Method for detecting breast cancer recurrence and metastasis monitoring indexes and application thereof

Abstract

The invention discloses a breast cancer recurrence and metastasis monitoring index detection method and application thereof, and belongs to the technical field of biomedicine.The breast cancer recurrence and metastasis monitoring index detection method comprises the following steps that an EL ISA detection kit is adopted for conducting EL ISA detection on a serum sample and a standard substance of a breast mass patient, and absorbance values of the breast mass patient sample and the standard substance are measured; and drawing a standard curve according to the absorbance values of the breast lump patient sample and the standard substance and the concentration of the standard substance, obtaining the MMP-2 content, the MMP-9 content and the Twi st content of the breast lump patient sample according to the standard curve, and respectively taking the digits as the Cut-off value for breast cancer recurrence and metastasis monitoring. The median of the contents of MMP-2, MMP-9 and Twi st in preoperative serum of a breast cancer patient is used as a breast cancer recurrence and metastasis monitoring index, so that the content accuracy of the three detection indexes is remarkably improved, and the risk of recurrence and metastasis of the breast cancer can be further evaluated.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to a detection method for monitoring indicators of breast cancer recurrence and metastasis and an application thereof. Background Art

[0002] In recent years, although the diagnosis and treatment technologies and methods of breast cancer have developed to a certain extent, the invasive growth and metastasis of breast cancer is still one of the important causes of death. How to detect clinical recurrence and metastasis early is of great significance to improving the diagnosis and treatment of breast cancer. Tumor invasion and metastasis is a dynamic, complex and multi-step process. Effective monitoring and timely detection of tumor recurrence and metastasis is the key to adopting a reasonable comprehensive treatment plan.

[0003] Matrix metalloproteinases (MMPs) are currently known to be important proteolytic enzymes for tumor invasion and metastasis, and can degrade all extracellular matrices. They are mainly divided into six categories, of which MMP-2 and MMP-9 belong to one of them. After being activated, they can form type IV collagenase (gelatinase), which can promote tumor growth and spread by participating in the formation of new blood vessels; they can also specifically degrade the extracellular matrix and basement membrane, break through the matrix barrier, and allow tumor cells to infiltrate the surrounding tissues, promoting tumor invasion and metastasis. The peripheral blood MMP levels of various malignant tumors are closely related to the biological behavior of tumors. Tumor cells can enter the blood through autocrine secretion or induce host cells to secrete a large amount of MMP. Twist is an important tumor metastasis factor that can encode apoptosis inhibitory proteins and has the characteristics of oncogenes. It can participate in regulating the epithelial-mesenchymal transition of tumor cells by affecting the expression of the target gene E-box promoter, and thus play an important role in tumor invasion and metastasis. Studies have shown that Twist regulates the expression of MMP-2 and MMP-9 at a certain level. Therefore, the levels of MMP-2, MMP-9 and Twist in the blood are closely related to the recurrence and metastasis of breast cancer.

[0004] At present, the methods for detecting MMP-2, MMP-9 and Twist in breast cancer are mainly for postoperative (tumor) tissue specimens. The histological test results are only of certain guiding significance for short-term postoperative treatment and prognosis judgment, but there are certain defects in the dynamic monitoring of tumor metastasis and recurrence during preoperative unclear diagnosis, postoperative patient follow-up and radiotherapy and chemotherapy. In addition, breast cancer tissue detection needs to be taken from postoperative mass specimens, which is highly invasive, and the detection method is time-consuming and complicated. Compared with tissue detection, blood detection is less invasive, low-cost, repeatable and fast, and has important auxiliary significance in the diagnosis of patients with no obvious palpable masses in the clinic, so that targeted and effective treatment can be carried out on breast cancer patients as soon as possible. However, the sensitivity of blood detection is lower than that of tissue detection, which results in reduced diagnostic accuracy. Summary of the invention

[0005] The purpose of the present invention is to provide a detection method for monitoring indicators of breast cancer recurrence and metastasis and its application to solve the following technical problems: improve the accuracy of detecting breast cancer MMP-2, MMP-9 and Twist, and thus improve the accuracy of breast cancer recurrence and metastasis diagnosis.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] A method for detecting indicators for monitoring recurrence and metastasis of breast cancer comprises the following steps:

[0008] Step 1: Use an ELISA test kit to perform ELISA tests on serum samples of patients with breast lumps and standard products (provided by the ELISA test kit) to measure the absorbance values ​​of the samples of patients with breast lumps and the standard products;

[0009] Step 2: Draw a standard curve based on the absorbance values ​​of the breast mass patient samples and the standard products and the concentrations of the standard products, and calculate the contents of MMP-2, MMP-9 and Twist in the breast mass patient samples based on the standard curve;

[0010] Step 3: Take the median values ​​of MMP-2, MMP-9 and Twist levels in samples from patients with breast tumors as indicators for monitoring the recurrence and metastasis of breast cancer.

