Multiple biomarkers for diagnosing biliary tract cancer and pharmaceutical composition for preventing or treating biliary tract cancer

Abstract

The present invention relates to a multi-biomarker for diagnosing biliary tract cancer and a use thereof, and, more specifically, to: a composition for diagnosing biliary tract cancer, a kit and a method for diagnosing biliary tract cancer, which comprise an agent capable of measuring the levels of TAGLN2 and CA19-9 proteins or the expression levels of genes encoding same; a pharmaceutical composition for preventing or treating biliary tract cancer, comprising a TAGLN2 inhibitor and a CA19-9 inhibitor; and a method for preventing or treating biliary tract cancer. The biomarker TAGLN2 according to the present invention enables biliary tract cancer to be diagnosed with high specificity and sensitivity, and enables biliary tract cancer to be diagnosed with increased accuracy when combined with CA19-9. Furthermore, TAGLN2 expression in the stroma can predict the prognosis of biliary tract cancer patients.

Description

Multiple biomarkers for diagnosing biliary tract cancer and pharmaceutical compositions for preventing or treating biliary tract cancer

[0001] The present invention relates to a multiple biomarker for diagnosing bile duct cancer and its use, and more particularly, to a multiple biomarker for diagnosing bile duct cancer including Transgelin-2 (TAGLN2) and carbohydrate antigen 19-9 (CA19-9), a composition for diagnosing bile duct cancer including an agent capable of measuring the level of TAGLN2 or CA19-9 protein or the expression level of a gene encoding the same, a kit, a method for diagnosing bile duct cancer, and a method for screening drugs for preventing or treating bile duct cancer.

[0002]

[0003] Biliary tract cancer (BTC) is a relatively rare but malignant gastrointestinal cancer with a high mortality rate (Khan SA, et al., Gut. 2012;61(12):1657-69), and surgical resection is the only treatment (Nimura Y., HPB (Oxford). 2008;10(3):183-5). Even after complete resection, the recurrence rate is high in resected patients with BTC (Igami T, et al., J Hepatobiliary Pancreat Sci. 2010;17(4):449-54), and the 5-year survival rate is low at 20-30% (Klempnauer J, et al., Cancer. 1997;79(1):26-34). Furthermore, most tumors are metastatic at the time of diagnosis, making standard treatment difficult (Zhu AX., Best Pract Res Clin Gastroenterol. 2015;29(2):355-61). Therefore, for patients with unresectable or metastatic biliary tract cancer, adjuvant treatment other than systemic chemotherapy or radiotherapy is the only option. For these patients, the combination of gemcitabine and cisplatin is recommended as first-line treatment. This combination improves progression-free survival and overall survival, but the median overall survival for metastatic biliary tract cancer does not exceed 1 year (Valle JW, et al., Ann Oncol. 2014;25(2):391-8).

[0004] Cancer stem cells (CSCs) play a crucial role in tumor initiation, progression, and recurrence. Because most CSCs exhibit epithelial-mesenchymal transition (EMT), CSCs in biliary tract cancer are characterized by high levels of desmoplasia (fibrosis), morphological heterogeneity, aggressiveness, and chemotherapy resistance. Previous studies have demonstrated that CSCs in biliary tract cancer contribute to poor response to chemotherapy and high recurrence rates (Cardinale V, et al., Am J Pathol. 2015;185(6):1724-39). The discovery of specific CSC markers for biliary tract cancer is a dynamic area of ​​research. Several potential markers have been proposed, including CD24, CD44, EpCAM, CD133, and aldehyde dehydrogenase 1. However, the typical heterogeneity and cellular plasticity of CSCs hinder the application of these markers (Olempska M, et al., Hepatobiliary Pancreat Dis Int. 2007;6(1):92-7). Therefore, identifying representative CSC markers for biliary tract cancer and understanding the characteristics of CSCs (particularly EMT) in biliary tract cancer are crucial for developing targeted therapies to improve chemosensitivity and inhibit EMT.

[0005] Several studies have explored the potential of sphere formation, a well-established method for maintaining cells with stem cell-like properties (Leng Z, et al., PLoS ONE. 2013;8(2):e56082). In a previous study, we performed cDNA microarray analysis using adherent and spheroid cells from the human cholangiocarcinoma cell lines SNU1196 and SNU245 to assess the unique molecular patterns of cholangiocarcinoma CSCs and identified 70 genes (with a fold change (FC) of 1.2 in spheroids and 2 in cholangiocarcinomas) (Jung DE, et al., Sci Rep. 2017;7(1):10921). Among the identified genes, Transgelin-2 (TAGLN2) was overexpressed in both cholangiocarcinoma and CSCs. TAGLN2, an actin-binding protein highly expressed in tumor cells, plays a crucial role in determining cell morphology and transformation (Shapland C, et al., J Cell Biol. 1993;121(5):1065-73) and is implicated in various human malignancies (Wang L, et al., Endocr Relat Cancer 2023, 30(1)). Several studies have reported that it is involved in metastasis through direct interaction with cytoplasmic actin or induction of expression of metastasis-related genes (Ji C, et al., Clin Transl Oncol. 2023;25(12):3405-19). A recent study exploring the role of TAGLN2 in cancer demonstrated an association with multidrug resistance and metastasis in breast cancer, highlighting the potential of TAGLN2-targeted therapy (Jo JH, et al., Int J Mol Sci 2019, 20(3)). Additionally, TAGLN2 has been identified as an oncogene associated with prognosis and immunity in various cancers (Pan T, et al., J Cancer. 2023;14(10):1809-36), and has also been shown to be involved in tumor proliferation and migration in colon cancer (Zhao Z, et al., Oncol Lett. 2021;22(4):737).

