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Diagnostic value of Doppler ultrasound parameters combined with MMP-11 in early breast cancer and benign breast diseases

Diagnostic value of Doppler ultrasound parameters combined with matrix metalloproteinase-11 (MMP-11) in early breast cancer and benign breast diseases were investigated. A total of 72 patients who underwent color Doppler ultrasound examination in Liaocheng Third People's Hospital from March 2015 to August 2018 were collected as research subjects, and the blood of 60 healthy subjects who underwent physical examinations was collected. The expression level of MMP-11 in serum of breast cancer patients was evaluated, and the diagnostic value of color Doppler ultrasound combined with MMP-11 in breast cancer was assessed. The diagnostic results of color Doppler ultrasound and the imaging characteristics of breast cancer patients were recorded. The results of biopsy and ultrasound were compared. The expression level of MMP-11 in serum of breast cancer patients was significantly higher than that of healthy subjects (P<0.05). The AUC of MMP-11 was 0.735, the sensitivity was 66.67%, and the specificity was 86.11%. Among the 72 patients, there were 41 patients diagnosed with breast cancer by serum MMP-11 examination, 38 patients diagnosed by ultrasound examination, 33 patients diagnosed by combined diagnosis, and 30 patients diagnosed by pathology biopsy. The pathological biopsy was used as the gold standard. The diagnostic efficacy of ultrasound combined with mammography examination was significantly better than the other two single examinations (P<0.050). In conclusion, Doppler ultrasound parameters combined with MMP-11 has a high diagnostic accordance rate in the diagnosis of breast cancer. At the same time, different diagnostic methods combined with the clinical manifestations of patients can improve the diagnostic accuracy, which is worthy of providing reference and advice for future clinical practice.


Introduction
Breast cancer is the most common malignant tumor among women, and its incidence has remained high for many years (1), accounting for 8-12% of the malignant tumors in the whole body (2). According to the data, there are more than one million cases of breast cancer diagnosed globally every year, and approximately 410,000 deaths (3). Breast cancer is more common in Europe, North America and other developed cities, among which the United States has the highest incidence in the world (4). According to Turner et al (5), in the next 50 years, the incidence of breast cancer will exceed 50%, becoming the second most common malignant tumor after gastric cancer. Furthermore, as early breast cancer has no obvious characteristics, it is often ignored by patients, making them miss the best treatment period, leading to high mortality. The fatality of breast cancer in situ is not high, but once the cancer cells fall off, free cancer cells can be transferred to any place through blood circulation and lymph circulation, and the threat of breast cancer is greatly increased (6). Therefore, 'early detection and early treatment' is advocated for the occurrence of breast cancer in clinical practice, and timely intervention treatment is conducted to ensure the health of patients before in situ cancer has spread (7).
Color Doppler ultrasound is a commonly used method for detection and diagnosis of breast cancer. It can assess the shape, direction, internal structure and edge of lesions from multiple planes, and has high resolution in the fat-dominated breast and the compact gland structure (8). Matrix metalloproteinase (MMP) is a family of zinc endopeptidases which can lyse almost all components of the extracellular matrix and many other soluble and cell-related proteins. In the study of Cheng et al (9), MMP-11 was found as a possible prognostic marker, and the expression reflects the differentiation stage and LNM of breast cancer.
Therefore, by studying the diagnostic value of color Doppler ultrasound parameters combined with MMP-11 in early breast cancer and benign breast diseases and comparing the diagnostic accuracy of single diagnosis of breast cancer, this study provides reference and guidance for clinical practice.

Patients and methods
Basic patient data. A total of 72 patients underwent color Doppler ultrasound examination in Liaocheng Third People's Blood sample processing. On an empty stomach in the morning, venous blood was extracted and stored at 4˚C for 30 min. The serum samples were centrifuged at 1,500 x g and 25˚C for 10 min to extract the supernatant and stored in a refrigerator at -80˚C.

