Biomarkers for discriminating hepatocellular carcinoma and intrahepatic cholangiocarcinoma and applications thereof

Abstract

The application discloses biomarkers for identifying hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) and application thereof, and belongs to the technical field of biological medicine. The biomarkers are BRD4 and EEF2. Multicolor immunofluorescence experiments show that the co-expression cells of BRD4 and EEF2 are significantly increased in intrahepatic cholangiocarcinoma tissues, are 4-10 times higher than normal liver tissues, and have no significant difference in hepatocellular carcinoma and normal liver tissues; BRD4 + EEF2 + The median survival time of the double-positive cell high-expression group of ICC patients is less than 20 months. Based on the clear cause-and-effect logic of specific transcriptional regulation and combined expression activation of translation elongation factors caused by intrahepatic cholangiocyte proliferation, the application can accurately identify intrahepatic cholangiocarcinoma and hepatocellular carcinoma, distinguish different pathological subtypes and tumor heterogeneity of ICC, has the value of prognosis evaluation, is simple to operate, controllable in cost, and is suitable for popularization and application in clinical pathology departments.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to biomarkers for differentiating hepatocellular carcinoma and intrahepatic cholangiocarcinoma, and their applications. Background Technology

[0002] Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are two primary liver cancers with different cellular origins (hepatic parenchymal cells or bile duct epithelial cells). The incidence of ICC has been increasing in recent years. Due to the lack of accurate differential diagnostic methods, 70%–80% of ICC patients are already out of the surgical window when diagnosed by imaging. Although both HCC and ICC share common changes such as inflammation and liver fibrosis, their pathogenic mechanisms differ. HCC is mainly caused by abnormally active glycolysis (Warburg effect) and fatty acid metabolism in the liver parenchyma, activating signaling pathways such as Wnt / β-catenin, PI3K / Akt / mTOR, and MAPK. ICC, on the other hand, is mainly caused by abnormal cell proliferation and differentiation leading to abnormal cellular metabolism, such as amino acid metabolism. It is often accompanied by specific molecular mutations in isocitrate dehydrogenase 1 (IDH) and IDH1 / 2, which are associated with mitochondrial function, and presents with glandular or tubular structures. High-throughput sequencing technology reveals significant individual differences in the mutated molecular characteristics of different pathological subtypes of ICC, indicating that ICC tumor cell subtypes exhibit high heterogeneity in the pathological process. This leads to the diversity of precise targeted biomarker immunotherapy, as well as the complexity of early diagnosis of ICC and responses to targeted drugs and immune checkpoint inhibitors.

[0003] Existing methods for detecting and identifying hepatocellular carcinoma (ICC), such as immunohistochemistry (IHC) for fibroblast growth factor receptor 2 (FGFR2), serum or pathological tissue IHC for CK19 molecular markers, next-generation sequencing (NGS), and fluorescence in situ hybridization (FISH), suffer from unclear causal logic. The frequency of high-frequency mutant cell lines of different ICC subtypes varies between primary and metastatic lesions and among different individuals, thus affecting the precise efficacy of targeted drugs. For example, the efficacy of FGFR inhibitors is only effective in some individuals sensitive to FGFR signals. Although imaging examinations such as enhanced CT and MRI can capture features like arterial phase enhancement and portal venous phase washout in HCC, and serum marker AFP also shows high sensitivity in HCC, inflammatory factors such as IL-6 and TNF-α are highly expressed in the HCC microenvironment, while immunosuppressive cell infiltration is more pronounced in ICC. CK19 and CEA, as commonly used auxiliary diagnostic methods for ICC, show different results in predicting the clinical prognosis of ICC subtypes of large and small bile ducts and different pathological subtypes. Meanwhile, the mutation rate of the essential cellular respiratory enzymes IDH1 / 2 in ICC is only 4.9%–20.0%. In conclusion, there is an urgent need to establish accurate differential diagnostic techniques for HCC and ICC in clinicopathological diagnosis.