[0006] In one embodiment of the present invention, it was confirmed that TAGLN2 expression was increased in both cancer tissues and serum of patients with biliary tract cancer, suggesting that TAGLN2 is a diagnostic marker and therapeutic target for biliary tract cancer targeting EMT and CSC.

[0007]

[0008] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0009]

[0010] Summary of the invention

[0011] The purpose of the present invention is to provide a biomarker capable of diagnosing bile duct cancer with high specificity and sensitivity.

[0012] Another object of the present invention is to provide a composition and kit for diagnosing bile duct cancer, a method for diagnosing bile duct cancer, and a method for providing information for diagnosing bile duct cancer, which can diagnose bile duct cancer with high specificity and sensitivity.

[0013] Another object of the present invention is to provide a method for screening drugs for preventing or treating bile duct cancer and a composition for predicting the prognosis of bile duct cancer through measurement of the level of the biomarker.

[0014]

[0015] To achieve the above purpose, the present invention provides a composition and kit for diagnosing bile duct cancer, including a preparation capable of measuring the level of TAGLN2 protein or the expression level of a gene encoding it, and a preparation capable of measuring the level of CA19-9 protein or the expression level of a gene encoding it.

[0016] The present invention also provides a method for diagnosing bile duct cancer and a method for providing information for diagnosing bile duct cancer, comprising the following steps: (a) measuring the level of TAGLN2 protein or the expression level of the gene encoding it and the level of CA19-9 protein or the expression level of the gene encoding it from a sample isolated from a subject; and (b) comparing the level of the protein or the expression level of the gene encoding it with a control group.

[0017] The present invention also provides a method for screening drugs for preventing or treating biliary tract cancer, comprising the steps of: (a) treating a sample isolated from a subject or an animal model of biliary tract cancer with a candidate drug; and (b) measuring the level of TAGLN2 protein or the expression level of a gene encoding it and the level of CA19-9 protein or the expression level of a gene encoding it in the sample or animal model treated with the candidate drug.

[0018] The present invention also provides a composition for predicting the prognosis of biliary tract cancer, comprising a preparation capable of measuring the level of TAGLN2 protein or the expression level of a gene encoding it and a preparation capable of measuring the level of CA19-9 protein or the expression level of a gene encoding it.

[0019]

[0020] Figure 1 shows the results of confirming TAGLN2 expression in serum samples from patients with bile duct cancer.

[0021] Figure 2 shows the results confirming that TAGLN2 is overexpressed in the blood of patients with biliary tract cancer. (A) Dot plots of serum CA19-9 levels in normal controls and patients with biliary stones and biliary tract cancer. (B) Dot plots of TAGLN2 expression in the serum of normal controls and patients with biliary stones and biliary tract cancer. (C) Receiver Operating Characteristic (ROC) curves for TAGLN2, CA19-9, and their mathematical combinations in patients with biliary tract cancer and benign disease. (D) Distribution of TAGLN2 and CA19-9 levels in patients with benign disease (black dots) and patients with biliary tract cancer (red dots). The optimal cutoff levels for CA19-9 and TAGLN2 were 37 U / mL and 1061.9 pixels, respectively.

[0022] Figure 3 shows the results of confirming TAGLN2 expression in biliary tract cancer patient tissues and cancer-associated fibroblasts. (A) TAGLN2 expression in normal human tissues assessed using immunohistochemistry (IHC). (B) Images showing TAGLN2 expression in cancer cells and stromal regions of biliary tract cancer surgical tissues. (C) Kaplan-Meier analysis of disease-free survival (DFS) and overall survival (OS) according to TAGLN2 IHC expression indices in cancer and stroma, respectively. (D) Immunofluorescence staining shows TAGLN2 overexpression in the stroma of biliary tract cancer tissues, and its location coincides with α-SMA expression. (E) TAGLN2 expression in cancer-associated fibroblasts (CAFs) of biliary tract cancer assessed using immunofluorescence staining. Typical morphology of fibroblasts in bright field, and immunofluorescence staining studies show expression of α-SMA and FAP, and underexpression of the epithelial marker CK-7.

[0023] Figure 4 shows the expression of TAGLN2 in normal and bile duct cancer patient tissues. (A) TAGLN2 expression in normal human tissues assessed by immunohistochemical staining (40x magnification). (B) TAGLN2 expression in cancerous and adjacent normal tissues from bile duct cancer surgery.

[0024] Figure 5 shows the results of TAGLN2 expression in cancer tissue and stroma of bile duct cancer patients (100x, 200x magnification).

