KR102219112B1 - A method of diagnosing cholangiocarcinoma - Google Patents

Abstract

The present invention relates to a method for determining benign biliary disease patients, pancreatic cancer and biliary tract cancer by measuring GCA (glycocholic acid) and TCDCA (Taurochenodeoxycholic acid) in patients with biliary tract cancer, and in detail, in patients with benign biliary tract disease, pancreatic cancer, and biliary tract cancer. As a result of measuring GCA (glycocholic acid) and TCDCA (Taurochenodeoxycholic acid) in bile, it was confirmed that biliary cancer patients had high GCA (glycocholic acid) and low TCDCA (Taurochenodeoxycholic acid), and GCA (glycocholic acid) and TCDCA (Taurochenodeoxycholic acid). ) Is a biomarker that can distinguish patients with biliary tract cancer among patients with benign biliary tract disease, pancreatic cancer, and biliary tract cancer. It relates to the screening method of.

Description

{A method of diagnosing cholangiocarcinoma}

The present invention relates to a method for determining benign biliary disease patients, pancreatic cancer and biliary tract cancer by measuring GCA (glycocholic acid) and TCDCA (Taurochenodeoxycholic acid) in patients with biliary tract cancer, and in detail, in patients with benign biliary tract disease, pancreatic cancer, and biliary tract cancer. As a result of measuring GCA (glycocholic acid) and TCDCA (Taurochenodeoxycholic acid) in bile, it was confirmed that biliary cancer patients had high GCA (glycocholic acid) and low TCDCA (Taurochenodeoxycholic acid), and GCA (glycocholic acid) and TCDCA (Taurochenodeoxycholic acid). ) Is a biomarker that can distinguish patients with biliary tract cancer among patients with benign biliary tract disease, pancreatic cancer, and biliary tract cancer. It relates to the screening method of.

Biliary duct cancer (Cholangiocarcinoma; CCA) is one of the malignant tumors of the bile duct epithelium. It is difficult to diagnose because it is location-specifically located deep in the body, and the initial symptoms are minimal. Therefore, it is difficult to diagnose biliary tract cancer early and it belongs to carcinoma with a very high mortality rate. According to a recent statistical survey, the 5-year survival rate of biliary tract cancer is only 25%, and if the patient is in stage 3 or 4, it is 10% or 0%, respectively. In the United States, more than 7,000 patients with biliary tract cancer have died each year since 2013, and the mortality rate has also increased by 36% from 1999 to 2014. Therefore, it is essential to develop a biomarker that can accurately diagnose biliary tract cancer.

Carbohydrate antigen 19-9 (CA19-9) or carcinoembryonic antigen (CEA) are the most commonly used biliary tract cancer biomarkers in clinical practice. However, by using the biomarker, the ability to distinguish between biliary cancer patients and normal persons is excellent, but it is difficult to distinguish between biliary tract cancer, benign biliary disease and pancreatic cancer. In order to solve the above problems, biomarker studies related to biliary tract cancer have been conducted. For example, MicroRNA-21 (miR-21) has been studied as a representative transcriptional biomarker for biliary tract cancer. MicroRNA-21 is highly sensitive and is associated with tumor growth in biliary tract cancer, but is tissue specific and increases in various diseases. Among the proteome biomarkers that distinguish biliary tract cancer, include insulin-like growth factor-1 and elastase. However, these biomarkers are not specific to other diseases, or most of them have not been investigated in the mechanism of the invention of biliary tract cancer. Therefore, there are few studies related to metabolic biomarkers in biliary tract cancer compared to transcriptional or proteomic biomarkers. The new metabolic biomarkers are more powerful than other markers because they accurately express the molecular phenotype. The present inventors discovered a metabolic biomarker related to this while studying a specific metabolic biomarker that specifically occurs more or less in the bile of biliary tract cancer patients than in patients with benign biliary tract disease and pancreatic cancer.

Unlike other biological fluids such as serum or urine, bile is located near a biliary tumor, which increases the probability of detection of biliary cancer-specific biomarkers. Certain bile acids act as signaling molecules involved in the invention of biliary tract cancer. Therefore, in order to discover potential biomarkers of biliary tract cancer, bile acids have been analyzed in several studies.

