Correlation between ultrasonic features and expression levels of C-erbB-2, VEGF and nm23 in breast cancer.

Breast cancer is one of the most common female malignancies in clinical practice, which ranks number one in terms of its high incidence. We investigated the relevance of ultrasonic features of breast cancer and expression levels of C-erbB-2, vascular endothelial growth factor (VEGF) and nm23 and its clinical significance. A total of 76 patients with breast cancer were recruited who were admitted to The Affiliated Hospital of Qingdao University from January, 2016 to August, 2017. All patients underwent color Doppler ultrasonic imaging, and expression levels of C-erbB-2, VEGF and nm23 in their tumor tissues were measured by immunohistochemistry. The ultrasonic features were evaluated and compared with the expression levels of C-erbB-2, VEGF and nm23 for each patient. Ultrasonography showed a tumor mass with spiculated margins, abnormal vasculature, and a diameter no less than 3 cm, as well as lymph node metastasis. The above signs were associated with high expression of C-erbB-2, VEGF and nm23 (p<0.05), but calcification was not associated with high expression of these biomarkers (p>0.05). For patients with highly expressed C-erbB-2 and VEGF, the time to peak (TTP) of the time-intensity curve obtained by contrast enhanced ultrasound was shorter, while the peak intensity (PI) was higher. On the contrary, for patients with highly expressed nm23, the TTP was apparently longer, while the PI was lower (p<0.05). The ultrasonic features of breast cancer were relevant to the expression levels of C-erbB-2, VEGF and nm23. Thus, the expression levels of C-erbB-2, VEGF and nm23 were predictable indirectly according to the ultrasonic features of the patient, which can be used as a reference for breast cancer treatment and prognosis prediction.

Introduction

Breast cancer is one of the most common female malignancies in clinical practice, which ranks number one in terms of its high incidence (1). The clinical symptoms of breast cancer mainly include non-gestational nipple discharge, nipple and/or areola abnormalities, breast lumps, enlarged axillary lymph nodes, and skin changes (2). At present, ultrasound is a major tool for diagnosing breast cancer, and has been widely used in clinic (3). Clinical treatments of breast cancer include surgery, chemotherapy, radiation, targeted and traditional Chinese medicine adjuvant therapy. Surgery is the most commonly used treatment, and a radical mastectomy is the preferred type of surgery (4). The C-erbB-2 protein is a member of the epidermal growth factor receptor family, which is closely associated with onset and progression of breast cancer (5). Vascular endothelial growth factor (VEGF) is a growth factor of vascular endothelial cells that promotes the growth of new blood vessels, and it plays a major role in invasion and metastasis of breast cancer (6). The nm23 gene is a tumor suppressor gene, and is associated with inhibition of metastasis in many tumors, such as hepatocellular carcinoma, melanoma, breast cancer and gastric cancer. Patients with breast cancer who were diagnosed early and underwent effective treatments usually have a good prognosis, but those with advanced breast cancer still have a poor prognosis. So, early screening of breast cancer is of great clinical significance (7). In this study, ultrasonography was performed for patients with breast cancer, and the expression levels of C-erbB-2, VEGF and nm23 were measured, in order to explore the relevance of ultrasonic features of breast cancer to expression levels of C-erbB-2, VEGF and nm23. The results may serve as a reference for diagnosis and treatment of breast cancer.

Materials and methods

Subjects

A total of 76 patients with breast cancer were enrolled in this study who were admitted to The Affiliated Hospital of Qingdao University (Qingdao, China) from January, 2016 to August, 2017. Inclusion criteria included patients meeting the diagnostic criteria (female patients, Han ethnicity) for breast cancer (8), undergoing ultrasound examination, without mental disorders, and were not previously treated with surgery, radiotherapy and chemotherapy. Exclusion criteria excluded patients during pregnancy, with moderate to severe anemia, during menstrual cycle or with unexplained vaginal bleeding, and previously treated with drugs and surgery.

This study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University and informed consents were signed by the patients or guardians.

