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Abstract

BACKGROUND:

Molecular marker analysis has become important in breast cancer diagnosis and treatment and may reveal new mechanisms in breast cancer pathogenesis. Aside from the commonly used hormonal receptors and HER2, VEGF-A has been increasingly shown to be important in breast cancer diagnosis and pathogenesis.

OBJECTIVE:

This study aimed to determine the relationship between VEGF-A expression on ER and PR and HER2 hormonal status in patients with late-stage breast cancer (locally advanced or with distant metastases).

METHODS:

This observational, cross-sectional study examined VEGF-A expression and molecule markers (ER, PR, and HER2) of breast cancer tissue using immunohistochemistry. The Chi-square test was used to determine whether two categorical variables were correlated. Statistical significance was set at p < 0.05.

RESULTS:

VEGF-A showed no significant correlation with demographic characteristics, TNM staging, pathological grading, luminal or non-luminal type, or hormonal receptor markers but showed a significant positive correlation with HER2 receptors (p = 0.036).

CONCLUSIONS:

VEGF-A was positively correlated with HER2 expression in breast tumor tissue but showed no significant correlation with other breast cancer markers, including luminal typing or hormonal receptors. Further study is needed to understand the mechanistic interplay between VEGF and HER2 in breast cancer pathogenesis.

Keywords

Breast cancer locally advanced breast cancer metastatic breast cancer

1. Introduction

Vascular endothelial growth factor A (VEGF-A) is one of the most important factors controlling angiogenesis [1]. Breast cancer is characterized by angiogenesis, which is mediated by the vascular endothelial growth factor (VEGF) signaling axis [2]. A study found a significant relationship between serum VEGF and prognostic factors. Breast cancer has very heterogeneous biological behavior, so many parameters are needed to determine prognostic factors [3]. Estrogen receptor (ER) was the first biomarker used to select for patients who will show a good response to hormone inhibition therapy. This was followed by progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), which is expressed in 25% of breast cancer patients and can predict a good response to the monoclonal antibody trastuzumab [4]. The St. Gallen Consensus of 2013 further groups breast cancers according to these intrinsic subtypes into luminal A, luminal B, HER2-overexpressed, and basal-like [5]. All these molecular subtypes are connected to the expression of cellular growth factors that affect the tumor proliferative activity, apoptosis, and migration of cancer cells [5,6].

Aside from mediating neovascularization into the tumor, VEGF is a potent mitogen for endothelial vascular cells: as a mediator for degradation of the extracellular matrix, which facilitates metastasis; as a survival factor through inhibition of apoptosis; and as a modulator of cellular migration into the angiogenic site [7,8]. Since the link between neovascularization and tumor growth was discovered, many discoveries have focused on angiogenesis (and VEGF expression) as an independent prognostic factor. VEGF expression has independent prognostic results that are significant and related to lower overall survival in breast cancer [7]. However, most studies have reported inconsistent results, perhaps due to the molecular heterogeneity in breast cancer [9]. Thus, the question of whether VEGF is a reliable prognostic factor, or a predictor of response to adjuvant or hormonal therapy, remains unanswered [10].

Further, no study has established whether the lower prognosis manifested by other molecular markers, such as HER2, hormone receptors, or triple-negative breast cancers, is related to VEGF expression and angiogenesis. We hypothesized that circulating serum VEGF may be related to other, more widely accepted, prognostic parameters and that discovered correlations may bring new understanding of the role of angiogenesis in tumor development. This study aimed to determine the relationship between VEGF-A expression on ER and PR and HER2 hormonal status in patients with locally advanced or distantly metastatic breast cancer.

2. Methods

2.1. Setting and subjects

This was an observational cross-sectional study conducted at Dr. Wahidin Sudirohusodo Hospital, Makassar, Indonesia, and its network. Our pathology anatomy laboratory examined VEGF-A expression and intrinsic subtypes (ER, PR, and HER2) of breast cancer tissue. The Ethical Committee of the Faculty of Medicine, Hasanuddin University Makassar, Indonesia, approved this study (number: 200/UN4.6.4.5.31/PP36/2022). The inclusion criteria were (a) women with late-stage breast cancer (TNM stages III & IV) who had never received prior therapy; (b) no diagnosis of underlying diseases, including metabolic diseases or diabetes mellitus, ischemic heart disease, or other ischemic cerebrovascular diseases; and (c) consent to participate in this study. The exclusion criteria were (a) damaged or unrepresentative samples taken and (b) patients with another type of cancer (synchronous cancer).

