Use of sox10 antibodies, kits

Abstract

The application discloses application of SOX10 antibodies in preparation of products for identifying UDH and ADH / low-grade DCIS. The application also provides a kit for identifying UDH and ADH / low-grade DCIS. The application discloses application of SOX10 antibodies in preparation of products for identifying IDPoma with UDH and IPDC. The application also provides a kit for identifying IDPoma with UDH and IPDC. The kit of the application can accurately identify UDH and ADH / low-grade DCIS and IDPoma with UDH and IPDC based on the research finding that SOX10 antibodies are differentially expressed in UDH and ADH / low-grade DCIS and in IDPoma with UDH and IPDC, and can eliminate or reduce defects such as false positive, over-diagnosis, missed diagnosis and misdiagnosis caused by single use of CK5 / 6 and ER antibodies for diagnosis, and has important clinical significance and clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to the application of an SOX10 antibody and a kit for the differential diagnosis of intraductal lesions in the breast. Background Technology

[0002] In clinical practice, accurate diagnosis of precursor / precancerous lesions of breast cancer is crucial for subsequent treatment. Intraductal proliferative lesions (IPLs) and intraductal papillary lesions (IPDs) are necessary conditions driving the occurrence and development of some breast cancers. Studies have found that IPLs and IPDs have their own independent neoplastic disease progression spectrums, with different clinical treatment approaches corresponding to different stages of progression. Therefore, the clinicopathological differential diagnosis of IPLs and IPDs at various stages of progression is particularly important, as accurate pathological diagnosis will guide subsequent clinical treatment.

[0003] Our previous research has shown that the challenge in clinicopathological diagnosis of intraductal proliferative lesions and intraductal papillary lesions lies in... Figure 1 , Figure 2 The differential diagnoses indicated by the "*Clinical Treatment Boundary Line" are: ① Differential diagnosis between common ductal hyperplasia (UDH) and atypical ductal hyperplasia (ADH) / low-grade ductal carcinoma in situ (DCIS); ② Differential diagnosis between intraductal papilloma with common ductal hyperplasia (IDPoma with UDH) and intraductal papillary carcinoma (IPDC). Specific clinical problems in these differential diagnoses are as follows: ① Due to tumor heterogeneity, the same disease may exhibit morphological and biological behavior differences in tissues of different patients. Relying solely on morphology for differential diagnosis carries a degree of subjectivity and the risk of misdiagnosis or missed diagnosis; ② Diseases of the same spectrum may show morphological overlap at different stages of progression, and even overlap in immunophenotype and semi-quantitative immunohistochemical counting. Current immunomarkers cannot objectively and accurately differentiate between UDH and the morphologically deceptive low-grade DCIS, as well as between IDPoma with UDH and IPDC.

[0004] In order to differentiate diseases on both sides of the "clinical treatment boundary", we have previously used immunomarkers including CK5 / 6 and ER in clinical practice. However, these two immunomarkers each have certain limitations. In clinical work, we have found that: (1) CK5 / 6 is usually positively expressed in UDH and IDPoma with UDH, but it is also positively expressed in some DCIS and IPDC, especially in basal-like subtype DCIS where CK5 / 6 is highly expressed. At the same time, although CK5 / 6 is a mature and stable clinical antibody, its positive staining is located in the cytoplasm and is inevitably affected by experimental conditions, which may sometimes lead to false positive results, thus affecting the clinical pathological diagnosis. (2) ER is an important immunomarker for the diagnosis and treatment of breast cancer. ER is usually monoclonal positive in DCIS and IPDC (i.e., diffuse and consistent positive expression). However, due to the homology of the occurrence and development of breast lesions (i.e., most originate from the terminal duct lobule unit and are mostly related to estrogen and progesterone receptors), the expression pattern of ER overlaps in some breast lesions. Sometimes the expression of ER in breast tumors deviates. Sometimes the expression level of ER in UDH and IDPoma with UDH is close to or even exceeds the expression level in DCIS and IPDC, which may lead to misdiagnosis, overdiagnosis, or overtreatment.