[0004] While cancer cells share commonalities in cellular metabolic characteristics such as uncontrolled proliferation and activation of immune escape signaling pathways across different cell cycles, they exhibit unique spatiotemporal differences in the regulatory mechanisms of cell proliferation and differentiation across different tissues. Tumor cell-associated mutation features obtained from comparisons between cancerous and adjacent tissues, amplified using PCR technology, can rapidly identify individual genome-specific mutations in normal individuals and cancer patients, such as single nucleotide variants (SNVs) and short insertion / deletion variants (Indels). However, a drawback is that if the number of these cancerous mutated cells is below the detection threshold, false negatives can easily lead to missed diagnoses. Furthermore, the arrest of proliferation in these mutated cells, or the presence of numerous apoptotic cells, even with positive cancer-associated biomarkers, indicates individual variability in the dynamic changes of cancer progression. Epigenetically activated tumor cells alter their function through post-translational modifications of proteins, without changing their original DNA sequence. These false negatives, false positives, and epigenetic phenotypes can interfere with accurate diagnosis and delay treatment. Therefore, it is necessary to develop a combination of biomarkers that can accurately capture specific changes in cancer-like tumor stem cells. These biomarkers can be used for monitoring extracellular cell-free DNA and RNA (cell-free DNA (cfDNA) and cell-free RNA (cfRNA)) derived from specific tumor stem cell fragments in peripheral blood, and / or for the accurate monitoring of circulating tumor cells (CTCs).

[0005] By employing a set of biomarkers with a clear causal relationship to reduce non-specific background interference such as individual differences, it is helpful to detect and accurately identify different pathological subtypes of HCC and ICC at an early stage, and provide new specific molecular markers for precise auxiliary diagnosis in order to effectively evaluate surgery and targeted therapy and formulate personalized diagnosis and treatment plans. Summary of the Invention

[0006] The purpose of this invention is to provide biomarkers for differentiating hepatocellular carcinoma and intrahepatic cholangiocarcinoma, and their applications, in order to solve the problems existing in the prior art. By detecting the expression of BRD4 and EEF2 in tumor cells, the number of BRD4 and EEF2 positive tumor cells can be determined to differentiate hepatocellular carcinoma and intrahepatic cholangiocarcinoma. This method solves the problems of unclear causal logic and low accuracy in the prior art, and overcomes the shortcomings of the prior art in that it is easy to miss low-abundance cancer cells and that the existing biomarkers cannot reflect the heterogeneity of tumor cells.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a biomarker for the auxiliary diagnosis of hepatocellular carcinoma and intrahepatic cholangiocarcinoma, wherein the biomarker is BRD4 and EEF2.

[0008] The present invention also provides the use of the biomarkers described herein in the preparation of kits for differentiating hepatocellular carcinoma from intrahepatic cholangiocarcinoma.

[0009] The present invention also provides a kit for differentiating hepatocellular carcinoma from intrahepatic cholangiocarcinoma, the kit comprising: a) A primary diagnostic reagent for the specific detection of BRD4 expression levels; b) A second detection reagent specifically for detecting EEF2 expression levels.

[0010] Preferably, the first detection reagent is an anti-BRD4 antibody, and the second detection reagent is an anti-EEF2 antibody.

[0011] Preferably, the kit further includes immunohistochemical detection reagents or immunofluorescence detection reagents.

[0012] Preferably, the expression of BRD4 and EEF2 in the subject samples is used to determine whether it is hepatocellular carcinoma or intrahepatic cholangiocarcinoma.

[0013] Preferably, the method for determination is as follows: when BRD4 and EEF2 are both highly expressed in the subject's sample, the subject is determined to have intrahepatic cholangiocarcinoma; when BRD4 and EEF2 are not both highly expressed, the subject is determined to have hepatocellular carcinoma.

[0014] Preferably, the common high expression refers to the expression levels of BRD4 and EEF2 in intrahepatic cholangiocarcinoma tissues being 4-10 times higher than those in adjacent or normal tissues.

[0015] The present invention also provides the use of the biomarkers or kits described herein in the preparation of diagnostic products for evaluating the efficacy of postoperative targeted therapy or for assessing survival in patients with intrahepatic cholangiocarcinoma.

[0016] Preferably, the evaluation includes detecting BRD4 in the intermediate region of intrahepatic cholangiocarcinoma tissue. + EEF2 + Percentage of double-positive cells, BRD4 + EEF2 + Patients with a high percentage of double-positive cells had a median survival of less than 20 months.

