×10⁶ mononuclear cells per 240/mm² from each aspiration side were directly spun onto glass slides (400×g for 5 min) coated with poly-L-lysine (Sigma, Deisenhofen, Germany) using a Hettich cytocentrifuge (Tuttlingen, Germany) for the detection of CK-positive cells. The slides were air-dried overnight at room temperature.

Immunocytochemistry. Staining for CK-positive cells was performed using the murine monoclonal antibody Mab A45-B/B3 (Micromet, Germany), directed against a common epitope of CK polypeptides including the CK heterodimers 8/18 and 8/19. The protocol has been described in detail elsewhere (9). Briefly, the method includes: a) permeabilization of the cells with a detergent (5 min), b) fixation with a formaldehyde-based solution (10 min), c) binding of the conjugate Mab A45-B/B3-alkaline phosphatase to cytoskeletal CKs (45 min), and d) formation of an insoluble red reaction product at the site of binding of the specific conjugate (15 min) using the DAKO-APAAP detection kit (DakoCytomation, Denmark) according to the manufacturer's instructions. Subsequently, the cells were mounted with Kaiser's glycerol/gelatine (Merck, Darmstadt, Germany) in Tris EDTA buffer (Sigma, Deisenhofen, Germany). A control antibody (conjugate of Fab-fragment; Micromet) served as negative control. For each test, a positive control slide with the breast carcinoma cell line MCF-7 (ATCC, Rockville, MD, USA) was treated under the same conditions.

Evaluation of CK-positive cells. Microscopic evaluation of the slides was carried out using the ARIOL system (Applied Imaging International, Newcastel upon Tyne, UK) according to the ISHAGE evaluation criteria and the DTC consensus (8, 9). This automated scanning microscope and image analysis system consists of a slide loader, camera, computer and software for the detection and classification of cells of interest based on particular color, intensity, size, pattern, and shape.

Immunohistochemical analysis of the primary tumor. For each of the 54 patients, the tumor type, TNM staging and grading were assessed according to the WHO Classification of tumors of the breast (10) and the sixth edition of the TNM Classification System (11). The estrogen receptor (ER) and progesterone receptor (PR) status were determined by immunohistochemistry. Sections of 5-μm-thickness were cut and mounted on SuperFrost® Plus slides (Menzel, Braunschweig, Germany). Following individually optimized heat-based antigen retrieval for each antibody, each glass slide was immunostained with commercially available antibodies. The following antibodies were used: anti-ER [clone SP1, DCS (Hamburg, Germany), dilution 1:300, antigen retrieval: 30 min 95°C], citrate buffer, pH 6.0 and anti-PR [clone 16, DCS (Hamburg, Germany), dilution 1:200, antigen retrieval: 30 min 95°C, citrate buffer, pH 6.0]. Automated immunohistochemistry was performed using a Dako Autostainer Plus System (DakoCytomation, Carpinteria, CA, USA) with the anti-mouse IgG EnVision Plus detection kit (DakoCytomation) for secondary and tertiary immunoreactions. Reaction products were developed with diamino benzidine (DAB), according to general protocols. Positive and negative control sections were included in each run, which showed appropriate results.

The DAKO score for the expression of HER2 was determined by the pathologists using the HercepTest® and the results were interpreted as follows: 0=no membranous staining, 1+=faint, partial membranous staining, 2+=faint, complete membranous staining in more than 10% of invasive cancer cells, 3+=intense, complete membranous staining in more than 10% of invasive cancer cells. Fluorescence in situ hybridization (FISH) analyses in cases of 2+-staining were determined by the HercepTest and performed as described (12). For patients with first diagnosis between 1998-2003, the DAKO score was determined retrospectively by the pathologists.