Apparatus, method, system for diagnosing lymph node low volume metastasis of endometrial cancer and applications thereof

Abstract

The application provides a device, a method, a system and an application thereof for diagnosing endometrial cancer lymph node low volume metastasis, and belongs to the technical field of medical evaluation. Through bioinformatics analysis methods such as multivariate statistical analysis, difference screening, random forest and ROC analysis, three biomarkers CK19, EMA and EPCAM for evaluating endometrial cancer lymph node low volume metastasis are obtained. Based on the biomarkers of the application, an evaluation device, a computer execution method, a computer system, a kit and the like for evaluating endometrial cancer lymph node low volume metastasis are constructed, which are used for accurately judging whether endometrial cancer patients have lymph node low volume metastasis.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to devices, methods, systems, and applications for diagnosing or evaluating low-volume lymph node metastases in endometrial cancer. Background Technology

[0002] Endometrial cancer is one of the most common malignant tumors of the female reproductive system, and its incidence is increasing year by year, seriously threatening women's health. Lymph node metastasis is an important prognostic factor and staging basis for endometrial cancer; once metastasis occurs, the patient's survival rate is significantly reduced. Among them, low-volume lymph node metastases, including micrometastases and isolated tumor cells, have a significant impact on patient prognosis and treatment options due to their small size and low number of cancer cells.

[0003] Intraoperative frozen section hematoxylin and eosin (HE) staining is currently a routine and rapid pathological diagnostic method for assessing lymph node metastasis during endometrial cancer surgery, providing a basis for adjusting the surgical plan in a short time. However, in clinical practice, intraoperative frozen sections have limitations such as cell deformation, tissue folding, cauterization artifacts, and difficulty in identifying micrometastases, resulting in a very high rate of missed diagnoses of low-volume lymph node metastases. This can easily lead to inaccurate intraoperative pathological diagnosis, preventing patients from receiving timely and standardized lymph node dissection, and even requiring a second surgery, increasing trauma and medical costs.

[0004] Currently, there is still a lack of a rapid, accurate, intraoperative device, method, or system in clinical practice that can effectively identify low-volume lymph node metastases in endometrial cancer. Summary of the Invention

[0005] This invention relates to the field of tumor pathological diagnosis and intraoperative rapid assessment technology, specifically to an assessment device, computer execution method, and computer system for intraoperative assessment of low-volume lymph node metastasis in endometrial cancer.

[0006] To address the shortcomings of existing technologies, such as insufficient ability of intraoperative frozen section hematoxylin and eosin (HE) staining for identifying lymph node micrometastases and isolated tumor cells, resulting in a high rate of missed diagnoses, and the time-consuming nature of conventional immunohistochemical testing, which fails to meet the needs of rapid intraoperative diagnosis, this invention provides a rapid intraoperative assessment technology based on the combined determination of multiple biomarkers. This solution improves the detection sensitivity and assessment accuracy of low-volume lymph node metastases in endometrial cancer by introducing a combination of specific biomarkers and constructing corresponding data processing and risk assessment mechanisms, without significantly prolonging intraoperative detection time.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides an assessment device for intraoperative evaluation of low-volume lymph node metastasis in endometrial cancer, comprising a data acquisition device and a data processing device. The data acquisition device acquires detection data of the assessment subject, which originates from the detection results of markers for low-volume lymph node metastasis in endometrial cancer in human lymph node samples. These markers include two or a combination of three of CK19, EMA, and EPCAM. The data processing device processes the detection data to determine the expression status of each marker and outputs corresponding risk assessment results based on preset risk determination rules.

[0008] In a preferred embodiment, the expression status of the biomarker is determined based on the results of rapid immunohistochemical detection of intraoperative frozen sections, with positive or negative results used as the interpretation criteria. When the biomarker combination is determined to be positive, a high-risk assessment result is output; when it is determined to be negative, a low-risk assessment result is output.

[0009] Furthermore, this invention introduces a compensation judgment mechanism based on the complementary characteristics of biomarker expression: when the expression intensity of the CK19 biomarker is lower than a preset threshold, if the EPCAM biomarker is positive, the EPCAM detection result is used to compensate for the CK19 detection result, and the overall detection result is thus judged as positive. This compensation mechanism effectively solves the problem of weakened or even undetectable CK19 expression signals due to factors such as tissue folding, detachment, cauterization, or uneven staining under intraoperative frozen section conditions, thereby reducing the risk of missed detection and improving the identification stability of low-volume transfers.

[0010] The present invention also provides a computer execution method and computer system, which reads the detection data of the marker, processes the marker expression state, and outputs risk assessment results based on the above-mentioned joint judgment and compensation mechanism, thereby realizing the automated assessment of low-volume lymph node metastasis of endometrial cancer.

[0011] This invention further relates to the application of a combination of CK19 antibody and EMA antibody in the preparation of a reagent for diagnosing low-volume lymph node metastasis in endometrial cancer. The detection is based on intraoperative frozen sections and employs rapid immunohistochemistry to quickly determine the lymph node metastasis status. The low-volume metastasis includes micrometastases and isolated tumor cells.

[0012] In the technical solution of the present invention, CK19 antibody and EMA antibody in the form of enzyme-labeled primary antibody are preferably used. By directly labeling the target antigen, the detection process is simplified and the detection time is shortened, thereby meeting the needs of rapid intraoperative diagnosis.

[0013] This invention further provides a rapid immunohistochemical detection process based on intraoperative frozen sections, including: preparing frozen sections of lymph node specimens for HE staining and immunohistochemical staining; sequentially performing fixation, blocking, primary antibody incubation, signal enhancement, staining, counterstaining, and mounting on the immunohistochemical sections; and determining the marker expression status by observing whether a specific color signal appears on the cell membrane or cytoplasm. Preferably, a positive result is determined when the cell membrane or cytoplasm shows a brownish-yellow stain (this color can vary depending on the staining system and can be other colors).

[0014] In practice, the fixation step is preferably performed using 10% neutral formalin buffer for 30–60 seconds for rapid fixation, and the primary antibody incubation time is preferably 3–5 minutes. The entire detection process can be completed within 20–33 minutes, thereby achieving efficient detection without significantly prolonging the intraoperative pathological diagnosis time.

[0015] This invention is applicable to the detection of lymph nodes in multiple sites in patients with endometrial cancer, including pelvic lymph nodes, para-aortic lymph nodes, and presacral lymph nodes. By combining HE staining results with rapid immunohistochemical results of CK19 and EMA, a comprehensive interpretation of the presence of tumor metastasis within lymph nodes can be achieved, and macrometastases, micrometastases, and isolated tumor cells can be further distinguished. Macrometastases are defined as metastatic lesions with a maximum diameter greater than 2 mm; micrometastases are defined as metastatic lesions with a maximum diameter of 0.2–2 mm and / or a tumor cell count greater than 200; isolated tumor cells are defined as metastatic lesions with a maximum diameter not exceeding 0.2 mm and a tumor cell count not exceeding 200.

[0016] Furthermore, the rapid immunohistochemical reaction of the present invention is preferably performed under humidified chamber conditions at 40–42°C to further improve reaction efficiency and colorimetric quality. Based on the above technical solution, the present invention also provides an immunohistochemical kit suitable for rapid intraoperative detection, which includes components such as CK19 enzyme-labeled primary antibody, EMA enzyme-labeled primary antibody, tissue blocking agent, signal enhancement solution, DAB chromogenic solution, and hematoxylin counterstaining solution, enabling rapid, sensitive, and specific detection of low-volume lymph node metastases in endometrial cancer.

[0017] Based on the above technical solutions, the present invention further provides an extended scheme for the joint detection of three biomarkers, namely, introducing EPCAM antibody on the basis of the combination of CK19 antibody and EMA antibody to construct a joint detection system of CK19, EMA and EPCAM, which is used to prepare reagents for diagnosing low-volume lymph node metastasis of endometrial cancer, thereby further improving the stability and reliability of detection.

