Molecular marker for detecting, identifying and diagnosing breast cancer and benign breast tumor and application of molecular marker

Abstract

The invention discloses a molecular marker for detecting, identifying and diagnosing breast cancer and benign breast tumor and application thereof, relates to the field of disease diagnosis marker detection, and aims to solve the problem of lack of molecular markers for identifying and diagnosing breast cancer and benign breast tumor at present. The breast cancer marker and the kit are developed by taking blood related markers of a breast cancer patient as a combination, so that differential diagnosis of breast cancer and benign breast tumor is realized, and the early diagnosis rate of breast cancer is improved. It is found that the Cyst C content level in the blood of a breast cancer patient is remarkably lower than that of a benign breast tumor patient, the beta2-m content level in the blood of the breast cancer patient is remarkably lower than that of the benign breast tumor patient, and the specificity and sensitivity of independently detecting the Cyst C and beta2-m levels in the blood for differential diagnosis of the breast cancer and the benign breast cancer are not high; however, when Cyst C and beta2-m are jointly applied to differential diagnosis of breast cancer and benign breast cancer, good specificity and sensitivity are achieved.

Description

Technical Field

[0001] This invention belongs to the field of disease diagnosis, specifically to the use of molecular markers to distinguish whether an individual has breast cancer and to differentiate it from benign breast tumors. Background Technology

[0002] Breast tumors account for about two-thirds of breast diseases, with benign breast tumors, malignant breast tumors, and other breast diseases each accounting for about one-third. Among them, breast cancer (BCa) is the leading cause of cancer-related deaths in women. [Bray F, et al [CA Cancer J Clin. 2018] Early detection of breast cancer has a good long-term prognosis, with an overall 5-year survival rate of over 90% [Bray F, et al [CA Cancer J Clin. 2018]. However, advanced breast cancer has a poor prognosis, with an overall 5-year survival rate approaching 20% ​​[Bray F, et al [CA Cancer J Clin. 2018]. Therefore, early diagnosis of BCa may lead to better treatment response and more favorable clinical outcomes [Bray F, et al [CA Cancer J Clin. 2018], On the other hand, there is a consensus that timely diagnosis can significantly reduce the cost and complexity of clinical treatment, with the aim of increasing the clinical stage of disease diagnosis [Bray F, et al[CA Cancer J Clin. 2018. Sun L, et al. PLoS One. 2018]. Currently, clinical examinations for breast tumors include visual inspection and palpation by clinicians, breast ultrasound, and mammography. Breast ultrasound examination primarily uses the BI-RADS (Breast Imaging Reporting Data system) grading system to determine whether a tumor is malignant or benign. The BI-RADS grading system ranges from 0 to 6, with higher grades generally indicating a higher degree of malignancy. A grade of 0 typically indicates a slow-growing, relatively regular-shaped breast tumor with little infiltration of surrounding tissues and relatively low vascularity, usually a benign tumor. A grade of 6 typically indicates a malignant breast tumor with abundant surrounding blood vessels, an uneven surface, and potential infiltration of surrounding tissues or metastasis. Mammography, also known as breast X-ray, is one of the simplest and non-invasive imaging methods for diagnosing breast diseases. The images captured allow for comparison of changes in breast tissue at different times, and it can detect breast lumps that clinicians cannot palpate. However, regardless of clinical examination, ultrasound, or mammography, the most accurate method for differentiating breast tumors is pathological examination, typically using a breast biopsy. While these examinations significantly improve the early diagnosis rate of breast cancer, subjective factors such as the individual skill level of clinicians can still lead to some discrepancies in the differential diagnosis between benign breast tumors and breast cancer, necessitating more scientific and reliable laboratory indicators. Therefore, one of the important directions of current breast cancer research is the discovery of new molecular markers that can help clinicians detect early-stage BCa patients and provide accurate prognostic estimates and predictions of clinical treatment response [Nicolini A, et al. Semin CancerBiol. 2018. Leto G, et al. Clin Exp Pharmacol Physiol. 2019]. However, the complex heterogeneity of breast cancer makes it difficult to discover biomolecular markers with high specificity and sensitivity.