[0005] Therefore, providing a kit for the accurate differential diagnosis of common ductal epithelial hyperplasia (UDH) and atypical ductal epithelial hyperplasia (ADH) / low-grade ductal carcinoma in situ (DCIS), as well as intraductal papilloma with common ductal epithelial hyperplasia (IDPoma with UDH) and intraductal papillary carcinoma (IPDC) of the breast is of great clinical significance. Summary of the Invention

[0006] The technical problem to be solved by this invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a kit for the application of SOX10 antibody and for the accurate differentiation and diagnosis of common ductal hyperplasia (UDH) and atypical ductal hyperplasia (ADH) / low-grade ductal carcinoma in situ (DCIS) of the breast, as well as intraductal papilloma with common ductal hyperplasia (IDPoma with UDH) and intraductal papillary carcinoma (IPDC). To solve the above technical problem, the technical solution proposed by this invention is as follows:

[0007] Application of SOX10 antibody in the preparation of products for identifying UDH and ADH / low-grade DCIS.

[0008] As a general technical concept, the present invention also provides a kit for identifying UDH and ADH / low-grade DCIS, comprising a first immunohistochemical staining reagent containing an SOX10 antibody.

[0009] In the above kit, preferably, the first immunohistochemical staining reagent is a ready-to-use SOX10 immunohistochemical antibody.

[0010] Preferably, the above-mentioned kit further includes a second immunohistochemical staining reagent and a third immunohistochemical staining reagent, wherein the second immunohistochemical staining reagent and the third immunohistochemical staining reagent contain CK5 / 6 antibody and ER antibody, respectively, and the first immunohistochemical staining reagent, the second immunohistochemical staining reagent and the third immunohistochemical staining reagent are provided separately.

[0011] In the above kit, preferably, the second and third immunohistochemical staining reagents are ready-to-use CK5 / 6 immunohistochemical antibody and ready-to-use ER immunohistochemical antibody, respectively.

[0012] As a general technical concept, the present invention also provides the use of SOX10 antibody in the preparation of products for identifying IDPoma with UDH and IPDC.

[0013] As a general technical concept, the present invention also provides a kit for identifying IDPoma with UDH and IPDC, comprising a first immunohistochemical staining reagent containing an SOX10 antibody.

[0014] In the above kit, preferably, the first immunohistochemical staining reagent is a ready-to-use SOX10 immunohistochemical antibody.

[0015] Preferably, the above-mentioned kit further includes a second immunohistochemical staining reagent and a third immunohistochemical staining reagent, wherein the second immunohistochemical staining reagent and the third immunohistochemical staining reagent contain CK5 / 6 antibody and ER antibody, respectively, and the first immunohistochemical staining reagent, the second immunohistochemical staining reagent and the third immunohistochemical staining reagent are provided separately.

[0016] In the above kit, preferably, the second and third immunohistochemical staining reagents are ready-to-use CK5 / 6 immunohistochemical antibody and ready-to-use ER immunohistochemical antibody, respectively.

[0017] Breast stem cells exist in normal breast tissue and some breast proliferative lesions, and have bidirectional differentiation capabilities. They can differentiate from glandular intermediate cells into glandular terminal cells, or from epithelial intermediate cells into myoepithelial terminal cells. Therefore, the following five types of cells exist in breast tissue: (1) directed stem cells, which express only high molecular weight keratin such as CK5 / 6 and CK14; (2) glandular intermediate cells, which express both high molecular weight and low molecular weight keratin; (3) glandular terminal cells, which express only low molecular weight keratin; (4) myoepithelial intermediate cells, which express both high molecular weight keratin and myoepithelial markers such as SMA and p63; and (5) myoepithelial terminal cells, which express only myoepithelial markers.