[0025] Figure 6 shows the expression of TAGLN2 in biliary tract cancer patient tissues and cancer-associated fibroblasts. (A) Immunofluorescence staining of biliary tract cancer tissues revealed overexpression of TAGLN2 in the stroma of the tissues, and its location coincided with α-SMA expression (magnifications of 100x and 200x). (B) TAGLN2 expression in biliary tract cancer cell aggregates (CAF) assessed using immunofluorescence staining (magnifications of 400x); α-SMA, alpha-smooth muscle actin.

[0026]

[0027] Detailed description of the invention and preferred embodiments

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0029]

[0030] In the concentration range described herein, “within” is used to mean including (above and below) both critical ranges, and when both critical ranges are not included, the concentration range is described as “over” and “below.” As used in numerical values ​​herein, “about” is used to mean including a range that is expected to exhibit substantially the same effect as the numerical value described by a person skilled in the art, and may be, for example, ±20%, ±10%, ±5%, etc. of the described numerical value, but is not limited thereto.

[0031]

[0032] In this study, we evaluated TAGLN2 expression using Western blotting and immunohistochemistry in samples obtained from patients with biliary tract cancer to verify its clinical applicability. TAGLN2 was highly expressed in biliary tract cancer tissues, particularly in tumor-associated fibroblasts within the stroma. Patients with low stromal immunohistochemical indices had longer disease-free survival compared to those with high stromal indices (11.5 months vs. 74 months, P=0.013). TAGLN2 expression was higher in the serum of patients with biliary tract cancer than in the serum of patients with benign disease. TAGLN2 had a higher area under the curve (0.901) than the tumor marker CA19-9 (0.799; P<0.001). TAGLN2 expression in patient tissues and serum suggests its potential as a secretory biomarker for biliary tract cancer.

[0033] Several markers expressed in CAFs have been reported as prognostic factors associated with biliary tract cancer survival. Previous studies have shown that high expression of IL-33 in both cancer cells and stromal CAFs is associated with improved 2-year survival in patients with biliary tract cancer (Yangngam S, et al., J Cancer. 2020;11(22):6571-81). Periostin, a matricellular protein expressed in α-SMA+ CAFs, has been reported as a poor prognostic factor in post-resection biliary tract cancer (Sirica AE, et al., Hepatology. 2014;59(6):2397-402). Stromal cell-derived factor-1, another marker associated with tumor fibrosis and EMT, has also been shown to be associated with reduced median survival in patients with biliary tract cancer (Okamoto K, et al., Int J Oncol. 2012;41(2):573-82). According to the present invention, TAGLN2 expression was increased in the stromal tissue surrounding cancer cells in patient tissues, and TAGLN2 expression in the stroma was associated with patient prognosis. Immunofluorescence staining analysis of patient tissues confirmed that TAGLN2 expression coincided with the expression of α-SMA, suggesting that TAGLN2 is expressed in CAFs. Furthermore, TAGLN2 expression in CAFs derived from biliary tract cancer patients was confirmed through immunofluorescence staining. TAGLN2 has been reported as a myCAF marker (Elyada E, et al., Cancer Discov. 2019;9(8):1102-23), and this study reported the association between TAGLN2 expression in CAFs and CAFs in biliary tract cancer. In the present invention, increased TAGLN2 expression in CAFs was associated with a poor prognosis in biliary tract cancer patients.

[0034] In the present invention, we demonstrated that serum TAGLN2 levels were significantly increased in patients with biliary tract cancer. Compared with the tumor marker CA19-9, TAGLN2 showed a higher receiver operating characteristic (ROC) area under the curve (AUC) in patients with biliary tract cancer than in patients with normal or benign disease (TAGLN2 vs. CA19-9: 0.901 vs. 0.799, P=0.026). When combined with CA19-9, the ROC AUC increased to 0.948. These results suggest that TAGLN2 can be used as a diagnostic marker for biliary tract cancer. Notably, TAGLN2 levels were elevated above the threshold in 37 of 44 CA19-9-negative patients (84.1%), suggesting that TAGLN2 can overcome the low sensitivity of CA19-9.

[0035]

[0036] Accordingly, the present invention relates, in one aspect, to a combination of biomarkers for diagnosing bile duct cancer, including TAGLN2 and CA19-9.

[0037] From another aspect, the present invention relates to a composition for diagnosing bile duct cancer, comprising a preparation capable of measuring the level of TAGLN2 protein or the expression level of a gene encoding it and a preparation capable of measuring the level of CA19-9 protein or the expression level of a gene encoding it.

[0038] In the present invention, the agent capable of measuring the protein level may be characterized by being selected from the group consisting of antibodies, oligopeptides, PNA (peptide nucleic acid), metal nanoparticles, and aptamers that specifically bind to each protein, but is not limited thereto.