2.2. Stages of breast cancer

According to the American Joint Committee on Cancer, the staging division of breast cancer is based on tumor size (T), metastasis to regional lymph nodes (N), and the presence or absence of distant metastases (M). Cancers are grouped into stages I, II, III, and IV [11,12].

2.3. Histopathological grading

Histopathological grading is the division of degrees of differentiation of breast cancer based on the Nottingham modification of the Bloom–Richardson breast cancer grading system recommended by the World Health Organization [13,14]. The grading is determined based on the scoring of nuclear pleomorphism, tubular formation, and mitotic index, which are set as low, moderate, and high [13].

2.4. Immunohistochemistry

Tissue samples obtained from each patient’s cancer tissue according to the clinical stage were taken to the Anatomic Pathology Laboratory at Hasanuddin University Hospital for the immunohistochemistry examination of the expression of VEGF-A. The immunohistochemistry staining technique was examined by primary monoclonal antibody to VEGF (catalog number sc7269, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The results of VEGF-A staining were interpreted semiquantitatively by examining the percentage of colored cell groups and the staining intensity. The percentage was calculated by adding all positive cells within the field of view of the tumor preparation examined under a light microscope.

2.5. Interpretation of the immunohistochemistry results

The immunohistochemistry distribution of VEGF-A was classified as focal (5–10%) value 1, regional (11–<25%) value 2, small diffuse (25–50%) value 3, and diffuse (>50%) value 4. The staining intensity was classified as weak value 1, medium value 2, and strong value 3. When the accumulated intensity and extent of the staining distribution in the nucleus and cytoplasm gave a result, values >2 were considered positively expressed. Values less than 2 were considered negatively expressed.

2.6. Statistical analysis

Statistical analyses were carried out using SPSS software version 22 (IBM Corp., Armonk, NY, USA). The frequency distribution and Chi-square statistical tests were used as statistical methods. The Chi-square test is used to determine whether two categorical variables are correlated (nominal or ordinal scale). If the P-value of the test was <0.05, the results were statistically significant.

3. Results

3.1. Characteristics

The 41 enrolled female patients with diagnosed and treated late-stage breast cancer met the study inclusion criteria. The participants had different characteristics, grouped according to their age, hormonal status, and pathologic grading in Table 1. The average age was 49.12 ± 8.84 years, with most in the 41–60 years age range (68.3%), premenopausal (70.7%), and with a moderate pathologic grading (73.2%). The characteristics of the sample are presented in Table 1.

Table 1
Characteristics of participants

Characteristic n %

Age (years)

21–40 7 17.1

41–60 28 68.3

>60 6 14.6

Hormonal status

Premenopausal 29 70.7

Post menopause 12 29.3

Histopathological grading

Low 3 7.3

Moderate 30 73.2

High 8 19.5

Characteristic	n	%
Age (years)
21–40	7	17.1
41–60	28	68.3
>60	6	14.6
Hormonal status
Premenopausal	29	70.7
Post menopause	12	29.3
Histopathological grading
Low	3	7.3
Moderate	30	73.2
High	8	19.5

The tumor cells of patients underwent immunohistochemical staining through the expression of ER, PR, HER2, and VEGF-A and molecular subtype grouping according to the expression of ER, PR, and HER2 (Table 2). Most patients had negative ER and PR expression and positive HER2 expression. These two markers were used to group them further into luminal and non-luminal molecular subtypes. Most late-stage patients had a non-luminal molecular subtype (53.7%), and the remaining 46.3% had a luminal molecular subtype. VEGF-A expression analysis revealed that most patients (82.9%) had positive VEGF-A expression, and only 17.1% had negative VEGF-A expression.

Table 2

Immunohistochemical analysis results of late-stage breast cancer patients

Molecular marker	n	%
ER
Positive	18	43.9
Negative	23	56.1
PR
Positive	16	39.0
Negative	25	61.0
HER2
Positive	22	53.7
Negative	19	46.3
Molecular subtype
Luminal	19	46.3
Non-luminal	22	53.7
VEGF-A
Positive	34	82.9
Negative	7	17.1

We found no significant correlation between luminal and non-luminal molecular subtype grouping and positive VEGF-A expression (p = 1.000). The results of individual molecular markers (ER, PR, and HER2) and VEGF-A expression are explained in Table 3. Chi-square analysis revealed that ER and PR expression were not significantly correlated with VEGF-A expression (p = 1.000 and p = 0.819, respectively). However, patients with positive VEGF-A expression mostly had positive HER2 expression, and Chi-square analysis revealed a significant correlation between HER2 expression and VEGF-A expression in late-stage breast cancer patients (p = 0.036, p > 0.05).