[0018] Studies show that UDH and IDPoma are diseases originating from breast-directed stem cells, expressing high-molecular-weight keratins such as CK5 / 6, CK14, and 34βE12. Therefore, CK5 / 6 antibodies can serve as immunomarkers for the differential diagnosis of UDH and ADH / low-grade DCIS, as well as IDPoma with UDH and IPDC. ER antibodies also typically play a role in the differential diagnosis of these two groups of diseases because ER tends to show monoclonal positive expression as the grade of intraductal lesions increases, which is indicative of the diagnosis. SOX10 antibody is a transcription factor. Our research shows that SOX10 is specifically expressed in breast stem cells and is positive in UDH and IDPoma lesions, but it is not expressed in most breast tumor stem cells. Utilizing this characteristic, SOX10 can be used to differentiate between UDH and ADH / low-grade DCIS, as well as IDPoma with UDH and IPDC. Furthermore, to increase specificity, this invention proposes using a combination of three antibodies—SOX10, ER, and CK5 / 6—for semi-quantitative immunohistochemical diagnosis. This method is more precise and accurate than previous diagnostic methods, capable of specifically distinguishing between UDH and ADH / low-grade DCIS, as well as IDPoma with UDH and IPDC. Each set of immunohistochemical results corresponds to one pathological diagnosis, resulting in more accurate and objective differentiation. Figure 1 , Figure 2 Lesions on both sides of the "clinical treatment boundary line" in the middle.

[0019] In this invention, ready-to-use immunohistochemical antibodies (an integrated reagent consisting of antibody and antibody diluent) can be used directly during immunohistochemical staining. This invention does not have special requirements for the concentration of the antibody. The amount used during staining can be found in the manufacturer's instructions for use of the ready-to-use antibody. The amount of antibody is sufficient to perform normal immunohistochemical staining.

[0020] The specific methods and identification processes for using the kit of the present invention are illustrated below:

[0021] 1. First, the pathologist will determine under a microscope whether the disease is an intraductal proliferative lesion (UDH and ADH / low-grade DCIS) or an intraductal papillary lesion (IDPoma with UDH and IPDC) based on the morphological characteristics of the H&E stained sections.

[0022] 2. For intraductal proliferative lesions or intraductal papillary lesions, immunohistochemical staining is first performed on the sections before observation under a microscope. The specific steps for immunohistochemical staining using SOX10 antibody are illustrated below: Prepare paraffin-coated white slides with a thickness of 3-4 μm; place the slides in a 65°C oven for 12 hours; dewax with turpentine oil for 15 minutes; soak in anhydrous ethanol for 5 minutes; soak in 95% alcohol for 5 minutes; soak in 75% alcohol for 5 minutes; rinse with tap water for 5 minutes; rinse once with distilled water; perform high-pressure heat repair with EDTA for 5 minutes, then allow to cool naturally; rinse with running water for 5 minutes; block with 3% hydrogen peroxide (reagent 1) for 20 minutes; wash three times with PBS, 5 minutes each time; incubate with primary antibody (i.e., the first immunohistochemical staining reagent containing SOX10 antibody) at 37°C for 2 hours. Wash three times with PBS, 5 minutes each time; incubate with immunoreaction enhancer (reagent 2) at 37°C for 20 minutes; wash three times with PBS, 5 minutes each time; incubate with polymeric secondary antibody (reagent 3) at 37°C for 30 minutes; wash three times with PBS, 5 minutes each time; develop with DAB for 2-5 minutes; rinse with running water for 5 minutes; rinse once with distilled water; stain with hematoxylin for 20 seconds; rinse with running water for 5 minutes; stain with saturated lithium carbonate for 2 seconds; rinse with running water; dry and cover with neutral resin; observe under a microscope. The immunohistochemical staining steps for ER, CK5 / 6 antibodies and SOX10 antibody are the same, except that the second immunohistochemical staining reagent containing CK5 / 6 antibody and the third immunohistochemical staining reagent containing ER antibody are used instead of the first immunohistochemical staining reagent. For a single pathological tissue, three immunohistochemical staining processes are required: one staining each of SOX10, CK5 / 6, and ER.