[0039] In the present invention, the antibody includes all "antibodies" such as polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, and the term "antibody" refers to a specific protein molecule directed against an antigenic site. Polyclonal antibodies can be produced by methods well known in the art, including injecting an animal with the diagnostic biomarker protein for ovarian cancer of the present invention as an antigen and collecting blood from the animal to obtain serum containing the antibody. Such polyclonal antibodies can be produced from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, mice, rats, cows, and dogs. Monoclonal antibodies can be produced using hybridoma methods well known in the art (see Kohler and Milstein (1976) European Jounral of Immunology 6:511-519) or phage antibody library techniques (Clackson et al, Nature, 352:624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991). Antibodies produced by the above methods can be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. In addition, the antibody of the present invention includes not only a complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of antibody molecules. Functional fragments of antibody molecules mean fragments that have at least an antigen-binding function, such as Fab, F(ab'), F(ab') 2, and Fv.

[0040] These antibodies are generally quantitatively analyzed by colorimetric reaction using a secondary antibody conjugated with an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) and its substrate, or quantitative analysis can be performed directly using an AP or HRP enzyme conjugated to the protein monoclonal antibody.

[0041] In the present invention, the “PNA (Peptide Nucleic Acid)” is an artificially synthesized polymer similar to DNA or RNA, having an N-(2-aminoethyl)-glycine backbone linked by peptide bonds. PNA has enhanced binding affinity and stability to DNA or RNA, and is used in diagnostic analysis.

[0042] In the present invention, the “aptamer” may be an oligonucleotide or peptide molecule, and may be characterized by specifically binding to a target.

[0043] In the present invention, the measurement of the protein level means confirming the presence and expression level of TAGLN2 and CA19-9, which are diagnostic markers for bile duct cancer of the present invention, in a biological sample in order to diagnose the disease of the present invention.

[0044] In the present invention, methods for measuring or comparing protein levels include, but are not limited to, western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complete fixation assay, FACS, protein chip, mass spectrometry, etc.

[0045] In the present invention, the mass spectrometry can be classified into, for example, FAB, CI, APCI, ESI, DESI, MALDI, SELDI, ICP, DESI, SESI, LAESI, FD, FAB, DIOS, DART, SIMS, TIMS, etc. according to the ionization method, into, for example, a quadrupole mass filter, an ion trap, an electron multiplier, etc. according to the mass selection method, and into, for example, gas chromatography (GS), liquid chromatography (LC), etc. according to the combination with the separation technology, and data derived from the mass spectrometry can be expressed in a method such as SIM, TIC, SRM, and each classification is combined and named. Specific examples include, but are not limited to, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDITOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, liquid chromatography-mass spectrometry (LC-MS), and LCMS / MS (liquid chromatography-mass spectrometry / mass spectrometry).

[0046] In the present invention, the agent capable of measuring the expression level of the gene may be characterized by being selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically bind to a gene encoding each protein, but is not limited thereto.

[0047] In the present invention, the term "primer" refers to a short nucleic acid sequence having a short free 3'-terminal hydroxyl group, which can form base pairs with a complementary template and functions as a starting point for template replication. In the present invention, the occurrence of biliary tract cancer can be diagnosed by performing PCR amplification using sense and antisense primers and determining whether a desired product is produced. PCR conditions and the lengths of the sense and antisense primers can be modified based on those known in the art.

[0048] The term "probe" in the present invention refers to a nucleic acid fragment, such as RNA or DNA, ranging from a few bases to several hundred bases in length, capable of specifically binding to mRNA, and labeled to enable detection of the presence or absence of a specific mRNA. The probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc.

[0049] In the present invention, the term "diagnosis" refers to accurately determining the condition of a subject with respect to a specific disease or condition. For example, the condition of a subject with respect to a specific disease or condition is used in a broad sense, including not only susceptibility to a specific disease or condition, determination of the disease the subject is currently suffering from, but also confirmation of the characteristics of the disease, such as prognosis, identification of cancer status, determination of cancer stage, or prediction of cancer sensitivity and responsiveness to treatment, obtaining a basis for appropriate treatment according to the patient's disease and condition, such as confirming the condition of the subject to confirm the therapeutic effect of a specific drug, and further predicting and confirming the presence or absence of recurrence in a subject who has been cured of a specific disease or condition. In the present invention, the diagnosis is preferably to confirm whether or not a disease has developed or is likely to develop.

[0050] In the present invention, the term "prognosis" means an expectation of medical outcome (e.g., long-term survival possibility, disease-free survival rate, etc.), and includes a positive prognosis (positive prognosis) or a negative prognosis (negative prognosis), wherein the negative prognosis includes disease progression or mortality such as recurrence, tumor growth, metastasis, drug resistance, etc., and the positive prognosis includes disease remission such as disease-free state, improvement or stabilization such as tumor regression, etc.

[0051] In the present invention, the term "prediction" means to guess in advance about the medical outcome, and for the purpose of the present invention, it means to guess in advance the course of a patient diagnosed with bile duct cancer (progression, improvement, recurrence, tumor growth, drug resistance).

[0052]

[0053] In another aspect, the present invention relates to a kit for diagnosing bile duct cancer, comprising a preparation capable of measuring the level of TAGLN2 protein or the expression level of a gene encoding it and a preparation capable of measuring the level of CA19-9 protein or the expression level of a gene encoding it.

[0054] In the context of the present invention, definitions and implementation examples of terms not described may share the same characteristics as those described in the context of the composition for diagnosing bile duct cancer, unless otherwise stated.