Ultrasonography

All patients underwent ultrasound examination of the whole breasts and the axilla area with a Philips iU22 Color Doppler Ultrasound System (The Philips Foundation, Amsterdam, The Netherlands) at a probe frequency of 7.5–12.0 MHz. Patients lay down in a supine position with the hands up. The probe scanned an area with the nipple, the lower edge of the breast, and the axilla on the boundary. The cancer lesion was checked for its morphology, size, margins, echogenicity, presence of calcification, and blood flow signal. Axillary lymph nodes were also checked for accompanied enlargement. SonoVue was used as the contrast agent (registration no. H20080059; Bracco SpA, Milan, Italy). The ultrasound probe was positioned on the region of interest, followed by injection of the contrast agent through the peripheral vein. Time was recorded from the point of injection. When the contrast agent passed the imaging window, a sector scan was performed slowly on the lesion, and the contrast-enhanced ultrasound images were stored continuously for about 3 min (Table I).

Table I.

General infromation.

Items	Subjects (n=76)
Age range (years)	25–65
Average age (years)	43.72±5.73
BMI (kg/m3)	21.67±1.31
Mean tumor diameter (cm)	3.06±1.73
The average diameter
Clinical stage (n,%)
I	12 (15.79)
IIA	19 (6.32)
Beyond IIA	45 (59.21)
Category (n,%)
Familial	9 (11.84)
Sporadic	67 (88.16)
Ultrasound signs (n,%)
Burr	49 (64.47)
Abnormal vascular	48 (63.16)
Lymphatic metastasis	32 (42.11)
Calcification	33 (43.42)

BMI, body mass index.

Measurement of expression levels of C-erbB-2, VEGF and nm23

The expression levels of C-erbB-2, VEGF and nm23 in tumor tissues were measured by immunohistochemistry (IHC). Rabbit anti-human VEGF polyclonal antibody was purchased from Beijing Bioss Biotechnology Co., Ltd. (Beijing, China). Rabbit anti-human C-erbB-2 polyclonal antibody and mouse anti-human nm23 monoclonal antibody were all purchased from Fuzhou Maixin Biotechnology Co., Ltd. (Fuzhou, China). The paraffin-embedded tumor tissue was cut into 4 µm sections using a microtome (Leica Microsystems GmbH, Wetzlar, Germany). The slides with paraffin sections were baked overnight in an incubator (Shanghai Medical Instrument Co., Ltd., Shanghai, China) at 60°C. With xylene dewaxed, the slides were put in ethanol of 100, 95, 80, and 75% in this order for 10 min each, followed by a soak in distilled water for 5 min. Endogenous peroxidases were inactivated by adding 3% hydrogen peroxide solution 50 µl and incubation at 20°C for 10 min. After washing with PBS 3 times (5 min each), the primary antibody 50 µl was added, followed by incubation at 4°C overnight. The secondary antibody was added, incubation at 20°C for 10 min, followed by DAB staining (DAB kit was purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). The staining progress was monitored under a microscope (Olympus Corporation, Tokyo, Japan). After staining, the slides were washed with distilled water to terminate staining. After counterstaining with hematoxylin for 2 min, the slides were mounted with natural balsam.

Evaluation indicators

Two senior ultrasound specialists performed the double-blind diagnostic imaging examinations. Based on the ultrasonic features, patients were divided into the following groups: Group with tumor diameter ≥3 cm and group with tumor diameter <3 cm; group with spiculated tumor mass and group with non-spiculated tumor mass; group with abnormal tumor vascularity and group without abnormal tumor vascularity; group with lymph node metastasis and group without lymph node metastasis; group with calcification and group without calcification. The ultrasonography time-intensity curve (TIC) was recorded. Relevant parameters include the time to peak (TTP) and the peak intensity (PI). TTP is the time between start of contrast signal and PI on the region of interest. PI is the maximum signal intensity on region of interest.

The expression levels of C-erbB-2, VEGF and nm23 in breast cancer tissues were measured by IHC

Five fields of view of high magnification, ×400 were randomly selected from each slide. Brownish yellow cells were regarded as positive expression of C-erbB-2 and VEGF. The percentage of positive cells was calculated and a score (PP) was given according to the percentage. A PP score of 0, 1, 2 and 3 points was given if the percentage of positive cells was 0, <5, >5 but ≤20 and >20%, respectively. Apart from the PP scores, the intensity of staining was scored as well (SI). An SI score of 0, 1, 2 and 3 points were given if the nuclei color was colorless, light yellow, brown and dark brown, respectively. An immunoreactivity score (IRS) was calculated by the product of the percentage of positive cells (PP score) and the intensity of staining (SI score): IRS=PP × SI. IRS >4 was defined as high expression, whereas IRS ≤4 was defined as low expression (9). Expression of nm23 was observed in cytoplasm, according to SI score: ≥2 represented high expression, while <2 represented low expression.

