Application of LAMC2 expression detection reagent in preparation of kit for identifying esophageal cancer in-situ cancer and invasive cancer

Abstract

The invention discloses an application of an LAMC2 expression detection reagent in preparation of a kit for identifying esophageal carcinoma in situ cancer and invasive cancer. The invention finds that the expression of mRNA or protein of LAMC2 in the early invasion stage of esophageal squamous cell carcinoma is obviously up-regulated, and the mRNA or protein of LAMC2 can be used as a marker to identify infiltrating cancer and carcinoma in situ and identify the position of infiltration leading edge, so that the sensitivity and specificity of early diagnosis are obviously improved. In clinical application, endoscopic examination and tissue biopsy are combined with an immunohistochemical technology to detect the expression of LAMC2, assist pathologists to judge the development stage of esophageal cancer and identify esophageal squamous epithelial carcinoma in situ and invasive cancer, so that the early diagnosis rate is improved, clinical doctors can make more accurate decisions when making therapeutic schedules, metastasis and relapse are reduced, and the treatment efficiency is improved. And a better prognosis result is obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of cancer detection, and more specifically, to the use of a detection reagent for LAMC2 expression in preparing a kit for distinguishing carcinoma in situ and invasive carcinoma of esophageal cancer. Background Art

[0002] Esophageal cancer is a cancer that originates from the esophageal epithelium between the beginning of the hypopharynx and the esophagogastric junction. It includes two main types, esophageal squamous cell carcinoma and esophageal adenocarcinoma, as well as other rare types of malignant tumors. It is a relatively common cancer.

[0003] It is known that the onset of esophageal cancer is closely related to eating and living habits, including hot food, hot tea, drinking, smoking, etc., in addition to factors such as food mold, charcoal grilling or smoking preparation methods, drinking water, soil composition or environmental microbial flora. Promoting a healthy lifestyle and changing bad eating habits can help prevent the occurrence of esophageal cancer; screening and early diagnosis and treatment of high-risk groups for esophageal cancer can effectively increase the early diagnosis rate of esophageal cancer and reduce the mortality rate.

[0004] Esophageal cancer is mainly divided into two subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). ESCC is the main type of esophageal cancer in China. Its development process is: normal squamous cells → squamous epithelial basal cell proliferation → mild atypical hyperplasia → moderate atypical hyperplasia → severe atypical hyperplasia → carcinoma in situ → invasive cancer.

[0005] Among them, the invasive front is the area where the cancerous tissue grows and infiltrates and intersects with the non-cancerous tissue. In the diagnosis and treatment of cancer, accurately distinguishing carcinoma in situ from invasive carcinoma is of great significance for formulating appropriate treatment plans and improving patient survival rates. Accurately judging the stage of the case in which the tumor is located is a prerequisite for reasonable treatment decisions, which will seriously affect the patient's prognosis. Therefore, pathology is the gold standard for diagnosing esophageal cancer, and endoscopic biopsy is required. However, clinical pathologists currently rely mainly on pathological histological features for differential diagnosis, mainly including cell morphology, fibrous interstitial reaction, and basement membrane integrity. Given the lack of guidelines for esophageal endoscopic evaluation and the ease of being affected by the endoscopist's operation and experience level, the diagnosis of different doctors may vary. As a result, pathologists have certain difficulties in accurately distinguishing esophageal squamous cell carcinoma in situ from invasive carcinoma.

[0006] At present, the tumor markers commonly used for auxiliary diagnosis of esophageal cancer include cytokeratin fragment 19 (CYFRA21-1), carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC) and tissue polypeptide specific antigen (TPS), etc. However, the above markers are mainly used for the diagnosis and prognosis of advanced esophageal cancer, and there is little difference in expression between squamous cell carcinoma in situ and invasive carcinoma. Type IV collagen staining can clearly show the integrity of the basement membrane, thereby distinguishing carcinoma in situ from invasive carcinoma, but this indicator can only be diagnosed indirectly and has poor specificity (PMID: 3909566 and PMID: 36253088). Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiency of the prior art that there is no diagnostic marker with high specificity and sensitivity for differential diagnosis of early invasive esophageal squamous cell carcinoma and carcinoma in situ, and to provide a detection reagent for the expression of LAMC2 (Gene ID: 3918 in NCBI) for use in the preparation of a kit for distinguishing carcinoma in situ and invasive carcinoma of esophageal cancer.