[0023] 3. Observation Results. For intraductal proliferative lesions, whether they belong to UDH or ADH / low-grade DCIS, please refer to Table 1 below. Figure 3 Make a judgment. For intraductal papillary lesions, whether they belong to IDPoma with UDH or IPDC, please refer to Table 2 below and... Figure 4 Make a judgment.

[0024] Table 1: Identification parameters for UDH and ADH / low-grade DCIS

[0025]

[0026] Table 2: Identification parameters of IDPoma with UDH and IPDC

[0027]

[0028] In the table above, for ER antibody staining results, "less than 80%" and "greater than 80%" refer to the ratio of ER-positive target cells (e.g., for IPDC, where the target cells are tumor cells) to all target cells in the entire immunohistochemically stained section being less than 80% or greater than 80%. Combining the staining characteristics of ER antibodies with the purpose of ER antibody staining in this invention, our research shows that using the above-mentioned criteria helps ensure the accuracy of the identification results.

[0029] Compared with the prior art, the advantages of the present invention are as follows:

[0030] The kit of the present invention is based on research findings that SOX10 antibody has different specific expressions in UDH and ADH / low-grade DCIS, as well as in IDPoma with UDH and IPDC. It can accurately identify UDH and ADH / low-grade DCIS, as well as IDPoma with UDH and IPDC. It can eliminate the defects such as false positives and overdiagnosis that exist when using CK5 / 6 or ER antibodies alone, and has important clinical significance. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This section outlines the disease progression spectrum of intraductal proliferative lesions and the corresponding clinical treatments for each stage.

[0033] Figure 2 This section outlines the disease progression spectrum of intraductal papillary tumors and the corresponding clinical treatments for each stage.

[0034] Figure 3 A roadmap for the differential diagnosis of UDH and ADH / low-grade DCIS.

[0035] Figure 4 This is a roadmap for the differential diagnosis of IDPoma with UDH and IPDC.

[0036] Figure 5The staining results are as follows: A is H&E staining; B is ER < 80% positive; C is CK5 / 6 positive; D is SOX10 positive.

[0037] Figure 6 The staining results are as follows: A is H&E staining; B is ER>80% positive; C is CK5 / 6 negative; D is SOX10 negative.

[0038] Figure 7 The staining results for IDPoma with UDH are shown below; A is H&E staining; B is ER < 80% positive; C is CK5 / 6 positive; D is SOX10 positive.

[0039] Figure 8 The staining status of IPDC is as follows: A is H&E staining; B is ER>80% positive; C is CK5 / 6 negative; D is SOX10 negative. Detailed Implementation

[0040] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0041] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0042] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0043] Example 1:

[0044] Application of SOX10 antibody in the preparation of products for identifying UDH and ADH / low-grade DCIS. This example utilizes the SOX10 antibody to prepare a kit for identifying UDH and ADH / low-grade DCIS.

[0045] A kit for identifying UDH and ADH / low-grade DCIS includes a first immunohistochemical staining reagent, a second immunohistochemical staining reagent, and a third immunohistochemical staining reagent, which are separately configured. The first immunohistochemical staining reagent is an SOX10 antibody (ready-to-use SOX10 immunohistochemical antibody), and the second and third immunohistochemical staining reagents are an CK5 / 6 antibody (ready-to-use CK5 / 6 immunohistochemical antibody) and an ER antibody (ready-to-use ER immunohistochemical antibody), respectively.

[0046] In this embodiment, the details of the SOX10 antibody are as follows:

[0047] Manufacturer: Fuzhou Maixin Biotechnology Development Co., Ltd.; Product Number: RMA-0726; Clone Number: EP268; Applicable Tissue: Paraffin Section; Positive Site: Cell Nucleus; Specification: 6ml; Source: Rabbit Anti-Human Monoclonal Antibody.

[0048] In this embodiment, the details of the ER antibody are as follows:

[0049] Manufacturer: Fuzhou Maixin Biotechnology Development Co., Ltd.; Product Number: Kit-0012; Clone Number: SP1; Applicable Tissue: Paraffin Section; Positive Site: Cell Nucleus; Specification: 6ml; Source: Rabbit Anti-Human Monoclonal Antibody.

