**4.4. Association between calpain-1 expression, cell proliferation and apoptosis in TNBC tissues**

received neoadjuvant treatment and 5 (19.2%) achieved complete pathological response. Anthracyclines and taxanes were the most commonly used chemotherapeutic agents as frontline treatment. Breast cancer related overall survival (OS) was defined as the time interval (in months) from the date of diagnosis until death from breast cancer. Similarly, recurrence-free survival (RFS) was defined as the time interval (in months) between the start of primary treat-

Immunostained tissues with calpain-1 were significantly expressed and demonstrated cytoplasmic and membranous staining with some granularity and heterogeneity between adjacent tumor cells varying from weak to intense staining in which low staining was detected in 32.7% (18 of 55), intermediate staining in 38.2% (21 of 55) and high staining in 29.6% (16 of 55) of the cases analyzed. The cut off value was determined by screening the stained tissue under light microscope where the staining intensity of calpain-1 in tumor cells was assessed as none (0), weak (1), medium (2), and strong (3) using an immunohistochemical *H*-score. The *H*-scores were calculated by multiplying the percentage area by the intensity grade (*H*-score range 0–300).

**4.3. Correlation between calpain-1 expression and clinicopathological variables and** 

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

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 clinicopatho-

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

ment and date of cancer relapse.

154 Breast Cancer and Surgery

**outcome of TNBC patients**

logical variables [40].

(0–105 months) in the total patient cohort.

**4.2. Calpain-1 expression in TNBC tissues**

Calpains have been reported to be involved in the proliferation of breast cancer cells [48, 49]. However, the role of the calpain family in proliferation of TNBC cells has not been reported yet. Ki-67, a nuclear antigen is a protein encoded by Ki-67 on 10q25 and considered to be a proliferation marker for predicting tumor development [53]. It is expressed during all active phases of the cell cycle except the resting phase, thus being present only in dividing cells. Ki-67 is detected by monoclonal antibody MIB-1 which can be a useful marker of proliferation and of prognostic value [53]. The quantitative assessment of Ki-67 staining on paraffin embedded tumor sections has been reported as an accurate estimate of the proliferation index of individual tumors [53].

Therefore, proliferative fractions of paraffin embedded breast cancer tissues were determined by immunohistochemical staining for Ki-67 antibody. The cellular proliferative activity was estimated as the percentage of tumor cells stained per field ×40. Statistical analysis showed no significant correlation between calpain-1 expression and proliferation ( = 0.29). Possible theories of the presence and absence of the hormonal receptors, differences in the genetic makeup, and other members of calpains involvement may also influence the correlation with proliferation.

Cell proliferation along with cell death are both phenomena responsible for control of cell number in normal tissues and tumors. Since chemotherapy induces programmed cell death by apoptosis, hence, the apoptotic tumor cells can be morphologically identified using the conventional hematoxylin and eosin (H&E) method and cells are counted using light microscopy. Therefore, there has been interest in the application of the apoptotic index in malignant growths as a putative prognostic marker. The percentage of apoptotic cells in tumor sections may also be measured by a molecular-based approach, labeling of fragmented DNA breaks and calculating the apoptotic index (AI) using the terminal transferase-uridyl nick-end labeling (TUNEL) assay.

Therefore, in order to determine whether the frequency of apoptosis was related to tumorigenesis, two approaches; the conventional H&E staining method and the apoptotic TUNEL assay were both used to detect apoptotic cells and to prove that the two methods comparatively correlate with each other. H&E detects apoptosis in its degradation phase and can be subjective whereas the TUNEL assay detects apoptosis in its early phase and is more objective. Apoptotic cells were counted per 100 invasive tumor cells using ×40 objective. Apoptotic counts using either method, were significantly correlated ( < 0.001, = 0.547). Although both assays tested apoptosis from different aspects, but the results were the same, indicating the reliability of both assays. These findings were also consistent with a previous study by Watanabe et al. [54]. In addition, the relationship between apoptosis and proliferation was investigated in TNBC tissues. For all of the patients, high apoptotic counts significantly correlated with increased cell proliferation ( = 0.045). The positive correlation between proliferative and apoptotic indices seen in this study is also consistent with other types of cancers such as colorectal cancers [54].