[0003] In recent decades, the application of advanced technologies such as high-throughput genomics, transcriptomics, quantitative proteomics, metabolomics, and single-cell sequencing has greatly promoted a deeper understanding of the molecular mechanisms driving tumor cell growth, invasion, and metastasis. Numerous experimental and clinical studies have also provided reliable scientific evidence, including research demonstrating that lysosomal cysteine ​​proteases are upregulated in breast cancer, and that their expression levels are significantly correlated with malignant progression. Research has also found that altered regulation of specific endogenous inhibitors of lysosomal cysteine ​​proteases in breast cancer (i.e., cysteine ​​protease inhibitors, or cystatin) is one of the possible reasons for elevated cysteine ​​protease levels in breast cancer. Among cysteine ​​protease inhibitors, Cyst C is the most effective inhibitor of cysteine ​​cathepsins. New research suggests that Cyst C may be a potential new therapeutic target for breast cancer, and may also serve as a potential neonatal biomarker for early detection, prognosis, and predictive treatment response in BCa patients. Human cystatin C (Cyst C) is a 13.4 kDa non-glycosylated basic protein belonging to the cystatin family of type 2 cysteine ​​protease inhibitors. This small protein is the tightest-binding inhibitor of lysosomal cysteine ​​cathepsins B, H, K, L, and S, as well as the aspartate endopeptidase legumain. Cyst C is encoded by the housekeeping gene CST3 (20p11.21) located on the short arm of chromosome 20. This molecule is widely distributed in all human body fluids and most organs, although its distribution is uneven. The primary site of catabolism for Cyst C is the kidneys, where it is filtered by the glomeruli and then reabsorbed and enzymatically broken down in the proximal tubular cells. Under normal physiological conditions, the level of Cyst C in human blood is relatively constant, serving as an endogenous biomarker. Cyst C is almost undetectable in the urine of healthy individuals; therefore, an increase in urinary Cyst C levels is considered a substitute for creatinine, a marker of proximal tubular damage, used to assess renal function under normal and pathological conditions. However, besides its role as a regulator of the intracellular and extracellular activity of lysosomal cysteine ​​cathepsins, Cyst C has also been shown to regulate other important biological functions, particularly cell proliferation, differentiation, migration, angiogenesis, immune regulation, and bone remodeling. Furthermore, a growing body of preclinical and clinical studies in recent years have provided evidence regarding Cyst C's involvement in the pathogenesis of various human non-malignant and malignant pathological conditions, including breast cancer. Experimental and clinical observations have shown that Cyst C is widely expressed in breast cancer cells and other tumor tissues; however, compared to healthy breast tissue, the expression level of Cyst C in BCa tissue has been generally reduced [Leto G, et al. Life Sci. 2018], while the level of cysteine ​​cathepsins is increased.Consistent with these observations, studies have reported that an increased cathepsin / Cyst C ratio, particularly increased cathepsin expression accompanied by decreased Cyst C expression, is associated with a more aggressive tumor phenotype. These data further support the findings of in vitro studies that Cyst C's ability to interfere with breast cancer development appears to be primarily mediated through its inhibitory effect on cathepsin activity.