[0017] The present invention discloses the following beneficial effects: (1) This invention provides BRD4 and EEF2 as biomarkers for the identification of ICC / HCC. The two have clear synergy in transcription and translational elongation and a causal logical relationship with ICC pathogenesis, which can effectively eliminate non-specific background interference and overcome the inconsistency in judgment caused by unclear causal logic in the prior art.

[0018] (2) The biomarkers of the present invention are expressed 4-10 times higher in ICC cancer tissue than in normal tissue, while the difference is not significant in HCC. Compared with CK19 (2.5 times), they have a higher signal-to-noise ratio, which significantly improves the accuracy and sensitivity of differential diagnosis.

[0019] (3) The present invention can identify different pathological subtypes and heterogeneous tumor stem cells of ICC, and realize the visual assessment of the highly heterogeneous characteristics of ICC bile duct endothelium, making up for the shortcomings of existing markers that cannot capture the heterogeneity of tumor cells.

[0020] (4) The present invention uses multicolor immunofluorescence technology, which can simultaneously detect BRD4 (nucleus), EEF2 (plasma) and CK19 (cholecystic epithelial cell cytoplasm) in situ in tissues, distinguish cells in different differentiation states in the parenchymal and interstitial regions, and provide spatial conformation information that cannot be obtained by traditional methods.

[0021] (5) This invention has prognostic value, BRD4 + EEF2 + The median survival of patients in the high expression group was less than 20 months, and the positive rate remained at 60% in CK19-negative poorly differentiated cells, providing a basis for postoperative evaluation and personalized treatment of ICC.

