BCL3 expression is strongly associated with the occurrence of breast cancer relapse under tamoxifen treatment in a retrospective cohort study

Abstract

Patients with estrogen receptor positive breast cancer are usually receiving an anti-estrogen therapy by either aromatase inhibitors or selective estrogen receptor mediators such as tamoxifen. Nevertheless, acquired resistance to tamoxifen under treatment frequently hampers therapy. One proposed explanation for this phenomenon is the interaction of the tumor cells with cells of the tumor microenvironment via the Insulin-like growth factor RNA binding protein 5/B-cell lymphoma 3 (IGFBP5/BCL3) axis. Here we investigated whether a high expression of BCL3 either cytoplasmic or nuclear is associated with the occurrence of a relapse under anti-estrogen therapy in patients. Formaldehyde-fixed, paraffin-embedded samples of 180 breast cancer patients were analyzed for BCL3 expression by immunohistochemistry. An immunoreactive score (IRS) was calculated from staining intensity in cytoplasm and nucleus as well as the percentage of positive tumor cells. These scores were correlated with clinico-pathological parameters using cross-tabulation analysis and patients' relapse free and overall survival by Kaplan-Meier analysis and Cox regression. A tamoxifen-adapted MCF-7 derived cell line was investigated for BCL3 localization by immunofluorescence. The cytosolic BCL3-IRS significantly correlated with the proliferation marker Ki-67, and with the occurrence of a relapse under tamoxifen treatment. Nuclear score correlated only with tamoxifen-relapse. In survival analysis, both scores were highly significant prognostic factors for relapse free, but not for overall survival. This was especially obvious for estrogen receptor positive and HER2/NEU negative cases as well as lobular breast cancer. Tamoxifen-treated, but not aromatase-treated patients had a poor survival when BCL3 scores were high. A tamoxifen adapted cell line exhibited a reduced expression and mainly nuclear localization of BCL3, compared to the parental estrogen receptor positive cell-line MCF-7. Altogether, these data strongly support a function of BCL3 in tamoxifen resistance and its potential use as a predictive biomarker for tamoxifen resistance.

Keywords: BCL3; Immunohistochemistry; Biomarker; Breast cancer; Tamoxifen; Survival.

Fig. 1

A Indirect immunofluorescent staining of BCL3 in MCF-7 and MCF7-TamR cells. Cells were stained using the BCL3 antibody and a secondary fluorescent antibody. The nuclei were counter-stained using propidium iodine (PI). Images were obtained using a laser scanning microscope as described in “Materials and methods.” The scale bar represents 50 µm. B Western blot analysis of BCL3 in protein extracts of MCF-7 compared to three Tam-adapted cell lines (TamR) derived from this cell line. BCL3 Western blot signals as well as poinceau red protein stain (PR) are shown. The bar graph indicates the BCL3 signal normalized to the PR staining result averaged for each cell line with standard deviation

Fig. 2

Representative results of the IHC of BCL3 in BC samples. Scale bars indicate 50 or 250 µm, respectively. A, C, E, G, I, K, M, and O show low magnification; B, D, F, H, J, L, N, and P high magnification. Intensity- and %-scores: A, B: cyt. 0, 0 nucl. 1, 3 (I, %); I, J (cibriform DCIS): cyt. 0, 0 nucl. 1, 15 (I, %); C, D: cyt. 1, 90 nucl. 3, 95 (I, %); K, L: cyt. 1, 40 nucl. 1, 3 (I, %); E, F: cyt. 2, 90 nucl. 3, 80 (I, %); M, N: cyt. 2, 30 nucl. 0, 0 (I, %); G, H: cyt. 3, 40 nucl. 0, 0 (I, %); O, P: cyt. 3 100 nucl. 3, 70 (I, %)

Fig. 3

Determination of a cut-off value for BCL3 IRS based on Kaplan–Meier survival analysis. A Data for cytosolic BLC3. Log-rank p for RFS, OS, the relative number of BCL3 high cases and the average relapse-free and overall-survival time depending on the cut-off value are shown. B Data for nuclear BCL3 IRS are shown as described for A. C Receiver-operator-curves (ROC) for cytosolic and nuclear IRS and relapse-free survival for all cases and tamoxifen-treated cases

Fig. 4

Kaplan–Meier plots for relapse-free survival (RFS) depending on BCL3-IRS_nuc and BCL3-IRS_cyt or a combined score. (0: negative in both locations; 1: positive in one location; 2: positive in both locations)

Fig. 5

Kaplan–Meier plots for relapse-free survival depending on BCL3 nuclear and cytoplasmic score stratified for estrogen status (A), HER2 status (B), ductal and lobular histology (C), and treatment with tamoxifen or aromatase inhibitor (D)

Fig. 6

Distribution of BCL3 mRNA in public datasets. The METABRIC dataset was stratified according to the 3-gene classifier subtypes. Cases are labelled according to genomic BCL3 alterations such as mutations or copy number. Significance was determined with one-way ANOVA and Tamhane T2 post hoc analysis: *: p < 0.05, ** p < 0.01, *** p < 0.001. Results obtained from the GEPIA2 database are shown on the right