**4.3. Correlation between calpain-1 expression and clinicopathological variables and outcome of TNBC patients**

In order to investigate the possibility of using calpain-1 protein as a prognostic biomarker in TNBC, its expression was assessed for association with a number of clinicopathological variables. We determined that calpain-1 expression displayed a significant positive association to the lymph node status (*P* = 0.02) but not with other clinicopathological variables. Kaplan– Meier survival curves were plotted with significance determined using the log-rank test in order to determine the relationship between calpain-1 protein expression in the recurrencefree survival (RFS) and in the overall survival (OS) patients. The expression of calpain-1 in the triple-negative tissues was not significantly associated with breast cancer RFS ( = 0.71) or OS ( = 0.88) in which the median RFS was 18 months (3–77 months) and OS was 41 months (0–105 months) in the total patient cohort.

TNM classifies lymph node status as a tumor-related prognostic factor, therefore, our results suggest that calpain-1 might be used as a prognostic factor in TNBC. Calpain-1 was also found to be associated with lymph node status in other types of cancer, such as renal cell carcinoma [51]. The observation of the lack of association of calpain-1 with other clinicopathological variables is consistent with a study conducted by Storr et al. in which they demonstrated a correlation between calpain-1 expression and tumor grade but not with other clinicopathological variables [40].

The variations among the presence or absence of association with lymph node status or tumor grade which are essential in determining its prognosis can be explained by several theories; (i) the majority of patient samples were of intermediate grade tumor and therefore calpain-1 activity may have started at later stages as suggested by its correlation with the lymph node status, (ii) the lack of wide range of sample collection in regards to tumor grades may have created a diversion in the statistical analysis, (iii) the insufficiency of samples might have contributed to lack of significant correlations, (iv) the possibility of genetic differences between the populations in the current study and the ones already published may be the cause of differences on the expression of calpain-1 in breast cancer cells [40] and finally (v) the presence or absence of the hormonal receptors such as ER, PR, and HER2 that determine breast cancer behavior and thus treatment can influence the outcome. Storr *et al*. (2011) reported that there was no association between the expression of calpain-1 in HER2-positive breast cancer patients treated with trastuzumab following adjuvant chemotherapy with any of the clinicopathological variables [52]. Hence, their observation is consistent with our data but may differ in terms of the positivity of HER2.