[0022] (6) This invention is based on conventional immunohistochemistry / immunofluorescence technology, which is simple to operate and cost-controllable. It can be completed using commercial antibodies and standard pathology equipment, and is suitable for clinical promotion and use in pathology departments of hospitals at all levels. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 To validate the differential expression of BRD4 and EEF2 in tumor and normal tissues using immunohistochemical detection; A: TCGA public database analysis showed significant differential expression of EEF2 in tumor and normal tissues (left panel), and BRD4 antibody immunohistochemical analysis validated its significant differential expression in intrahepatic cholangiocarcinoma (ICC) and adjacent normal tissues (right panel); B: TCGA public database analysis showed significant differential expression of EEF2 in tumor and normal tissues (left panel), and EEF2 antibody immunohistochemical analysis validated its significant differential expression in intrahepatic cholangiocarcinoma (ICC) and adjacent normal tissues (right panel), where a1-a4 represent ICC tissues from different sources, and b1-b4 represent the corresponding adjacent normal tissues; in the immunohistochemical analysis results, the scale bar is 200 μm; CHOL represents intrahepatic cholangiocarcinoma, and LIHC represents hepatocellular carcinoma. Figure 2 The results of fluorescence immunoassay for anti-EEF2 and anti-BRD4 in three pathological tissue sections from HCC (A) and ICC (B); scale bar: 50 μm; Figure 3 Multicolor immunofluorescence assay was used to identify clinicopathological subtypes of hepatocellular carcinoma (HCC) using BRD4 and EEF2 dual antibodies. "AA": HCC tissue sample from a 53-year-old male patient with T2 stage HCC (HCC D1); "BB": HCC tissue sample from the same patient as "AA" (HCC D2); "CC": HCC tissue sample from a different patient with T1 stage HCC (HCC B1); Red indicates anti-BRD4 (nucleus) cells in HCC D1, and green indicates anti-EEF2 positive epithelial and stromal cells (cytoplasm). HCC staging was based on AJCC 7th edition; CV: central vein branch; PV: portal vein branch; BRD4 / EEF2 expressing cells showed heterogeneous proliferation and distribution near CV and PV; BRD4 expressing cells near PC were small cells with large nuclei, while those near CV were large hepatic lobular cells with large nuclei; bile duct cells were mostly distributed near PV, and their size and morphology differed significantly from hepatocytes. Figure 4 This shows the distribution of BRD4 / EEF2 positive cells in liver tissue; BRD4 / EEF2 positive cells are mostly located in prometaphase and / or anaphase (BB), with partial or complete disappearance of the nuclear membrane, and sister chromosomes paired and aligned at the equatorial plate in the center of the cell by centromere traction. Bile duct epithelial cells are shown in the figure. BRD4 is highly expressed in the nucleus (red), while EEF2 is expressed in the cytoplasmic endoplasmic reticulum and cytoskeleton; morphologically distinct from adjacent hepatocytes (mostly in the prophase, with chromatin condensed in the nucleus and chromosomes scattered throughout the nucleus, see figure ""). (A1-A2 are anatomical diagrams of the liver in cross-section and longitudinal section, respectively). Figure 5The phenotype and distribution of BRD4 and EEF2 double antibody-positive cells in pathological sections of six different ICC cases; ICC-A1-59F (ICC cancer tissue of a 59-year-old female), ICC-A1 is a partial view of the small bile ducts and stroma of ICC-A1-59F, and ICC-A2 is the adjacent tissue of the same patient, in which BRD4 / EEF2 double antibody-positive cancerous cells are aggregated and scattered around many necrotic empty lumens (ICC-A2a is a magnified view of the boxed area of ​​ICC-A2). ICC-B1-45M (cancer tissue from a 45-year-old male): Hyperplastic intrahepatic microcholedochal epithelial cells showed high BRD4 / EEF2 double antibody positivity; B2 was adjacent tissue from the same patient (BRD4 / EEF2 double antibody positive cells scattered in the interstitial region of the liver lobules); ICC-C1-73F was T1 cholangiocarcinoma of the bile duct cystadenocarcinoma (cancer tissue from a 73-year-old female): Numerous atrophic and necrotic bile ducts (hollowed-out bile duct epithelium, necrosis not well-formed); ICC-C2 was from the same patient... The patient's adjacent tissue; BRD4 / EEF2 double antibody-positive cancerous cells in liver lobules and stromal cells; ICC-D1-75M is chronic cholangitis T1 (cancer tissue from a 75-year-old male); D1 is an enlarged view of the boxed area of ​​ICC-D1-75M chronic cholangitis T1 (shrunken and necrotic bile duct epithelial cells, some of which are BRD4 / EEF2 double antibody-positive); ICC-D2 is