Determination of Serum Angiopioetin-2 levels in Breast cancer patients at Damascus University Hospitals

 

Majd Haidar, Jumana Al Saleh

Department of Microbiology, Hematology and Immunology, Collage of Pharmacy, Damascus University, Damascus Syria.

*Corresponding Author E-mail: majd.ha1992@gmail.com

 

ABSTRACT:

Background: Breast cancer has increased universally. Angiogenesis is an essential step for cancer growth, progression and metastasis. Recent studies have focused on Angiopoietin-2 as a promising biomarker in prospecting the prognosis of the cancer. Objective: Determine the relation between serum concentrations of Angiopoietin-2 and clinicopathological data in breast cancer patients. Methodology: This study is prospective, cohort type. It includes 30 women with breast cancer, who met the study criteria. The serum Ang-2 value was calibrated using ELISA technique. The data was processed using SPSS program. Results: Ang-2 serum levels were statistically significant associated with ER status (P = 0.036). However, no signifi­cant correlation was observed between serum Ang-2 level and the other clinicopathologic param­eters tested, including Histological type (P = 0.417), Clinical grade G (P = 0.91), Clinical stage T (P = 0.913), Clinical stage N (P = 0.086), PR status (P = 0.467), and HER-2 status (P = 0.791). Serum levels of Ang-2 for breast cancer patients was significantly higher than healthy control (20411.33± 6283.19 vs 1731.70± 368.35 pg/ml, P< 0.000). our data indicates that serum levels of Ang-2 has no correlation between the tumor grade and stage in breast cancer patients.

 

KEYWORDS: Breast cancer, Angiogenesis, Angiopoietin-2.

 

 

INTRODUCTION:

Breast cancer is the most common cancer and the second leading cause of cancer-related deaths in females worldwide1. Over 1.5 million women (25% of all women with cancer) are diagnosed with breast cancer every year throughout the world2.

 

Angiogenesis plays an important role in carcinogenesis. it is regulated by a complex balance of proangiogenic and antiangiogenic factors.3 Therefore, the development of angiogenic characteristics is vital for cancer growth in a number of tumor types including breast cancer4. There are a large number of angiogenic mediators including the angiopoietin (Ang) family which play an important role in both physiological and pathological angiogenesis.

 

Four Ang have been identified; termed Ang 1-4. Of these, Ang-1 and Ang-2 are the most widely studied and function as ligands for Tie-2, which is a receptor tyrosine kinase specifically expressed on endothelial cells5. Angiopoietin-2 (Ang2) is a growth factor belonging to the angiopoietin/Tie (tyrosine kinase with Ig and EGF homology domains) signaling pathway, one of the main pathways involved in angiogenesis6. Ang-2 was first identified as a natural antagonist for Tie-2, which is only up-regulated at sites of active vascular remodeling, released from endothelial cells, binds Tie2 and contributes to vessel permeability, instability, and vascular remodeling resulting in vessel destabilization and regression7,8.

 

Ang2 is highly expressed in the vasculature of many tumor types, including breast. it appears to play an important role in tumor angiogenesis, possibly as a Tie2 agonist9,10. In fact, Ang-2 role is highly context-dependent, shows proangiogenic and antiangiogenic activities6,11 that explains why its role in tumor is controversial. In other words, Ang-2 expression is upregulated in tumor-associated endothelium, and its expression has been shown to induce tumor growth and tumor invasiveness, while Ang-2 also has been found to reduce angiogenesis, to retard tumor growth and to enhance apoptosis cancer types12. overexpression of Ang-2 is associated with advanced disease and poor prognosis in several tumor entities such as breast cancer, gastric cancer, melanoma, lung cancer, colorectal cancer, and hepatocellular cancer13.

 

Circulating Ang-2 levels have been associated with tumor angiogenesis in several cancers, but still the role of Ang-2 is controversial because elevated and reduced circulating levels of angiopoietins have been reported14. Accumulating evidence demonstrated a link of increased expression of Ang-2 with invasive and metastatic phenotypes of various types of human cancers including breast cancers1,12. A study found that increased Ang-2 expression was strongly associated with histological grade, lymph node metastasis, and clinical stage in breast cancer patients1.

 

In the present study, we aimed to assess the association between the serum levels of Angipoietin-2 and clinicopathological data from breast cancer patients.

 

MATERIALS AND METHODS:

Blood samples were collected from Al- Biruni University Hospital, Damascus, Syria, between 2018-2019. The prospective cohort study included 46 participants: 30 female patients with a breast cancer (65.2%),these samples were taken twice: immediately prior to surgery (tₒ) and after 6 months (t₆) following the ending of chemotherapy according to the applied protocol at Al-Biruni University Hospital, 6 females with benign breast disease also requiring surgery (13%) and 10 sex and age matched healthy controls (21.8%) with no previous history of any cancer and free of inflammatory, neoplastic, atherosclerotic or connective tissue disease. The histological samples from breast cancer patients were examined. Subsequently, the grade and the stage of the tumor were detected.

