Biomarker combination for grading diagnosis of esophageal squamous intraepithelial neoplasia and application thereof

Abstract

The invention discloses a biomarker combination for grading diagnosis of esophageal squamous intraepithelial neoplasia and application of the biomarker combination, and relates to the technical field of biological medicines. The biomarker combination comprises p53, Ki-67 and Statmin-1, and is characterized in that the biomarker combination comprises p53, Ki-67 and Tissue specimens of esophageal squamous intraepithelial neoplasia patients and esophageal squamous cell carcinoma patients are subjected to retrospective analysis, the expression conditions of p53, Ki-67 and Statmin-1 are detected by using an immunohistochemical technology, and the application value of Statmin-1, p53 and Ki-67 protein joint detection in grading diagnosis of esophageal squamous intraepithelial neoplasia is discussed. The result shows that the Statmin-1, p53 and Ki-67 protein combined detection has certain diagnosis and prediction values on esophageal squamous intraepithelial neoplasia grading, and the diagnosis accuracy of esophageal squamous intraepithelial neoplasia can be improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a biomarker combination for grading diagnosis of esophageal squamous intraepithelial neoplasia and an application thereof. Background Art

[0002] Esophageal intraepithelial neoplasia is a type of human squamous intraepithelial neoplasia (SIN). It specifically refers to the tumor-like changes in the esophageal mucosal epithelium. This change covers the entire process from mild dysplasia to severe dysplasia and is an important precancerous lesion of esophageal cancer. According to the classification of the World Health Organization (WHO), esophageal intraepithelial neoplasia is further subdivided into esophageal high-grade squamous intraepithelial neoplasia (EHGSIN) and esophageal low-grade squamous intraepithelial neoplasia (ELGSIN). ELGSIN refers to epithelial cells with mild dysplasia that do not exceed 1 / 2 of the entire layer. EHGSIN refers to epithelial cells with moderate to severe dysplasia that exceed 1 / 2 of the entire layer. Since there are large differences between the observations and judgments of pathologists, it is very important to find a set of objective immunohistochemical examination indicators to provide an objective basis for standardized pathological diagnosis. In addition, conventional grading standards also have certain limitations in guiding the treatment of esophageal intraepithelial neoplasia. For ELGSIN, an observation and follow-up strategy is usually adopted, but for EHGSIN, in addition to endoscopic treatment, a personalized treatment plan needs to be formulated based on the patient's specific situation. Therefore, finding a new grading standard can also provide sufficient basis for the formulation of treatment plans. Summary of the invention

[0003] The purpose of the present invention is to provide a biomarker combination for grading and diagnosing esophageal squamous intraepithelial neoplasia and its application to solve the problems existing in the above-mentioned prior art. The biomarker combination has a certain diagnostic and predictive value for grading esophageal squamous intraepithelial neoplasia, which helps to improve the diagnostic accuracy of esophageal squamous intraepithelial neoplasia.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a biomarker combination for grading diagnosis of esophageal squamous intraepithelial neoplasia, wherein the biomarker combination comprises p53, Ki-67 and Stathmin-1.

[0006] The present invention also provides the use of a reagent for detecting the above-mentioned biomarker combination in the preparation of a product for grading diagnosis of esophageal squamous intraepithelial neoplasia.

[0007] Furthermore, the product is a kit.

[0008] Furthermore, the reagent is a reagent for immunohistochemical staining.

[0009] The present invention also provides a product for grading diagnosis of esophageal squamous intraepithelial neoplasia, comprising a reagent for detecting the above-mentioned biomarker combination.

[0010] Furthermore, the product is a kit.

[0011] Furthermore, the reagents include monoclonal antibodies to Stathmin-1, p53 and Ki-67.

[0012] Furthermore, the reagent also includes enzyme-labeled secondary antibodies labeled with different enzymes and used to detect Stathmin-1, p53 and Ki-67 respectively.

[0013] Furthermore, the reagents also include a color developing solution, a buffer solution, an immunohistochemical antigen repair buffer solution and / or a hematoxylin staining solution.