[0055] In the present invention, the bile duct cancer diagnostic kit may be characterized by including the bile duct cancer diagnostic composition of the present invention.

[0056] In the present invention, the bile duct cancer diagnostic kit may include one or more types of other component compositions, solutions, or devices suitable for the analysis method. For example, it may be an RT-PCR kit, a DNA chip kit, a protein chip kit, a rapid kit, or an SRM (selected reaction monitoring) / MRM (multiple reaction monitoring) kit.

[0057] The above RT-PCR kit may include, in addition to each primer pair specific for a marker gene, a test tube or other appropriate container, a reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor, DEPC water, sterile water, etc. In addition, it may include a primer pair specific for a gene used as a quantitative control. The DNA chip kit includes a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative structural gene or a fragment thereof.

[0058] In addition, the kit according to the present invention may be a diagnostic kit including a preparation for measuring the protein level, wherein the preparation for measuring the protein level may preferably be an antibody specific for the protein. Accordingly, the diagnostic kit including the preparation for measuring the protein level may be, for example, a diagnostic marker detection kit including the essential elements necessary for performing an ELISA, and such a kit may also include reagents capable of detecting antibodies that have formed an "antigen-antibody complex", such as a labeled secondary antibody, chromophores, enzymes (e.g., conjugated to antibodies), and substrates thereof. In addition, an antibody specific for a quantitative control protein may be included.

[0059] Additionally, the amount of antigen-antibody complex formed can be quantitatively measured through the size of the signal of the detection label. Such detection labels can be selected from the group consisting of, but not necessarily limited to, enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioisotopes.

[0060] The above-mentioned SRM (selected reaction monitoring), also called MRM (multiple reaction monitoring), is a method used in tandem mass spectrometry and is used for targeted quantitative proteomic analysis. SRM used for targeted quantitative proteomic analysis is described in detail in Nature Methods. 9 (6): 555-566.

[0061]

[0062] In another aspect, the present invention relates to a method for diagnosing bile duct cancer or a method for providing information for diagnosing bile duct cancer, comprising the steps of: (a) measuring the level of TAGLN2 protein or the expression level of the gene encoding it and the level of CA19-9 protein or the expression level of the gene encoding it from a sample isolated from a subject; and (b) comparing the protein level or the expression level of the gene encoding it with a control group.

[0063] In the present invention, it may be characterized by further including a step of determining that it is bile duct cancer when the protein level or the expression level of the gene encoding it is higher than that of the control group.

[0064] In the present invention, the protein level is determined by protein mass spectrometry, protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry), Western blot, ELISA (Enzyme Linked Immunoassay) The expression level of the gene may be measured by a method selected from the group consisting of, but not limited to, PCR, RT-PCR, NGS (Next Generation Sequencing), Northern blot, and DNA chip, and the expression level of the gene may be measured by a method selected from the group consisting of, but not limited to, immunosorbent assay, fluorescence amplification method, and Raman spectroscopy.

[0065] In the present invention, the increase in the expression level of each protein level or the gene encoding it is derived based on values ​​confirmed and verified in tissue samples of 41 biliary tract cancer patients, blood samples of 89 biliary tract cancer patients, 10 gallstone patients, and 40 normal control subjects in the examples of the present invention. It will be apparent to those skilled in the art that the increase in level may vary for each individual subject. Therefore, in the present invention, a step of interpreting the increase in the expression level of the protein level or the gene encoding it may be additionally included to accurately diagnose biliary tract cancer or to provide information for diagnosis.

[0066] In one embodiment of the present invention, if the expression level of TAGLN2 is 1061.9 pixels or more by Western blot analysis and the expression level of CA19-9 is 37 U / mL or more, it can be determined to be bile duct cancer.

[0067] In the present invention, the sample may be characterized by being selected from the group consisting of biliary tract tissue, biliary tract tissue-derived cells or cultures thereof, whole blood, serum, plasma, bile, lymph, extracellular vesicles, and urine, but is not limited thereto.

[0068]

[0069] In one embodiment of the present invention, it is apparent to those skilled in the art that the combination of biomarkers for diagnosing bile duct cancer can be used to diagnose whether a subject is suffering from bile duct cancer, and based on this, it can also be used to determine whether bile duct cancer is improved or treated in a patient already diagnosed with bile duct cancer.

[0070]

[0071] Therefore, from another aspect, the present invention relates to a method for screening drugs for preventing or treating biliary tract cancer, comprising the steps of (a) treating a sample isolated from a subject or an animal model of biliary tract cancer with a candidate drug; and (b) measuring the level of TAGLN2 protein or the expression level of a gene encoding it and the level of CA19-9 protein or the expression level of a gene encoding it in the sample or animal model treated with the candidate drug.

[0072] In the present invention, it may be characterized by further including a step of selecting a drug for preventing or treating bile duct cancer when the protein level or gene expression level decreases.

[0073] In terms of the drug screening method for preventing or treating biliary tract cancer of the present invention, definitions and implementation examples of terms not described may share the same characteristics as those described in terms of the above-described biliary tract cancer diagnosis or information providing method for diagnosis, unless otherwise described.