[0004] Clinical studies have demonstrated the potential impact of Cyst C at various stages of breast cancer progression, and have hypothesized its potential clinical role as a novel biomarker that could help improve current clinical treatment outcomes and management for patients with breast cancer (BCa) [Leto G, et al. Life Sci. 2018]. However, to date, studies supporting this hypothesis have yielded controversial results [Kwon WS, et al. Oncol Lett. 2018]. Several factors could contribute to these discrepancies, particularly differences in the number and clinical characteristics of patients in different studies, variations in analytical conditions and methods, and / or the different cutoff values ​​considered in these studies [Kwon WS, et al. Oncol Lett. 2018]. For example, multiple studies have found higher circulating Cyst C levels in patients with various malignant diseases, including breast cancer [Decock J, et al. Int JBiol Markers. 2008. Leto G, et al. Cancer Invest. 2016]. Compared with healthy subjects, patients with early-stage breast cancer and inflammatory breast cancer had higher serum Cyst C levels, and serum Cyst C levels were significantly correlated with tumor size, but not with other clinicobiological parameters [Leto G, et al. Cancer Invest. 2016]. Furthermore, studies have found higher Cyst C levels in patients with bone metastases (BCa) compared to patients with primary tumors, and receiver operating characteristic (ROC) curve analysis showed good diagnostic performance of Cyst C in distinguishing between cancer and non-cancer patients. Recent studies have reported that serum Cyst C levels in BCa patients significantly decreased after tumor surgery, but remained elevated even with the presence of metastatic lesions [Kwon WS, et al. Oncol Lett. 2018]. While these studies identified serum Cyst C levels as a potential predictive biomarker for BCa presence and used them to assess treatment response and clinical outcomes in these patients, no specific studies have been reported on changes in serum Cyst C levels between patients with breast cancer and those with benign breast tumors.

[0005] β2-microglobulin (β2-M) is a non-glycosylated protein with a molecular weight of 12 kDa, synthesized in all nucleated cells. β2-M is a component of the human leukocyte antigen (HLA) light chain. β2-M can be separated from HLA molecules and exists in free form in extracellular fluid, and can also be detected in blood. Under normal physiological conditions, the free soluble β2-M in serum, urine, and other body fluids remains at a relatively constant low level. However, when kidney function is impaired, glomerular filtration and tubular reabsorption and breakdown are affected, leading to increased β2-M levels in blood and urine. Therefore, β2-M in blood and urine is commonly used as an indicator of kidney function. Furthermore, studies have found increased synthesis and release of β2-m in some malignant diseases, including solid tumors such as multiple myeloma, lymphoma, and breast cancer. These patients exhibit elevated β2-m concentrations in their serum or urine, and serum β2-m can serve as a marker for advanced tumors at diagnosis or during post-treatment efficacy monitoring [Jongvilaikasem, S. et al. Clinical Oncology, 2021]. Patients with metastatic breast cancer have higher serum β2-m levels than those with early or locally advanced breast cancer [Petekkaya I, et al. J BUON, 2017]. Recent research has also found that breast cancer patients with low serum β2-m levels have significantly higher overall survival (OS) than those with high serum β2-m levels (P < 0.01) [Jongvilaikasem, S. et al. Clinical Oncology, 2021]. Mechanistic studies have demonstrated that circulating soluble β2-M is an important autocrine and paracrine growth stimulant that promotes cancer cell metastasis or entry into and colonization of the bone. β2-M interacts with its receptor, human hemoglobin (HFE), regulating epithelial-mesenchymal transition (EMT) through the iron-responsive pathway, thereby promoting tumor cell invasion and migration. While these studies have established serum β2-M levels as a potential predictive biomarker for BCa progression from both clinical and mechanistic perspectives, specific research reports on changes in serum β2-M levels between breast cancer and benign breast tumor patients have not yet been published. Summary of the Invention

[0006] The purpose of this invention is to address the current lack of effective molecular markers for the differential diagnosis of benign breast tumors and breast cancer in clinical practice, and to provide a molecular marker and its application. This invention provides a diagnostic kit for detecting Cyst C and β2-M proteins in blood samples from patients with breast tumors.

[0007] One marker of the present invention is Cyst C and β2-M.

[0008] The present invention relates to a molecular marker for the differential diagnosis of breast cancer and benign breast tumors, and its application in establishing a method for the differential diagnosis of breast cancer and benign breast tumors.

[0009] Furthermore, the method for establishing a differential diagnosis between breast cancer and benign breast tumors involves quantitatively detecting the levels of the aforementioned molecular markers in blood samples from patients with breast lumps, and comparing and analyzing the levels of the aforementioned biomarkers among different patients.