[0014] The present invention discloses the following technical effects:

[0015] The present invention retrospectively analyzes tissue specimens of patients with esophageal squamous intraepithelial neoplasia and esophageal squamous cell carcinoma, uses immunohistochemistry to detect the expression of p53, Ki-67 and Stathmin-1, and explores the application value of combined detection of Stathmin-1, p53 and Ki-67 proteins in the diagnosis of esophageal squamous intraepithelial neoplasia grading. The results show that combined detection of Stathmin-1, p53 and Ki-67 proteins has certain diagnostic and predictive value for the grading of esophageal squamous intraepithelial neoplasia, which helps to improve the diagnostic accuracy of esophageal squamous intraepithelial neoplasia, thereby providing reference information for the formulation of clinically relevant diagnosis and treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1Comparison of the positive expression rates of Stathmin-1 in esophageal squamous intraepithelial neoplasia tissue and normal esophageal mucosal tissue; A is the immunohistochemical staining of Stathmin-1 in normal esophageal mucosal tissue (200×); B is the immunohistochemical staining of Stathmin-1 in esophageal squamous intraepithelial neoplasia tissue (200×); C is the immunohistochemical staining of Stathmin-1 in esophageal squamous cell carcinoma tissue (200×);

[0018] Figure 2 Immunohistochemical staining of p53, Ki-67 and Stathmin-1 in ELGSIN and EHGSIN tissues (200×);

[0019] Figure 3 The receiver operating characteristic curve for the combined detection of Stathmin-1, p53 and Ki-67 proteins in the diagnosis of esophageal squamous intraepithelial neoplasia was presented;

[0020] Figure 4 This is the receiver operating characteristic curve for the combined detection of Stathmin-1, p53 and Ki-67 proteins in the classification of EHGSIN. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] Esophageal squamous intraepithelial neoplasia, especially those with a long longitudinal diameter and extensive involvement of the esophageal circumference, is affected by multiple factors in terms of disease progression and outcome. The clinical treatment strategy for this type of lesion is directly related to the patient's disease course, recovery speed, and possible complications. Therefore, early diagnosis and treatment of esophageal squamous intraepithelial neoplasia and accurate diagnosis and treatment are crucial to reducing the incidence of esophageal cancer and improving the cure rate and survival rate of patients with esophageal squamous cell carcinoma. The present invention aims to explore the application value of combined detection of Stathmin-1, p53 and Ki-67 proteins in the graded diagnosis of esophageal squamous intraepithelial neoplasia. The results showed that the positive expression rates of Stathmin-1, p53 and Ki-67 were closely related to the grade of esophageal squamous intraepithelial neoplasia, and the combined detection of these three proteins could significantly improve the sensitivity and specificity of diagnosis, providing new ideas and methods for the graded diagnosis of esophageal squamous intraepithelial neoplasia.

[0027] Clinical pathologists accurately identify the cell atypia and tissue structural atypia of esophageal squamous intraepithelial neoplasia, which is the foundation for early diagnosis, early treatment and precise diagnosis and treatment. At present, esophageal squamous intraepithelial neoplasia is mainly divided into two categories: low-grade and high-grade. The characteristic of low-grade intraepithelial neoplasia is that the tumor cells are limited to the lower 1 / 2 of the epithelial layer; high-grade intraepithelial neoplasia refers to tumor cells involving more than 1 / 2 to the entire layer of the epithelium, and the atypia is more significant. The key to identifying intraepithelial neoplasia is that it has recognizable morphological characteristics, showing a tendency to malignancy, but no interstitial infiltration. As an important tumor suppressor gene, p53 plays a key role in the occurrence and development of esophageal squamous intraepithelial neoplasia and esophageal cancer. p53 mutations are very common in esophageal squamous cell carcinoma, reaching 59% to 93%. Wild-type p53 shows nuclear positivity of varying strengths, while p53 missense mutations often lead to accumulation of mutant proteins in the nucleus, showing strong and consistent nuclear positivity. When p53 undergoes nonsense mutations, p53 protein expression is completely absent in tumor cells. Ki-67 is a nuclear antigen, which is related to ribosomal RNA transcription and is a reliable marker for cell proliferation. In the cell cycle, the expression of Ki-67 is closely related to the proliferation activity of cells, especially in the late G1 phase, S phase, G2 phase and M phase, and is not expressed in the G0 phase. Therefore, the positive expression rate of Ki-67 is often used as an important indicator for evaluating the proliferation activity of tumor cells. The present invention confirms that the positive expression rates of Ki-67 and p53 in low-grade squamous intraepithelial neoplasia (ELGSIN) tissues are relatively low, while the positive expression rates of Ki-67 and p53 in high-grade squamous intraepithelial neoplasia (EHGSIN) tissues are significantly increased. This trend suggests that the positive expression rates of Ki-67 and p53 are closely related to the grade of esophageal squamous intraepithelial neoplasia.