[0050] In this embodiment, the details of the CK5 / 6 antibody are as follows:

[0051] Manufacturer: Fuzhou Maixin Biotechnology Development Co., Ltd.; Product Number: MAB-0744; Clone Number: MX040; Applicable Tissue: Paraffin Section; Positive Site: Cytoplasm; Specification: 6ml; Source: Mouse anti-human monoclonal antibody.

[0052] Once the pathological tissue is determined to be an intraductal proliferative lesion, the method for differentiating UDH and ADH / low-grade DCIS using the above-mentioned kit in this embodiment may include the following steps:

[0053] SOX10 antibody immunohistochemical staining: Prepare paraffin-coated slides with a thickness of 3 to 4 μm; place slides in a 65°C oven for 12 hours; dewax with turpentine for 15 minutes; soak in anhydrous ethanol for 5 minutes; soak in 95% ethanol for 5 minutes; soak in 75% ethanol for 5 minutes; rinse with tap water for 5 minutes; rinse once with distilled water; perform EDTA autoclaving for 5 minutes, then allow to cool naturally; rinse with running water for 5 minutes; block with 3% hydrogen peroxide (reagent 1) for 20 minutes; wash 3 times with PBS, 5 minutes each time; incubate with 1 drop of primary antibody (i.e., the first immunohistochemical staining reagent) at 37°C for 2 hours; wash 3 times with PBS, 5 minutes each time; incubate with immunoreaction enhancer (reagent 2) at 37°C for 20 minutes; wash 3 times with PBS, 5 minutes each time; incubate with polymeric secondary antibody (reagent 3) at 37°C for 30 minutes; Wash 3 times with PBS, 5 minutes each time; develop with DAB for 2-5 minutes; rinse with running water for 5 minutes; rinse with distilled water once; stain with hematoxylin for 20 seconds; rinse with running water for 5 minutes; stain with saturated lithium carbonate for 2 seconds; rinse with running water; dry and cover with neutral resin; observe under a microscope.

[0054] ER antibody immunohistochemical staining: Same as SOX10 antibody immunohistochemical staining, but use ER antibody instead of SOX10 antibody.

[0055] Immunohistochemical staining with CK5 / 6 antibody: Same as immunohistochemical staining with SOX10 antibody, but CK5 / 6 antibody can be used instead of SOX10 antibody.

[0056] Based on the above three immunohistochemical staining results, using Table 1 and... Figure 3 The identification scheme in the document is used to identify UDH and ADH / low-grade DCIS.

[0057] Example 2:

[0058] Application of SOX10 antibody in the preparation of a product for identifying IDPoma with UDH and IPDC. This example utilizes the SOX10 antibody to prepare a kit for identifying IDPoma with UDH and IPDC.

[0059] A kit for identifying IDPoma with UDH and IPDC includes a first immunohistochemical staining reagent, a second immunohistochemical staining reagent, and a third immunohistochemical staining reagent, which are separately configured. The first immunohistochemical staining reagent is an SOX10 antibody (ready-to-use SOX10 immunohistochemical antibody), and the second and third immunohistochemical staining reagents are an CK5 / 6 antibody (ready-to-use CK5 / 6 immunohistochemical antibody) and an ER antibody (ready-to-use ER immunohistochemical antibody), respectively.

[0060] In this embodiment, the SOX10 antibody, ER antibody, and CK5 / 6 antibody are the same as in Example 1.

[0061] Once the pathological tissue was determined to be an intraductal papillary lesion, the method used in this embodiment to differentiate between IDPoma with UDH and IPDC using the above-mentioned kit was the same as in Example 1, using Table 2 and... Figure 4 The identification scheme in the text is used to identify IDPoma with UDH and IPDC.

[0062] Immunohistochemical staining analysis was performed on 159 clinical paraffin-embedded samples using the kits and identification methods described in Examples 1 and 2. These 159 samples were confirmed to contain UDH (n=10), ADH / low-grade DCIS (n=23), IDPoma with UDH (n=76), and IPDC (n=50). Specific immunohistochemical staining results are shown in Tables 3-4. Figures 5-8 As shown.

