Biomarker for accurate typing of esophageal squamous carcinoma lesion and application of biomarker

Abstract

The invention discloses a biomarker for accurate typing of esophageal squamous carcinoma lesion and application of the biomarker, and belongs to the technical field of molecular biology, the biomarker is SOX2 protein and KDM4B protein which are co-localized in nuclei, and the biomarker is applied to accurate typing of esophageal squamous carcinoma lesion with a non-diagnostic purpose. The cell proportion and distribution conditions of the SOX2 protein and the KDM4B protein are simultaneously displayed on the same section by adopting a double-label immunofluorescence method, and tests prove that the intranuclear co-localization of the SOX2 and the KDM4B can be used as an early warning mark and is used for identifying high-risk patients or early stages of precancerous lesions or distinguishing tumor patients, so that the missed diagnosis rate is reduced, and the diagnosis time is shortened. Therefore, a reliable basis is provided for accurate typing of esophageal squamous carcinoma lesions.

Description

Technical Field

[0001] This invention relates to the field of molecular biotechnology, and in particular to a biomarker for the precise subtyping of esophageal squamous cell carcinoma lesions and its application. Background Technology

[0002] In the field of pathological diagnosis, the traditional hematoxylin and eosin (H&E) staining technique has significant limitations in the diagnosis of precancerous lesions of esophageal squamous cell carcinoma, especially in the identification of early lesions.

[0003] First, H&E staining has a high rate of missed diagnoses for both low-grade and high-grade dysplasia. Dysplasia is a key pathological change in the development of esophageal squamous cell carcinoma, with low-grade dysplasia (LGIN) and high-grade dysplasia (HGIN) representing different degrees of cellular and structural abnormalities. However, H&E staining often struggles to accurately identify subtle changes in the cell nucleus and abnormal cell arrangement when differentiating these early lesions, leading to insufficient sensitivity and specificity in diagnosis. Particularly in low-grade dysplasia, cellular atypia may be mild, and the morphological features under H&E staining are not readily apparent, easily confused with reactive hyperplasia or inflammatory lesions, thus increasing the risk of missed diagnoses.

[0004] Secondly, basal cell hyperplasia is another common phenomenon in precancerous lesions of the esophagus, but its morphological changes are difficult to accurately identify in routine pathological examinations. Basal cell hyperplasia typically manifests as thickening of the basal layer and increased cell density, changes that may be mistaken for physiological hyperplasia or other nonspecific changes in normal tissue under H&E staining. Because the morphological characteristics of basal cell hyperplasia overlap with those of early precancerous lesions, routine pathological examinations often struggle to distinguish these subtle differences, leading to decreased diagnostic accuracy. Furthermore, basal cell hyperplasia may be accompanied by varying degrees of inflammatory response, further complicating pathological diagnosis.

[0005] However, current supplementary interpretation methods for H&E staining, such as molecular marker detection, still cannot meet the accuracy requirements. To improve the diagnostic accuracy of early esophageal squamous cell carcinoma precancerous lesions, pathologists have explored molecular marker detection methods. For example, immunohistochemical staining (IHC) can identify cell proliferation markers (such as Ki-67) or cell cycle regulatory proteins (such as p53) through specific antibody labeling, thereby aiding in the differentiation between dysplasia and basal cell proliferation. However, due to the subjectivity of staining result interpretation and the insufficient universality of molecular markers, the limitations in early screening, early diagnosis, and diagnosis of esophageal squamous cell carcinoma lesions remain significant. Summary of the Invention

[0006] One of the objectives of this invention is to provide a biomarker for the precise subtyping of esophageal squamous cell carcinoma lesions, in order to solve the above-mentioned problems.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a biomarker for precise subtyping of esophageal squamous cell carcinoma lesions, wherein the biomarker is SOX2 protein and KDM4B protein co-localized in the nucleus.

[0008] SOX2, or SRY-box transcription factor 2, is a transcription factor containing a SRY (sex-determining region Y)-associated high-mobility family (HMG) cassette. It plays a crucial role in various cellular biological processes, particularly in embryonic development, maintenance of stem cell pluripotency, and cell fate determination. SOX2 protein is known to be aberrantly expressed in various cancers, including breast cancer, prostate cancer, pancreatic cancer, cervical cancer, lung cancer, and esophageal cancer. One study analyzed the expression of SOX2 and OCT3 / 4 in 162 patients with esophageal squamous cell carcinoma, finding that high expression of these two markers was associated with higher histological grade or TNM stage (p < 0.001), demonstrating their involvement in dedifferentiation in these tumors. Forghanifard et al. found that stemness state regulators SALL4 and SOX2 were overexpressed in 64 esophageal cancer samples, and co-overexpression was associated with the depth of tumor invasion and metastasis.