[0018] Specifically, in this three-marker technical solution, CK19 antibody and EMA antibody remain as core detection markers, used for specific labeling of epithelial tumor cells derived from endometrial cancer; simultaneously, EPCAM antibody is introduced as a supplementary marker to assist in the judgment and compensation of detection results under specific circumstances. Preferably, the CK19 antibody, EMA antibody, and EPCAM antibody are all enzyme-labeled primary antibodies to further shorten the detection time and simplify the operation procedure.

[0019] In terms of detection methods, the combined three-marker protocol is also based on the rapid immunohistochemical detection process of intraoperative frozen sections, including frozen section preparation, fixation, blocking, primary antibody incubation, enhancement, staining, counterstaining, and mounting. The overall detection time is preferably controlled within 20–33 minutes. The detection results are interpreted through colorimetric signals, where the appearance of specific color signals in the cytoplasm or cell membrane (e.g., brownish-yellow staining) indicates a positive result for the corresponding marker.

[0020] Regarding the interpretation strategy, this invention further proposes a stratified judgment mechanism based on three biomarkers: Under normal circumstances, CK19 and EMA are used together to determine whether there is tumor metastasis in lymph nodes; when both CK19 and EMA are positive, it can be directly determined that there is a risk of low-volume lymph node metastasis of endometrial cancer; when both are negative, it is determined to be low risk. Furthermore, in some complex samples, when the expression intensity of the CK19 biomarker is lower than a preset threshold (weakly positive or close to negative) and the judgment is uncertain, the EPCAM biomarker is introduced for compensatory judgment: if EPCAM is positive, the overall detection result is corrected to positive, thereby improving the detection ability of micrometastases and isolated tumor cells.

[0021] By combining the above three biomarkers, we can maintain rapid detection while effectively compensating for the shortcomings of the dual biomarker system in cases of low expression or tissue damage (such as cauterization, folding, detachment, etc.), further reducing the false negative rate and improving the stability and consistency of intraoperative assessment.

[0022] In addition, the present invention also provides an intraoperative rapid immunohistochemical detection kit containing CK19 enzyme-labeled primary antibody, EMA enzyme-labeled primary antibody and EPCAM enzyme-labeled primary antibody, and may further contain components such as blocking agent, signal enhancement solution, color development system and counterstaining reagent, for the purpose of realizing the simultaneous or stepwise detection of the three markers.

[0023] The CK19, EMA, and EPCAM described in this invention are briefly introduced below: (1) CK19 (Cytokeratin 19): CK19 is a low-molecular-weight acidic keratin belonging to the cytokeratin family. It is an important component of the epithelial cytoskeleton and is widely expressed in various epithelial-derived tissues and tumor cells. In this invention, CK19 is used as a specific marker for epithelial-derived tumor cells to identify tumor cells derived from endometrial cancer, and is particularly suitable for detecting micrometastases and isolated tumor cells in lymph nodes. Its positive expression is usually manifested as specific staining signals in the cytoplasm.

[0024] (2) EMA (Epithelial Membrane Antigen): This is a transmembrane glycoprotein mainly expressed on the surface of epithelial cells and has strong epithelial origin specificity. In this invention, EMA is used as a broad-spectrum epithelial marker to assist in the identification of endometrial cancer cells and can compensate for the insufficient detection sensitivity of CK19 in some cases (such as when the tissue structure is damaged or the expression is reduced). Its positive expression is usually manifested as a specific staining signal on the cell membrane.

[0025] (3) EPCAM (Epithelial Cell Adhesion Molecule): A transmembrane glycoprotein mainly involved in intercellular adhesion, it is highly expressed in various epithelial tumor cells. In this invention, EPCAM is used as a supplementary marker to assist or compensate for the detection results when the expression intensity of CK19 is reduced or uncertain, thereby improving the detection rate of low-volume lymph node metastases in endometrial cancer. Its positive expression can manifest as specific staining signals on the cell membrane or cytoplasm.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects: 1. By combining CK19 and EMA, highly specific identification of endometrial cancer cells can be achieved, significantly improving the detection sensitivity of low-volume metastases; 2. By introducing EPCAM markers to construct a compensation judgment mechanism, an effective supplement is provided when CK19 expression is weakened or difficult to interpret, thereby improving the stability and reliability of the detection results; 3. It can perform rapid immunohistochemical detection and interpretation under intraoperative frozen section conditions, with the overall detection time controlled within a clinically acceptable range, meeting the needs of real-time decision-making during surgery; 4. Effectively reduces the missed diagnosis rate of micrometastases and isolated tumor cells, provides surgeons with more accurate information on lymph node status, helps optimize surgical plans and reduce the risk of secondary surgery; 5. The technical solution can be automated through equipment and computers to process and output results, and has a high degree of standardization and clinical application value.

[0027] In summary, this invention, by constructing a multi-marker joint judgment and compensation mechanism, achieves highly sensitive and accurate assessment of low-volume lymph node metastasis in endometrial cancer in a rapid intraoperative detection scenario, and has significant clinical application value and promotion prospects. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The following are microscopic observations of intraoperative frozen HE-stained sections in this embodiment of the invention: Macrometastatic lesions within lymph nodes, with tumor tissue nested within the lymph nodes and proliferating fibrous tissue between the cell nests; the maximum diameter of the metastatic lesions is greater than 2 mm, and the original structure of the lymph node is destroyed, magnified 4 times (a); Micrometastatic lesions within lymph nodes (circled in red) are mainly distributed under the lymph node capsule, with tumor cells distributed in small nests; the maximum diameter of the metastatic lesions is ≤2 mm but greater than 0.2 mm, and does not significantly destroy the original structure of the lymph node, magnified 10 times (b); In short: a) Macrometastatic lesions within lymph nodes, with tumor tissue distributed in nests, and the maximum diameter of the metastatic lesions is >2 mm; b) Micrometastatic lesions within lymph nodes, with tumor cells distributed in small nests under the capsule, and the maximum diameter of the metastatic lesions is 0.2–2 mm.

[0030] Figure 2These are comparative images of micrometastases missed by intraoperative frozen section HE staining and confirmed by rapid immunohistochemistry using CK19 and EMA in embodiments of the present invention. Specifically: In the HE section of case 1, tumor cells appear as elongated glandular structures within the lymphoid tissue, magnified 10x (a). Intraoperative frozen section immunohistochemistry using EMA (b) and CK19 (c) clearly shows the metastatic lesions within the lymphoid tissue. In the HE section of case 2, tumor cells with burn marks appear as small nests within the lymphoid tissue, magnified 10x (d). Intraoperative frozen section immunohistochemistry using EMA (e) and CK19 (f) clearly shows the metastatic lesions. In the HE section of case 3, only a very small number of nuclearly atypia cell nests are seen under the lymph node capsule, magnified 10x (g). Intraoperative frozen section immunohistochemistry using EMA (h) and CK19 (i) clearly shows multifocal metastatic lesions located under the capsule. In case 4, the HE section showed tissue detachment and folding artifacts, with only a few cells showing enlarged nuclei. At 10x magnification (j), intraoperative frozen section immunohistochemistry (EMA (k)) clearly revealed tumor cells scattered within the lymphoid tissue (k). CK19 labeling, due to tissue detachment, only showed scattered brown signals on the tissue surface (l). In short: a) Intraoperative frozen section with hematoxylin and eosin (HE); b) EMA rapid immunohistochemical staining of sections; c) CK19 rapid immunohistochemical staining sections; d) Intraoperative frozen section with hematoxylin and eosin (HE) for patients with burns; e) Corresponding EMA rapid immunohistochemical staining sections; f) Corresponding CK19 rapid immunohistochemical staining sections; g) Intraoperative frozen HE sections with tissue folds; h) Corresponding EMA rapid immunohistochemical staining section; i) Corresponding CK19 rapid immunohistochemical staining sections; j) Intraoperative frozen HE sections with tissue detachment; k) Corresponding to EMA rapid immunohistochemical staining sections; l) Corresponding CK19 rapid immunohistochemical staining section.