Statistical analysis

Data were processed using SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA). Measurement data are expressed as mean ± standard deviation (SD) using t-test. Enumeration data are expressed as a number or percentage (%) using χ2 test. Pearson's analysis was usde for correlation of C-erbB-2, VEGF and nm23 expression. Difference was statistically significant at p<0.05.

Results

Positive expression of C-erbB-2, VEGF and nm23 was observed in patients with breast cancer (Fig. 1A-C). Ultrasound examination showed different signs of breast cancer, including burr sign, abnormal vascular sign, lymphatic metastasis sign and calcification sign (Fig. 2A-D).

Figure 1.

IHC images of breast cancer. (A) C-erbB-2 positive in breast cancer tissues (×400). (B) VEGF positive in breast cancer tissues (×400). (C) nm23 positive in breast cancer tissues (×400). IHC, immunohistochemistry; VEGF, vascular endothelial growth factor.

Figure 2.

Typical ultrasound image in breast cancer. (A) Calcification sign. (B) Lymphatic metastasis sign. (C) Burr sign. (D) Abnormal vascular sign.

Analysis of accuracy of ultrasonic examinations in the diagnosis of breast cancer

There was no significant differences in the accuracy of diagnosis of breast cancer between the two methods (p>0.05) (Table II).

Table II.

Comparison of accuracy of ultrasonic examinations and pathological examinations in the diagnosis of breast cancer (n, %).

Methods	Diagnosis (%)	Misdiagnosis (%)	Error diagnosis (%)	Accuracy (%)
Pathological examinations	76 (100.00)	0 (0.00)	0 (0.00)	76 (100.00)
Enhanced CT	72 (94.47)	2 (2.63)	2 (2.63)	72 (94.47)
χ2				2.311
p-value				0.129

CT, computed tomography.

Relevance of tumor characteristics of ultrasonic signs to expression levels of C-erbB-2, VEGF and nm23

Apparently, the high expression rates of C-erbB-2 and VEGF in the group with tumor diameter ≥3 cm were higher than those in the group with tumor diameter <3 cm, whereas the high expression rate of nm23 was higher in the group with tumor diameter <3 cm. (p<0.05) (Table III).

Table III.

Correlation between tumor diameter and high expression rates of C-erbB-2, VEGF and nm23 (n, %).

Groups	Cases	C-erbB-2 high expression	VEGF high expression	nm23 high expression
Tumor diameter ≥3 cm	41	40 (97.56)	39 (97.56)	22 (53.66)
Tumor diameter <3 cm	35	19 (54.29)	19 (54.29)	34 (97.14)
χ2		17.947	15.234	16.238
p-value		<0.001	<0.001	<0.001

VEGF, vascular endothelial growth factor.

The high expression rates of C-erbB-2 and VEGF were higher in the group with spiculated tumor margins than those in the group with non-spiculated tumor margins, whereas the high expression rate of nm23 was higher in the group with non-spiculated tumor margins (p<0.05) (Table IV). The high expression rates of C-erbB-2 and VEGF were higher in the group with abnormal tumor vascularity than those in the group without abnormal tumor vascularity, whereas the high expression rate of nm23 was higher in the group without abnormal tumor vascularity (p<0.05) (Table V).

Table IV.

Correlation between spiculated tumor margins and high expression rates of C-erbB-2, VEGF and nm23 (n, %).

Groups	Cases	C-erbB-2 high expression	VEGF high expression	nm23 high expression
Spiculated tumor margins	49	43 (87.76)	42 (85.71)	21 (42.86)
Non-spiculated tumor margins	27	16 (59.25)	16 (59.25)	25 (92.59)
χ2		6.582	5.356	16.001
p-value		0.010	0.021	<0.001

Table V.

Correlation between abnormal tumor vascularity and high expression rates of C-erbB-2, VEGF and nm23 (n, %).