[0028] Stathmin-1 (also known as Op18 or STMN) is an important intracellular protein that is mainly involved in the regulation of microtubules. As a key regulator of the cell cycle and microtubule dynamics, Stathmin-1 plays a vital role in cell division, migration and other physiological processes. The present invention found that compared with normal esophageal mucosal tissue, the positive expression rate of Stathmin-1 in esophageal squamous intraepithelial neoplasia tissue was significantly increased, and the positive expression rate of Stathmin-1 in esophageal squamous cell carcinoma tissue was further increased, suggesting that Stathmin-1 may be involved in the onset of esophageal squamous intraepithelial neoplasia and the gradual development of esophageal squamous cell carcinoma. Further analysis showed that the positive expression rates of Stathmin-1, p53 and Ki-67 in EHGSIN tissue were higher than those in ELGSIN tissue, indicating that with the increase in the grade of neoplasia, the abnormal expression trend of these proteins is more obvious, which may be closely related to the malignant biological behaviors such as proliferation, invasion and metastasis of tumor cells. In addition, the present invention also found that in p53 and Ki-67 positive esophageal squamous intraepithelial neoplasia tissue, the positive expression rate of Stathmin-1 was also significantly higher than that of p53 and Ki-67 negative tissue. This result further confirmed the synergistic effect of Stathmin-1, p53 and Ki-67 in esophageal squamous intraepithelial neoplasia, and they may jointly participate in the occurrence and development of the tumor.

[0029] The research process of the present invention is described in detail as follows:

[0030] Example 1

[0031] Development process of a biomarker combination for grading and diagnosing esophageal squamous intraepithelial neoplasia:

[0032] 1 Materials and methods

[0033] 1.1 Materials

[0034] Methods: From January 2019 to May 2024, esophageal squamous intraepithelial neoplasia tissues (ESIN group) of 80 patients who were pathologically diagnosed with ESIN and underwent endoscopic submucosal dissection (ESD) in Nanjing Hospital of Traditional Chinese Medicine and Nanjing Tongren Hospital, normal esophageal mucosal tissues (normal group) of people who underwent endoscopic examination in the health management center during the same period, and cancer tissues of patients with esophageal squamous cell carcinoma were collected. General clinical data were collected, and the lesion data were recorded in detail. This included the range of lesion involvement, longitudinal diameter of the lesion, lesion site classified according to the eighth edition of the TNM staging criteria for esophageal cancer, presence or absence of white fur-like changes, typing of intrapapillary capillary loops (IPCL), presence or absence of erosion, ulceration or bleeding and mucosal color change, preoperative endoscopic biopsy (EFB) and postoperative pathological results, etc.

[0035] Patient inclusion criteria: 1) All patients received comprehensive imaging examinations before surgery; 2) All patients were diagnosed with esophageal EHGSIN or ELGSIN by EFB before surgery and received ESD treatment; or patients diagnosed with esophageal squamous cell carcinoma before surgery; 3) All patients were examined by white light endoscopy or magnifying endoscopy with narrow-band imaging (ME-NBI) before surgery; 4) All required clinical data were complete; 5) All patients had no serious underlying diseases.

[0036] Exclusion criteria: 1) postoperative pathological results suggest esophageal adenocarcinoma, papilloma, sarcomatoid carcinoma and other diseases; 3) combined with a history of other tumors.

[0037] The experimental study of the present invention has been approved by the Ethics Committee of Nanjing Tongren Hospital (approval number: 2022-03-005), and all patients gave informed consent to the present invention.

[0038] The pathological specimens were fixed, dehydrated, embedded, sliced, and stained with H&E. 24 cases of ELGSIN and 56 cases of EHGSIN were screened under the microscope.

[0039] 1.2 Immunohistochemistry

[0040] Monoclonal antibodies of stathmin-1, p53 and Ki-67 were purchased from Abcam and Myxin Biotech, respectively. Immunohistochemical staining was performed using the Roche Benchmark XT fully automatic immunohistochemical staining instrument.

[0041] 1.3 Result determination

[0042] Low-grade squamous intraepithelial neoplasia is characterized by the presence of diseased cells confined to the lower third of the epithelium, with mild cellular disorganization and mild nuclear atypia. In contrast, high-grade squamous intraepithelial neoplasia is characterized by significant polarity disorganization of the basal and parabasal cells of the epithelium, and more pronounced nuclear atypia.

[0043] The positive expression of Ki-67 is localized in the cell nucleus. When the positive expression is limited to the basal layer of the epithelium, it is judged as (-); if it exceeds the basal layer and occupies 10% to 25% of the whole layer, it is judged as (+); when it occupies 26% to 75% of the whole layer, it is judged as (++); and when it exceeds 75% of the whole layer, it is judged as (+++).