[0031] Figure 3 This is a comparison of isolated tumor cells missed by intraoperative frozen section HE staining and detected by rapid immunohistochemistry using CK19 and EMA in an embodiment of the present invention: HE sections exhibit artifacts of tissue detachment and folding, making it almost impossible to observe metastatic tumor cells. At 10x magnification (a), scattered isolated tumor cells can be clearly observed by intraoperative frozen section immunohistochemistry using EMA (b) and CK19 (c). In short: a) Intraoperative frozen section with hematoxylin and eosin (HE); b) EMA rapid immunohistochemical staining of sections; c) CK19 rapid immunohistochemical staining sections.

[0032] Figure 4 This is a schematic diagram of the integrated evaluation device described in Embodiment 6 of the present invention.

[0033] like Figure 1 As shown, the device includes a sample processing unit, a biomarker detection unit, a data acquisition unit, a data processing unit, and a result output unit. These units can be integrated into a single device or interconnected via a communication interface. The sample processing unit is used for lymph node tissue sampling, cryopreservation, and sectioning; the biomarker detection unit is used for detecting CK19, EMA, and optionally EPCAM; the data acquisition unit is used to acquire the detection data; the data processing unit is used to execute biomarker joint determination and compensation determination logic; and the result output unit is used to output the risk assessment results for low-volume lymph node metastasis in endometrial cancer.

[0034] Figure 5 This is a schematic diagram of the computer-executed evaluation method flow described in Embodiment 7 of the present invention.

[0035] like Figure 5 As shown, the method includes the following steps: Step S1, reading biomarker detection data; Step S2, determining the expression status or expression intensity of each biomarker; Step S3, performing a basic joint determination of CK19 and EMA; Step S4, performing a compensation determination when the CK19 expression intensity is lower than a preset threshold T1 and EPCAM is positive; Step S5, outputting the evaluation results; Optionally, it also includes Step S6, storing and feeding back the evaluation results.

[0036] Figure 6 This is a schematic diagram of the computer system structure described in Embodiment 8 of the present invention.

[0037] like Figure 6 As shown, the system includes a processor, a memory, an input / output interface, and an optional image acquisition module. The memory stores biomarker detection data, judgment rules, and threshold parameters. The processor executes data processing, biomarker joint judgment, and compensation judgment logic. The input / output interface receives data from the detection device and outputs the evaluation results to a display terminal or medical information system, thereby achieving automated evaluation of low-volume lymph node metastasis in endometrial cancer. Detailed Implementation

[0038] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Other implementation methods obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the protection scope of the present invention.

[0039] This invention provides an assessment technique for intraoperative evaluation of low-volume lymph node metastasis in endometrial cancer. It acquires and processes the detection results of markers in lymph node samples and outputs risk assessment results based on preset judgment rules.

[0040] The markers include two or three combinations of CK19, EMA and EPCAM, preferably a combination of CK19 and EMA, and EPCAM can be further introduced as a supplementary marker.

[0041] Based on dual biomarker (CK19+EMA) detection, this invention introduces an EPCAM construction compensation judgment mechanism to solve the problem of missed detection caused by unstable biomarker expression under intraoperative rapid detection conditions.

[0042] In one embodiment, the evaluation device of the present invention includes: 1. Data acquisition equipment The data acquisition device is used to acquire detection data of the evaluation subject. The detection data comes from the detection results of biomarkers in lymph node samples from patients with endometrial cancer.

[0043] Specifically, the detection data may include: The expression level or expression status of CK19 markers; The expression level or expression status of EMA markers; The expression level or expression status of EPCAM markers.

[0044] The data acquisition methods include, but are not limited to: Test results are manually entered using an input device; The test results are read from external testing equipment or data storage devices via a communication interface; The integrated testing equipment automatically outputs the test data.

[0045] 2. Data processing equipment The data processing device is used to process the detection data and output risk assessment results.

[0046] Specifically, it includes: (1) Determination of indicator values Based on the detection data, the expression status of each marker is determined, including but not limited to: Positive; Negative; Weak positive (or expression intensity below the preset threshold).

[0047] In a preferred embodiment, the expression state is obtained based on the results of rapid immunohistochemical detection of intraoperative frozen sections, wherein: CK19 positive: Specific detection signal appears in the cytoplasm, preferably brownish-yellow staining; EMA positive: Specific detection signal appears on the cell membrane, preferably brownish-yellow staining; EPCAM positive: Specific detection signal appears on the cell membrane or cytoplasm, preferably brownish-yellow staining.

[0048] The positive determination is based on the presence or intensity of a detection signal, wherein the detection signal includes a colorimetric signal, a fluorescence signal, or other visual signals.

[0049] (2) Basic Judgment Rules In one embodiment, the data processing device makes a preliminary determination based on the following rules: When the combination of biomarkers is determined to be positive, a high-risk assessment result is output. When the combination of markers is determined to be negative, a low-risk assessment result is output.

[0050] The “combination determination” can be based on the combined results of two markers (CK19+EMA) or three markers.

[0051] (3) Compensation determination mechanism In a preferred embodiment, the present invention further introduces a compensation determination mechanism: When the following conditions are met: The expression intensity of the CK19 biomarker was lower than the preset threshold T1 (i.e., it was weakly positive or close to negative). The EPCAM marker was positive. but: The CK19 marker was compensated and determined using the detection results of the EPCAM marker; The test result that might have been initially judged as negative was corrected to positive; Output high-risk assessment results.

[0052] In this invention, "the expression intensity of the CK19 marker is lower than a preset threshold T1" means: The CK19 marker detection signal obtained from the testing of lymph node samples from patients with endometrial cancer had an intensity lower than the preset threshold for distinguishing between positive and negative states.

[0053] In one embodiment, the expression intensity can be characterized in the following way: Qualitative judgment method Based on the pathologist's visual interpretation of the staining results, the CK19 expression status was classified as: negative, weakly positive, and positive, among which: Negative: No clear specific detection signal was observed; Weak positive: Only focal, weak or discontinuous specific detection signals are observed, making it difficult to form a clear basis for positive interpretation; Positive: A continuous, clear, and diagnostically significant specific detection signal is observed.

[0054] In this case, "expression intensity below the preset threshold T1" includes the weak positive state and the near negative state.

[0055] Semi-quantitative scoring method In another embodiment, the expression intensity can be semi-quantitatively assessed using an immunohistochemical scoring system, including but not limited to a comprehensive score based on staining intensity and the proportion of positive cells. For example: Staining intensity rating: 0 (no signal), 1 (weak), 2 (moderate), 3 (strong); Positive cell percentage score: 0 (<5%), 1 (5%–25%), 2 (26%–50%), 3 (>50%). When the overall score is lower than the preset score (e.g., ≤1 or ≤2), it is determined to be lower than the preset threshold T1.

[0056] Quantitative detection methods In a further embodiment, the expression intensity can also be quantitatively determined using image analysis software or nucleic acid quantification methods, for example: Image grayscale value or optical density (OD value); Fluorescence intensity value; Ct value or relative expression level (ΔΔCt) in qPCR detection; When the detected value is lower than the preset threshold range, it is determined to be lower than the threshold T1.

[0057] How to determine the threshold T1 The preset threshold T1 can be determined in the following way: Determined based on a control experiment comparing normal lymph node tissue with metastatic positive tissue; The optimal cutoff value was determined through statistical analysis of clinical samples; Experienced pathologists will formulate the guidelines based on staining results. The threshold T1 can be a fixed value or it can be adjusted according to different detection methods, sample types or experimental conditions.

