Prediction of breast cancer sensitivity to neoadjuvant chemotherapy based on status of DNA damage repair proteins

Abstract

Introduction: Various agents used in breast cancer chemotherapy provoke DNA double-strand breaks (DSBs). DSB repair competence determines the sensitivity of cells to these agents whereby aberrations in the repair machinery leads to apoptosis. Proteins required for this pathway can be detected as nuclear foci at sites of DNA damage when the pathway is intact. Here we investigate whether focus formation of repair proteins can predict chemosensitivity of breast cancer.

Methods: Core needle biopsy specimens were obtained from sixty cases of primary breast cancer before and 18-24 hours after the first cycle of neoadjuvant epirubicin plus cyclophosphamide (EC) treatment. Nuclear focus formation of DNA damage repair proteins was immunohistochemically analyzed and compared with tumor response to chemotherapy.

Results: EC treatment induced nuclear foci of gammaH2AX, conjugated ubiquitin, and Rad51 in a substantial amount of cases. In contrast, BRCA1 foci were observed before treatment in the majority of the cases and only decreased after EC in thirteen cases. The presence of BRCA1-, gammaH2AX-, or Rad51-foci before treatment or the presence of Rad51-foci after treatment was inversely correlated with tumor response to chemotherapy. DNA damage response (DDR) competence was further evaluated by considering all four repair indicators together. A high DDR score significantly correlated with low tumor response to EC and EC + docetaxel whereas other clinicopathological factors analyzed did not.

Conclusions: High performing DDR focus formation resulted in tumor resistance to DNA damage-inducing chemotherapy. Our results suggested an importance of evaluation of DDR competence to predict breast cancer chemosensitivity, and merits further studying into its usefulness in exclusion of non-responder patients.

Figure 1

Immunohistochemistry controls and antibody specificity. (a) Immunohistochemical staining with control IgG for tumors after the first cycle of epirubicin plus cyclophosphamide (EC) treatment (upper panel). Lower panel shows Rad51 staining for morphologically diagnosed non-cancerous breast tissues (left upper part) and tumor (right lower part) after the first cycle of EC treatment. Although non-cancerous breast cells also expressed nuclear foci formation the number and intensity was significantly lower than that in tumor cells. (b and c) DNA damage-induced nuclear foci formations detected by antibodies used in the study. HeLa cells were either untreated (-), treated with 5 Gy ionizing radiation (IR) or 0.2 μg/ml epirubicin (Epi), incubated for three hours and fixed. Cells were then subjected either to immunofluorescent analyses with the indicated primary antibodies and (b) FITC- (green) or Rhodamine- (red) conjugated secondary antibodies, or (c) to the same protocol as that used in the tissue stain. For immunofluorescent analyses the nucleus was counterstained with DAPI. Ub, conjugated ubiquitin.

Figure 2

Nuclear focus formation in response to chemotherapy. Tumor specimens were obtained by core needle biopsy before and 18 to 24 hours after the first cycle of epirubicin plus cyclophosphamide (EC) treatment. Immunohistochemical findings from representative cases for γH2AX, conjugated ubiquitin (Ub), BRCA1, and Rad51 are shown. Graphs at right demonstrate changes in nuclear foci score after EC treatment in all cases analyzed. The red, blue, and black lines indicate cases with increased, decreased, and unchanged scores, respectively. The thickness of the lines proportionally reflects the number of cases. The thinnest line (γH2AX score 3 to 3) and the thickest line (Ub, score 0 to 0) represent 1 and 34 cases, respectively. n, number of cases analyzed.

Figure 3

Mean tumor volume reductions after (a) EC or (b) EC and DOC according to DDR score. Error bars represent standard deviation. Significance was analyzed by Tukey-Kramer test setting P < 0.05 as significance threshold. DDR, DNA damage response; DOC, docetaxel; EC, epirubicin plus cyclophosphamide.