[0011] As a further embodiment of the present invention, the serum sample of the patient with breast mass is prepared by the following steps:

[0012] The blood of the patient with breast mass is placed at 18-22°C for 2-4 hours, and then centrifuged at 1000×g for 20 minutes after standing for 12-16 hours. 3 ml of the supernatant is taken and diluted 100-200 times with PBS (phosphate buffer) with a molar concentration of 0.1 M to obtain the serum sample of the patient with breast mass.

[0013] As a further embodiment of the present invention, the breast lump patients include breast cancer patients and benign breast lesion patients.

[0014] As a further embodiment of the present invention, the patient with the breast mass has not undergone chemotherapy, radiation or immunotherapy, and has no other special signs and diseases.

[0015] As a further embodiment of the present invention, the ELISA test specifically comprises the following steps:

[0016] Step a1: The serum sample of the patient with breast mass and the reagent of the ELISA detection kit are balanced to 18-22°C for use; blank wells, standard wells and test sample wells are set up on a 96-well ELISA plate, 100 μL of sample diluent is added to the blank well, 100 μL of standard is added to the standard well, 100 μL of serum sample of the patient with breast mass is added to the test sample well, and then a film is added, and incubated at 37°C for 2 hours;

[0017] Step a2: discard the liquid in the wells, spin dry, add 100 μL of detection solution A to each well, add the film, incubate at 37°C for 1 hour, discard the liquid in the wells, spin dry, add 400 μL of concentrated washing solution to each well, soak for 1-2 minutes, wash the plate, spin dry, repeat the washing and spin drying 3 times;

[0018] Step a3: Add 100 μL of detection solution B to each well and then cover with a film, incubate at 37°C for 1 hour, discard the liquid in the well, spin dry, add 400 μL of concentrated washing solution to each well, soak for 1-2 minutes, wash the plate, spin dry, and repeat the washing and spin drying 5 times;

[0019] Step a4: Add 90 μL of substrate solution to each well and then cover with film. After color development at 37°C in the dark for 15-30 min, add 50 μL of stop solution to each well to terminate the reaction. The order of adding the stop solution is the same as that of the substrate solution. Immediately measure the absorbance of each well at a wavelength of 450 nm using an ELISA reader.

[0020] As a further embodiment of the present invention, the ELISA detection kit is purchased from BioSource International Inc. (BIOI).

[0021] As a further solution of the present invention, the application of the detection method of the breast cancer recurrence and metastasis monitoring index is applied to assess the risk of breast cancer recurrence and metastasis.

[0022] Beneficial effects of the present invention:

[0023] The present invention uses an ELISA detection method to perform preliminary detection of the levels of MMP-2, MMP-9 and Twist in the preoperative serum of patients with breast cancer and benign breast lesions. On the one hand, using preoperative serum samples for detection overcomes the problems that the detection tissue needs to be taken from postoperative tumor specimens, which is highly invasive, and the detection method is time-consuming and complicated. In addition, using preoperative serum samples for detection as a non-invasive detection method is repeatable, fast and timely, and can perform real-time dynamic tracking and monitoring.

[0024] The present invention adopts the median of the detected MMP-2, MMP-9 and Twist contents as the cut-off value (critical value) of the breast cancer recurrence and metastasis monitoring index, significantly improves the accuracy of the contents of the three detection indicators of MMP-2, MMP-9 and Twist, can further evaluate the risk of breast cancer recurrence and metastasis according to the median of the MMP-2, MMP-9 and Twist contents, so that targeted treatment can be carried out for patients with breast masses, greatly improving the treatment effect, improving the prognosis of patients, and further improving the quality of life of patients. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The embodiment of the present invention uses 77 paraffin specimens of tumor tissue and preoperative serum specimens of breast tumor patients who have not undergone chemotherapy, radiation or immunotherapy in recent years and have no other special signs and diseases in the pathology department of a certain hospital (including 31 paraffin specimens of tumor tissue and preoperative serum specimens in the benign breast lesion group, and 46 paraffin specimens of tumor tissue and preoperative serum specimens in the breast cancer group); the median age of breast cancer patients was analyzed and sorted out, and breast cancer was graded and staged according to the standards of WHO (2020) breast tumors; all breast cancer patients were followed up after surgery, and the number of recurrences, survivals and deaths was sorted out.

[0027] Serum sample preparation includes the following steps:

[0028] The blood of the above-mentioned breast mass patient was placed at 18°C ​​for 2 hours, and then centrifuged at 1000×g for 20 minutes after standing for 12 hours. 3 ml of the supernatant was taken and diluted 100 times with PBS having a molar concentration of 0.1 M to obtain the serum sample of the breast mass patient.