[0008] LAMC2 is highly expressed at the edge of invasive esophageal squamous cell carcinoma nests, and invasive esophageal squamous cell carcinoma can be directly identified by IHC staining.

[0009] The first object of the present invention is to provide a detection reagent for LAMC2 mRNA expression for use in preparing a kit for pathological staging of esophageal cancer.

[0010] The second object of the present invention is to provide a detection reagent for the expression of LAMC2 protein for use in preparing a kit for pathological staging of esophageal cancer.

[0011] The third object of the present invention is to provide a use of a detection reagent for LAMC2 mRNA expression in the preparation of a kit for identifying the invasive front of esophageal cancer.

[0012] The fourth object of the present invention is to provide the use of a detection reagent for the expression of LAMC2 protein in the preparation of a kit for identifying the invasive front of esophageal cancer.

[0013] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0014] The present invention claims the following applications:

[0015] Application of a detection reagent for LAMC2 mRNA expression in the preparation of a kit for pathological staging of esophageal cancer.

[0016] Application of a detection reagent for LAMC2 protein expression in the preparation of a kit for pathological staging of esophageal cancer.

[0017] Preferably, the pathological staging is to distinguish between carcinoma in situ and invasive carcinoma.

[0018] More preferably, the invasive cancer is early invasive cancer.

[0019] More preferably, the invasive cancer is microinvasive cancer and / or microinvasive cancer.

[0020] Use of a detection reagent for LAMC2 mRNA expression in preparing a kit for identifying the invasive front of esophageal cancer.

[0021] Application of a detection reagent for LAMC2 protein expression in the preparation of a kit for identifying the invasive front of esophageal cancer.

[0022] Preferably, the reagent is an antibody against LAMC2 protein.

[0023] More preferably, the antibody against LAMC2 protein is a monoclonal antibody against LAMC2 protein.

[0024] As a specific example,

[0025] More preferably, the kit is an immunohistochemistry kit, further comprising an immunohistochemistry reagent.

[0026] More preferably, the kit is an enzyme-linked immunosorbent assay kit, further comprising an enzyme-linked immunosorbent assay reagent.

[0027] Preferably, the reagent is a primer for LAMC2 mRNA.

[0028] More preferably, the kit is a fluorescent quantitative PCR kit, and further contains a fluorescent quantitative PCR reagent.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] Currently, there is no accurate method for diagnosing and distinguishing invasive cancer from carcinoma in situ for esophageal cancer. The present invention found that the expression of LAMC2 mRNA or protein was significantly upregulated in the early invasion stage of esophageal squamous cell carcinoma. Using it as a marker, invasive cancer and carcinoma in situ can be distinguished, and the location of the invasive front can be identified, which significantly improves the sensitivity and specificity of early diagnosis.

[0031] The detection of cancer nest edges by HE staining and IHC staining was consistent; and because LAMC2 was highly expressed at the edge of cancer nests in invasive esophageal squamous cell carcinoma, the edge of cancer nests stained by IHC was clearer, which was more conducive to the judgment of invasive esophageal squamous cell carcinoma or cancer nest edges of the sample.

[0032] Therefore, endoscopic examination and tissue biopsy are used clinically, combined with immunohistochemistry to detect the expression of LAMC2, to assist pathologists in determining the development stage of esophageal cancer and to differentiate esophageal squamous cell carcinoma in situ from invasive cancer, thereby improving the early diagnosis rate and enabling clinicians to make more accurate decisions when formulating treatment plans, reduce metastasis and recurrence, and obtain better prognosis results. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of hematoxylin-eosin (HE) and immunohistochemistry (IHC) staining of esophageal squamous cell invasive carcinoma tissues showed that LAMC2 was highly expressed at the invasive front.