We further analyzed the correlation between VEGF-A expression and demographic characteristics (age, hormonal status, histopathological grading) but did not find any statistically significant result (Table 4).

Table 3

Correlation between VEGF-A expression and molecular markers

Characteristic	VEGF-A expression				p-value
	Positive		Negative
	n	%	n	%
ER
Positive	15	44.1	3	42.9	1.000
Negative	19	55.9	4	57.1
PR
Positive	13	38.2	3	42.9	0.819
Negative	21	61.8	4	57.1
HER2
Positive	21	61.8	1	14.3	0.036
Negative	13	38.2	6	85.7

Note: Chi-square test.

Table 4

Correlation between VEGF-A expression and patient characteristics

Characteristic	VEGF-A expression				p-value
	Positive		Negative
	n	%	n	%
Age (years)
21–40	4	11.8	3	42.9	0.130
41–60	25	73.5	3	42.9
>60	5	14.7	1	14.2
Hormonal status
Premenopausal	24	70.6	5	71.4	1.000
Post menopause	10	29.4	2	28.6
Histopathological grading
Low	3	8.8	0	0.0	0.213
Moderate	23	67.6	7	100.0
High	8	23.5	0	0.0

Note: Chi-square test.

4. Discussion

In this study, most patients had negative ER expression (56.1%), negative PR expression (62.0%), and positive HER2 expression (53.7%). The low expression of ER and PR and the positive expression of HER2 may signal a worse prognosis. Negative ER expression is associated with higher proliferative activity [15]. Progesterone is a risk factor for breast cancer, and alterations in progesterone signaling pathways contribute to the early stages of tumor development. PR signaling stimulates epithelial cell proliferation through an unknown mechanism in pre-neoplastic lesions and mammary tumors. However, primary tumors with negative PR expression are associated with a less differentiated, more invasive phenotype and a poorer prognosis than tumors expressing PR [16].

HER2 is an epidermal growth factor on the cell surface that transmits growth signals to the cell nucleus. HER2 receptor overexpression is associated with a poor prognosis. HER2 expression has also been significantly correlated with lymph node involvement. Lymph node status is important in determining the stage of cancer and treatment options. As the number of positive axillary lymph nodes increases, the survival rate decreases and the recurrence rate increases so that HER2 expression is often associated with lymph node metastasis. The number of positive lymph nodes and expression of HER2 are associated with prognosis [17]. Higher HER2 expression is associated with a higher degree of breast cancer [18].

This study also measured VEGF-A expression and found that most patients with advanced breast cancer had positive VEGF-A expression (82.9%). Previous studies also reported that breast cancer patients had higher VEGF-A expression than healthy controls [19]. The high positive VEGF-A expression in this study was associated with advanced breast cancer. This is because VEGF has a major role in angiogenesis and tumor development in breast cancer [9,20]. VEGF overexpression often occurs before the invasion of breast cancer cells. Serum VEGF levels are correlated with advanced stages of breast cancer [21].

Sahana et al. [22] measured circulating VEGF in patients as a marker of angiogenesis and metastasis. VEGF is a potent angiogenic cytokine in normal and tumor tissues, stimulates endothelial cell proliferation in vitro, and induces angiogenesis in vivo. The highest serum VEGF concentrations were found in metastatic breast cancer, especially among patients who did not receive metastatic cancer therapy. A study of 29 invasive breast carcinomas revealed that VEGF expression in peritumoral endothelial cells correlated with angiogenesis, lymphangiogenesis, and higher pathological stages [22,23]. In a study of 50 invasive ductal carcinomas, VEGF mRNA and protein expression correlated with TNM stage, lymph node metastases, and tumor size. The total microvessel density correlates with VEGF expression and axillary lymph node metastasis [22,24]. High VEGF levels and low soluble VEGFR-1 (VEGF-A) levels are significantly associated with poor prognosis of breast cancer [22]. The high expression of VEGF family members in breast cancer is associated with a more aggressive cancer phenotype [10].