[0010] Furthermore, the quantitative detection of the molecular marker content levels in blood samples of breast cancer patients, and the analysis of the molecular marker content in different patients, the comparative analysis refers to the fact that the Cyst C content in the blood of breast cancer patients is lower than the molecular marker content in benign breast tumors, and the β2-M content in the blood of breast cancer patients is higher than the molecular marker content in benign breast tumors.

[0011] The present invention relates to a kit for detecting molecular markers of breast tumors, the kit comprising nitrocellulose membranes (NC) separately coated with Cyst C and β2-M antibodies, separately immobilized on a solid carrier labeled with Cyst C and β2-M antibodies, and a sample diluent.

[0012] The beneficial effects of this invention are: This invention utilizes a combination of breast tumor-related molecular markers and reagent kits to differentiate between breast cancer and benign breast tumors, facilitating early diagnosis of breast cancer. The invention reveals that breast cancer patients have significantly lower Cyst C levels than benign breast tumor patients, while their β2-M levels are significantly higher. While detecting Cyst C or β2-M levels alone has low sensitivity and specificity for differentiating between breast cancer and benign breast tumors, combining Cyst C and β2-M detection demonstrates better sensitivity and specificity.

[0013] The molecular detection kit for breast tumors of this invention uses serum, plasma or whole blood as the detection sample. The detection is rapid and convenient, the operation is simple, and it has good specificity and sensitivity. Clinical verification shows that it has high discrimination accuracy. Attached Figure Description

[0014] Figure 1 The graph shows the sensitivity and specificity of Cyst C alone in patients with breast cancer and benign breast tumors.

[0015] Figure 2 The graph shows the sensitivity and specificity of β2-M alone in patients with breast cancer and benign breast tumors.

[0016] Figure 3The graph shows the sensitivity and specificity of the combined detection of Cyst C and β2-M in patients with breast cancer and benign breast tumors.

[0017] Figure 4 The graph shows the sensitivity and specificity of the Cyst C and β2-M immunochromatographic gold standard quantitative kit in combination in patients with breast cancer and benign breast tumors.

[0018] Figure 5 The diagram shows the structure of the test card in the kit.

[0019] 1—Test card 2—Sample application port 3—Observation port Figure 6 The diagram shows the composition and structure of the test strip inside the kit's test card.

[0020] 4—Absorbent layer at the sample application end; 5—Gold-labeled antibody layer; 6—Control line 7—Detection line; 8—Detection layer; 9—Water-absorbing end, water-absorbing layer 10—Liner Specific implementation methods

[0021] To make the objectives, technical solutions, and advantages of the embodiments of this invention clearer, the spirit of the contents disclosed in this invention will be described in detail below. Any modifications and alterations made by those skilled in the art based on the techniques taught in this invention after understanding the embodiments of this invention will not depart from the spirit and scope of this invention.

[0022] The illustrative examples and descriptions of this invention are used to explain the invention, but are not intended to limit the invention.

[0023] This invention utilizes immunochromatographic quantitative detection methods to analyze the levels of molecular markers in blood samples from breast cancer and benign breast tumors. Furthermore, this invention uses a Cyst C immunochromatographic quantitative detection kit (colloidal gold method) and a β2-M immunochromatographic quantitative detection kit (colloidal gold method) to detect the levels in serum samples from 517 breast cancer patients and 254 benign breast tumor patients, respectively. The results showed significant differences in Cyst C levels (p<0.05) and β2-M levels (p<0.01) between breast cancer and benign breast tumor patients, thus establishing Cyst C and β2-M as reliable molecular markers for the differential diagnosis of breast cancer and benign breast tumors.

[0024] A detection kit was prepared using Cyst C and β2-M as detection target molecules. The kit includes colloidal gold-labeled antibodies, antibody-coated NC membranes, and clinical sample validation.