[0074]

[0075] In another aspect, the present invention relates to a composition for predicting the prognosis of biliary tract cancer, comprising a preparation capable of measuring the level of TAGLN2 protein or the expression level of a gene encoding it and a preparation capable of measuring the level of CA19-9 protein or the expression level of a gene encoding it.

[0076] In another aspect, the present invention relates to a method for predicting the prognosis of biliary tract cancer or a method for providing information for predicting the prognosis of biliary tract cancer, comprising the steps of (a) measuring the level of TAGLN2 protein or the expression level of the gene encoding it and the level of CA19-9 protein or the expression level of the gene encoding it from a sample isolated from a subject; and (b) comparing the level of the protein or the expression level of the gene encoding it with a control group.

[0077] In the present invention, the TAGLN2 and CA19-9 may preferably be characterized as being overexpressed in the stroma and / or CAF (cancer-associated fibroblast) of cancer tissue.

[0078] In terms of the method for predicting the prognosis of bile duct cancer or the method for providing information for predicting the prognosis of the present invention, definitions and implementation examples of terms that are not described may share the same characteristics as those described in terms of the above-mentioned method for diagnosing bile duct cancer or providing information for diagnosis, unless otherwise described.

[0079]

[0080] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0081]

[0082] Example 1: Materials and Methods

[0083] Example 1-1: Western blotting

[0084] Western blotting was performed as described in a previous study (Jung DE, et al., Sci Rep. 2017;7(1):10921). Patient blood samples were collected in 10 mL BD serum tubes and centrifuged at 3,000 × g for 20 min at 4°C. 3 μL of the supernatant serum sample was loaded. The primary antibody against TAGLN2 (Sigma-Aldrich, HPA001925) was used. The secondary antibody was goat anti-rabbit immunoglobulin G (IgG)-HRP (Santa Cruz Biotechnology; 1:5000). Proteins were visualized using Super Signal® West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). The expression of TAGLN2 protein in serum was expressed as a pixel area occupied by the visualized western blot bands in a given area based on densitometry using ImageJ software.

[0085]

[0086] Example 1-2: Patient

[0087] We analyzed tissue samples from 41 patients with biliary tract cancer obtained through surgical resection at Severance Hospital, Yonsei University College of Medicine, and 139 human blood samples from 89 patients with biliary tract cancer, 10 patients with biliary stones, and 40 normal controls. The study protocol was approved by the Institutional Review Board and Institutional Review Board of Severance Hospital, Yonsei University College of Medicine, Seoul, Korea (IRB approval code: 4-2011-0625; November 24, 2011). All procedures involving human participants were performed in accordance with the ethical standards of the IRB and the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Written informed consent was obtained from all subjects. Information on patient demographics and clinical data, including age at diagnosis, sex, tumor stage at diagnosis, and serum carcinoembryonic antigen (CEA) levels, was obtained from electronic medical records. Tumors were classified according to the 7th edition of the American Joint Committee on Cancer (AJCC) staging classification.

[0088]

[0089] Example 1-3: Immunohistochemical and immunofluorescence staining

[0090] Immunohistochemical and immunofluorescent staining were performed as described in a previous study (Jo JH, et al., Int J Mol Sci 2019, 20(3)). For immunohistochemical staining, tissue slides were deparaffinized in xylene and rehydrated in graded alcohols. Endogenous peroxidase activity was blocked with 0.3% (v / v) hydrogen peroxide in methanol. Antigen retrieval was performed by microwaving the slides in sodium citrate buffer (0.01 M, pH 6.0) for 5 minutes. To block nonspecific staining, sections were incubated with 10% (v / v) normal donkey serum for 1 hour, and then incubated overnight at 4°C with the appropriate antibodies. Subsequent reactions were performed using the Envision kit (Dako Cytomation California, Inc., Carpinteria, CA, USA) according to the manufacturer's instructions. Immunoreactions were developed with the DAKO Liquid diaminobenzidine substrate-chromogen system (DAB+) and counterstained with Harris hematoxylin (Sigma-Aldrich). Subsequently, the reaction was performed with the LSAB+ Kit (Dako), and the sections were counterstained with Mayer's hematoxylin, dehydrated, and observed under a BX51 microscope (Olympus, Tokyo, Japan). For immunofluorescence staining, tissue slides were visualized using Cy5-goat anti-rabbit IgG dissolved in antibody diluent and incubated for 30 min at room temperature. Three washes were performed with PBS on a rocking platform for 5 min each between each step. The slides were coverslipped using mounting medium for observing fluorescence with DAPI (Vecta shield H-1200; Vector Laboratories, Inc. Burlingame, CA, USA).Primary antibodies against TAGLN2 (Sigma-Aldrich), alpha-smooth muscle actin (α-SMA), fibroblast-associated protein (FAP), and cytokeratin-7 (CK-7) (Santa Cruz Biotechnology) were used.