the adjacent tissue of the same patient (BRD4 / EEF2 in the shrunken bile duct and surrounding fibrotic stroma). Distribution of F2 double antibody positive cells); ICC-E1-66F moderately differentiated ICC-T2-II (66-year-old female cancer tissue), E1 is a magnified view of the boxed area of ​​ICC-E1-66F moderately differentiated ICC-T2-II, with bile duct necrosis forming irregular cavities; ICC-E2 is a high-power magnified view of the adjacent tissue of the same patient (heterogeneously expressed BRD4 / EEF2 double antibody positive cells in the melted area of ​​necrotic micro-bile ducts); ICC-G1-46M T1III / IV (46-year-old male cancer tissue), G1 is a magnified view of the boxed area of ​​ICC-G1-46M T1 III / IV (liver parenchyma and interstitium filled with a large number of proliferating micro-bile ducts), ICC-G2 is the adjacent tissue of the same patient (the magnified image also shows a large number of BRD4 / EEF2 double antibody positive cells). Figure 6 This is the BRD4 / EEF2 immunofluorescence assay result for late-stage ICC; the sample came from patient ICC-J1, a 63-year-old male (T3, clinical stage III / IV), with intrahepatic bile duct hyperplasia covering the entire pathological section, and the bile duct endothelium exhibiting high heterogeneity. Figure 6 High magnification of box A: A' and A"), the bile duct epithelium of the adjacent tissue also shows high heterogeneity; B1-B2, B1'-B2' in the figure: BRD4 negative (only DAPI blue) or BRD4 positive (red cell nucleus), and the cell nuclei have different shapes and are in different cell cycles (different chromosome density). Figure 7 Diagnostic value of BRD4 / EEF2 expressing cells in the proliferation of cholangiocarcinoma-adjacent tissues; A: Proportion of BRD4 and EEF2 expressing cells in whole cells of cholangiocarcinoma and related cluster analysis of clinical survival; B: Proportion of BRD4 and EEF2 expressing cells in stromal cells of cholangiocarcinoma and related cluster analysis of clinical survival. Figure 8 The overall distribution of each cell phenotype is shown; different colors represent different cell types, and the length of the band represents the number of cells; A: the composition of markers in different cells; B: the expression of different markers in different cells. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Example 1 This invention is based on the highly evolutionarily conserved TGFb / BMP signaling in vertebrates, which plays a crucial role in regulating cell proliferation and nuclear DNA replication, primarily by initiating DNA mismatch-induced cell checkpoint activation, and triggering a series of immune activation mechanisms in the stem cell microenvironment, including cellular immunity and apoptosis. Regnases (Zc3h12 family factors) and eukaryotic translation elongation factors (eEF family factors) are one of the important switch combinations for initiating stem cell proliferation and differentiation and regulating the stem cell nuclear genome STEM-loop. Building upon previous research on the relationship between the Zc3h12b gene or protein and the occurrence of primary liver tumors (see "Invention Patent ZL201911399060.4: Uses of ZC3H12b Gene or Protein and a Method for Establishing an Animal Model of Liver Disease" for the construction of animal models of liver diseases), this study, through comparative analysis of the RNA transcriptome of liver tissues from individuals with normal and absent Zc3h12b, focused on significantly differentially expressed factors shared by large-scale liver data analysis from the tumor public databases TCGA and GEPIA (http: / / gepia.cancer-pku.cn). It was found that the combination of BRD4 and EEF2, while not showing significant differences in hepatocellular carcinoma (HCC), exhibited significant differences in intrahepatic cholangiocarcinoma (ICC) (the difference was 4-10 times higher in cancerous tissue than in the normal group). Figure 1 (A and B) indicate BRD4 activation. Its bromodomain specifically binds to H3K27ac (acetylated lysine of histone 3, an important marker of transcriptional activation), regulating the dissociation or release of RNA polymerase II into productive elongation at the transcription promoter region. The elongation factor EEF2 also participates in regulating the dynamic balance between transcription and translation and is associated with the degree of retrograde differentiation. Therefore, the activation of these factors presents a clear and reliable causal relationship with tumor stem cell carcinogenesis. Immunohistochemical analysis showed that this group of target molecules can distinguish different subtypes of ICC cancer tissue (…). Figure 1 A and B; ICC cancer tissue a1-a4; a1 & a3: T2 clinical stage III / IV; a2 & a4: T1 clinical stage II; corresponding adjacent normal tissue b1-b4).