In connecting VEGF expression and molecular markers, we found no significant differences except for HER2 expression. Previous studies have varied in their results. The lack of positive correlation between VEGF and molecular profiles in this study may be explained by the existence of luminal A and B subtypes within the luminal grouping; the luminal B subtype is known to be related to high VEGF expression.

Regarding the expression of HER2, this study found a significant correlation, in accordance with previous studies [10]. VEGF in the aggressive phenotype shown by excess HER2 expression further suggests the possible benefits of hormonal therapy. Research in Iraq reported that VEGF is correlated positively with the degree, size, nodal involvement, and HER2 expression of the tumor, which partly agrees with the results of this study. VEGF overexpression plays an important role in the evolution pathogenesis of breast carcinoma [25].

This study has some limitations. The VEGF-A analysis was limited to luminal and non-luminal subtypes, not further subtypes such as luminal A and B, HER2, and basal-like. The study was also only performed before chemotherapy, with no analysis of the effect of chemotherapy on the changes in VEGF-A expression.

5. Conclusions

This study found that VEGF-A was significantly associated with HER2 expression as one of the prognostic molecular markers of breast cancer. This was associated with the level of progression in breast cancer that had positive HER2 expression. Existing evidence suggests that HER2 activation is one of several mechanisms that promote angiogenesis and that HER2-amplified breast cancers have increased angiogenesis.

Footnotes

Acknowledgements

The authors would like to thank all staff in the Division of Oncology, Department of Surgery, Faculty of Medicine, Hasanuddin University – Dr. Wahidin Sudirohusodo Hospital, Makassar, Indonesia, who helped with conducting the research study.

References

, Cao

, Bhattacharya

, Dutta

, Wang

, Mukhopadhyay

, Endogenous vascular endothelial growth factor-A (VEGF-A) maintains endothelial cell homeostasis by regulating VEGF receptor-2 transcription, J Biol Chem , 287: 3029–3041, 2012. doi:10.1074/jbc.M111.293985.

Balalis

, Tsakogiannis

, Kalogera

, Kokkali

, Tripodaki

, Ardavanis

, Serum concentration of selected angiogenesis-related molecules differs among molecular subtypes, body mass index and menopausal status in breast cancer patients, J Clin Med , 11: 4079, 2022. doi:10.3390/jcm11144079.

Ali

, Sheta

, El Mohsen

, Elevated serum and tissue VEGF associated with poor outcome in breast cancer patients, Alexandria J Med , 47: 217–224, 2011. doi:10.1016/j.ajme.2011.07.003.

Pavlou

, Diamandis

, Blasutig

, The long journey of cancer biomarkers from the bench to the clinic, Clin Chem , 59: 147–157, 2013. doi:10.1373/clinchem.2012.184614.

Yersal

, Biological subtypes of breast cancer: Prognostic and therapeutic implications, World, J Clin Oncol , 5: 412, 2014. doi:10.5306/wjco.v5.i3.412.

Inic

, Zegarac

, Inic

, Markovic

, Kozomara

, Djurisic

, Difference between luminal A and luminal B subtypes according to Ki-67, tumor size, and progesterone receptor negativity providing prognostic information, Clin Med Insights Oncol , 8: 107–111, 2014. doi:10.4137/CMO.S18006.

Gisterek

, Szewczyk

, Matkowski

, Sedlaczek

, Staszek

, Zolnierek

, Serum vascular endothelial growth factor A165 levels in patients with various histological types of breast cancer and benign tumors, J Clin Oncol , 26: 22166, 2008. doi:10.1200/jco.2008.26.15_suppl.22166.

Indra

, Sampepajung

, Haryasena

, Faruk

, Correlation between vascular endothelial growth factor-A (VEGF-A) serum with clinical stage, histopathologic grading and estrogen receptor status breast cancer patient in Makassar, Int J Surg Med , 7: 44–48, 2020. doi:10.5455/ijsm.vegf-a-breast-cancer.

Byrne

, McDowell

, Agarawal

, Sinha

, Kumar

, Bundred

, Serum vascular endothelial growth factor in breast cancer, Anticancer Res , 27: 3481–3487, 2007.

10.