[0034] Figure 2 The results of hematoxylin-eosin (HE) and immunohistochemistry (IHC) staining of esophageal squamous cell carcinoma in situ tissue showed low expression of LAMC2.

[0035] Figure 3 These are the results of hematoxylin-eosin staining (HE) and immunohistochemistry (IHC) staining of esophageal squamous cell carcinoma tissues with both carcinoma in situ and invasive carcinoma. LAMC2 is highly expressed in the invasive front, while carcinoma in situ expresses LAMC2 at a low level.

[0036] Figure 4 These are the results of hematoxylin-eosin staining (HE) and immunohistochemistry (IHC) staining of esophageal squamous cell carcinoma tissues with both carcinoma in situ and invasive carcinoma. LAMC2 is highly expressed in the invasive front, while carcinoma in situ expresses LAMC2 at a low level.

[0037] Figure 5 To score LAMC2 expression in esophageal squamous cell carcinoma in situ and invasive carcinoma.

[0038] Figure 6 To present the sensitivity and specificity statistics of immunohistochemical staining (IHC) results of esophageal squamous cell carcinoma pathological sections.

[0039] Figure 7 These are the immunohistochemical results of LAMC2 and type IV collagen in esophageal squamous cell carcinoma in situ tissue.

[0040] Figure 8 These are the immunohistochemical results of LAMC2 and type IV collagen in esophageal squamous cell carcinoma tissues showing both carcinoma in situ and invasive carcinoma.

[0041] Fig. 9 These are the immunohistochemical results of LAMC2 and type IV collagen in esophageal squamous cell invasive carcinoma tissue. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0043] Example 1 A method for detecting the expression of LAMC2 protein in esophageal tissue using immunohistochemistry

[0044] 1. Experimental Reagents

[0045] LAMC2 monoclonal antibody (Abcam, catalog number: ab210959), distilled water or deionized water, xylene, anhydrous ethanol, ethanol aqueous solution (volume fraction of 50%, 75%, 85% or 95%), endogenous peroxidase blocker (Zhongshan Jinqiao, catalog number: PV-6000), 5% BSA solution (5g / 100ml), PBS solution (pH7.2-7.4), citric acid buffer (pH6.0), enzyme-labeled goat anti-mouse / rabbit IgG polymer (Zhongshan Jinqiao, catalog number: PV-6000), hematoxylin staining solution (catalog number: 7211), DAB color developer, neutral resin, immunohistochemistry pen.

[0046] 2. Experimental Methods

[0047] (1) The paraffin-embedded surgical specimens were fixed with 10% (v / v) formalin buffer and the tissue sections were 4 μm / sheet.

[0048] (2) Dewaxing and hydration: The paraffin sections containing the esophageal tissue to be tested were baked in an oven at 70°C for 2 h, and then immersed in the following order: xylene (10 min each time, immersion twice) → anhydrous ethanol (5 min each time, immersion twice) → 95% ethanol aqueous solution (5 min each time, immersion once) → 85% ethanol aqueous solution (5 min each time, immersion once) → 75% ethanol aqueous solution (5 min each time, immersion once) → 50% ethanol aqueous solution (5 min each time, immersion once) → H2O (3 min each time, immersion twice), and the solution was changed each time.

[0049] (3) Antigen repair: Preheat 300 ml of citric acid buffer in a steamer for 10 min, then immerse the slice rack containing the slices in the buffer and repair at high temperature for 25 min. After heating, take out and cool naturally for 1 h (the slices are still in the citric acid buffer), and then wash with H2O for 5 min.

[0050] (4) Draw circles: Wipe off the moisture around the tissue and use an immunohistochemistry pen to draw circles on the slide, completely encircling the tissue section on the slide.

[0051] (5) Removal of peroxidase: Add endogenous peroxidase blocker to the slices and treat for 15 min. Then wash them in PBS solution three times, each for 8 min.

[0052] (6) Blocking: Add 5% BSA solution to the slices to completely cover the tissue slices and block at room temperature for 1 h.