[0029] Example 1

[0030] A method for detecting indicators for monitoring recurrence and metastasis of breast cancer comprises the following steps:

[0031] Step 1: ELISA test kit (purchased from BioSource International Inc.) was used to perform ELISA test on preoperative serum samples and standard products (provided by ELISA test kit) of 77 patients with breast masses, and the absorbance values ​​of samples and standard products of patients with breast masses were measured, which specifically included the following steps:

[0032] Step a1: The serum sample of the patient with breast mass and the reagent of the ELISA detection kit are balanced to 18-22°C for use; blank wells, standard wells and test sample wells are set up on a 96-well ELISA plate, 100 μL of sample diluent is added to the blank well, 100 μL of standard is added to the standard well, 100 μL of serum sample of the patient with breast mass is added to the test sample well, and then a film is added, and incubated at 37°C for 2 hours;

[0033] Step a2: discard the liquid in the wells, spin dry, add 100 μL of detection solution A to each well, add the film, incubate at 37°C for 1 hour, discard the liquid in the wells, spin dry, add 400 μL of concentrated washing solution to each well, soak for 1-2 minutes, wash the plate, spin dry, repeat the washing and spin drying 3 times;

[0034] Step a3: Add 100 μL of detection solution B to each well and then cover with a film, incubate at 37°C for 1 hour, discard the liquid in the well, spin dry, add 400 μL of concentrated washing solution to each well, soak for 1-2 minutes, wash the plate, spin dry, and repeat the washing and spin drying 5 times;

[0035] Step a4: Add 90 μL of substrate solution to each well and then cover with film. After color development at 37°C in the dark for 15-30 min, add 50 μL of stop solution to each well to terminate the reaction. The order of adding the stop solution is the same as that of the substrate solution. Immediately measure the absorbance of each well at a wavelength of 450 nm using an ELISA reader.

[0036] Step 2: Draw a standard curve based on the absorbance values ​​of the breast mass patient samples and the standard products and the concentrations of the standard products, and calculate the contents of MMP-2, MMP-9 and Twist in the breast mass patient samples based on the standard curve;

[0037] Step 3: Take the median values ​​of MMP-2, MMP-9 and Twist levels in samples from patients with breast tumors as indicators for monitoring the recurrence and metastasis of breast cancer.

[0038] The measured content distribution and median distribution of MMP-2, MMP-9 and Twist are shown in Table 1:

[0039] Table 1

[0040]

[0041] As shown in Table 1, the cut-off values ​​of MMP-2, MMP-9 and Twist are 21.2 ng / mL, 47.5 ng / mL and 39.8 ng / mL, respectively. By comparing with the critical values, we divide the serum levels into high or low. When the serum MMP-2 content of breast cancer patients after treatment is higher than 21.2 ng / ml, or the MMP-9 content is higher than 47.5 ng / ml, or the Twist content is higher than 39.8 ng / ml, it indicates that the patient has recurred or metastasized; when the serum MMP-2 content is lower than 21.2 ng / ml, or the MMP-9 content is lower than 47.5 ng / ml, or the Twist content is lower than 39.8 ng / ml, it indicates that the patient has not recurred or metastasized.

[0042] Comparative Example 1

[0043] The expression and distribution of MMP-2, MMP-9 and Twist proteins in paraffin specimens of breast tumor tissues of 77 patients with breast tumors were detected by immunohistochemistry, as shown in Table 2:

[0044] Table 2

[0045]

[0046] Application Testing

[0047] The test data tables of Example 1 and Comparative Example 1 were statistically processed using SPSS 13.0 software. The sample rate was compared using x 2 Test; t test was used to compare the means between the two groups, and the data were expressed as x±s; the correlation of count data was analyzed using the non-parametric Spearman rank correlation test.

[0048] The results of testing tissue specimens and serum specimens of breast cancer and benign breast lesions showed that:

[0049] The expression of MMP-2, MMP-9 and Twist proteins in the tumor tissue of the breast cancer group was higher than that in the benign breast lesion group, indicating that they are all involved in the occurrence of breast cancer and are helpful for the identification of benign and malignant breast lesions and the monitoring of recurrence and metastasis after treatment. The positive rates of MMP-2, MMP-9 and Twist proteins in the tumor tissue of the breast cancer group increased with the increase of TNM stage, and were higher in the axillary lymph node metastasis group than in the non-metastasis group, indicating that the high expression of MMP-2, MMP-9 and Twist in breast cancer (enhancing the ability of tumor cells to destroy the extracellular matrix) is related to the earlier occurrence of axillary lymph node metastasis, and the TNM stage is closely related to the development of the tumor and adverse biological behavior, indicating that the positive expression of MMP-2, MMP-9 and Twist in breast cancer can warn of the recurrence and metastasis of breast cancer.