[0091]

[0092] Example 1-4: Statistical Analysis

[0093] Categorical data are χ 2 and Fisher's exact tests were used for analysis. Student's t-test and Mann-Whitney U test were used for continuous variables. Survival rates were estimated and compared using Kaplan-Meier analysis with log-rank test. Serum TAGLN2 and CA19-9 levels were compared between patients with benign and biliary tract cancer using the nonparametric Kruskal-Wallis test. Cutoff values, receiver operating characteristic (ROC) curves, area under the ROC curve (AUC), and 95% confidence intervals (CI) were determined. All statistical analyses were performed using IBM SPSS Statistics for Windows (version 25.0; IBM Corp., Armonk, NY). A P value less than 0.05 was considered statistically significant.

[0094]

[0095] Example 2: TAGLN2 Overexpressed in Blood Samples from Patients with Biliary Tract Cancer

[0096] To confirm the potential of TAGLN2 as a secretory biomarker for biliary tract cancer, TAGLN2 was detected in serum samples from patients using Western blot analysis (Fig. 1). Baseline characteristics of the patients are shown in Table 1. A total of 139 participants (89 patients with biliary tract cancer, 10 patients with choledocholithiasis, and 40 normal controls) were included in the Western blot analysis. Among the biliary tract cancer patients, 29 (32.6%) had intrahepatic cholangiocarcinoma (IHCC), 28 (31.5%) had perihilar cholangiocarcinoma (PHCC), and 32 (36.0%) had distal common bile duct cancer (distal CBD cancer). At the time of blood sampling, the clinical stage was stage I in 6 patients (6.7%), stage II in 35 patients (39.3%), stage III in 29 patients (32.6%), and stage IV in 19 patients (21.3%).

[0097]

[0098] To evaluate the diagnostic significance of TAGLN2 compared with CA19-9, serum CA19-9 levels were compared between normal controls, patients with biliary stones, and biliary tract cancer. There was no significant difference in the mean serum CA19-9 level between normal controls (9.0±6.3 U / mL) and patients with biliary stones (12.1±6.7 U / mL) (P=0.194, Fig. 2A). In addition, patients with biliary tract cancer had significantly higher serum CA19-9 levels than patients with benign cancer (9.6±6.4 vs. 2417±5377 U / mL; P=0.0021, Fig. 2A). As shown in Fig. 2B, the mean level of serum TAGLN2 expression did not differ between normal controls (538±776 pixels) and patients with biliary stones (953±1087 pixels, P=0.171). Patients with biliary tract cancer had significantly higher serum TAGLN2 expression than patients with benign disease (622±851 vs. 3710±2568 pixels, P<0.0001). These data confirm that TAGLN2 is a secreted biomarker protein detectable in human blood and is overexpressed in patients with biliary tract cancer compared to healthy individuals or patients with benign disease.

[0099] To evaluate the diagnostic performance of serum TAGLN2 and CA19-9 levels in differentiating benign and biliary tract cancer samples, the area under the curve (AUC) was calculated using receiver operating characteristic (ROC) curves. For TAGLN2, the AUC was 0.901 (95% confidence interval (CI): 0.849–0.952), and for CA19-9, the AUC was 0.799 (95% CI: 0.725–0.872), showing a significant difference (P=0.026, Fig. 2C). When the optimal threshold was 1061.9 pixels, the sensitivity and specificity of TAGLN2 for differentiating biliary tract cancer from benign conditions were 0.865 and 0.857, respectively. For CA19-9, the sensitivity and specificity were 0.506 and 1.000, respectively, at a threshold of 37 U / mL. The ROC AUC (0.948; 95% CI: 0.914–0.983) of the combination of TAGLN2 and CA19-9 was significantly higher than that of CA19-9 alone (P<0.0001, Fig. 2C). Fig. 2D shows the distribution of TAGLN2 and CA19-9 levels in patients with benign disease and biliary tract cancer.

[0100]

[0101] Example 3: Confirmation of the correlation between TAGLN2 overexpression in biliary tract cancer tissue and TAGLN2 expression in stroma and patient survival.

[0102] TAGLN2 is weakly expressed in the small and large intestine, but its expression is rarely observed in other major organs. Therefore, before analyzing TAGLN2 expression in biliary tract cancer tissues, we analyzed TAGLN2 expression in normal organ tissues using immunohistochemistry (IHC; Figs. 3A and 4A). We also analyzed normal tissues adjacent to patient cancer tissues, and relatively high expression was observed in cancer tissues (Fig. 4B). Subsequently, we evaluated TAGLN2 expression in cancer cells and surrounding stroma in 41 surgical samples from biliary tract cancer patients using IHC. The intensity score was defined as no staining (0), light brown (1), brown (2), or dark brown (3), and the percentage score was defined as <10% (0), 10-25% (1), 25-50% (2), 50-75% (3), or >75% (4) (Figs. 3B and 5). The IHC score was calculated by multiplying the intensity and percentage scores. Patients were divided into low and high IHC score groups based on TAGLN2 expression in the tumor and stromal compartments. Patients with an IHC score >7 were classified into the high IHC group, and those with a score <7 were classified into the low IHC group.