Groups	Cases	C-erbB-2 high expression	VEGF high expression	nm23 high expression
Abnormal tumor vascularity	48	43 (89.58)	42 (87.50)	22 (45.83)
Without abnormal tumor vascularity	28	16 (57.14)	16 (57.14)	24 (85.71)
χ2		8.931	7.451	10.162
p-value		0.003	0.007	0.001

The high expression rates of C-erbB-2 and VEGF were higher in the group with lymph node metastasis than those in the group without lymph node metastasis, whereas the high expression rate of nm23 was higher in the group without lymph node metastasis (p<0.05) (Table VI). There was no significant difference in the high expression rates of C-erbB-2, VEGF and nm23 between the group with calcification and the group without calcification (p>0.05) (Table VII).

Table VI.

Correlation between lymph node metastasis and high expression rates of C-erbB-2, VEGF and nm23 (n, %).

Groups	Cases	C-erbB-2 high expression	VEGF high expression	nm23 high expression
Lymph node metastasis	30	30 (100.00)	30 (100.00)	11 (34.38)
Without lymph node metastasis	46	37 (80.43)	26 (56.52)	45 (97.83)
χ2		15.304	16.500	35.664
p-value		<0.001	<0.001	<0.001

VEGF, vascular endothelial growth factor.

Table VII.

Correlation between calcification and high expression rates of C-erbB-2, VEGF and nm23 (n, %).

Groups	Cases	C-erbB-2 high expression	VEGF high expression	nm23 high expression
Calcification	31	21 (63.64)	21 (63.64)	23 (69.70)
Without calcification	45	38 (84.44)	37 (82.22)	33 (73.33)
χ2		3.416	2.543	0.009
p-value		0.064	0.111	0.922

VEGF, vascular endothelial growth factor.

Comparison of TIC parameters in different expression levels of C-erbB-2, VEGF and nm23

The TTP was shorter in patients with high C-erbB-2, high VEGF and low nm23 expression, the corresponding PI was higher (p<0.05) (Tables VIII, IX and X).

Table VIII.

Correlation between TIC parameters and expression level of C-erbB-2.

Groups	Cases	TTP (sec)	PI (dB)
C-erbB-2 high expression	59	6.25±2.04	3.57±0.75
C-erbB-2 low expression	17	9.56±2.52	1.69±0.46
t-value		5.585	9.790
p-value		<0.001	<0.001

TTP, time to peak; PI, peak intensity; TIC, time-intensity curve.

Table IX.

Correlation between TIC parameters and expression level of VEGF.

Group	Cases	TTP (sec)	PI (dB)
VEGF high expression	58	6.23±2.03	3.57±0.73
VEGF low expression	18	9.54±2.54	1.64±0.47
t-value		5.685	10.533
p-value		<0.001	<0.001

VEGF, vascular endothelial growth factor; TTP, time to peak; PI, peak intensity; TIC, time-intensity curve.

Table X.

Correlation between TIC parameters and expression level of nm23.

Groups	Cases	TTP (sec)	PI (dB)
nm23 high expression	56	9.57±2.52	1.97±0.32
nm23 low expression	20	6.26±2.04	2.79±0.58
t-value		5.281	6.005
p-value		<0.001	<0.001

TIC, time-intensity curve.

Correlation analysis of C-erbB-2, VEGF and nm23 expression

Pearson's correlation analysis showed that there was a positive correlation between C-erbB-2 and VEGF, VEGF and nm23, nm23 and C-erbB-2 (r=0.463, 0.452, 0.478, P<0.05) (Fig. 3).

Figure 3.

Correlation analysis of C-erbB-2, VEGF and nm23 expression. VEGF, vascular endothelial growth factor.

Discussion

Breast cancer is the most common cancer among women, which affects women's physical health as well as their psychological well-being in a negative way. According to statistical analysis, breast cancer incidence can be as high as 25%. The current incidence in China is still low, but with the development of China's society, it has been increasing at a higher rate than in developed countries (10). Breast cancer ranks the highest in incidence of cancers diagnosed among Chinese women. The incidence varies between urban and rural areas in China, which is higher in urban areas than that in rural areas. The pathogenesis of breast cancer is currently still under debate. There is consensus that onset and progression of breast cancer results from multi-step synergies of various factors, involving mutation and proliferation of tumor cells, infiltration of the basement membrane, inactivation of tumor suppressor genes, and imbalance of protease secretion (11). High risk factors of breast cancer may include: Age, family history, unmarried late pregnancy, early menarche, late menopause, frequent chest X-rays, excessive drinking, long-term use of contraceptives, abortion and inhibition of breastfeeding (12). Early breast cancer symptoms are invisible and not noticeable without a professional screening, thus the disease is easily overlooked and missed. It can slow down deterioration of breast cancer to a certain extent and reduce mortality if early screening is performed and patients receive comprehensive treatments (13).