[0044] The positive expression of p53 is also located in the cell nucleus, and its judgment criteria are similar to those of Ki-67: limited to the basal layer of the epithelium is (-), exceeding the basal layer and occupying 5% to 25% of the entire layer is (+); occupying 26% to 75% of the entire layer is (++); and exceeding 75% of the entire layer is (+++).

[0045] As for the positive expression of Stathmin-1, it is localized in the cytoplasm. The criteria are: limited to the basal layer of the epithelium is (-); exceeding the basal layer and occupying 10% to 25% of the entire layer is (+); occupying 26% to 75% of the entire layer is (++); exceeding 75% of the entire layer is (+++).

[0046] In the above determination, (-) and (+) were regarded as negative expression, and (++) and (+++) were regarded as positive expression.

[0047] 1.4 Statistical analysis

[0048] SPSS 22.0 statistical software was used for data analysis, and the quantitative data were expressed as mean ± standard deviation. The data were expressed in the form of , and the t test was used to compare the data between groups. For count data, they were expressed in the form of number of cases and rate (%), and the chi-square test was used to compare the data between groups. 2 The categorical data between the two groups were compared and analyzed by the test. In order to evaluate the detection efficiency of the combined examination of Stathmin-1, p53 and Ki-67 for the graded diagnosis of esophageal squamous intraepithelial neoplasia, the receiver operating characteristic (ROC) curve was drawn and the area under the curve (AUC) was calculated. P<0.05 was considered statistically significant.

[0049] 2 Results

[0050] 2.1 Expression of Stathmin-1 in esophageal squamous intraepithelial neoplasia and esophageal squamous cell carcinoma tissues

[0051] In the normal group, the expression of Stathmin-1 was limited to the lower 1 / 2 of the epithelial thickness ( Figure 1 In the esophageal squamous intraepithelial neoplasia group, the expression range of Stathmin-1 extended to between the lower 1 / 2 and upper 1 / 3 of the epithelial thickness ( Figure 1 In esophageal squamous cell carcinoma tissue, the expression range of Stathmin-1 protein was further expanded, and the staining intensity was significantly increased ( Figure 1 In addition, the positive expression rate of Stathmin-1 in the esophageal squamous intraepithelial neoplasia group was significantly higher than that in the normal group (χ 2 =12.52, P < 0.01, Table 1), while the positive expression rate of Stathmin-1 in esophageal squamous cell carcinoma tissues was significantly higher than that in esophageal squamous intraepithelial neoplasia group (χ 2 =10.46, P<0.01, Table 1).

[0052] Table 1 Positive expression of Stathmin-1 in esophageal squamous intraepithelial neoplasia and esophageal squamous cell carcinoma tissues [n (%)]

[0053]

[0054] Note: P1<0.01, vs. normal esophageal mucosa group; P2<0.01, vs. esophageal squamous intraepithelial neoplasia group.

[0055] 2.2 Comparison of positive expression of Stathmin-1, p53 and Ki-67 in EHGSIN and ELGSIN

[0056] The expressions of p53, Ki-67 and Stathmin-1 in the EHGSIN group were significantly higher than those in the ELGSIN group, and the differences were statistically significant (p53:χ 2 =6.923, P<0.01; Ki-67: χ 2 =5.334, P<0.05; Stathmin-1: χ 2 =6.715, P<0.01) (Table 2, Figure 2 ).

[0057] Table 2 Expression of Stathmin-1, p53 and Ki-67 in high-grade squamous intraepithelial neoplasia of the esophagus [n (%)]

[0058]

[0059]

[0060] 2.3 Comparison of Stathmin-1 positive expression in esophageal squamous intraepithelial neoplasia tissues with different p53 and Ki-67 expressions

[0061] The positive expression rate of Stathmin-1 in the p53 and Ki-67 positive expression samples of esophageal squamous intraepithelial neoplasia tissue was higher than that in the negative expression group (Table 3).

[0062] Table 3 Comparison of Stathmin-1 positive expression rate in esophageal squamous intraepithelial neoplasia tissues with different p53 and Ki-67 expressions [n (%)]

[0063]

[0064] 2.4 ROC curve analysis of combined detection of Stathmin-1, p53 and Ki-67 proteins for esophageal squamous intraepithelial neoplasia grading Combined detection of Stathmin-1, p53 and Ki-67 proteins has diagnostic value for esophageal squamous intraepithelial neoplasia and EHGSIN grading ( Figure 3-Figure 4 ), the AUCs were 0.914 and 0.919, respectively, which were higher than the AUCs of p53+Ki-67 protein combined detection (0.852 and 0.851).