[0031] Example 2 This study employed mIHC multiplex antibody immunohistochemical fluorescence labeling technology to perform quantitative immunofluorescence detection of pathological microarray tissue samples with multiple target combinations. It included in-situ qualitative analysis of cell phenotypes, comparison of qualitative and quantitative information between cells in in-situ, and follow-up and statistical analysis of overall survival for postoperative high-expression and low-expression groups. The main experimental reagents, instruments, and materials involved in the immunohistochemical fluorescence labeling technology were: (1) Main experimental reagents and instruments Table 1 Antibodies Primary antibody incubation conditions: room temperature (RT), 1 h; Secondary antibody incubation conditions: room temperature (RT), 10 min.

[0032] Table 2 Experimental Instruments Table 3 Experimental Reagents (2) Experimental materials: Tissue microarrays were provided by Shanghai Chipchao Biotechnology Co., Ltd. The liver cancer tissue microarray (HLivH150CS06) and intrahepatic cholangiocarcinoma microarray (HIBDA180SU02) were collected from paraffin blocks of pathological tissues (cancer and adjacent tissues) diagnosed in the pathology departments of various cooperating hospitals. The patient groups and sample information corresponding to the collected paraffin blocks of pathological tissues are shown in Table 4.

[0033] Table 4 Patient Groups and Sample Information The experimental procedures for immunohistochemical fluorescent labeling are further explained below: 1) Baking the slides: Place the tissue chips in an oven, set the temperature to 63℃, and bake with wax for 1 hour; 2) Dewaxing: Completed in a fully automatic dyeing machine. The dewaxing times are as follows: xylene 2 tanks, 15 min each; 100% alcohol 2 tanks, 7 min each; 90% alcohol 1 tank, 5 min; 80% alcohol 1 tank, 5 min; 70% alcohol 1 tank, 5 min. 3) Antigen retrieval: Dilute 10×AR6 antigen retrieval solution to 1× working solution, microwave on high for 3 minutes to boil, then place a glass slide in the microwave, microwave on low for 15-20 minutes (ensure the tissue is immersed in the liquid throughout the process), and allow to cool naturally at room temperature. 4) Blocking: Remove the slide, wash it with TBST, place it in a humidified chamber, add blocking buffer, and incubate for 10 minutes; 5) Primary antibody incubation: Remove blocking buffer, place in a humidified chamber, add primary antibody, incubate at room temperature for 1 h, and wash slides with TBST. 6) Secondary antibody incubation: Remove the slide, place it in a humidified chamber, add secondary antibody (included in the Opal 7-color Manual IHC Kit, catalog number NEL801001KT), incubate at room temperature for 10 min, and then wash the slide with TBST. 7) Opal dye development: Remove the slide, place it in a humidified chamber, add Opal dye dilution solution (1:100 dilution), incubate at room temperature for 10 min, and then clean the slide with TBST. 8) Microwave treatment (removal of primary and secondary antibodies): Repeat step 3) and then clean the slides with TBST. 9) For subsequent indicators after re-infection, repeat steps 4)-8) until all indicators are marked; 10) DAPI staining and mounting: Remove the slide, place it in a humidified chamber, add DAPI working solution, incubate at room temperature for 10 min, and wash the slide with TBST. Remove the slide, add fluorescent anti-quenching mounting medium, and mount. 11) Panoramic multispectral scanning of slides was performed using the TissueFAXS system, and the data was imported into StrataQuest analysis software. The multispectral scanned samples were spectrally split using a spectral library built from the monochromatic samples to obtain clean single-channel fluorescence signals. Cell nuclei were identified using the DAPI channel, and cell debris and adherent cells were removed by plotting the nuclear area and fluorescence intensity to screen for effective single cells. 12) Using the effective cell nucleus as the center, set a distance radius according to the staining of each protein channel, and look for protein fluorescence signals in the cell nucleus and / or cytoplasm; 13) Set thresholds based on the fluorescence intensity of each channel protein, divide the positive cell population, and calculate the positive rate; 14) Co-labeling of Protein A and Protein B: Based on effective single cells, a scatter plot is created, with the fluorescence intensity of protein A as the x-axis and the fluorescence intensity of protein B as the y-axis. The threshold values ​​of both proteins divide the scatter plot into four quadrants: upper right, upper left, lower right, and lower left, corresponding to A and B respectively. + B + A - B + A + B - A - B - Four types of cells; 15) Protein A, B, and C co-labeling: using A as the protein labeling standard. + Based on the cell, a scatter plot was created with the fluorescence intensity of protein B as the x-axis and the fluorescence intensity of protein C as the y-axis. The thresholds of B and C divided the scatter plot into four quadrants: upper right, upper left, lower right, and lower left, corresponding to A, B, and C respectively. + B + C + A + B - C + A + B + C - A + B - C - Four types of cells; 16) By analogy, more protein co-labeled cells can be obtained; 17) Through CK19 + Channel fluorescence identifies epithelial cell regions, which are tumor regions; 18) Cell distribution in tissues: More than half are located in CK. + Cells in the tumor region are classified as tumor region cells, and those not in the tumor region are classified as stromal region cells; the gray value of the tumor region is set to 255, and the gray value of the stromal region is 0; the horizontal axis is the cell nucleus area, and the vertical axis is the average position intensity of the pixels in the cell nucleus; with 255 / 2=127.5 as the threshold, cells above the CUT OFF line are tumor region cells, and cells below it are stromal region cells; 19) By plotting a scatter plot based on total cell count, the total cell count in CK19-positive cells (CK19) can be obtained. + ) region and CK19 negative cells (CK19 - The distribution of cells in the CK19 region can be obtained by plotting a scatter plot based on a specific cell type. + Region and CK19 - Distribution of regions; 20) Observe and photograph low-magnification x200 and high-magnification x630 images using a confocal microscope (Leica TCSSP8, Germany); 21) Bioinformatics analysis was performed using the statistical and plotting packages in the R language; the R language version was R4.5.0, and the differential analysis and plotting were performed using the ggpubr and ggplot2 packages; survival-related analyses were performed using the survminer and survival packages; specifically: Kaplan-Meier survival analysis: Based on each marker expression value as a cutoff value, the samples were divided into high-expression and low-expression groups, and prognostic significance analysis was performed between the two groups. The best cutoff value was selected from all results, and the corresponding prognostic analysis plot was plotted. Figure 7 In the graph, `low_expression` and `high_expression` represent low and high expression, respectively; the curve for high expression is below the curve, indicating a poor prognosis for high gene expression; `p < 0.05` is considered statistically significant; the number of cells at different levels was plotted using the `ggplot2` package in R. Figure 8 The distribution of cell phenotypes in each sample. Each vertical bar represents a sample, and different colors represent different cells. The X-axis represents the sample, and the Y-axis represents the number of cells; by analyzing the proportion of different cell types, the cell types with a high overall proportion are identified.

