Calpain-1 Expression in Triple-Negative Breast Cancer: A Potential Prognostic Factor Independent of the Proliferative/Apoptotic Index

Abstract

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer in which calpain system plays an important role in its cellular processes including apoptosis and proliferation. Although such roles have been assessed in tumor pathogenesis, the correlation of its expression to the proliferating/apoptotic index has not been studied yet. Immunohistochemical staining of calpain-1 was performed on paraffin-embedded tissues to correlate its expression with clinicopathological variables and outcome. The proliferation activity was determined by calculating the percentage of cells expressing the Ki-67 antigen. The apoptotic index was assessed morphologically and biochemically using Haematoxylin & Eosin method and Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, respectively. Calpain-1 was significantly expressed in TNBC tissues varying from low to high with a significant correlation to lymph node status but not with the other clinicopathological variables, suggesting its role as a prognostic factor. In addition, a positive correlation was found between both apoptotic counts assays (P < 0.001, r = 0.547) as well as with proliferation (P = 0.045). Calpain-1 expression had no significant correlation with either proliferation (P = 0.29) or apoptotic indices (P = 0.071 and P = 0.100). Determining calpain-1 expression may provide relevant prognostic value for TNBC cancer patients.

Figure 1

Detection of calpain-1 protein in TNBC tissues. Representative immunostaining photomicrographs of high, medium, and low calpain-1 expression [(a) high expression, (b) medium expression, (c) low expression, and (d) negative control (non-TNBC tissue)]. Photomicrographs are shown at ×40 magnification and a scale bar of 20 μm.

Figure 2

Kaplan-Meier analysis of TNBC patients' overall survival. (a) Recurrence-free survival and (b) the impact of high, intermediate, and low calpain-1 protein expression. Calpain-1 expression is not significantly associated with recurrence-free survival (P = 0.71) with significance determined using the log-rank test.

Figure 3

Kaplan-Meier analysis of TNBC patients' survival and calpain-1 expression. (a) Overall survival and (b) the impact of high, intermediate, and low calpain-1 protein expression. Calpain-1 expression is insignificantly associated with overall survival (P = 0.88) with significance determined using the log-rank test.

Figure 4

Immunostaining detection of Ki-67 protein in TNBC tissues. Cellular proliferative activity of breast cancer tissue was evaluated as high (a) and low (b) percentages using anti-Ki-67 antibody by Immunohistochemistry. Photomicrographs are at ×40 magnification where scale bar shows 20 μm.

Figure 5

The correlation between calpain-1 expression and proliferation (CI = 95%).

Figure 6

Evaluation of apoptotic index using H&E and TUNEL staining. ((a) human colon mucosa, positive control, (b) human colon mucosa, negative control, (c) highly expressed apoptotic cells stained by TUNEL method, triple-negative breast cancer tissue, and (d) apoptotic cells stained with H&E). Photomicrographs are at ×40 magnification and a scale bar of 20 μm.

Figure 7

The correlation between calpain-1 expression and TUNEL-based apoptotic ((a) CI = 95%) and H&E-based apoptotic ((b) CI = 95%) staining.

Figure 8

Representative photomicrographs of series of specimen from the same patient. (a) Calpain-1 (medium), (b) Ki-67 (70%), (c) apoptosis-based H&E satin (60%), and (d) apoptotic-based TUNEL assay (60%). Photomicrographs are at ×40 magnification and a scale bar equals 20 μm.