 

Breast cancer patients were staged according to the 8th ed. American Joint Committee on Cancer (AJCC) TNM Staging Classification for Breast cancer. According to AJCC staging, 15 patients had stage 0, 2 patients had stage I, 6 patients had stage II, 7 patients had stage III disease. 15 patients had in situ ductal carcinoma (50%) and 15 invasive ductal carcinoma (50%).

 

Exclusion criteria for breast cancer group:

Women with previous tumor story, or any woman had previous successfully treated breast cancer. Women who already had chemotherapy or radio-therapy were excluded. Also, we excluded women with known diseases affecting serum Ang-2 values such as another cancer type, acute infections, immune diseases, diabetes, obese, atherosclerosis, connective tissue diseases and pregnancy.

 

Ang-2 assay:

5 ml blood samples were collected from informed consented women prior to surgery (before any other primary treatments like radiation or chemotherapy) in the morning between 7:00 and 8:00 a.m., after an overnight fasting. Processed within one hour of collection, Serum was separated after centrifugation (2,500 rpm, 10 min) and stored at -80°C. Sandwich- Enzyme-linked immunosorbent assays were used to measure the serum levels of Ang-2 according to man­ufacturer’s instructions (Elabscience®; Wuhan, China). All samples were examined in duplicate, and the mean values were used for statistical analysis. The detection range was: 46.88-3000 pg/ml. serum sample were diluted with assay buffer 10-fold. The optical density (OD) was measured at a wavelength of 450 nm. A standard curve was constructed using the standards’ concentration provided in the kit, and the OD values of the samples applied on it to calculate the corresponding samples’ concentration.

 

Data analysis:

Data were entered into the computer and analyzed using SPSS- 13 (SPSS, Inc., Chicago, IL). The data were presented in descriptive form as percentage, mean± standard deviation. The analysis was performed on the t-student test with the variable considered statistically significant when the value of the significance level is less than 0.05.

 

RESULTS:

The study involved 30 women with breast cancer. Their Ages range from (25-62 years). The association between the expression of serum levels of Ang-2 and the clini­copathological characteristics of patients with breast cancer were investigated.

Table 1: correlation between hormones receptors status and levels of Ang-2 (pg/ml)

Clinicopathological data

Case number (%)

Ang-2 pg/ml (mean ± SD)

P value

ER Status

Negative

9 (30%)

24034.44±3945.64

0.036

Positive

21 (70%)

18858.57±6525.16

PR Status

Negative

11 (36.7%)

21532.73±6086.04

0.467

Positive

19 (63.3%)

19762.11±6466.30

HER2 Status

Negative

21 (70%)

20615.24±5649.07

0.791

Positive

9 (30%)

19935.56±7953.08

KI67 Status

KI67< 14%

13 (43.3%)

20020±4863.44

0.771

KI67 ≥ 14%

17 (56.7%)

20710.59±7320.88

ER: estrogen receptor, PR: progesterone receptor, HER2: human epidermal growth factor receptor2

 

Table 2: correlation between tumor grade and stage and serum levels of Ang-2

Clinicopathological data

Case number (%)

Ang-2 pg/ml (mean ± SD)

P value

Histological type (Ductal 100%)

In Situ carcinoma

15 (50%)

19416 ± 6160.84

0.395

Invasive carcinoma

15 (50%)

21406.67 ± 6457.39

Clinical grade

0/Ⅰ/Ⅱ

23 (76.7%)

19886.52± 6748.66

0.417

Ⅲ/Ⅳ

7 (23.4%)

22135.71± 4392.22

Clinical stage

T

Tis: 15(50%)

20346.67± 6110.38

0.913

T1: 6 (20%)

T2: 3 (10%)

T3: 4 (13.3%)

20670± 7558.14

T4: 2 (6.7%)

N

N0: 20 (66.7%)

18842.50± 6507.26

0.086

N1: 7 (23.3%)

24902.86± 4779.64

N2: 3 (10%)

20390± 2531.64

M

No metastasis

 

 

Ang-2 levels were measured and the mean value for its concentrations in each group was calculated (30 breast cancer patients and10 sex and age matched healthy controls).