[0009] KDM4B, or Lysine-Specific Demethylase 4B, is a histone demethylase belonging to the JmjC subfamily of enzymes. It primarily removes H3K9me3 (an inhibitory histone marker) and activates gene expression. LeiQiu et al.'s paper, "Histone lysine demethylase 4B regulates general and unique gene expression signatures in hypoxic cancer cells," reported that high expression of KDM4B was associated with better recurrence-free survival (RFS) in esophageal adenocarcinoma (EAC) patients; however, no reports have been found regarding its association with esophageal squamous cell carcinoma.

[0010] The dynamic regulation of gene expression is highly dependent on epigenetic modifications. Studies have shown that the histone demethylase KDM4B regulates the expression of the SOX2 gene by specifically clearing methylation modifications at the H3K9 site through its enzymatic activity, and helps establish and maintain the euchromatin state required for embryonic stem cells (ES cells), which is crucial for the self-renewal and differentiation balance of stem cells.

[0011] Normally, KDM4B is primarily located in the cytoplasm, while SOX2, as a transcription factor, mainly functions in the nucleus. However, the inventors of this invention have demonstrated through extensive experiments that KDM4B and SOX2 exhibit significant co-localization in tumor tissues. Notably, their protein expression levels are not significantly correlated with tumor stage or patient survival. This finding suggests that the co-localization of KDM4B / SOX2 may be independent of changes in their expression levels and possesses unique biological significance.

[0012] The second objective of this invention is to provide an application of the above-mentioned biomarkers for the precise subtyping of esophageal squamous cell carcinoma lesions for non-diagnostic purposes.

[0013] The non-diagnostic purposes mentioned include, but are not limited to: risk assessment of esophageal squamous cell carcinoma lesions, lesion severity grading, and progression monitoring, such as identifying high-risk patients, distinguishing between LGIN (low-grade dysplasia) and HGIN (high-grade dysplasia), and predicting whether the lesion will progress to cancer.

[0014] As a further preferred technical solution, the esophageal squamous cell carcinoma lesion is a precancerous lesion and carcinogenesis of esophageal squamous cell carcinoma.

[0015] As a further preferred technical solution, the esophageal squamous cell carcinoma lesion is at least one of LGIN or HGIN.

[0016] As a preferred technical solution, the method for accurate subtyping of esophageal squamous cell carcinoma lesions is to use double-label immunofluorescence to simultaneously display the cell ratio and distribution of SOX2 protein and KDM4B protein on the same slice; the degree of nuclear colocalization is quantified by Pearson correlation coefficient (PCC).

[0017] Compared with the prior art, the advantages of the present invention are: the intranuclear colocalization of SOX2 and KDM4B can serve as an early warning marker to identify high-risk patients or the early stage of precancerous lesions; the present invention significantly reduces the missed diagnosis rate for low-grade and high-grade dysplasia, thus providing a reliable basis for the accurate classification of esophageal squamous cell carcinoma lesions. Attached Figure Description

[0018] Figure 1 Immunofluorescence images of ESCC tumor tissue and adjacent normal tissue;

[0019] Figure 2 HE staining comparison with immunofluorescence of this invention for different stages of esophageal squamous cell carcinoma evolution (from low-grade to high-grade dysplasia). Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments.

[0021] It should be noted that in the following embodiments and figures, "A protein" refers to "SOX2 protein"; "B protein" refers to "KDM4B" protein; and "AB protein" refers to "SOX2-KDM4B protein".

[0022] Sources of reagents: KDM4B: Catalog No.: R389135, Manufacturer: Chengdu Zhengneng Biotechnology Co., Ltd.; SOX2: Catalog No.: R30004, Manufacturer: Chengdu Zhengneng Biotechnology Co., Ltd. Unless otherwise specified, all other reagents described below are commercially available reagents commonly known to those skilled in the art.

[0023] Unless otherwise specified, the "%" in the following examples refers to mass percentage.