[0025] The preparation process of the reagent kit is as follows: Preparation of Cyst C antibody and β2-M antibody Cyst C antibody 1, Cyst C antibody 2, β2-M antibody 1 and β2-M antibody 2 are murine monoclonal antibodies prepared by Lanzhou Yahua Biotechnology Co., Ltd. and used as labeling antibodies and NC membrane coating antibodies for the next step.

[0026] Preparation of labeled antibodies The preparation method for labeled antibodies includes the following steps: i. Preparation of colloidal gold: Add 1.3%~2% (by volume) of trisodium citrate (1%~3%) to chloroauric acid (0.004%~0.012%), boil for 10~20 minutes to obtain a colloidal gold solution of 15~50 nm; ii. Labeling Cyst C monoclonal antibody with colloidal gold: Adjust the pH of the colloidal gold solution to 8.0~8.4 with 0.2M potassium carbonate solution, add Cyst C monoclonal antibody 1 at 4~12 mg / 100 ml, stir, and then add animal serum protein at 0.1~0.6 g / 100 ml. Let stand at 4℃ for 2~4 hours; iii. Centrifuge the above colloidal gold solution at 2000 rpm for 10~15 minutes, remove the precipitate, and obtain the supernatant; iv. Centrifuge the supernatant at 10000 rpm for 60~80 minutes to obtain the precipitate; v. Dissolve the precipitate in 0.02 MPa h7.4 Tris-HCl buffer at a ratio of 4-10 ml / 100 ml to obtain a colloidal gold solution containing 0.2%-0.6% animal serum protein and 0.01%-0.06% sodium azide; vi. Immerse the above colloidal gold solution in glass fiber or non-woven fabric until the liquid begins to seep out, and dry at 37°C to form a specific gold-labeled antibody layer for the immunochromatographic kit.

[0027] The preparation of the antibody-coated NC membrane involves adding a fixative to the purified rabbit or goat anti-mouse IgG antibody and Cyst C monoclonal antibody 2. A dedicated membrane spraying machine is used to coat the cellulose membrane with the Cyst C monoclonal antibody 2 detection line, and the rabbit or goat anti-mouse IgG antibody is coated to form the control line. Alternatively, the preparation method involves spraying the antigen or antibody onto the NC membrane using a dedicated membrane spraying machine at a concentration of 0.8-1.2 mg / ml. After spraying, the membrane is dried at 37°C, blocked for 30 minutes with 0.01 mM pH 7.0 PBS containing 10% fetal bovine serum, washed with 0.01 mM pH 7.0 PBS, and dried at 37°C to form the specific detection membrane for the immunochromatographic assay kit.

[0028] The kit consists of a test card and a sample diluent. The structure of the test card is shown in the attached figure. Figure 5 As shown, the test card is a plastic card holder. The front of the holder has a sample application hole for adding the test sample and an observation hole for viewing the test results. Inside the test card is a test strip, the structure of which is shown in the attached diagram. Figure 6As shown, the specific structure includes a sample application end absorbent layer 4, a detection layer 8, and an absorbent end absorbent layer 9 on the liner 10. A gold-labeled Cys C antibody layer 1 5 is disposed between the detection layer 8 and the absorbent end absorbent layer 9. Detection lines 7 and control lines 6 are coated on the detection layer 8. The sample application end absorbent layer 4 is made of multiple layers of glass fiber or non-woven fabric; the absorbent end absorbent layer 9 is made of multiple layers of filter paper; the detection layer 8 is a cellulose membrane, which can be a nitrocellulose membrane or a cellulose acetate membrane; and the gold-labeled antibody layer is made of glass fiber or non-woven fabric impregnated with colloidal gold-labeled antibody.

[0029] The preparation process of the β2-M immunochromatographic quantitative detection kit is similar to that of the Cyst C immunochromatographic quantitative detection kit.