 

Table 3: levels of Ang-2 pg/ml (mean ± SD) in breast cancer patients and controls

Studied group

Case number

Ang-2 pg/ml (mean ± SD)

P-value

Breast cancer patients

30

20411.33±6283.19

<0.000

Healthy control

10

1731.70±368.35

 

DISCUSSION:

A linkage between the degree of angiogenesis and metastasis was reported in a variety of malignant tumors3. Ang-2 has been found to be highly expressed in some cancer tissues, including breast cancer. Previously, Sfiligoi C et al found that increased expression of Ang-2 in breast cancer tissues correlated with lymph node invasion and short survival. Li, P., et al. found that serum Ang-2 level expression were significantly positively correlated with histological grade, lymph node involvement, and clinical stage, suggesting that Ang-2 might be involved in the carcinogenesis and metastasis of breast cancer1.

 

Our Study, showed that serum Ang-2 values were not statistically significantly different in breast cancer patients with different tumor stages and grades (as shown in Table 2). On the other hand, these values were significantly correlated to ER Status, that means, serum levels of Ang-2 in breast cancer patients with negative estrogen receptor were higher than the other patients with positive ER (Table1). Furthermore, we found that serum concentrations of Ang-2 were significantly increased in breast cancer patients compared with healthy controls (Table 3). According to the study, the serum values of Ang-2 can distinguish between breast cancer patient and healthy controls. So, it might be use as a biomarker to predict the disease, but could not reflect the disease progression.

 

Our study was limited to detect serum levels of Ang-2 in different group and whether the difference of these values considered statistically significant or not. For further investigations, the study recommended a prospective cohort study with a larger number of the participants of each group in order to determine the diagnostic and prognostic importance of serum Ang-2 titration in breast cancer. We suggest to titer serum levels of VEGF (Vascular Endothelial Growth Factor) with serum Ang-2 because of their overlapping role in angiogenesis.

 

CONFLICT OF INTEREST:

The author declares no conflict of interest.

 

ACKNOWLEDGMENT:

I would like to thank Dr. Jumana Al- Saleh for her advices and supervision.

 

REFERENCES:

1.      Li, P., et al., Diagnostic and prognostic potential of serum angiopoietin-2 expression in human breast cancer. International Journal of Clinical and Experimental Pathology, 2015; 8(1): p. 660.

2.      Sun, Y.-S., et al., Risk factors and preventions of breast cancer. International Journal of Biological Sciences, 2017; 13(11): 1387.

3.      Xu, Y., et al., The role of serum angiopoietin-2 levels in progression and prognosis of lung cancer: A meta-analysis. Medicine, 2017; 96(37).

4.      Barron, G.A., et al., Circulating levels of angiogenesis-related growth factors in breast cancer: A study to profile proteins responsible for tubule formation. Oncology Reports, 2017; 38(3): 1886-1894.

5.      Srirajaskanthan, R., et al., Circulating angiopoietin-2 is elevated in patients with neuroendocrine tumours and correlates with disease burden and prognosis. Endocrine-Related Cancer, 2009; 16(3): 967-976.

6.      Souma, T., et al., Context-dependent functions of angiopoietin 2 are determined by the endothelial phosphatase VEPTP. Proceedings of the National Academy of Sciences, 2018; 115(6): 1298-1303.

7.      Gillen, J., D. Richardson, and K. Moore, Angiopoietin-1 and Angiopoietin-2 inhibitors: Clinical development. Current oncology reports, 2019; 21(3): 22.

8.      Engin, H., et al., Plasma concentrations of Ang-1, Ang-2 and Tie-2 in gastric cancer. European Cytokine Network, 2012; 23(1): 21-24.

9.      Kim, H., et al., Oncogenic function of angiopoietin-2 in vitro and its modulation of tumor progression in colorectal carcinoma. Oncology Letters, 2017; 14(1): 553-560.

10.    Daly, C., et al., Angiopoietins bind thrombomodulin and inhibit its function as a thrombin cofactor. Scientific Reports, 2018; 8(1): 1-12.

11.    Zhang, Y., et al., Angiopoietin-Tie signaling pathway in endothelial cells: a computational model. iScience, 2019; 20: 497-511.

12.    Fagiani, E. and G. Christofori, Angiopoietins in angiogenesis. Cancer Letters, 2013; 328(1): 18-26.

13.    Detjen, K.M., et al., Angiopoietin-2 promotes disease progression of neuroendocrine tumors. Clinical Cancer Research, 2010; 16(2): 420-429.

14.    Sallinen, H., et al., Preoperative angiopoietin-2 serum levels: a marker of malignant potential in ovarian neoplasms and poor prognosis in epithelial ovarian cancer. International Journal of Gynecologic Cancer, 2010; 20(9).

 

 

 

Received on 12.10.2020           Modified on 16.11.2020

Accepted on 14.12.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(9):4856-4858.

DOI: 10.52711/0974-360X.2021.00843