[0024] Example 1:

[0025] The application of SOX2 / KDM4B co-localization in improving the sensitivity of esophageal squamous cell carcinoma biopsy and reducing the false negative rate was demonstrated in the following trial:

[0026] 1. Preparation of cancer tissue microarrays:

[0027] 1.1 Esophageal squamous cell carcinoma tissue specimens were obtained, fixed in formalin, embedded in paraffin, and numbered;

[0028] 1.2 Esophageal squamous cell carcinoma tissue blocks were collected by using a tissue microarray fabrication machine with fine needle perforation to create cylindrical cancer tissue blocks;

[0029] 1.3 Then, the esophageal squamous cell carcinoma tissue was neatly arranged onto a blank paraffin block and embedded in paraffin to create a tissue chip paraffin block;

[0030] 1.4 The tissue chip paraffin block was then serially sectioned to a thickness of 4 μm;

[0031] 2. Staining process:

[0032] 2.1 Dewaxing paraffin sections to water, followed by peroxidase inhibition (3% (w / v) H2O2, 10 min);

[0033] 2.2 After antigen retrieval, nonspecific binding was blocked with 5% (w / v) BSA (30 min, RT).

[0034] 2.3 Primary antibody incubation (overnight at 4°C), secondary antibody (HRP polymer, catalog number: 511203, manufacturer: Chengdu Zhengneng Biotechnology Co., Ltd.) and Tyramide signal amplification (TSA) reagent incubation, following the procedure of TSAPLus fluorescence double labeling three-color staining kit (catalog number: G1226; manufacturer: Wuhan Saiweier Biotechnology Co., Ltd.);

[0035] 2.4 Microwave-peel the antibody (98℃, 20 min), and repeat step 3 for a second round of staining;

[0036] 2.5. DAPI counterstaining, anti-fluorescence quenching mounting, to obtain sample sections (or smears) that can be used for fluorescence microscopy observation, clearly showing cell nuclei (or chromosomes) and with stable fluorescence signals.

[0037] 3. Data Processing:

[0038] Taking the data processing of sample slices obtained by the above method as an example, the specific processing steps include:

[0039] 1) Acquire multi-channel fluorescence images of sample sections using a fluorescence microscope;

[0040] 2) Preprocess the fluorescence image; this can be done using software such as ImageJ, Zen, Imaris, etc.

[0041] 3) Determine the “Region of Interest (ROI)”; for example, randomly select 5 fields of view (200×200μm²) from each slice, focus on the basal cell nucleus region, and exclude necrotic or folded areas;

[0042] 4) Extract pixel intensity data: Extract the raw intensity value of each pixel in the ROI region of both channels one by one to form two sets of data; this can be done using professional software (such as Fiji, Imaris, Zen);

[0043] 5) Background subtraction (critical correction): If there is still a weak background signal within the ROI (such as fluorescence background in protein-free areas), the background needs to be subtracted first;

[0044] 6) Calculate the coefficient r using the Pearson coefficient formula;

[0045] 7) Determine the degree of colocation based on the coefficient r.

[0046] The following examples will illustrate this in more detail:

[0047] 1) Use PannoracicScanner software and select the "Fluorescence Scan Mode" to image the sample. Open the MRXS format scan file using CaseViewer 2.4 software to achieve full-view browsing of the digital slides;

[0048] 2) Co-location quantification:

[0049] Pearson colocalization coefficients were calculated using the ImageJ plugin Coloc for two-line fluorescence colocalization analysis.

[0050] 3) Region selection strategy:

[0051] Five fields of view (200×200μm²) were randomly selected from each slice, focusing on the basal cell nucleus region and excluding necrotic or folded areas.

[0052] See results Figure 1 .from Figure 1 As can be seen from the immunofluorescence staining results, in adjacent normal tissue, KDM4B protein (i.e., "B protein") is mainly expressed in the cytoplasm, while SOX2 protein (i.e., "A protein") is expressed in the nucleus, with a clear subcellular separation between the two, and no co-localization was observed. However, compared with the aforementioned adjacent normal tissue, the subcellular localization of KDM4B changed in tumor tissue: its expression pattern shifted from the cytoplasm to the nucleus. Importantly, within the nucleus of tumor cells, KDM4B protein and SOX2 exhibited significant spatial co-localization. This co-localization relationship was quantitatively verified using the Pearson correlation coefficient (PCC), which yielded PCC = 0.0365 (p < 0.05), and statistical analysis confirmed the significant existence of this co-localization phenomenon.