With continuous improvement of ultrasound technology, color Doppler ultrasound has become one of the most important diagnostic methods for breast cancer. The morphology, size and blood flow of tumor mass can be clearly viewed, thus it provides a reliable way to effectively distinguish benign and malignant tumors (14). The C-erbB-2 is an oncogene that indicates tumor prognosis (15). VEGF is one of the most potent and specific angiogenic factors, and it belongs to the family of platelet-derived growth factors, including VEGF-A (namely VEGF), VEGF-B, VEGF-C and VEGF-D. VEGF-A stimulates the vascular endothelial cells, promoting its division and proliferation, and ultimately forming new blood vessels. VEGF-B can promote vascular endothelial growth and migration, tumor growth and metastasis. VEGF-C and VEGF-D can promote lymphangiogenesis and thus promote lymphatic metastasis of tumors. VEGF is highly expressed in many kinds of tumor tissues (16,17). The nm23 gene is a tumor suppressor gene. It was first found in an animal experiment that the nm23 gene can inhibit tumor growth and metastasis (18). The results in this study showed that tumor diameter ≥3 cm, spiculated tumor margin, lymph node metastasis and abnormal tumor vascularity were all associated with higher expression of C-erbB-2 and VEGF and lower expression of nm23 (p<0.05). There were no significant differences in expression of C-erbB-2, VEGF and nm23 between tumor with calcification and tumor without calcification (p>0.05). Thus, bigger tumor size, spiculated tumor margins, lymph node metastasis and abnormal tumor vascularity were specific ultrasonic features of breast cancer. Under pathological conditions, activation of CerbB-2 led to expression of VEGF activity. The balance between angiogenic factors and inhibitory factors was broken, which promoted microvascular formation, leading to uncontrollable vascular endothelial cell proliferation. As indicators, expression levels of C-erbB-2 and VEGF measured by IHC were high. As tumor advanced and grew bigger, expression of nm23 was hindered. Especially in the lymph nodes metastasis, the expression level of nm23 was significantly low.

Tumor onset, progression and metastasis all rely on microvascular formation. Recently, there have been continual advances in ultrasound technology. Currently ultrasound imaging is widely used in clinical diagnosis of various cancers due to merits such as convenience, no radiation exposure and good reproducibility in evaluation of tumor microvascular network. Ultrasound is especially highly sensitive in assessing tumor blood flow. Tumor blood flow pattern can be quantified by analyzing TTP and PI from the TIC, which may help improve cancer diagnosis (19). The results in this study showed that the TTP of patients with high expression of C-erbB-2 and VEGF was shorter than that of patients with low expression of C-erbB-2 and VEGF, and the corresponding PI was higher. On the contrary, the TTP of patients with high expression of nm23 was longer than that of patients with low expression of nm23, and the corresponding PI was lower (p<0.05). Tumor was in continual progression when expression levels of C-erbB-2 and VEGF were high. At this stage, cancer cell proliferation was fast, and the tumor mass became larger, leading to lymphatic and hematogenous metastasis. In tumor tissue, blood supply was abundant, and blood flow was fast as well, thus the average blood volume was large, leading to high PI in the contrast-enhanced ultrasound imaging. When nm23 expression level was high, tumor cells were well differentiated, and were less likely to spread to lymph nodes. At this stage, the tumor blood flow was slow, leading to low PI in the contrast-enhanced ultrasound imaging. When nm23 expression level became low, suppression of tumor metastasis was compromised, which promoted onset and progression of breast cancer (20).

In summary, ultrasound can show morphological changes in breast cancer. The ultrasound features and blood flow parameters were closely associated with expression levels of C-erbB-2, VEGF and nm23. Their correlations can serve as a reference for diagnosis and prognosis of breast cancer.