[0058] 3. The mechanism of operation of the compensation mechanism Under intraoperative frozen section conditions, due to the short tissue processing time and the significant impact of the freezing process on tissue structure, the following situations often occur: Tissue folding; Tissue smearing; tissue cauterization or compression; Uneven staining or weakened signal.

[0059] Under the above circumstances, CK19, as a cytoplasmic marker, may exhibit the following: The staining signal is discontinuous; Reduced expression intensity; Local missing.

[0060] EPCAM, as a biomarker related to epithelial origin, has the following characteristics: It is stably expressed in tumor cells; The staining localization is clear (mostly cell membrane expression); It has relatively low sensitivity to damage to tissue morphology.

[0061] Therefore, when the CK19 signal is weakened or difficult to interpret, introducing EPCAM can provide an independent and stable positive signal, thereby: To avoid missed detections due to the failure of a single marker; Improve the ability to identify low-volume transfers; Enhance the reliability of overall test results.

[0062] In another embodiment, the present invention also provides a computer-executed method for outputting risk assessment results of low-volume lymph node metastasis in endometrial cancer, comprising the following steps: Step a) Data reading Read biomarker detection data, including the expression status or level of CK19, EMA, and EPCAM.

[0063] Step b) Data processing Processing the detection data includes: Determine the expression status of each marker; Determine whether the basic positive criteria are met; Determine whether the compensation criteria are met (low CK19 expression + positive EPCAM).

[0064] Step c) Output the result Based on the above determination results, the risk assessment result is output as follows: A positive result indicates a high-risk warning. If the result is negative, a low-risk warning will be issued.

[0065] In one embodiment, the computer system of the present invention includes: At least one processor; Memory; The computer program stored in the memory; When the computer program is executed on the processor, it implements each step of the above-described computer execution method, including: Obtain test data; Execute the marker determination logic; Implement a compensation determination mechanism; Output the risk assessment results.

[0066] It should be noted that the detection method for the biomarker in this invention is not limited to rapid immunohistochemistry, but may also include: Immunofluorescence detection; Western blot; Flow cytometry; Nucleic acid amplification methods (such as RT-PCR, qPCR, digital PCR); In situ hybridization (ISH); Gene sequencing, etc.

[0067] When using nucleic acid detection, the status of biomarkers can be determined by detecting the expression levels of the corresponding genes of CK19, EMA, and EPCAM, and the same rules and compensation mechanisms of this invention apply.

[0068] Through the above technical solution, the present invention has at least the following technical effects: 1. Improve the intraoperative detection sensitivity for low-volume lymph node metastases in endometrial cancer; 2. Significantly reduces the false negative rate of micrometastases and isolated tumor cells; 3. Achieve rapid and reliable detection and evaluation within the limited time window during surgery; 4. Enhance the stability of test results through a compensation judgment mechanism; 5. It possesses good engineering feasibility and clinical application value.

[0069] The present invention will further demonstrate the selection principle, process and effect of the above-mentioned biomarkers through a specific embodiment, so as to enable those skilled in the art to understand the essence of the present invention.

[0070] Example 1: Screening of specific biomarkers for lymph node metastasis in endometrial cancer To obtain specific biomarkers that are highly expressed in endometrial cancer tissues, lowly expressed in normal lymph nodes, and highly correlated with lymph node metastasis, this invention employs a strategy of joint screening using multiple omics databases and experimental verification to gradually identify the optimal combination from a large number of candidate biomarkers.

[0071] 1. Differentially expressed genes (DEG) analysis from the TCGA database A custom R script was used to analyze differentially expressed genes between normal endometrial tissue and endometrial cancer (EC) tissue in the Cancer Genome Atlas (TCGA) database, identifying a total of 2450 genes with significant differential expression.

[0072] The screening criteria were set as follows: expression change fold change (FC) > 2, resulting in 61 genes.

[0073] 2. Screening for genes highly expressed in cancer tissue (HumanProteinAtlas) The RNA expression levels (pTPM median) of the above 61 genes in endometrial cancer tissues were analyzed in The Human Protein Atlas database. A pTPM > 100 was defined as high expression in cancer tissues, and 13 genes were obtained.

[0074] 3. Screening for lymph node metastasis-related genes (Genecards) A search was conducted in the Genecards database for genes related to "lymph node metastasis," and genes with an association score >1.5 were selected, yielding a total of 518 results.

[0075] Taking the intersection of the above 13 genes with lymph node metastasis-related genes yields 9 genes.

[0076] 4. Screening for genes with low expression in normal lymph nodes The expression levels (nTPM) of the above nine genes in normal lymph nodes were analyzed using The Human Protein Atlas database. An nTPM < 2 was defined as low expression in normal lymph nodes, ultimately resulting in five genes: CLDN7, CLDN4, MUC1, EPCAM, RAB25.

[0077] 5. Expand the candidate pool and finally determine the optimal combination. To ensure the specificity, sensitivity, and staining applicability of intraoperative diagnosis, this invention, combining database screening results, literature review, epithelial-derived marker characteristics, and rapid immunohistochemical compatibility, further selected seven of the most promising genes for parallel experimental verification: CK19, EPCAM, RAB25, MUC1, CLDN7, MMP7, PLAP.

[0078] 6. Experimental Verification and Final Selection Experiments using intraoperative frozen rapid immunohistochemistry parallel comparison, staining intensity, background interference, and low volume metastasis detection rate confirmed that: CK19: Strongly positive for endometrial cancer cells, completely absent in normal lymph nodes, and extremely sensitive to the identification of small lesions; EMA: A broad-spectrum epithelial marker, strongly positive in endometrial cancer cell membranes, but not expressed in normal lymph nodes, which can significantly compensate for the shortcomings of CK19 detection alone; When used together, the staining is clear, the interpretation is easy, and the time required is short (20-33 minutes). The detection rate of low-volume transfer is significantly higher than that of other candidate genes.

[0079] Although EMA was not included in the aforementioned high-throughput screening gene set, this invention, during the systematic experimental screening and pairing optimization of epithelial biomarkers, discovered that EMA possesses highly complementary labeling characteristics to CK19: it exhibits specific high expression in endometrial cancer cells, but no expression in normal lymph node tissue, and its staining is clear and easy to interpret against a frozen section background. Combined use with CK19 significantly improves the detection rate of low-volume metastases, increasing both sensitivity and specificity. This combined effect is unattainable with a single biomarker and represents an unexpected technical effect obtained through experimental screening. This invention ultimately determined the CK19 and EMA combination as the optimal dual-labeling scheme. Therefore, this invention ultimately selected the CK19 and EMA combination as the core detection biomarker for low-volume lymph node metastases in endometrial cancer.

[0080] Example 2: Clinical Validation I. Methods 1. Clinical data: All samples were collected from patients with endometrial cancer who underwent surgery at Zhejiang Cancer Hospital between November 6, 2024 and March 29, 2025 and met the inclusion criteria. Inclusion criteria: (1) Female, 18-75 years old; (2) Pathologically confirmed endometrial cancer (histochemical type not limited), and scheduled for systematic lymph node dissection or sentinel lymph node biopsy; (3) Rapid pathological examination of lymph nodes was performed during the operation to determine whether there was metastasis of endometrial cancer.

[0081] 2. Intraoperative frozen specimen processing: The surgeon collecting the specimen bisects the extended axis of the clinically submitted lymph nodes. Half of the specimen is used to prepare intraoperative frozen sections (including HE staining and rapid immunohistochemical staining), and the other half is used to prepare routine postoperative pathological sections. The tissue used for intraoperative frozen sections is placed on a sample holder containing OCT (Optimum Cutting Temperature Compound) embedding medium and placed on the cryostat stage for rapid freezing at a low temperature of -20°C to -26°C to allow the tissue to solidify quickly. The frozen tissue block is then sliced ​​into 4μm thick sections on a microtome and attached to a glass slide, producing a total of 3 sections: 1 for intraoperative frozen HE staining and 2 for IR-IHC staining.