Diagnostic value of Doppler ultrasound parameters combined with MMP-11 in early breast cancer and benign breast diseases
Color ultrasound detection and main reagents. Color Doppler ultrasound was performed on subjects using Acuson Sequoia 512 (Siemens AG) with supine position and upper limb lift, fully exposing chest and both axilla. The couplant was applied around the nipple, and the quadrants of the breast were sequentially detected to both axilla. If a lump was found, it was classified according to the number of lactiferous ducts, composition of adipose stroma, and proportion of fibrous glandular tissue in the image under naked eye observation, and the classification standard was referred to (11). enzyme-linked immunosorbent assay (ELISA) was used to determine the expression of MMP-11 in serum. MMP-11 was from wuhan Fine Biotech Co., Ltd., with the brand of FineTest and article number of EH0782. Operations were in strict accordance with the kit instructions.
Outcome measures. Main outcome measures: the expression level of MMP-11 in serum of breast cancer patients was observed, and the diagnostic value of color doppler ultrasound combined with MMP-11 in breast cancer was observed.
Secondary outcome measures: the diagnostic results of color Doppler ultrasound was observed, and the imaging characteristics of breast cancer patients were evaluated. The results of biopsy and ultrasound were compared.
Statistical method. In this study, SPSS 20.0 software package was used for statistical analysis of the collected data, GraphPad 7 software package was used to draw the required illustrations, and K-S test was used to analyze the distribution of measurement data, in which the normal distribution data was expressed as mean ± standard deviation (mean ± SD). Independent sample t-test was used for comparison between groups, and paired t-test was used for inter-group comparison analysis. Enumeration data was expressed by (%), qualified by Chi-square test and presented by χ 2 test. ROC was used to plot the diagnostic value of MMP-11 in breast cancer, and P<0.05 indicates a statistical difference.

Results
Clinical data of patients. The general data of the 72 cases of breast cancer patients, included age, height, ethnicity, education, residence, BMI and fertility circumstance, as shown in Table I.
Expression level and diagnostic value of MMP-11 in serum of breast cancer patients. The expression level of MMP-11 was 9.36±1.25 in breast cancer patients and 7.69±0.82 in serum of healthy subjects. The expression level of MMP-11 in serum of breast cancer patients was significantly higher than that of healthy subjects (P<0.05). Furthermore, by plotting ROC, the AUC of MMP-11 was 0.735, the sensitivity was 66.67%, and the specificity was 86.11%, P<0.05 (Table II and Fig. 1). Imaging characteristics. After the ultrasound diagnosis, all the data of the examination were read by 3 experienced clinicians in the hospital separately and the diagnosis results were jointly given after reaching an agreement. The main manifestations of ultrasound examination in breast cancer patients were: i) Unclear boundary and irregular shape, mostly showing burr shape and crab-like shape. ii) Increased anteroposterior diameter of the lesion, and the thickness/length was >1. iii) Posterior echo attenuation, multiple medullary cells, loose tissue structure, and no echo enhancement in the lesion posterior area. iv) Visible microscopic calcification within the lesion, with a size of 100-500 µm and acoustic shadowing in the rear. v) Abundant blood flow signals within the lesion, common high speed and high resistance blood flow. The main manifestations of mammography examination were: i) Irregular boundary, high density nodules, uneven density. ii) Burr shape edge, with visible small clusters of shape and gravel like calcification.
Diagnostic efficacy assessment. The pathology biopsy was taken as gold standard. According to the calculation, the sensitivity, specificity and diagnostic accordance rate of MMP-11 for breast cancer were 58.54, 80.65 and 68.06%, respectively. The sensitivity, specificity and diagnostic accordance rate of ultrasound for breast cancer were 68.42, 88.24 and 77.78%, respectively. The sensitivity, specificity and diagnostic accordance rate of combined examination were 87.88, 97.44 and 93.06%, respectively. The diagnostic efficacy of ultrasound combined with mammography examination was significantly better than the two single examinations (P<0.050) (Tables III-V).