[0034] 22) The differential expression of molecular markers with different protein compositions in different groups of ICC cancer (low expression and high expression groups, see Table 5) was detected by immunofluorescence on HLivH150CS06 hepatocellular carcinoma-adjacent tissue microarray and HIBDA180SU02 intrahepatic cholangiocarcinoma-adjacent tissue microarray produced by Shanghai Xinchao Biotechnology Co., Ltd.

[0035] Table 5 Composition of Molecular Markers (3) Results and Analysis: like Figure 2 As shown, the results indicated that the immunofluorescence signal of the BRD4 and EEF2 antibody combination in HCC cancer tissue pathological sections was significantly higher than that in adjacent normal tissues, consistent with TCGA prediction results. The positive signal intensity of the immunoreaction in cholangiocarcinoma (ICC patients, 91 cases) was also significantly higher than that in their adjacent normal tissues. The hepatocyte marker CK8 showed no significant difference between HCC cancer tissues and adjacent normal tissues; however, the expression of EEF2 in liver cancer tissues was significantly higher than that in adjacent normal tissues. Figure 2 The data (A) indicates that EEF2 is significantly activated in hepatocellular carcinoma cells; BRD4 and EEF2 are significantly differentially expressed in cancerous and adjacent tissues of ICC, consistent with the expression of the conventional ICC marker CK19. Figure 2 The results (B) indicate that EEF2 and BRD4 proteins are significantly activated in cholangiocarcinoma tissues. Immunohistochemical detection of the bile duct epithelial cell marker CK19, and targeting anti-BRD4 and anti-EEF2, can accurately capture HCC (HCC). Figure 2 Significant differential expression of cancerous tissue and adjacent normal tissue in ICC (intra-A) and ICC (intra-A) Figure 2 (B)

[0036] The activation levels and subtle differences of BRD4 and EEF2 positive cells in HCC and ICC pathological tissue sections are shown below. Figure 3 and Figure 4 The activation level of BRD4 and EEF2 positive cells in the liver parenchyma was consistent with the results of clinicopathological classification, namely the number of double-positive cells in the parenchyma of the clinical T2 stage. Figure 3 "AA": In clinical T2 stage HCC patients (D1), the number of BRD4 and EEF2 double antibody-positive cells in the liver parenchyma was significantly increased compared to T1 stage. Figure 3 "Zhong CC": Patient B1 in clinical T1 stage HCC. In the stroma adjacent to the non-cancerous tissue of patient D1, the BRD4 and EEF2 double antibody-positive cells of the microcholedochal epithelial cells have a morphology and tubular arrangement that are distinctly different from the prismatic cells and radial arrangement of the parenchyma in the liver lobule. Figure 3The immunofluorescence assay using BRD4 and EEF2 dual antibodies ("BBB") can identify the phenotypes of different differentiation stages of liver cells in situ, and can also detect subtle differences between hepatocytes (parenchyma) and bile duct epithelial cells (interstitial region). Figure 3 "AA" indicates that the hepatocytes in the liver lobule parenchyma have large, polygonal nuclei, abundant anti-EEF2 (green) cytoplasmic signal, and strong anti-BRD4 (red) nuclear signal. The cells are arranged in a trabecular radial pattern, and the nuclei of BRD4-positive cells are in prophase (with nucleoli or chromosome filamentation stage); while some cells in the interstitial region are in metaphase and / or anaphase (...). Figure 4 "Zhong BB" ). Schematic diagram of the transverse and longitudinal sections of the liver ( Figure 4 The A1-A2 sections show the corresponding portal areas (PV areas, containing interlobular arteries and veins), and the spatial conformation of cells at different stages of differentiation in the parenchymal and interstitial regions. BRD4 and EEF2 double antibody-positive cells in both the parenchymal and interstitial regions show different differentiation characteristics of hepatic lobular trabecular cells and bile duct epithelial cells (most parenchymal cells are in prophase; some interstitial cells are in mid-to-late mitosis). Figure 4 "Zhong BB" ).