[0082] 3. Intraoperative frozen section HE staining: Immediately place two sections for intraoperative frozen section HE staining into fixative for 1 minute, rinse briefly with tap water, immerse in hematoxylin staining solution for 1 to 2 minutes, rinse with water, immerse in blue solution for 3-5 seconds, rinse thoroughly with water, immerse in eosin staining for 2-5 seconds, dehydrate with graded alcohols (sequentially through 75% ethanol, 95% ethanol, and anhydrous ethanol), clear with xylene, and mount with neutral resin to complete the preparation of intraoperative frozen section HE staining.

[0083] 4. IR-IHC Staining: Immediately place two slides for IR-IHC staining in 10% neutral formalin buffer for 30-60 seconds, then rinse with running water for 3-5 seconds. Place the slides in a humidified chamber (water temperature 40-42℃), and add the blocking agent (incubate for 30 seconds), enzyme-labeled primary antibody (EMA or CK19, incubate for 3-5 minutes), enhancement solution (incubate for 3-5 minutes), DAB chromogenic solution (incubate for 2-3 minutes), and hematoxylin staining solution (incubate for 10-30 seconds) in sequence. Finally, rinse with TBS (Tris-Buffered Saline) for 3-5 seconds and mount the slides to complete the preparation of IR-IHC slides.

[0084] 5. Result Interpretation: Intraoperative frozen section HE and IR-IHC sections were simultaneously reviewed under a microscope by two senior pathologists. IR-IHC EMA was considered positive when the cell membrane stained brownish-yellow, and IR-IHC CK19 was considered positive when the cytoplasm stained brownish-yellow. Macrometastasis of lymph nodes was defined as the largest metastatic lesion in a lymph node with a maximum diameter >2 mm; micrometastasis was defined as the largest metastatic lesion in a lymph node with a maximum diameter of 0.2–2 mm and / or >200 cells; isolated tumor cells were defined as the largest metastatic lesion in a lymph node with a maximum diameter ≤0.2 mm and ≤200 tumor cells.

[0085] 6. Routine pathological lymph node hyperstaging: The lymph node tissue used for routine postoperative pathological examination is fixed, dehydrated, cleared, impregnated with paraffin, and embedded to prepare paraffin tissue blocks. Sections are prepared at 200μm intervals, with 5 sections cut from each paraffin block. One section is stained with IHC broad-spectrum cytokeratin (pan-CK), and the other 4 sections are stained with HE. After the 5 sections are prepared, they are observed and diagnosed under a microscope by the pathologist.

[0086] 7. Statistical methods: The formulas for calculating the sensitivity and specificity of intraoperative frozen section rapid pathological diagnosis before and after IR-IHC detection are as follows: Sensitivity = (Number of true positive cases / Total number of positive cases) × 100% Specificity = (Number of true negative cases / Total number of negative cases) × 100%.

[0087] II. Results 1. Clinicopathological characteristics of enrolled cases (Table 1): A total of 18 cases were collected, aged 47-78 years (median age 57 years), and staged according to the 2023 FIGO (International Federation of Gynecology and Obstetrics) classification, including IA1-IIIC1ii. Histological types included low-grade endometrioid adenocarcinoma (FIGO 1-2, 10 cases), high-grade endometrioid adenocarcinoma (FIGO 3, 3 cases), serous carcinoma (2 cases), clear cell carcinoma (1 case), mesonephric adenocarcinoma (1 case), and carcinosarcoma (2 cases). Tumor emboli were observed in 8 patients, of which 5 cases had a large number of intravascular tumor emboli (≥5 per section), and 3 cases had focal intravascular tumor emboli (≤4 per section). A total of 104 lymph nodes were submitted for examination, located in the pelvis (96), para-aortic region (5), and presacral region (3).

[0088]

[0089] 2. Intraoperative frozen section HE examination results: Microscopic examination of intraoperative frozen section HE revealed endometrial carcinoma metastases in 21 lymph nodes, including clear cell carcinoma (20 nodes) and mesonephric adenocarcinoma (1 node). 19 metastases were macrometastases. The metastatic tumor tissue was irregularly distributed in nest-like clusters within the lymph nodes, with proliferating fibrous tissue between the clusters. The original lymph node structure was destroyed, and the maximum diameter of the metastatic lesions was >2 mm. Figure 1 (a) Two micrometastases were observed, with the metastatic tumor tissue scattered in small nests within the lymph nodes, mainly located under the lymph node capsule. The original lymph node structure was not significantly destroyed. The maximum diameter of the metastatic lesions was ≤2mm, but >0.2mm. Figure 1 (b) in the middle.

[0090] 3. Intraoperative frozen section HE combined with IR-IHC section observation results: Microscopic observation of IR-IHC EMA and CK19 staining results showed that 26 lymph nodes contained varying degrees of brownish-yellow staining of cell membranes or cytoplasm, suggesting endometrial cancer metastases. Of these, 19 macrometastases were consistent with HE observation. Of the 6 micrometastases, 2 were consistent with HE observation, and 4 were missed by HE observation. The first micrometastase (case B12) showed tumor cells arranged in elongated glandular patterns within the lymphoid tissue in the HE section, with a maximum diameter of approximately 0.5 mm. Figure 2 (a) in IR-IHC marked EMA ( Figure 2 (b) and CK19 ( Figure 2 In section c), the brown staining of tumor cells was clearly visible; in the second section (case B13), the intraoperative frozen HE section showed burns, with only enlarged small nests of nuclei and atypical tumor cells accompanied by burns. The maximum diameter of the metastatic lesions in the HE section was approximately 0.2 mm. Figure 2 d), in the EMA marked with IR-IHC ( Figure 2 e) and CK19 ( Figure 2 In section f), in addition to the tumor cell nests observable in the HE slide, brown-stained tumor cells were also observed in the tissue with severe burns on the periphery, and the maximum diameter of the metastatic lesions increased to about 0.6 mm; the intraoperative frozen HE slide of the third case (case B13) showed tissue folding artifacts, with only a few atypical tumor cells with compression injuries seen under the lymph node capsule, which were easily confused with the tissue cells under the capsule in morphology, and the maximum diameter of the metastatic lesions was less than 0.2 mm. Figure 2 g), but in EMA marked with IR-IHC ( Figure 2 h) and CK19 ( Figure 2 In section i), multifocal brown-stained tumor cell nests were visible beneath the lymph node capsule, with the largest foci having a maximum diameter of approximately 0.3 mm; the intraoperative frozen section (case B17) also showed tissue folding artifacts, with a small number of enlarged, atypical tumor cells scattered throughout the lymphoid tissue near the folded areas, and the largest metastatic foci having a maximum diameter of approximately 0.3 mm. Figure 2 In the IR-IHC-labeled EMA sections, brown-stained tumor cells were observed not only near the tissue folds but also within the tissue folds themselves. Figure 2 In IR-IHC-labeled CK19 sections, only scattered brown-stained tumor cells were observed due to tissue detachment (k). Figure 2 (l) One isolated tumor cell was missed during HE observation (Case B17). The intraoperative frozen section for HE showed artifacts of tissue folding and tissue detachment, making it virtually impossible to observe clear metastatic tumor cells. Figure 3 (a) but three isolated brownish-yellow stained cell nests were visible in IR-IHC-labeled EMA sections. Figure 3 In section b), only one isolated brownish-yellow stained cell nest was observed in the CK19 section. Figure 3 (c) In this context, the maximum diameter of these cell nests is less than 0.2 mm, and the number of cells is less than 200.