[0053] (7) Primary antibody incubation: 5% BSA solution was used as the solution, LAMC2 monoclonal antibody was diluted 1:500, and incubated overnight at 4°C in a wet box; after incubation, the wet box was removed and allowed to stand for 10 min; then washed with PBS three times, 5 min each time;

[0054] (8) Secondary antibody incubation: Add enzyme-labeled goat anti-mouse / rabbit IgG polymer and incubate at 37°C for 30 min. After incubation, wash with PBS three times, each time for 8 min.

[0055] (9) Color development: Add DAB color developer and incubate at room temperature for 2.5 min. After incubation, wash twice with H2O, 3 min each time.

[0056] (10) Counterstaining: Hematoxylin staining for 1 min; then wash with H2O for 10 min.

[0057] (11) Dehydration and transparency: Soak in 75% ethanol for 3 min, 85% ethanol for 3 min, 95% ethanol for 3 min, anhydrous ethanol for 3 min, and xylene for 5 min; then dry thoroughly and seal with neutral gum.

[0058] When observing the stained esophageal cancer tissue sections under a microscope, positive results appear as brown-yellow granular staining.

[0059] Example 2 Immunohistochemical detection of LAMC2 protein expression in esophageal squamous cell invasive carcinoma tissue and carcinoma in situ tissue

[0060] 1. Experimental Methods

[0061] The method of Example 1 was used to detect the expression of LAMC2 protein in esophageal cancer tissue samples from patients with esophageal squamous cell invasive carcinoma (a total of 10 cases) and carcinoma in situ (a total of 7 cases). The samples were obtained from the Cancer Center of Sun Yat-sen University and were clinically and pathologically confirmed.

[0062] 2. Experimental Results

[0063] Results for patients with invasive cancer (partial) Figure 1 As shown, the results of patients with carcinoma in situ (partial) are as follows Figure 2The results showed that immunohistochemical detection of esophageal squamous cell carcinoma tissue showed that the expression of LAMC2 in the invasive front was higher than that in carcinoma in situ; immunohistochemical detection of esophageal squamous cell carcinoma tissue showed that the expression of LAMC2 in esophageal squamous cell carcinoma in situ tissue was lower than that in invasive carcinoma.

[0064] Example 3 Immunohistochemical detection of LAMC2 protein expression in esophageal tissues of esophageal cancer patients with both carcinoma in situ and invasive cancer

[0065] 1. Experimental Methods

[0066] The method of Example 1 was used to detect the expression of LAMC2 protein in the esophageal tissue of esophageal cancer patients with both carcinoma in situ and invasive cancer (7 cases in total). The samples were from the Cancer Center of Sun Yat-sen University, and the patients were clinically and pathologically diagnosed with both carcinoma in situ and invasive cancer.

[0067] 2. Experimental Results

[0068] The staining results of some patient samples are as follows Figure 3 and 4 As shown in the figure, immunohistochemistry showed that LAMC2 was highly expressed at the invasive front of invasive carcinoma, while LAMC2 was lowly expressed in carcinoma in situ. Both hematoxylin-eosin staining (HE) and immunohistochemistry staining (IHC) can show the edge of the cancer nest, and the edge of the cancer nest of HE is consistent with that of IHC; however, since LAMC2 is highly expressed at the edge of the cancer nest of invasive esophageal squamous cell carcinoma, the edge of the cancer nest in the IHC result is clearer.

[0069] Example 4 Immunohistochemical detection of LAMC2 protein expression in esophageal tissue of pathological specimens of patients with early esophageal cancer

[0070] 1. Experimental Methods

[0071] Pathological section specimens of 24 patients with early esophageal cancer were obtained from the Cancer Prevention and Treatment Center of Sun Yat-sen University, and the expression of LAMC2 protein was detected according to the method of Example 1.