[0063] Table 3: Expression of ER, CK5 / 6, and SOX10 in UDH and ADH / low-grade DCIS

[0064]

[0065] *A p-value less than 0.05 is statistically significant.

[0066] Table 4: Expression of ER, CK5 / 6, and SOX10 in IDPoma with UDH and IPDC

[0067]

[0068] *A p-value less than 0.05 is statistically significant.

[0069] When analyzing immunohistochemical staining results, if all indicators are consistent with Tables 1 and 2, then... Figure 3 , Figure 4 If the criteria in the table completely match, UDH and ADH / low-grade DCIS, IDPoma with UDH and IPDC can be clearly identified. However, immunohistochemical staining methods have a certain probability of deviation. In Table 3 above, for the staining results of ER, CK5 / 6, and SOX10 antibodies, if only two indicators match the results in Table 1, the results may be slightly different. Figure 3 The diagnostic criteria in the table are consistent, and a comprehensive judgment can be made based on the pathologist's experience and the results of the other two antibodies. For example, in the case of ADH / Low grade DCIS, CK5 / 6 staining results are partially negative and partially positive. If CK5 / 6 is positive at this time, the interpretation can be based on the results of ER and SOX10. For example, if ER is greater than 80% positive, SOX10 is negative, and the ductal epithelial cells show obvious atypia in morphology, or the ductal epithelial cells have a uniform morphology (with a clear morphological pattern of monoclonal proliferation of tumor cells), we can still judge it as ADH / Low grade DCIS. In the ER, CK5 / 6, and SOX10 antibody staining results, our research shows that the probability of deviation of a certain staining indicator is small, and it can be combined with other indicators for comprehensive analysis. However, if two or three indicators deviate simultaneously, the results may significantly differ from morphological findings. In such cases, comprehensive analysis combining other examinations is necessary, including color Doppler ultrasound (B-mode ultrasound), CT scans, MRI, mammography, breast palpation, surgical records, and even multidisciplinary team (MDT) consultations. The probability of two or three indicators deviating simultaneously is extremely low (a rare event). Immunohistochemical staining analysis of the 159 clinical paraffin samples mentioned above did not reveal any instances of two or three indicators deviating simultaneously. Overall, the method described in this embodiment can accurately differentiate between UDH and ADH / low-grade DCIS, as well as IDPoma with UDH and IPDC.

[0070] Figure 5-8Typical staining patterns of UDH, low-grade DCIS, IDPoma with UDH, and IPDC are shown respectively. Figure 5 In the table, A represents H&E staining; B represents ER < 80% positive; C represents CK5 / 6 positive; and D represents SOX10 positive. Figure 6 In the middle, A is H&E staining; B is ER>80% positive; C is CK5 / 6 negative; and D is SOX10 negative. Figure 7 In the middle section, A is H&E staining; B is ER < 80% positive; C is CK5 / 6 positive; and D is SOX10 positive. Figure 8 In the middle, A is H&E staining; B is ER>80% positive; C is CK5 / 6 negative; and D is SOX10 negative.

Claims

1. Use of SOX10 antibody in the preparation of a product for identifying IDPoma with UDH and IPDC.

2. Use according to claim 1, characterized in that, The product is a kit, which comprises a first immunohistochemical staining reagent containing SOX10 antibody; The kit further comprises a second immunohistochemical staining reagent and a third immunohistochemical staining reagent, which respectively contain CK5 / 6 antibody and ER antibody, and the first, second and third immunohistochemical staining reagents are separately arranged.

3. Use according to claim 2, characterized in that, The first immunohistochemical staining reagent is a ready-to-use SOX10 immunohistochemical antibody.

4. Use according to claim 2, characterized in that, The second and third immunohistochemical staining reagents are respectively a ready-to-use CK5 / 6 immunohistochemical antibody and a ready-to-use immunohistochemical ER antibody.

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