[0091] 4. Testing time: 104 lymph nodes were sent for testing in groups for a total of 41 times. Each test could detect 1-6 lymph nodes. The intraoperative frozen section rapid pathological diagnosis combined with IR-IHC testing took 20-33 minutes (median time 25 minutes).

[0092] 5. Postoperative routine pathological results: All lymph nodes underwent supra-staging examination postoperatively. Of the 104 lymph nodes examined, 16 showed macrometastases, 6 showed micrometastases, and 1 showed isolated tumor cells. The sensitivity of intraoperative frozen section hematologic resection (HE) for detecting low-volume lymph node metastases in endometrial cancer was 28.57% (2 / 7), with a specificity of 100%. The sensitivity and specificity of intraoperative frozen section hematologic resection combined with IR-IHC for detecting low-volume lymph node metastases in endometrial cancer were both 100%.

[0093] This invention, using IR-IHC technology, identified 7 low-volume metastases in lymph nodes from 104 endometrial cancer patients, 5 of which failed diagnosis by intraoperative frozen section HE staining (including 4 micrometastases and 1 isolated tumor cell). Using postoperative routine pathology as the gold standard, IR-IHC technology increased the sensitivity of rapid intraoperative frozen section pathology diagnosis from 28.57% to 100%, while the overall examination time was not significantly prolonged, demonstrating its significant advantages over traditional frozen section examination. Research on rapid intraoperative immunohistochemistry has been ongoing for over 20 years, with past studies primarily focusing on sentinel lymph node metastasis in breast cancer and the diagnosis of lung cancer. There have also been some studies on lymph node metastasis in malignant melanoma, the diagnosis of brain tumors, and the development of pan-cancer diagnostic kits. The detection methods used vary, and the required detection time is mostly around 20 minutes. The IR-IHC technology used in this invention is reported for the first time. It has high detection sensitivity and specificity, and the color development is the same as that of traditional immunohistochemistry, which allows diagnostic physicians to adapt more quickly. Moreover, its detection time is shorter, and the entire intraoperative frozen section diagnosis time is mostly controlled within 30 minutes. All of the above suggest that this detection method is more suitable for clinical application.

[0094] Traditional intraoperative frozen sections, lacking fixation with a fixative, undergo cell deformation during rapid freezing. Tumor cells metastasizing to lymph nodes exhibit diverse morphologies, with some specific cell types displaying atypical forms, easily confused with lymph node tissue cells or follicular dendritic cells. Furthermore, the number of tumor cells in lymph node micrometastases is typically small, and intraoperative frozen sections often require rapid assessment, leading to frequent missed diagnoses. Additionally, the preparation of intraoperative frozen sections differs from conventional tissue preparation. Lymphoid and adipose tissues have different requirements for freezing temperature and time, but lymph nodes may contain both, easily creating artifacts of tissue folding and detachment during slide preparation. If metastatic tumor cells are located at these folds or detachments, they are highly susceptible to being missed. IR-IHC labeling can alert the diagnosing physician to suspicious tumor cells at folds. Moreover, multiple IR-IHC slides may complement HE slides; folded tissue in HE slides may not be folded in the chromosomal section, and detached tissue in HE slides may not be detached in the chromosomal section, thus reducing missed diagnoses. Furthermore, the use of energy-based instruments to remove lymph nodes in clinical practice often results in the burning of lymph node tissue. Additionally, pathologists inevitably compress lymph node tissue during sampling, and the area near the lymph node capsule, where burning and compression most frequently occur, is precisely where lymph node micrometastases most commonly occur. Once metastatic lesions are burned or compressed, their morphology becomes difficult to identify, making them highly susceptible to missed diagnoses. IR-IHC technology can mark these deformed tumor cells, alerting diagnosticians to the presence of metastatic lesions and thus reducing missed diagnoses.

[0095] This invention demonstrates that labeling EMA and CK19 using IR-IHC technology can reduce the impact of factors such as insufficient cell quantity, atypical cell morphology, tissue compression, and tissue folding in traditional intraoperative frozen HE sections on the diagnosis of low-volume lymph node metastases. While not significantly prolonging the time for rapid intraoperative frozen pathological diagnosis, it greatly reduces the rate of missed diagnoses of low-volume metastases, helping surgeons to more accurately assess the lymph node status and adjust surgical plans in a timely manner, enabling patients to receive more precise treatment.

[0096] This clinical study was reviewed and approved by the Ethics Committee of Zhejiang Cancer Hospital, with ethics approval number IRB-2023-567 (Department).

[0097] Example 3: Detection of CK19 and EMA expression using a nucleic acid amplification method based on specific primers. To further expand the detection methods of the biomarkers of this invention, this embodiment provides a nucleic acid amplification detection scheme based on specific primers, which can realize the qualitative and quantitative detection of CK19 and EMA gene expression through RT-PCR, qPCR or in situ hybridization (ISH), and is also applicable to the auxiliary diagnosis of lymph node metastasis and low volume metastasis in endometrial cancer.

[0098] 1. Experimental Materials and Instruments Lymph node tissue samples, total RNA extraction kit, reverse transcription kit, qPCR kit, CK19 specific primers, EMA specific primers, real-time PCR instrument, in situ hybridization kit, etc.

[0099] 2. Experimental Methods (1) Total RNA extraction and reverse transcription Lymph node tissue was collected during the operation, and total RNA was extracted from the tissue according to the kit instructions. After the concentration and purity were found to be qualified, the RNA was reverse transcribed into cDNA using random primers or Oligo (dT) as primers and stored at -20℃ for later use.

[0100] (2) Real-time quantitative PCR (qPCR) amplification Using cDNA as a template, qPCR amplification was performed using CK19-specific primers and EMA-specific primers, with GAPDH as an internal control gene. Reaction conditions: 95℃ pre-denaturation for 30 s; 95℃ for 5 s, 60℃ for 30 s, for a total of 40 cycles. Each group was configured with 3 replicates.

[0101] (3) In situ hybridization (ISH) detection After dewaxing, digestion, and fixation, frozen or paraffin sections of lymph nodes are subjected to hybridization with CK19 and EMA-specific probes. Following incubation, staining, counterstaining, and mounting, positive signals are observed under a microscope. A positive result is defined as a brownish-yellow or purplish-red hybridization signal in the cytoplasm / nucleus.

[0102] 3. Result Determination qPCR: The relative expression levels were calculated using the ΔΔCt method. Significant upregulation of the relative expression levels of CK19 or EMA suggested the presence of endometrial cancer cell metastasis in lymph node tissue. In situ hybridization: The appearance of specific positive signals in tissue sections indicates metastasis positivity and can locate micrometastases or isolated tumor cells.

[0103] 4. Experimental Results Based on the results of Example 2, it can be seen that the expression levels of CK19 and EMA genes in endometrial cancer lymph node metastasis samples are significantly higher than those in normal lymph node tissue; the results of qPCR and in situ hybridization are consistent with the results of intraoperative rapid immunohistochemistry, and all of them can effectively identify micrometastases and isolated tumor cells.

[0104] Example 4: Kit for rapid diagnosis of low-volume lymph node metastasis in endometrial cancer This embodiment provides a kit that can be directly used for rapid intraoperative immunohistochemical detection, suitable for rapid detection of lymph node specimens from patients with endometrial cancer. It can achieve simultaneous labeling of CK19 and EMA, and accurately identify macrometastases, micrometastases, and isolated tumor cells.

[0105] 1. Kit Components This kit is specifically designed for rapid intraoperative diagnosis and contains the following components: 1) CK19 enzyme-labeled primary antibody working solution; 2) EMA enzyme-labeled primary antibody working solution; 3) Tissue blocking agents; 4) Signal enhancement fluid; 5) DAB colorimetric solution (solution A, solution B); 6) Hematoxylin counterstain solution; 7) 10% neutral formalin fixative; 8) Detergent (TBS); 9) Sealing adhesive; 10) Reagent kit instructions (including operating procedures, result interpretation criteria, and definition of metastatic lesions).