[0103] Baseline characteristics of cancer and stromal IHC scores are described in Tables 2 and 3. Most of the 41 surgical samples had high cancer IHC scores (n=31, 75.6%), and no significant differences in baseline characteristics were observed between the low and high cancer IHC groups. Survival analysis using Kaplan-Meier plots showed no significant differences in disease-free survival (DFS) (7.3 months vs. 9.9 months, P=0.182) and overall survival (OS) (26.9 months vs. 21.4 months, P=0.171) between the low and high cancer IHC groups. TAGLN2 expression in stromal tissue was detected in 22 patients (53.7%) in the high stromal IHC group and 19 patients (46.3%) in the low stromal IHC group. No significant differences in baseline characteristics were observed between the low-stromal IHC and high-stromal IHC groups. Survival analysis showed a significantly longer DFS in the low-stromal IHC group compared to the high-stromal IHC group (11.5 months vs. 7.4 months, P = 0.013). OS did not differ significantly between the low-stromal IHC index and high-stromal IHC index groups (23.4 months vs. 19.0 months, P = 0.220) (Fig. 3C).

[0104]

[0105]

[0106] To evaluate TAGLN2 expression in the stroma of surgical tissues, immunofluorescence (IF) staining was performed on surgical tissues using TAGLN2 and α-SMA, representative markers of cancer-associated fibroblasts (CAFs). TAGLN2 was overexpressed in the stroma of cancer tissues, and its localization coincided with α-SMA expression (Fig. 3D, Fig. 6A). IF analysis confirmed that TAGLN2 was expressed in CAFs of biliary tract cancer tissues (Fig. 3E, Fig. 6B). CAFs exhibited a typical morphology in bright field and were characterized by the expression of representative CAF markers such as α-SMA and FAP, and the absence of CK-7, an epithelial marker. These results suggest that TAGLN2 is expressed in CAFs of biliary tract cancer, and its overexpression is associated with patient survival.

[0107]

[0108] According to the present invention, the biomarker TAGLN2 enables the diagnosis of biliary tract cancer with high specificity and sensitivity, and when combined with CA19-9, the diagnosis can be further improved with increased accuracy. Furthermore, TAGLN2 expression in the stroma can predict the prognosis of biliary tract cancer patients.

[0109]

[0110] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for diagnosing bile duct cancer, comprising a preparation capable of measuring the level of TAG LN2 protein or the expression level of a gene encoding it and a preparation capable of measuring the level of CA19-9 protein or the expression level of a gene encoding it.

2. A composition for diagnosing bile duct cancer, characterized in that in paragraph 1, the agent capable of measuring the protein level is selected from the group consisting of antibodies, oligopeptides, PNA (peptide nucleic acid), metal nanoparticles, and aptamers that specifically bind to each protein.

3. A composition for diagnosing bile duct cancer, characterized in that in paragraph 1, the agent capable of measuring the expression level of the gene is selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically bind to a gene encoding each protein.

4. A kit for diagnosing bile duct cancer, comprising a preparation capable of measuring the level of TAG LN2 protein or the expression level of a gene encoding it and a preparation capable of measuring the level of CA19-9 protein or the expression level of a gene encoding it.

5. Information provision method for diagnosing bile duct cancer, including the following steps: (a) a step of measuring the TAGLN2 protein level or the expression level of the gene encoding it and the CA19-9 protein level or the expression level of the gene encoding it from a sample isolated from the subject; and (b) A step of comparing the protein level or the expression level of the gene encoding it with a control.

6. A method for providing information for diagnosing bile duct cancer, characterized in that in paragraph 5, it further includes a step of determining bile duct cancer when the protein level or the expression level of the gene encoding it is higher than that of the control group.

7. In the fifth paragraph, the protein level is determined by protein mass spectrometry, protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry), Western blot, ELISA (Enzyme Linked Immunoassay) An information providing method for diagnosing bile duct cancer, characterized in that the expression level of the gene is measured by a method selected from the group consisting of immunosorbent assay, fluorescence amplification method, and Raman spectroscopy, and the expression level of the gene is measured by a method selected from the group consisting of PCR, RT-PCR, NGS (Next Generation Sequencing), Northern blot, and DNA chip.

8. An information providing method according to claim 5, wherein the sample is selected from the group consisting of biliary tract tissue, biliary tract tissue-derived cells or cultures thereof, whole blood, serum, plasma, bile, lymph, extracellular vesicles, and urine.

9. A method for screening drugs for the prevention or treatment of bile duct cancer, comprising the following steps: (a) a step of treating a sample isolated from the subject or an animal model of biliary tract cancer with a candidate drug; and (b) a step of measuring the TAGLN2 protein level or the expression level of the gene encoding it and the CA19-9 protein level or the expression level of the gene encoding it in a sample or animal model treated with the candidate drug.

10. A screening method characterized in that, in claim 9, it further comprises a step of selecting a drug for preventing or treating bile duct cancer when the protein level or gene expression level decreases.

11. A composition for predicting the prognosis of biliary tract cancer, comprising a preparation capable of measuring the level of TAGLN2 protein or the expression level of a gene encoding it and a preparation capable of measuring the level of CA19-9 protein or the expression level of a gene encoding it.

12. A composition for predicting the prognosis of bile duct cancer, characterized in that in claim 11, TAGLN2 and CA19-9 are overexpressed in the stroma and / or cancer-associated fibroblast (CAF) of cancer tissue.

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