Ethical agreement

This study obtained approval from the College of Medicine/Al-Mustansiriyah University (1 Jun 2021, no.21), and consent was taken from patients to use their paraffin block samples.

Study design and sample criteria

In this retrospective study, breast tissue in paraffin-embedded blocks of a group of Iraqi women patients diagnosed with breast cancer was collected between the periods of January 2021 and June 2022 from archived materials from Al Yarmouk Teaching Hospital, Baghdad, Iraq. These paraffin blocks represented 40 cases of breast cancer; all cases were selected to be specimens of mastectomies. Cases included in this study had patients aged between 46 and 65 years with a mean of 54.7± 5.61 (SD) years. All cases were diagnosed as invasive ductal carcinoma of the breast. Clinicopathological parameters, including the age of patients, grade, and stage of the tumor, were obtained from the histopathologic reports and were independently reviewed by two histopathologists. Regarding tumor grade (according to the Nottingham Histological Grading System), ten cases were in grade Ⅰ, and fifteen cases were in grades 11 and Ⅲ. Concerning the tumor stage (according to the American Joint Committee on Cancer), the cases were divided into: nine cases in stage Ⅰ, eleven cases in stage 11, and 10 cases in each stage Ⅲ and stage Ⅳ, as shown in Tables 2 and 3. All cases were selected to be triple-negative (basal-like) breast cancer subtypes according to estrogen, progesterone, and her 2-receptors status at the time of diagnosis. Samples of normal breast tissue were taken from adjacent tissue from 10 cases of the same mastectomy specimens as a negative control. The positive control used in this study included tissue of a normal human liver for Annexin A2 and tissue of a normal human tonsil for Annexin A6 (according to kit leaflets) with each run. 

Immunohistochemical staining procedures

Selected paraffin blocks of breast tissue samples were sectioned into 4 µm. From each block, 3 sections were taken: one section for hematoxylin and eosin, and two sections were prepared on positively charged slides for immunohistochemical staining with Annexin A2 and Annexin A6. Immunohistochemical staining was achieved by using the following antibodies: Annexin A2 (dilution 1: 100, Abcam; Code: 178677, rabbit monoclonal antibody) and Annexin A6 (dilution 1: 2000, Abcam; Code: 201023, rabbit monoclonal antibody). An automated system from Leica was used for all staining steps. Specimens were deparaffinized, and then antigen retrieval was done by using a microwave for about 20 minutes. All slides were incubated for about 15 minutes with the primary antibody, then for 10 minutes with the post-primary polymer, and were blocked for 4 minutes by hydrogen peroxide 3% and with brown chromogen for 10 minutes, followed by staining with counterstain (hematoxylin) for 5 minutes. All these steps of incubation were achieved at room temperature. Slides were washed with buffered saline between these steps, and coverslipping by DPX was performed.

Assessment of immunohistochemical results

In Annexin A2 and Annexin A6, the location of cellular staining is cytoplasmic, and membranous or only cytoplasmic brown staining was regarded as a positive result. The two markers are also expressed in stromal cells, but this expression was not included in the evaluation. The assessment of the intensity of staining was as follows: 0 as none; 1 as mild (0% to 5% of epithelial cells); 2 as moderate (more than 5% to 50%); and 3 as strong (more than 50% staining) [18].

Statistical analysis

The Statistical Package of Social Sciences (SPSS version 25) program was used in the statistical analysis of the results. The student test (t-test) was used for handling the quantitative data: Annexin A2+, Annexin A2–, Annexin A6+, Annexin A6‑, and controls. If required, absolute variables were evaluated by a Pearson Chi-squared analysis test with an ANOVA test. The p-value was considered to be significant if it was less than (0.05).

Results

Figures & Tables

Discussion

Breast cancer represents the most frequent cancer in Iraqi women, and it is the first leading cause of cancer deaths among them. We investigate Annexin A2 and Annexin A6 due to their studied rules in the evolution and progression of different types of tumors, including breast cancer, selecting triple-negative (basal-like) breast cancer because of its aggressive behavior. The current study shows a positive expression of Annexin A2 in 72.5% of cases, the percentage of expression in the current study was higher in comparison with a previous study (positive in 54.54% of cases) by Lee, Kelsey M., Sayantan, et al. (2021) [19] and Lee and Jamboor (2017) [20].

Regarding Annexin A6 expression, this study shows positive expression in 67.5% of cases; this result agrees with a past study by Amos, Rainelli, Bobby, et al. (2010) [21]. The correlation between Annexin A2 and Annexin A6 expression in triple-negative breast cancer was significant in the investigated group of patients, suggesting the importance of these two markers in the development of this type of cancer. There was no previous study showing the correlation between these two markers in triple-negative breast cancer. There is a significant correlation between Annexin A2 expression and the tumor histological grade; we found that increases in expression were associated with a higher grade, while the correlation between Annexin A6 expression and the tumor histological grade was not significant, as shown in Table 2. There is a significant correlation between Annexin A2 expression and tumor stage; a positive expression is associated with advanced tumor stage, while no significant association was found between Annexin A6 expression and tumor stage, as shown in Table 3.