[0065] Example 2

[0066] A kit for grading and diagnosing esophageal squamous intraepithelial neoplasia, comprising the following components:

[0067] Primary antibody: includes two reagent packs, one is a mixture of two monoclonal antibodies Stathmin-1 and p53; the other is a mixture of two monoclonal antibodies Stathmin-1 and Ki-67;

[0068] Secondary antibody: Use HRP or AP enzyme-labeled mixed secondary antibody corresponding to the mixed primary antibody;

[0069] Color development solution: DAB and Fast-Red mixed;

[0070] Buffer solution (PBS);

[0071] Immunohistochemistry antigen retrieval buffer;

[0072] Hematoxylin staining solution.

[0073] Double dyeing technology principle:

[0074] 1. Antibodies and color development system:

[0075] (1) Antibody selection: Use primary antibodies from different hosts (such as mouse anti-p53 or Ki67 and rabbit anti-Stathmin-1), and then use the corresponding secondary antibody system to avoid cross-reaction.

[0076] (2) Color development strategy: Stathmin-1: Use red color development (such as Fast Red / alkaline phosphatase system). p53 or Ki67: Use brown color development (such as DAB / horseradish peroxidase system).

[0077] (3) Localization differences: The subcellular localization differences between Stathmin-1 (cytoplasmic localization) and p53 or Ki67 (nuclear localization) assist in differentiation.

[0078] 2. Experimental process optimization:

[0079] (1) Sequential staining: Usually p53 or Ki67 (nuclear localization) is stained first, followed by Stathmin-1 (cytoplasmic localization) to avoid color interference.

[0080] (2) Antigen retrieval: Expose antigen epitopes through heat repair or enzymatic digestion.

[0081] (3) Blocking step: Endogenous peroxidase and alkaline phosphatase are blocked to reduce background noise.

[0082] Result interpretation and diagnostic value:

[0083] 1. Double positive cells:

[0084] Staining in the same cell: When Stathmin-1 (cytoplasm) and p53 and Ki67 (nuclear) are co-expressed in the same cells in the middle to surface layers, it strongly suggests EHGSIN or cancer.

[0085] Staining in different cells: When Stathmin-1 (cytoplasmic) positive cells and p53, Ki67 (nuclear) positive cells are not expressed in the same cells at the same time, they need to be evaluated in combination with cell morphology.

[0086] 2. Differential diagnosis:

[0087] EHGSIN VS. ELGSIN: In EHGSIN, Stathmin-1, p53, and Ki67 are positive in the same cells in the middle to surface layers, while in ELGSIN, they are mostly confined to the lower 1 / 3.

[0088] Benign reactive lesions: Stathmin-1, p53, and Ki67 are mostly confined to the basal layer or the lower 1 / 3.

[0089] In summary, the combined detection of Stathmin-1, p53 and Ki-67 proteins has a high application value in the graded diagnosis of esophageal squamous intraepithelial neoplasia. This method not only helps to improve the diagnostic accuracy of esophageal squamous intraepithelial neoplasia, but also provides important reference information for the formulation of clinical diagnosis and treatment plans. In the future, the sample size will be further expanded to conduct in-depth research on the specific mechanism of action of these proteins in esophageal squamous intraepithelial neoplasia, so as to provide more scientific basis for the early detection, early diagnosis and early treatment of esophageal squamous intraepithelial neoplasia.

[0090] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A biomarker combination for grading and diagnosing esophageal squamous intraepithelial neoplasia, characterized in that: The biomarker panel includes p53, Ki-67 and Stathmin-1.

2. Use of a reagent for detecting the biomarker combination according to claim 1 in the preparation of a product for the graded diagnosis of esophageal squamous intraepithelial neoplasia.

3. The use according to claim 2, characterized in that: The product is a test kit.

4. The use according to claim 2, characterized in that: The reagent is a reagent for immunohistochemical staining.

5. A product for grading and diagnosing esophageal squamous intraepithelial neoplasia, characterized in that: Comprising reagents for detecting the biomarker combination of claim 1.

6. The product according to claim 5, characterized in that The product is a test kit.

7. The product according to claim 5, characterized in that The reagents include monoclonal antibodies to Stathmin-1, p53 and Ki-67.

8. The product according to claim 7, characterized in that The reagents also include enzyme-labeled secondary antibodies labeled with different enzymes and used for detecting Stathmin-1, p53 and Ki-67 respectively.

9. The product according to claim 8, characterized in that The reagents also include a color developing solution, a buffer solution, an immunohistochemical antigen repair buffer solution and / or a hematoxylin staining solution.

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