[0053] Example 2

[0054] The dual-label immunofluorescence assay for co-localization of SOX2 / KDM4B in different stages of esophageal squamous cell carcinoma evolution is as follows:

[0055] 1. Sample collection and grouping

[0056] 1.1 Sample Source and Inclusion Criteria

[0057] 1.1.1 Population selection: Samples were prospectively / retrospectively collected from the endoscopy center of a partner hospital, ensuring informed consent and ethical approval from patients;

[0058] 1.1.2 Group Design (Total Sample Size N=180, including 10% backup samples):

[0059] Normal esophageal tissue (N=50): normal mucosa at the endoscopic biopsy or surgical margin, confirmed by pathology to be free of inflammation or lesions;

[0060] Low-grade dysplasia (LGIN) (N=50): Pathological diagnosis meets WHO criteria, excluding cases with concurrent infection or ulceration;

[0061] High-grade dysplasia (HGIN) (N=50): includes cases of carcinoma in situ (Tis), where the lesion boundaries need to be clearly defined;

[0062] Early invasive carcinoma (T1a) (N=30): The tumor is confined to the mucosal layer and has no lymph node metastasis.

[0063] 1.2 Sample Types and Processing:

[0064] 1.2.1 Frozen tissue (50% of each group): Quick-frozen in liquid nitrogen within 30 minutes after surgical resection and stored at -80°C for single-cell sequencing and fresh tissue immunofluorescence;

[0065] 1.2.2 Paraffin-embedded samples (50% of each group): fixed in 10% (v / v) neutral formalin (within 24 hours), standardized dehydration and embedding, for immunofluorescence and retrospective verification;

[0066] 1.2.3 Clinical data matching: Record patient age, gender, smoking / drinking history, HPV infection status, pathological stage, and follow-up information (such as postoperative recurrence time).

[0067] 2. Multiplex immunofluorescence staining (mIHC):

[0068] (1) Dewax paraffin sections to water, and block peroxidase (3% (w / v) H2O2, 10 min);

[0069] (2) After antigen retrieval, nonspecific binding was blocked with 5% (w / v) BSA (30 min, RT).

[0070] (3) Primary antibody incubation (overnight at 4°C), secondary antibody (HRP polymer, catalog number: 511203, manufacturer: Chengdu Zhengneng Biotechnology Co., Ltd.) and Tyramide signal amplification (TSA) reagent incubation;

[0071] (4) Microwave-peel the antibody (98℃, 20min), and repeat step 3) for a second round of staining;

[0072] (5) DAPI counterstaining, anti-fluorescence quenching mounting.

[0073] 3. Data Processing

[0074] (1) Imaging parameters:

[0075] Using PannoracicScanner software, select the "Fluorescence Scan Mode" for sample imaging. Open the MRXS format scan file using CaseViewer 2.4 software to achieve full-view browsing of the digital slides;

[0076] (2) Co-location quantification:

[0077] Pearson colocalization coefficients were calculated using the ImageJ plugin Coloc for two-line fluorescence colocalization analysis.

[0078] (3) Region selection strategy:

[0079] Five fields of view (200×200μm²) were randomly selected from each slice, focusing on the basal cell nucleus region and excluding necrotic or folded areas.

[0080] The results are as follows Figure 2 As shown in the figure, the analysis of different stages of tumor evolution (from low-grade to high-grade dysplasia) reveals... Figure 2 The nuclear colocalization signal of KDM4B and SOX2 showed a progressively increasing trend. This indicates that the molecular event is closely related to the escalation of tissue lesions. Therefore, the nuclear colocalization of KDM4B and SOX2 can serve as an early warning marker to identify high-risk patients or the early stages of precancerous lesions.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A biomarker for precise subtyping of esophageal squamous cell carcinoma lesions, characterized in that, The biomarkers are SOX2 and KDM4B proteins co-localized in the nucleus.

2. The application of the biomarker according to claim 1, characterized in that, Precise classification of esophageal squamous cell carcinoma lesions for non-diagnostic purposes.

3. The application according to claim 2, characterized in that, The esophageal squamous cell carcinoma lesions mentioned refer to precancerous lesions and cancerous transformations of esophageal squamous cell carcinoma.

4. The application according to claim 3, characterized in that, The esophageal squamous cell carcinoma lesion is at least one of LGIN or HGIN.

5. The application according to claim 2, characterized in that, The method for precise subtyping of esophageal squamous cell carcinoma lesions is as follows: the proportion and distribution of SOX2 protein and KDM4B protein in cells are simultaneously displayed on the same slice using double-label immunofluorescence; the degree of intranuclear colocalization is quantified by Pearson correlation coefficient.

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