Discussion
As the most common malignant tumor among women in the world, the incidence and mortality of breast cancer are on the   rise year by year (12). In order to improve the diagnosis and treatment rate of breast cancer, clinical efforts are being made to explore the pathogenic mechanism of breast cancer from various perspectives for diagnosis and treatment, but no significant breakthrough has been made so far. Poortmans et al (13) considered that the pathogenesis of breast cancer was mainly due to the role of genetic factors, while Tutt et al (14) showed that the pathogenesis of breast cancer was closely related to tumor stem cells. At present, there is no accurate and reliable study that can indicate the exact cause of breast cancer, and the main clinical diagnosis is still imaging. with the development of the disease, increasingly difficult types of breast cancer cannot be effectively determined only through imaging (15). Therefore, it has become a very important research topic to summarize the risk factors of breast cancer for influencing the examination of medical means. Traditional ultrasound and mammography examination is simple, convenient, non-invasive, and has a long history of diagnosis, which is the advantage of breast cancer diagnosis. However, there are controversies about the determination of some benign tumors and cystic hyperplasia of the breast. MMP-11 belongs to matrix metalloproteins (16), and is closely related to many diseases such as atherosclerosis, rheumatoid arthritis and cancer in process of embryo implantation, organogenesis, tissue degeneration and repair (17)(18)(19)(20). MMP-11 typically acts during tissue remodeling that occurs at the epithelial/connective tissue interface to regulate epithelial homeostasis. In cancer, high MMP-11 level in primary tumors are associated with poor prognosis (21)(22)(23). In this study, we explored the diagnostic value and accuracy of Doppler ultrasound parameters combined with MMP-11 in early breast cancer and benign breast diseases, so as to provide information for future clinical practice.
In this study, we first observed the expression level of MMP-11 in serum of breast cancer patients, and found that the expression level of MMP-11 in serum of breast cancer patients was significantly higher than that of healthy subjects, indicating that MMP-11 might become a potential target for diagnosis and treatment of breast cancer. ROC curve was drawn and it was found that the AUC of MMP-11 curve was 0.735, the sensitivity was 66.67% and specificity was 86.11%, which was not high in sensitivity but high in specificity. This suggests that MMP-11 has certain predictive value for breast cancer patients. Biomarkers are used as tools in cancer diagnosis and treatment stratification. According to Hadler-Olsen et al (24), levels of one or more MMP members were elevated in most cancers. This family of proteolytic enzymes is involved in many stages of cancer development, including angiogenesis, invasion, and metastasis. Therefore, it is expected that MMP can be used as a diagnostic and prognostic marker in cancer patients. Benson et al (25) showed that MMPs are differentially regulated in breast cancer tissues, and they may play different roles in tumor invasion, metastasis and angiogenesis. Therefore, MMPs are of great research value as a diagnostic marker and drug target. The application of ultrasound and MMP-11 single examination has good specificity for breast cancer, but the combined diagnosis of the two is better for the diagnosis of breast cancer. It suggests that the early diagnostic rate of breast cancer can be improved by the combined examination of ultrasonography and mammography. Ultrasound is superior to MMP-11 in the diagnosis of tumor classification, tumor grade, tumor density, breast cystic hyperplasia and benign tumors, which is not available in MMP-11 detection. However, MMP-11 can play different roles in tumor invasion, metastasis and angiogenesis, and can be used as a diagnostic and prognostic marker and drug target. By using the two methods together, they can make up for each other's shortcomings and achieve the best diagnostic effect.
In conclusion, MMP-11 exhibits high expression in breast cancer patients. The ROC curve shows that MMP-11 has a good clinical diagnostic value and the use of ultrasound combined with MMP-11 examination can improve the early diagnostic rate of breast cancer. Moreover, different diagnostic methods combined with the clinical manifestations of patients can improve the diagnostic accuracy, which is worthy of providing reference and advice for future clinical practice.

Acknowledgements
Not applicable.

Funding
No funding was received.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions
HR conceived the study and drafted the manuscript. ZS and YuZ acquired the data. JS and YunhuaZ analyzed the data and revised the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate
The study was approved by the Ethics Committee of Liaocheng Third People's Hospital (Liaocheng, China). Patients who participated in this research had complete clinical data. Signed informed consents were obtained from the patients and/or the guardians.

Patient consent for publication
Not applicable.