[0030] The invention will be further illustrated below with specific implementation examples. Implementation Case 1: Validation of the specificity and sensitivity of serum molecular marker levels in breast tumor patients to differentiate between breast cancer and benign breast tumors. In Case Study 1, serum samples were collected from 517 breast cancer patients and 254 benign breast tumor patients. The inclusion criteria for breast cancer and benign breast tumor patients were histopathological diagnosis of either breast cancer or benign breast tumor, and no surgical treatment had been performed prior to blood sample collection. Serum Cyst C levels were measured using a Cyst C immunochromatographic gold standard quantitative assay kit, and serum β2-M levels were measured using a β2-M immunochromatographic gold standard quantitative assay kit. Nonparametric tests were used to analyze the differences in serum Cyst C and β2-M levels between breast cancer and benign breast tumor patients. The results showed significant differences in serum Cyst C and β2-M levels between the two groups (p<0.05, p<0.01). Based on these results, SPSS 23 statistical analysis software was used to analyze the sensitivity and specificity of Cyst C and β2-M levels in the differential diagnosis of breast cancer and benign breast tumors. The sensitivity and specificity of Cyst C and β2-M are as follows: Figure 1 , Figure 2 As shown, the areas under the AUC curves were 0.559 (sensitivity 90.90%, specificity 28.30%) and 0.616 (sensitivity 77.10%, specificity 43.30%), respectively. Using these two molecular markers alone as detection indicators was not ideal, but the combined detection of the two molecular markers showed a more ideal detection effect, achieving better specificity and sensitivity. The combined use of the two was significantly better than using them alone, with an AUC curve area of ​​0.966 (sensitivity 98.90%, specificity 90.90%). Therefore, this invention decided to use the combined detection of Cys C and β2-M as a molecular marker to differentiate between patients with breast cancer and benign breast tumors.

[0031] Case Study 2: Verification of the accuracy of Cyst C and β2-M quantitative detection kits. As mentioned above, Cyst C immunochromatographic gold nanoparticle quantitative detection kits and β2-M immunochromatographic gold nanoparticle quantitative detection kits were prepared separately. Case Study 2 collected serum from 75 patients with breast cancer and 123 patients with benign breast tumors. The accuracy of the kits was verified using both kits. The experimental results showed that the area under the AUC curve for the kits was 0.939 (sensitivity 83.60%, specificity 93.30%).

[0032] Validated on clinical samples, this invention demonstrates high accuracy in differentiating between breast cancer and benign breast tumors.

[0033] This invention can be used for the auxiliary differential diagnosis of breast cancer and benign breast tumors, and has high clinical application value.

Claims

1. A molecular marker for the detection and differential diagnosis of breast cancer and benign breast tumors, characterized by: The molecular marker is composed of Cyst C and β2-M proteins.

2. The molecular markers for the differential diagnosis of breast cancer and benign breast tumors as described in claim 1 are used in establishing a detection method for the differential diagnosis of breast cancer and benign breast tumors.

3. The application according to claim 2, characterized in that... The method for establishing a differential diagnosis between breast cancer and benign breast tumors involves detecting the levels of the aforementioned molecular markers in the blood of breast cancer patients and comparing them with the levels of the aforementioned molecular markers in the blood of benign breast tumor patients.

4. The application described in claim 3, characterized in that... The levels of the aforementioned molecular markers in the blood of breast cancer patients were detected and compared with those in the blood of patients with benign breast tumors. The comparison refers to the fact that the level of Cyst C in the blood of breast cancer patients was lower than that in the blood of patients with benign breast tumors, while the level of β2-M in the blood of breast cancer patients was higher than that in the blood of patients with benign breast tumors.

5. A kit comprising the molecular markers for the differential diagnosis of breast cancer and benign breast tumors as described in claim 1, characterized in that... The kit comprises colloidal gold-labeled Cyst C antibody, a nitrocellulose membrane coated with Cyst C antibody, and sample diluent, as well as colloidal gold-labeled β2-M antibody, a nitrocellulose membrane coated with β2-M antibody, and sample diluent, and is assembled according to the immunochromatographic detection kit to complete the Cyst C and β2-M kits.

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