Goussia

, Simou

, Zagouri

, Manousou

, Lazaridis

, Gogas

, Associations of angiogenesis-related proteins with specific prognostic factors, breast cancer subtypes and survival outcome in early-stage breast cancer patients. A Hellenic Cooperative Oncology Group (HeCOG) trial, PLoS One , 13: e0200302, 2018. doi:10.1371/journal.pone.0200302.

11.

Giuliano

, Edge

, Hortobagyi

, Eighth edition of the AJCC cancer staging manual: Breast cancer, Ann Surg Oncol , 25: 1783–1785, 2018. doi:10.1245/s10434-018-6486-6.

12.

Pranoto

, Haryasena

, Prihantono

, Rahman

, Sampepajung

, Indra

, The expression of programmed death-ligand 1 and its association with histopathological grade, stage of disease, and occurrence of metastasis in breast cancer, Breast Dis , 40: S71–S76, 2021. doi:10.3233/BD-219010.

13.

Bansal

, Singh

, Misra

, Sharma

, Tiwari

, Srivastava

, Comparative evaluation of the modified Scarff-Bloom-Richardson grading system on breast carcinoma aspirates and histopathology, Cytojournal , 9: 4, 2012. doi:10.4103/1742-6413.92550.

14.

van Dooijeweert

, van Diest

, Ellis

, Grading of invasive breast carcinoma: The way forward, Virchows Arch , 480: 33–43, 2022. doi:10.1007/s00428-021-03141-2.

15.

Fuckar

, Dekanić

, Stifter

, Mustać

, Krstulja

, Dobrila

, VEGF expression is associated with negative estrogen receptor status in patients with breast cancer, Int J Surg Pathol , 14: 49–55, 2006. doi:10.1177/106689690601400109.

16.

Morales

, Morimoto

, Vilchis

, Taniyama

, Bautista

, Robles

, Molecular expression of vascular endothelial growth factor, prokineticin receptor-1 and other biomarkers in infiltrating canalicular carcinoma of the breast, Oncol Lett , 12: 2720–2727, 2016. doi:10.3892/ol.2016.4961.

17.

Siadati

, Sharbatdaran

, Nikbakhsh

, Ghaemian

, Correlation of ER, PR and HER-2/Neu with other prognostic factors in infiltrating ductal carcinoma of breast., Iran, J Pathol , 10: 221–226, 2015.

18.

Jin

Y-H

, Hua

Q-F

, Zheng

J-J

, Ma

X-H

, Chen

T-X

, Zhang

, Diagnostic value of ER, PR, FR and HER-2-targeted molecular probes for magnetic resonance imaging in patients with breast cancer, Cell Physiol Biochem Int J Exp Cell Physiol Biochem Pharmacol , 49: 271–281, 2018. doi:10.1159/000492877.

19.

Hoar

, Lip

GYH

, Belgore

, Stonelake

, Circulating levels of VEGF-A, VEGF-D and soluble VEGF-A receptor (sFIt-1) in human breast cancer, Int J Biol Markers , 19: 229–235, 2004. doi:10.1177/172460080401900308.

20.

Sa-Nguanraksa

, Chuangsuwanich

, Pongpruttipan

, O-Charoenrat

, High vascular endothelial growth factor gene expression predicts poor outcome in patients with non-luminal A breast cancer, Mol Clin Oncol , 3: 1103–1108, 2015. doi:10.3892/mco.2015.574.

21.

Ayoub

, Jaradat

, Al-Shami

, Alkhalifa

, Targeting angiogenesis in breast cancer: Current evidence and future perspectives of novel anti-angiogenic approaches, Front Pharmacol , 13: 838133, 2022. doi:10.3389/fphar.2022.838133.

22.

Raghunathachar Sahana

, Akila

, Prashant

, Sharath Chandra

, Nataraj Suma

, Quantitation of vascular endothelial growth factor and interleukin-6 in different stages of breast cancer, Reports Biochem Mol Biol , 6: 33–39, 2017.

23.

Ghosh

, Sullivan

CAW

, Zerkowski

, Molinaro

, Rimm

, Camp

, High levels of vascular endothelial growth factor and its receptors (VEGFR-1, VEGFR-2, neuropilin-1) are associated with worse outcome in breast cancer, Hum Pathol , 39: 1835–1843, 2008. doi:10.1016/j.humpath.2008.06.004.

24.

Xie

X-D

, Qu

S-X

, Liu

Z-Z