[0037] ICC has numerous subtypes, stemming from anatomical differences in the heterogeneity of bile duct cells in the interlobular ducts and larger bile duct cells in the porta hepatis. Heterogeneity of the columnar nuclei of the bile duct endothelium in adjacent tissues is an important marker of high malignancy in ICC, but molecular markers are lacking for rigorous evaluation. Figure 5 The phenotype and distribution of BRD4 and EEF2 double antibody-positive cells were shown in pathological sections from six different ICC cases. Figure 5 In the study, ICC cancer tissues were categorized as follows: A1 - women aged 59 years; B1 - men aged 45 years; C1 - women aged 73 years; D1 - men aged 75 years; E1 - women aged 66 years; G1 - men aged 46 years. Different phenotypes were observed in cancerous and adjacent tissues (A2; B2; C2; D2; E2; G2). ICC-G1 (a 46-year-old male patient, T1 stage, clinical stage III / IV) exhibited a large number of asynchronous BRD4 red cell nuclei in its stroma. Figure 5 ICC-G1), with highly undifferentiated BRD4 and EEF2 double antibody-positive cells in the stroma adjacent to the cancerous tissue ( Figure 5 The patient, ICC-G2, was a male (T3, clinical stage III / IV) with intrahepatic bile duct hyperplasia covering the entire pathological section. The bile duct endothelium exhibited high heterogeneity. Figure 6 High magnification of box A in the middle: A' and A"), the bile duct epithelium of the large and small bile ducts in the adjacent tissue also showed high heterogeneity ( Figure 6The B1-B2 and B1'-B2' cells showed different nuclear types of BRD4 red cells, indicating high heterogeneity of BRD4 and EEF2 double antibody-positive cells, consistent with tumor stage, and possessing potential diagnostic value in detecting different cell subtypes and malignant progression of ICC tumors. Figure 6 ).

[0038] Figure 7 The results of statistical analysis of cell phenotypes and clinical Kaplan-Meier survival analysis in ICC tissues are shown. (BRD4) + EEF2 + Significantly different expression patterns were observed in both whole-cell and stromal cell phenotypes of ICC cancer tissue and adjacent normal tissue, similar to BRD4. + CK19 + Consistent with, or may have earlier hazard ratio (HR) predictive power (median in the high-expression group earlier than 20 months), Figure 7 A: Whole cell total; Figure 7 (B: Interstitial SA). Specifically, the proportion of BRD4 and EEF2-expressing cells was significantly increased in cholangiocarcinoma cells. BRD4 and EEF2-expressing cells mainly corresponded to EEF2-expressing cells in the interstitial region (blue), significantly higher than the proportion of cholangiocarcinoma epithelial cells expressed by CK19 (bright blue). The proportion of cholangiocarcinoma epithelial cells expressing CK19 was 2.5 times higher in intrahepatic cholangiocarcinoma and normal tissue compared to normal tissue. + EEF2 + Double-positive cells were 4-10 times higher in ICC than in normal / adjacent cells. Clinical survival-related cluster analysis showed that the T (median cutoff point of BRD4-EEF2 marker, which is closely related to primary tumors and has equivalent diagnostic value as a CK19 marker) was <20 months.

[0039] Figure 8 Display: BRD4 + EEF2 + Cells in whole tissues and CK19 + The proportion of cells (bile duct epithelial clusters) is similar to that of CK19, accounting for a similar percentage of BRD4. + 76.5% in isolated cells, while BRD4 was present in CK19- cells (poorly differentiated stem cell clusters). + EEF2 + The cell percentage is BRD4 + 60% alone, showing CK19 - The high heterogeneity of poorly differentiated cells provides a highly sensitive and specific molecular diagnostic approach for the early diagnosis of liver tumors, and offers a feasible basis for the formulation of targeted molecular immunotherapy regimens, the screening of targeted drugs, and the evaluation of treatment efficacy such as drug resistance.

[0040] Table 6. Results of ICC Clinical Sample Analysis The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of reagents for detecting BRD4 and EEF2 expression levels in the preparation of products for assessing postoperative survival in patients with intrahepatic cholangiocarcinoma.

Images