**References**

2000;**301**(1):143-152

2002;**50**(5):343-352

**2017**:10

2017;**18**(6):369-378

International. 2017;**2017**:3178794

Journal of Medicine. 2001;**344**(26):1997-2008

Drug Discovery. 2005;**4**(4):307-320

[1] Wahba HA, El-Hadaad HA. Current approaches in treatment of triple-negative breast

Triple-Negative Breast Cancer, Cisplatin and Calpain-1 http://dx.doi.org/10.5772/intechopen.74657 157

[2] Makki J. Diversity of breast carcinoma: Histological subtypes and clinical relevance.

[3] Cailleau R, Young R, Olive M, Reeves WJ Jr. Breast tumor cell lines from pleural effu-

[4] Sørlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications.

[5] Dai X, Li T, Bai Z, Yang Y, Liu X, Zhan J, et al. Breast cancer intrinsic subtype classification, clinical use and future trends. American Journal Of Cancer Research. 2015;**5**(10):2929-2943

[6] Hortobagyi GN. Toward individualized breast cancer therapy: Translating biological

[7] Makin G, Hickman JA. Apoptosis and cancer chemotherapy. Cell and Tissue Research.

[8] Kim R, Tanabe K, Uchida Y, Emi M, Inoue H, Toge T. Current status of the molecular mechanisms of anticancer drug-induced apoptosis. The contribution of molecularlevel analysis to cancer chemotherapy. Cancer Chemotherapy And Pharmacology.

[9] Al-Bahlani S, Al-Dhahli B, Al-Adawi K, Al-Nabhani A, Al-Kindi M. Platinum-based drugs differentially affect the ultrastructure of breast cancer cell types. BioMed Research

[10] Al-Bahlani SM, Al-Bulushi KH, Al-Alawi ZM, Al-Abri NY, Al-Hadidi ZR, Al-Rawahi SS. Cisplatin induces apoptosis through the endoplasmic reticulum-mediated, calpain 1 pathway in triple-negative breast cancer cells. Clinical Breast Cancer. 2017;**17**(3):e103-e112

[11] Al-Bahlani SM, Al-Rashdi RM, Kumar S, Al-Sinawi SS, Al-Bahri MA, Shalaby AA. Calpain-1 expression in triple-negative breast cancer: A potential prognostic factor independent of the proliferative/apoptotic index. BioMed Research International. 2017;

[12] Shapiro C, Recht A. Side effects of adjuvant treatment of breast cancer. The New England

[13] Jin, Jin J, Zhang W, Ji W, Yang F, Guan X. Predictive biomarkers for triple negative breast cancer treated with platinum-based chemotherapy. Cancer Biology & Therapy.

[14] Wang D, Lippard S. Cellular processing of platinum anticancer drugs. Nature Reviews

Proceedings of the National Academy of Sciences. 2001;**98**(19):10869-10874

cancer. Cancer Biology & Medicine. 2015;**12**(2):106-116

sions. Journal of the National Cancer Institute. 1974;**53**(3):661-674

concepts to the bedside. The Oncologist. 2012;**17**(4):577-5784

Clinical Medicine Insights Pathology. 2015;**8**:23-31

In experimental models the calpain system has been shown to influence apoptosis in breast cancer [48, 55, 56]. The relationship between calpain-1 expression and apoptosis using the two methods, H&E-based apoptotic counts and apoptotic counts derived from the apoptotic TUNEL assay was investigated in the TNBC tissues. Interestingly, the data revealed that there were no significant association between the apoptotic indices when compared to calpain-1 expression ( = 0.710 and 0.100), respectively. Such results suggest that the TNBC cells undergo apoptosis via other members of the calpain family such as calpain-2.

Taken together, these data have clearly demonstrated the absence of correlation between calpain-1 expression and the proliferating/apoptotic index or clinicopathological variables except with the lymph node status of TNBC patients. Hence, calpain-1 could be a useful prognostic marker in TNBC. More studies should be conducted in the future to evaluate the prognostic value of calpain-1 in TNBC.