[0072] The monoclonal antibody of LAMC2 protein was used to perform immunohistochemical staining on esophageal squamous cell carcinoma tissue to analyze the expression level of LAMC2. Specifically, the staining intensity of LAMC2 and the proportion of LAMC2 positive cells were scored according to the following standards. The specific scoring standards are as follows:

[0073] For the selected visual field, the scoring standard for staining intensity is: negative, 0 points; weak positive, 1 point; moderately positive, 2 points; strong positive, 3 points (scored by 2 senior pathologists);

[0074] For the selected visual field, the scoring criteria for the proportion of positive cells were: <25%, 1 point; 25%-50%, 2 points; 50%-75%, 3 points; >75%, 4 points;

[0075] Then, the expression score of LAMC2 was calculated using the staining intensity score and the positive cell ratio score according to the following formula: LAMC2 expression score = = staining intensity × positive ratio. According to the LAMC2 expression score, the samples were divided into LAMC2 high expression group and LAMC2 low expression group using the ROC curve method.

[0076] 2. Experimental Results

[0077] The results are as follows Figure 5 and 6 As shown, according to the expression score of LAMC2: the samples were divided into a low expression group (LAMC2 expression score ≤ 1) and a high expression group (LAMC2 expression score > 1). The results based on the immune combination were compared with the clinical diagnosis results, and it was determined that the overall prediction accuracy of this marker for the differential diagnosis of invasive esophageal squamous cell carcinoma and carcinoma in situ reached more than 95%.

[0078] Example 5 Comparison of immunohistochemical results using LAMC2 and type IV collagen

[0079] 1. Experimental Methods

[0080] Three samples came from the Cancer Center of Sun Yat-sen University, and were clinically and pathologically confirmed to be both carcinoma in situ and invasive carcinoma of the esophagus.

[0081] The expression of LAMC2 and type IV collagen in each sample was detected by immunohistochemistry. The detection method of LAMC2 was the same as that in Example 1, and the detection method of type IV collagen was the same as that in Example 1, except that the LAMC2 monoclonal antibody (Abcam, catalog number: ab210959) was replaced with the Collagen Type IV antibody (Zhongshan Jinqiao (ZS), catalog number: ZM-0081); and the antigen retrieval method was replaced with pepsin digestion solution (Zhongshan Jinqiao (ZS), catalog number: ZLI-9013) for repair.

[0082] 2. Experimental Results

[0083] Comparing the immunohistochemical results of LAMC2 and type IV collagen, LAMC2 can be directly used to differentiate invasive esophageal squamous cell carcinoma from carcinoma in situ, while type IV collagen can only be indirectly diagnosed through the integrity of the basement membrane. Figures 7 to 9 As shown, Figure 7 The immunohistochemistry result of collagen type IV in the basement membrane of carcinoma in situ was negative, which was inconsistent with the expectation (it should be a continuous and complete basement membrane). Figure 8In esophageal cancer tissue, both carcinoma in situ and invasive carcinoma exist. Fig. 9 The results showed that LAMC2 was significantly better than type IV collagen as a marker for distinguishing invasive esophageal squamous cell carcinoma from carcinoma in situ.

Claims

1. Application of LAMC2 mRNA expression detection reagent in the preparation of a kit for pathological staging of esophageal cancer.

2. Application of the detection reagent for LAMC2 protein expression in the preparation of a kit for pathological staging of esophageal cancer.

3. The use according to claim 1 or 2, characterized in that: The pathological staging is to distinguish between carcinoma in situ and invasive carcinoma.

4. Application of a detection reagent for LAMC2 mRNA expression in the preparation of a kit for identifying the invasive front of esophageal cancer.

5. Application of a detection reagent for LAMC2 protein expression in the preparation of a kit for identifying the invasive front of esophageal cancer.

6. The use according to any one of claims 2 or 5, characterized in that: The reagent is an antibody against LAMC2 protein.

7. The use according to claim 6, characterized in that: The kit is an immunohistochemistry kit and also contains immunohistochemistry reagents.

8. The use according to claim 6, characterized in that: The kit is an ELISA kit, and also contains an ELISA reagent.

9. The use according to any one of claims 2 or 5, characterized in that: The reagent is a primer of LAMC2 mRNA.

10. The use according to any one of claim 9, characterized in that: The kit is a fluorescence quantitative PCR kit, and also contains fluorescence quantitative PCR reagents.

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