[0106] 2. Scope of application of the reagent kit This method is suitable for rapid immunohistochemical detection of frozen sections of lymph nodes during surgery in patients with endometrial cancer, and can simultaneously detect pelvic lymph nodes, para-aortic lymph nodes, and presacral lymph nodes.

[0107] 3. Operating Procedures 1) Specimen processing: Lymph node specimens were bisected along their long axis, embedded in OCT, and 4 μm frozen sections were prepared; 2) Fixation: Fix sections with 10% neutral formalin for 30–60 seconds, then rinse with running water; 3) Blocking: Add tissue blocking agent and incubate at room temperature for 30 seconds; 4) Primary antibody incubation: Add CK19 enzyme-labeled primary antibody and EMA enzyme-labeled primary antibody respectively, and incubate at 40–42℃ for 3–5 minutes; 5) Enhancement: Add signal enhancement solution and incubate for 3–5 minutes; 6) Development: Add DAB developer and develop for 2–3 minutes in the dark; 7) Counterstaining and mounting: Counterstain with hematoxylin for 10–30 seconds, wash and then mount with mounting glue.

[0108] 4. Result Determination EMA positive: The cell membrane shows a brownish-yellow staining; CK19 positive: The cytoplasm shows brownish-yellow staining; A double positive result suggests the presence of lymph node metastasis in endometrial cancer. Macrometastasis, micrometastasis, and isolated tumor cells can be further distinguished based on the size of the lesion and the number of cells.

[0109] 5. Kit efficacy 1) The entire testing process takes only 20–33 minutes, meeting the needs for rapid intraoperative diagnosis; 2) Enzyme-labeled primary antibody directly develops color, eliminating the need for secondary antibody, simplifying the process and reducing background; 3) The accompanying enhancement solution significantly improves the detection rate of micrometastases and isolated tumor cells; 4) It has good stability and clear staining, making it suitable for routine use in pathology departments of hospitals at all levels.

[0110] Example 5: A joint judgment strategy for CK19 weak expression compensation based on EPCAM and its effectiveness verification When using CK19 and EMA for rapid immunohistochemical detection of intraoperative frozen sections, the inventors discovered that: Although the combination of CK19 and EMA can significantly improve the detection rate of low-volume lymph node metastases in endometrial cancer, the following problems still exist in some cases: CK19 is weakly or discontinuously expressed in some low-volume metastatic lesions; In areas of tissue detachment, cauterization, or folding, CK19 staining signals are easily weakened or absent; Even if EMA is positive, insufficient CK19 signal may still affect the overall interpretation.

[0111] Therefore, even with weak CK19 expression, the CK19+EMA combination may still present a risk of indeterminate interpretation or missed detection.

[0112] To address the above issues, this embodiment introduces the EPCAM marker and constructs the following compensation determination rule: When the following conditions are met: The expression intensity of the CK19 biomarker was lower than the preset threshold T1; The EMA marker was positive. The EPCAM marker was positive. The EPCAM marker was then used to compensate for the CK19 marker, and the test result was determined to be positive.

[0113] In this embodiment, the expression intensity of CK19 was semi-quantitatively assessed by immunohistochemical staining intensity: Grade 0: Unstained; Grade 1: Weakly positive (light-colored or discontinuous staining); Grade 2: Moderately positive; Level 3: Strongly positive; Wherein: when the staining intensity of CK19 is grade 0 or 1, it is defined as being below the threshold T1.

[0114] Experimental verification 1. Sample Source Intraoperative lymph node samples from Example 2 were selected for in-depth analysis of cases that were confirmed by postoperative pathology to have low-volume metastases.

[0115] 2. Comparison of Schemes Only the following two comparison groups are set up:

[0116] 3. Experimental Results In cases of low-volume metastases: CK19 + EMA combined detection: In cases with weak CK19 expression, there are difficulties in interpretation or missed detection. Invention solution (introducing EPCAM compensation): All cases with weak CK19 expression were correctly identified; No cases were missed during the inspection; The detection stability has been significantly improved.

[0117] Typical Case Analysis Case 1 (area of ​​tissue detachment) CK19: Weak positive (discontinuous signal) EMA: Positive EPCAM: Positive There was some uncertainty and hesitation in interpreting the CK19+EMA combination, while the compensation judgments were all clearly positive.

[0118] Case 2 (Burned area) CK19: Weakly expressed or nearly negative EMA: Positive EPCAM: Positive CK19+EMA: There is a risk of missed detection, while compensation determination can clearly identify metastatic lesions. Case 3 (isolated tumor cells) CK19: Extremely weak expression EMA: Positive EPCAM: Positive The CK19+EMA combination assessment carries the risk of being difficult to confirm, while the compensation assessment can clearly identify metastatic lesions.

[0119] Compared with the combined detection of CK19 and EMA alone, the present invention has the following significant advantages: 1. Improve detection capabilities in scenarios with weak expression. In cases of weak CK19 expression, EPCAM compensation can effectively prevent missed detections.

[0120] 2. Improve the stability of test results Reduce the risk of misjudgment due to section quality issues (detachment, folding, burning).

[0121] 3. Optimize the joint judgment mechanism The simple "dual marker superposition judgment" has been upgraded to a dynamic compensation judgment mechanism based on the expression state.

[0122] 4. Achieve unexpected technical effects Building upon the fact that CK19 and EMA have already significantly improved the detection rate, this invention further: A compensation mechanism is proposed to address the specific problem of weak expression; Further reduce the false negative rate without increasing the detection time.

[0123] This embodiment demonstrates that by introducing the EPCAM compensation judgment mechanism on the basis of combined CK19 and EMA detection, the accuracy or certainty of detecting low-volume lymph node metastases in endometrial cancer can be significantly improved, especially in the case of weak CK19 expression, thereby further enhancing the reliability of rapid intraoperative diagnosis.

[0124] Example 6: An integrated assessment device for evaluating low-volume lymph node metastasis in endometrial cancer This embodiment provides an integrated assessment device for rapid intraoperative assessment of low-volume lymph node metastasis in patients with endometrial cancer.

[0125] 1. Overall structure of the equipment The integrated assessment equipment includes: Sample processing unit Marker detection unit Data acquisition unit Data processing unit Result Output Unit The above units can be integrated into a single device, or they can be connected through a communication interface to form a system.

[0126] 2. Sample Processing Unit Used for processing lymph node tissue obtained during surgery, including: Lymph node sampling and segmentation Cryo-embedding (OCT) Frozen sections (preferably 3–5 μm thick) 3. Marker Detection Unit This is used for the detection of biomarkers associated with endometrial cancer, said biomarkers including: CK19 EMA Optional: EPCAM Intraoperative rapid immunohistochemistry (IR-IHC) is preferred, including: Add enzyme-labeled primary antibody (CK19, EMA, or EPCAM). Add enhancement liquid Add a color development system (such as DAB or other color development systems). Complete staining and counterstaining The overall testing time should ideally be controlled within 20–33 minutes.

[0127] The detection results are manifested as specific detection signals in tissue sections.

[0128] 4. Data Acquisition Unit Used to acquire marker detection data, including: Automatic acquisition of microscopic images; or Receive interpretation results input by the pathologist; or Read data from the storage module of the testing equipment. The detection data includes: Expression status of each marker (positive / negative / weakly positive); Or express intensity data (scores or quantitative values).

[0129] 5. Data Processing Unit Used to execute marker determination rules and output risk assessment results.

[0130] (1) Basic judgment rules If both CK19 and EMA are positive, the risk level is considered high. If all results are negative, the risk level is considered low.

