C–X–C ligand 10 and C–X–C receptor 3 status can predict tamoxifen treatment response in breast cancer patients

This retrospective cohort study was conducted using tumor material from a randomized tamoxifen trial conducted in 1976–1990 in Stockholm Sweden composed of 1,780 patients. Results and details of the trial were previously described [23]. All patients were postmenopausal with tumors ≤30 mm and were negative for axillary lymph node involvement (N₀). The patients received either breast conserving surgery followed by radiation treatment with a dose of 50 Gy with 2 Gy per fraction 5 days weekly, for about 5 weeks, or radical mastectomy. After surgery, patients were randomized to tamoxifen 40 mg daily or no endocrine treatment. After 2 years of tamoxifen treatment, most disease-free patients were randomized to tamoxifen for an additional 3 years or no further therapy. Tumor material from 912 women was available for the current investigation. The mean follow-up period for patients in the study was 18 years. The trial design and protocol was approved by the Research Ethics Committee at the Karolinska Institute (dnr 97–451, with amendments) [23]. In order to conduct tissue microarray analysis, a pathologist chose representative parts of the tumors. Three cores per patient with a diameter of 0.8 mm were chosen and transferred to a paraffin block using a manual arrayer (Beecher Instruments, Sun Prairie, WI). From the sample blocks sections were cut and placed on slides, forming the basis of the tissue microarray. A flow chart of patients included in the initial tamoxifen trial and further included in the current analysis is shown in Fig. 1. Estrogen receptor (ER) and progesterone receptor (PgR) status were determined with cut-off levels at 10 % of positively stained tumor cell nuclei, cytosol measurements were used in the case of missing immunohistochemical data, with a cut-off of 0.05 fmol/μg DNA [23]. HER2 expression score 0–3 was previously described [24].

Tissue microarray sample slides were heated to 60 °C for 12 h and stored at −70 °C. Following thawing, the sample slides were deparaffinized with Tissue Clear (HistoLab, Göteborg, Sweden). The sample slides were washed with decreasing concentrations of ethanol followed by water, after which they were placed in DIVA-buffer (BioCare, Concord, CA) inside a decloaking chamber (BioCare), heated to 120 °C, cooled to 90 °C and then left in room temperature. The sample slides were washed with PBS containing 0.1 % bovine serum albumin (BSA) and Protein Block (Spring Bioscience, Pleasanton, CA). Primary monoclonal anti-CXCR3 antibody [2Ar1] at 500 ng/ml (Abcam, Cambridge, UK), primary polyclonal rabbit anti-CXCL10 antibody at 111 ng/ml (ab9807, Abcam) or control IgG mouse antibody at 500 or 111 ng/ml (DAKO, Glostrup, Denmark) were added and the sample slides were incubated overnight at 4 °C. Envision secondary anti-mouse antibody conjugated to HRP (DAKO) was added, thereafter the sample slides were incubated in 3,3′-diaminobenzidine tetrahydrochloride (DAB) with hydrogen peroxide and counterstained with hematoxylin (BioRad, Hercules, CA), rehydrated and fixated with Mount-X (HistoLab) mounting solution. Grading was done using a Leica LB30T microscope (Leica Microsystems, Wetzlar, Germany). Sample scoring was done without evaluators’ knowledge of clinical or pathological data for patients, using a 0–3 scaling system, − indicating no staining, + indicating weak expression, ++ indicating moderate expression, and +++ indicating a strong expression. Only tumor cells were analyzed. Two individual evaluators judged all slides independently. Representative slides for each intensity were photographed using an Axio Lab A1 microscope with an AxioCam ICc5 camera (Zeiss, Oberkochen, Germany), all images were acquired using image acquire software Zen 2012 blue edition (Zeiss).

The relationships between grouped variables were analyzed using Spearman’s rank order correlation. To compensate for multiple testing P < 0.01 was set as significant. The survival curves were produced according to the lifetable method described by Kaplan and Meier and differences between groups were evaluated with log-rank tests. Patients with missing data were excluded. Univariate and multivariate analysis were conducted using Cox proportional hazards regression and P < 0.05 was considered significant. End points used were breast cancer-specific mortality, defined as when patients had a local or distant recurrence or when so stated by the Swedish cause of death registry. Local recurrence defined as a relapse on either chest wall or in a regional lymph node or distant recurrence, defined as the remaining metastatic events. The statistical package Statistica 10.0 (StatSoft Scandinavia, Uppsala, Sweden) was used for all calculations.