[0050] The preoperative serum MMP-2, MMP-9 and Twist levels of breast cancer patients were significantly positively correlated with the postoperative MMP-2, MMP-9 and Twist protein expressions in the tumor tissue of patients, indicating that the serum MMP-2, MMP-9 and Twist levels of breast cancer patients can reflect their protein expression levels in the tumor tissue, and the detection of serum MMP-2, MMP-9 and Twist levels in breast cancer patients after treatment can become an indicator for monitoring recurrence and metastasis.

[0051] According to the above research and experimental results, the present invention provides a method for detecting MMP-2, MMP-9 and Twist indicators in serum and its application in monitoring the recurrence and metastasis of breast cancer. By taking the Cut-off values ​​(critical values) of MMP-2, MMP-9 and Twist in the serum of breast cancer specimens obtained in the experiment as indicators, the MMP-2, MMP-9 and Twist levels in the serum of patients after treatment are monitored to assist in judging the recurrence and metastasis of breast cancer after treatment. The problem that the detection tissue needs to be taken again from the postoperative tumor specimen, which is highly traumatic, and the detection method is time-consuming and complicated is overcome. As a non-invasive detection method, serum detection is repeatable, fast and timely, and can perform real-time dynamic tracking and monitoring, so that targeted treatment can be taken in time for the recurrence and metastasis of breast cancer patients, which greatly improves the treatment effect and thus improves the quality of life of patients.

[0052] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0053] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting indicators for monitoring recurrence and metastasis of breast cancer, It is characterized in that The steps include: Step 1: Use an ELISA detection kit to perform ELISA detection on the serum samples of patients with breast masses and the standard products, respectively, and measure the absorbance values ​​of the samples of patients with breast masses and the standard products; Step 2: Draw a standard curve based on the absorbance values ​​of the breast mass patient samples and the standard products and the concentrations of the standard products, and calculate the contents of MMP-2, MMP-9 and Twist in the breast mass patient samples based on the standard curve; Step 3: Take the median values ​​of MMP-2, MMP-9 and Twist levels in samples from patients with breast tumors as indicators for monitoring the recurrence and metastasis of breast cancer.

2. A method for detecting indicators for monitoring recurrence and metastasis of breast cancer according to claim 1, It is characterized in that The serum sample of the breast mass patient is prepared by the following steps: The blood of the patient with breast mass is placed at 18-22°C for 2-4 hours, and then centrifuged at 1000×g for 20 minutes after standing for 12-16 hours. 3 ml of the supernatant is taken and diluted 100-200 times with PBS having a molar concentration of 0.1 M to obtain the serum sample of the patient with breast mass.

3. The method for detecting a breast cancer recurrence and metastasis monitoring indicator according to claim 1, It is characterized in that The breast lump patients include breast cancer patients and benign breast lesion patients.

4. The method for detecting a breast cancer recurrence and metastasis monitoring indicator according to claim 1, It is characterized in that The patient with the breast mass had not received chemotherapy, radiation or immunotherapy and had no other special signs and diseases.

5. The method for detecting indicators for monitoring recurrence and metastasis of breast cancer according to claim 1, It is characterized in that The ELISA test specifically comprises the following steps: Step a1: The serum sample of the patient with breast mass and the reagent of the ELISA detection kit are balanced to 18-22°C for use; blank wells, standard wells and test sample wells are set up on a 96-well ELISA plate, 100 μL of sample diluent is added to the blank well, 100 μL of standard is added to the standard well, 100 μL of serum sample of the patient with breast mass is added to the test sample well, and then a film is added, and incubated at 37°C for 2 hours; Step a2: discard the liquid in the wells, spin dry, add 100 μL of detection solution A to each well, add the film, incubate at 37°C for 1 hour, discard the liquid in the wells, spin dry, add 400 μL of concentrated washing solution to each well, soak for 1-2 minutes, wash the plate, spin dry, repeat the washing and spin drying 3 times; Step a3: Add 100 μL of detection solution B to each well and then cover with a film, incubate at 37°C for 1 hour, discard the liquid in the well, spin dry, add 400 μL of concentrated washing solution to each well, soak for 1-2 minutes, wash the plate, spin dry, and repeat the washing and spin drying 5 times; Step a4: Add 90 μL of substrate solution to each well and then cover with film. After color development at 37°C in the dark for 15-30 min, add 50 μL of stop solution to each well to terminate the reaction. The order of adding the stop solution is the same as that of the substrate solution. Immediately measure the absorbance of each well at a wavelength of 450 nm using an ELISA reader.

6. The method for detecting indicators for monitoring recurrence and metastasis of breast cancer according to claim 1, It is characterized in that The ELISA detection kit was purchased from BioSource International Inc. (BIOI).

7. Use of a method for detecting indicators for monitoring recurrence and metastasis of breast cancer according to any one of claims 1 to 6, It is characterized in that Used to assess the risk of recurrence and metastasis of breast cancer.

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