The results of the current study show that Annexin A2 expression in triple-negative breast cancer significantly correlates with progression, metastasis, and the poor clinical outcome and prognosis of the tumor, while Annexin A6 expression does not correlate with tumor grade or stage. This finding suggests that the investigation of Annexin A2 immunohistochemical expression may be a useful non-invasive biomarker that correlates with aggressive tumor behavior and poor prognosis and may introduce Annexin A2 as a possible new therapeutic target in this type of aggressive cancer. The limitation of this study is that we need a larger sample size and investigation of these two markers on other molecular types of breast cancer with follow-up of patients.

This study investigates the role of Annexin A2 and Annexin A6 in one of the most clinically aggressive subtypes of breast cancer, triple-negative breast cancer, and demonstrates the significant association of Annexin A2 and Annexin A6 expression in this type of cancer, and the significant association of Annexin A2 expression with tumor grade and stage. The expression is higher in the higher grade and advanced stage, which suggests the important role of Annexin A2 in the progression, metastasis, and prognosis of triple-negative breast cancer.

The authors declare that there is no conflict of interestregarding the publication of this paper.

References

1. World Health Organization. International Agency for Research on Cancer. The Global Cancer Observatory , March (2021).
2. Toss A, Cristofanilli M. Molecular characterization and targeted therapeutic approaches in breast cancer. Breast Cancer Research, (2015); 17:60.
3. Millis Z, Gatalica Z, Winkler J, Vranic S, Kimbrough J, Reddy S, et al. Predictive biomarker profiling of> over 6,000 breast cancer patients shows heterogeneity in TNBC, with treatment implications. Clinical breast cancer, (2015); 15(6):473–481.
4. Russo J. Molecular Classification of Breast Cancer. Breast Cancer Essentials, (2021); 33–38.
5. Ensenyat-Mendez M, Llinàs-Arias P, Orozco J, Íñiguez-Muñoz S, Salomon M, Sesé B, et al. Current Triple-Negative Breast Cancer Subtypes: Dissecting the Most Aggressive Form of Breast Cancer. Frontiers in oncology, (2021); 16:11:681476.
6. Tsang J, Tse G. Molecular classification of breast cancer. Advances in anatomic pathology, (2020); 27(1):27–35.
7. Xi Y, Ju R, Wang Y. Roles of Annexin A protein family in autophagy regulation and therapy. Biomedicine and pharmacotherapy, (2020); 130(8):110591.
8. Moreau K,Ghislat G ,Hochfeld W, Renna M ,Zavodszky E, Runwal G, et al. Transcriptional regulation of Annexin A2 promotes starvation-induced autophagy. Nature communication, (2015); 6(1): 1–12.
9. Chen Y, Fang Y, Cheng Y, Lin C, Hsu L, Wang S, et al. Exophagy of Annexin A2 via RAB11, RAB8A, and RAB27A in IFN-gamma-stimulated lung epithelial cells. Scientific reports, (2017); 7(1):5676.
10. Wang C, Lin C. Annexin A2: Its Molecular Regulation and Cellular Expression in Cancer Development. Disease markers, (2014); 4: 308976.
11. Sharma M. Annexin A2 (ANX A2): An emerging biomarker and potential therapeutic target for aggressive cancers. International Journal of Cancer, (2019); 1; 144(9):2074-2081.
12. Mussunoor S, Murray G. The role of annexins in tumor development and progression. Journal of Pathology, (2008); 216:131–140.
13. Enrich C, Rentero C, Grewal T. Annexin A6 in the liver: from the endocytic compartment to cellular physiology. Biochimica et Biophysica Acta (BBA): Molecular Cell Research, (2017); 1864 (6): 933–946.
14. Koese M, Rentero C, Kota B, Hoque M, Cairns R, Wood P, et al. Annexin A6 is a scaffold for PKC alpha to promote EGFR inactivation. Oncogene, (2013); 32: 2858–2872.
15. Creutz C, Hira J, Gee V, Eaton J. Protection of the membrane permeability barrier by Annexins. Biochemistry, (2012); 51:9966-9983.
16. Alvarez-Guaita A, Vilà de Muga S, Owen D, Williamson D, Magenau A, García-Melero A, et al. Evidence for Annexin A6 dependent plasma membrane remodeling of lipid domains. British Journal of Pharmacology, (2015); 172:1677-1690.
17. Boye T, Maeda K, Pezeshkian W, Sønder S, Haeger S, Gerke V, et al. Annexin A4 and A6 induce membrane curvature and constriction during cell membrane repair. Nature Communication, (2017); 8:1623.
18. Chand P, Garg A, Singla V, Rani N. Evaluation of the Immunohistochemical Profile of Breast Cancer for Prognostics and Therapeutic Use. Nigerian Journal of Surgery, (2018); 24(2): 100–106.
19. Gibbs L, Mansheim K, Maji S, Nandy R, Lewis C, Vishwanatha J, et al. Clinical Significance of Annexin A2 Expression in Breast Cancer Patients. Cancers, (2021); 13(1): 2.
20. Gibbs L, Vishwanatha J. Prognostic impact of AnxA1 and AnxA2 gene expression in triple-negative breast cancer, (2018); 9:2697-2704.
21. Sakwe A, Koumangoye R, Guillory B, Ochieng J. Annexin A6 contributes to the invasiveness of breast carcinoma cells by influencing the organization and localization of functional focal adhesions. Excremental cell research, (2001); 317(6): 823–837.