[0131] (2) Compensation determination mechanism When the following conditions are met: CK19 expression intensity was below the preset threshold T1; EPCAM positive; but: EPCAM was used to determine the compensation for CK19; The test result was corrected to positive. Output high-risk assessment results.

[0132] 6. Result Output Unit Used to output evaluation results, including: Displays high / low risk alerts; Provides a visual interface; Alternatively, it can be sent to the hospital information system (HIS / PACS) via an interface.

[0133] 7. Technical Effects The above structure achieves the following: Intraoperative rapid detection Multi-marker joint determination EPCAM compensation enhances stability Reduce the rate of missed diagnoses Example 7: Computer-Executed Assessment Method for Low-Volume Lymph Node Metastasis in Endometrial Cancer This embodiment provides a computer-executed method for automatically outputting risk assessment results.

[0134] Step S1: Data Reading Read marker detection data, including: CK19 expression data; EMA expression data; Optional: EPCAM expression data.

[0135] Step S2: Determine the expression state Based on the detection data, determine the expression status of each marker: Positive Negative Or express intensity level in: When the expression intensity is lower than the preset threshold T1, it is determined to be a low expression state.

[0136] Step S3: Basic Joint Decision Execute the following logic: If both CK19 and EMA are positive, the result is considered positive. Otherwise → Proceed to the compensation determination step.

[0137] Step S4: Compensation Determination (Key Step) When the following conditions are met: CK19 expression intensity <T1; And EPCAM was positive; but: The result was positive (compensation was awarded).

[0138] Step S5: Output the result Positive result → Output high-risk assessment result; Negative → Output low-risk assessment result.

[0139] Optional step S6: Results recording and feedback Store the evaluation results; Output report; Used for subsequent statistical analysis or model optimization.

[0140] This implementation method can achieve the following: Automated interpretation Marker logic rule execution Improve the consistency and stability of assessments Example 8: Computer System for Evaluating Low-Volume Lymph Node Metastasis in Endometrial Cancer This embodiment provides a computer system for implementing the above-described evaluation method.

[0141] 1. System Structure The system includes: Processor (CPU or GPU) memory Input / output interface Optional: Image acquisition module 2. Memory For storage: Marker detection data Judgment Rules and Procedures Threshold parameters (e.g., T1) 3. Processor Used to execute programs in memory, achieving: (1) Data processing module Receive CK19, EMA, and EPCAM data Parse the expression state (2) Judgment Module implement: Basic Joint Decision Logic Compensation determination logic (low CK19 expression + EPCAM positive) (3) Output module Output: Risk assessment results (high / low risk) Or visualize the analysis results 4. Input / Output Interfaces Used for: Receive testing equipment data Output to display terminal or medical system 5. System Implementation Method The system can be implemented as follows: Independent medical software system; Embedded device systems; Cloud computing platform; Or an integrated system that is integrated with pathological testing equipment.

[0142] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0143] Those skilled in the art will understand that all or part of the steps of the above embodiments can be implemented by hardware or by a program instructing related hardware. The program can be stored in a computer-readable storage medium, such as a read-only memory, a disk, or an optical disk.

[0144] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An assessment device for intraoperative evaluation of low-volume lymph node metastasis in endometrial cancer, characterized in that, include: The data acquisition device is used to acquire the detection data of the evaluation subject. The detection data is the level value of the marker among the markers of low volume lymph node metastasis of endometrial cancer detected in the human lymph nodes of the evaluation subject suspected of having low volume lymph node metastasis of endometrial cancer. The markers of low volume lymph node metastasis of endometrial cancer are a combination of CK19 and EMA, or a combination of all three markers, of the three markers CK19, EMA and EPCAM. The data processing equipment is used to calculate the index value of markers in low-volume lymph node metastasis markers of endometrial cancer based on the detection data of the evaluation object, and then calculate whether the index value of the obtained marker is within the risk range of the corresponding marker.

2. The evaluation device as described in claim 1, characterized in that: The indicator value of the biomarker is the result of rapid immunohistochemical detection of the biomarker based on intraoperative frozen sections of human lymph nodes of the evaluated subject: positive or negative; If the marker for low-volume lymph node metastasis in endometrial cancer is positive, a high-risk assessment result for low-volume lymph node metastasis in endometrial cancer will be given. If the marker for low-volume lymph node metastasis in endometrial cancer is negative, a low-risk assessment result for low-volume lymph node metastasis in endometrial cancer will be given.

3. The evaluation device as described in claim 1, characterized in that, When the expression intensity of the CK19 biomarker is lower than the preset threshold T1, if the EPCAM biomarker is positive, the detection result of the EPCAM biomarker is used to compensate for the detection result of the CK19 biomarker, and the detection data is determined to be positive, thereby determining the risk of low-volume lymph node metastasis of endometrial cancer.

4. The evaluation device as described in claim 1 or 2, characterized in that, The data acquisition device is an input device for inputting data or a communication device for reading data from an external data storage device or the storage device of the detection device via an interface.

5. The evaluation device as described in claim 4, characterized in that, When communication equipment is used, its corresponding external data storage device is the data storage device on the device used to measure biomarkers.

6. The evaluation device as described in claim 4, characterized in that, It is integrated into automated biomarker assay equipment, thereby directly outputting evaluation results after assay, realizing integrated detection and evaluation functions.

7. A computer-executed method for outputting risk assessment results of low-volume lymph node metastasis in endometrial cancer, the method comprising the following steps: a) Read the test data of patients with endometrial cancer, wherein the test data are the level values ​​of endometrial cancer lymph node low volume metastasis markers detected in the lymph nodes of human subjects suspected of having low volume lymph node metastasis of endometrial cancer, wherein the endometrial cancer lymph node low volume metastasis markers are a combination of CK19 and EMA, or a combination of all three markers, of the three markers CK19, EMA and EPCAM. b) Data processing to calculate the index value of the biomarker based on the level value in step a), wherein the index value of the biomarker is the result of rapid immunohistochemical detection of the biomarker based on intraoperative frozen sections of human lymph nodes of the evaluated subject: positive or negative; c) Output the evaluation results.

8. The execution method as described in claim 7, wherein in step c), if the marker for low-volume lymph node metastasis of endometrial cancer is positive, a high-risk assessment result for low-volume lymph node metastasis of endometrial cancer is given; if the marker for low-volume lymph node metastasis of endometrial cancer is negative, a low-risk assessment result for low-volume lymph node metastasis of endometrial cancer is given.

9. The execution method as described in claim 7, wherein in step c), when the expression intensity of the CK19 marker is lower than a preset threshold T1, the detection result of the EPCAM marker is called as a supplementary judgment basis, and the evaluation result is output based on the combined result of the two.

10. A computer system that performs the following steps: a) Obtain or receive the level values ​​of the biomarker in the lymph nodes of the assessment subject suspected of having low volume lymph node metastasis of endometrial cancer, wherein the biomarker for low volume lymph node metastasis of endometrial cancer is a combination of CK19 and EMA, or a combination of all three of the three biomarkers CK19, EMA and EPCAM. b) Determine whether the calculated indicator values ​​of the markers are within the preset risk range; c) Output the judgment result of step b), and optionally give a risk warning. If the marker for low-volume lymph node metastasis of endometrial cancer is positive, give the corresponding high-risk assessment result of low-volume lymph node metastasis of endometrial cancer. If the marker for low-volume lymph node metastasis in endometrial cancer is negative, a low-risk assessment result for low-volume lymph node metastasis in endometrial cancer will be given; if the EMA marker is positive, the CK19 marker is weakly positive (i.e., the expression intensity of the CK19 marker is below the preset threshold), and the EPCAM marker is positive, a high-risk assessment result for low-volume lymph node metastasis in endometrial cancer will be given.

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