The proportion of deoxycholic acid (DCA) in patients with biliary tract cancer was lower than those of biliary stones or normal subjects. In another study group, the ratio of cholic acid (CA) and kenodeoxy cholic acid (CDCA) was higher in patients with biliary tract cancer than in patients with benign biliary tract disease or hepatocellular carcinoma (HCC). Biliary tract cancer patients had higher levels of bile acids combined with taurine and glycine compared to patients diagnosed with benign biliary tract cancer. However, this study failed to analyze bile acid samples or types, and did not interpret the role of specific bile acid biomarkers in the development of biliary tract cancer.

While studying biomarkers that can distinguish patients with benign biliary disease, pancreatic cancer, and biliary tract cancer, the researchers analyzed bile acids in patients with biliary tract cancer. As a result, the bile acids of biliary tract cancer patients had different glycocholic acid (GCA). It was confirmed that the disease patients and pancreatic cancer patients were higher, and that TCDCA (Taurochenodeoxycholic acid) was lower, and the present invention was completed.

The present invention was conceived to solve the above problem, and an object of the present invention is for diagnosis of biliary tract cancer comprising an agent detecting glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA) It is to provide a composition.

Another object of the present invention is to provide a kit for diagnosing biliary tract cancer comprising a quantification device for glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA).

Another object of the present invention is to provide a method of providing information on biliary tract cancer by measuring the amount of glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA).

The present invention may include an agent that detects glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA).

The agent to be detected includes, but is not limited to, substances used in antibody analysis, chemiluminescence analysis, or liquid chromatography mass spectrometry.

The present invention may also provide a kit for diagnosis of biliary tract cancer, including a quantitative device for glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA).

The quantification device may be a chromatography/mass spectrometer. Chromatography used in the present invention is liquid-solid chromatography (LSC), paper chromatography (PC), thin-layer chromatography (TLC), gas-solid chromatography ( Gas-Solid Chromatography (GSC), Liquid-Liquid Chromatography, LLC, Foam Chromatography (FC), Emulsion Chromatography (EC), Gas-Liquid Chromatography (Gas- Liquid Chromatography (GLC), Ion Chromatography (IC), Gel Filtration Chromatograhy (GFC), Liquid chromatography-mass spectrometry (LC-MS/MS), or Gel Permeation Chromatography, GPC) including, but not limited to, all quantitative chromatography commonly used in the art may be used.

The present invention can also provide a method of providing information for diagnosing biliary tract cancer comprising the following steps.

(a) measuring the amount of glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA) from the biological sample isolated from the patient; And

(b) The amount of glycocholic acid (GCA; glycocholic acid) or taurochenodeoxycholic acid (TCDCA; Taurochenodeoxycholic acid) of the patient was determined as glycocholic acid (GCA; glycocholic acid) or taurokenodioxycholic acid (TCDCA) of the normal control group. ; Taurochenodeoxycholic acid) compared to the amount.

The amount of glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA) can be measured by chromatography/mass spectrometry.

The biological sample may be bile.

If the amount of the glycocholic acid (GCA) is greater than that of the sample of the normal control group, it may be determined to be biliary tract cancer.

If the amount of taurochenodeoxycholic acid (TCDCA) is less than that of a sample from a normal control patient, it may be determined that it is biliary tract cancer.

The present invention may also provide information for diagnosing biliary tract cancer comprising the following steps.

(a) measuring Carbohydrate antigen 19-9 (CA19-9) or Carcinoembryonic antigen (CEA) from the biological sample isolated from the patient;

(b) comparing the level of carbohydrate antigen 19-9 (CA19-9) or carcinoembryonic antigen (CEA) of the patient with the level of carbohydrate antigen 19-9 (CA19-9) or carcinoembryonic antigen (CEA) of a normal sample;

(c) If the level of Carbohydrate antigen 19-9 (CA19-9) or Carcinoembryonic antigen (CEA) of the patient is higher than the level of Carbohydrate antigen 19-9 (CA19-9) or Carcinoembryonic antigen (CEA) of a normal sample Diagnosing a patient suspected of cancer, pancreatic cancer or benign biliary tract disease;

(d) measuring the amount of glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA) from a biological sample isolated from a patient suspected of biliary tract cancer, pancreatic cancer or benign biliary tract disease; And

(e) The amount of glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA) in the suspected biliary tract cancer patient was determined as glycocholic acid (GCA; glycocholic acid) in a sample of a patient with pancreatic cancer or benign biliary tract disease. ) Or taurochenodeoxycholic acid (TCDCA; Taurochenodeoxycholic acid).

If the amount of the glycocholic acid (GCA) is greater than that of a patient with pancreatic cancer or benign biliary tract disease, it may be determined as biliary tract cancer.

If the amount of taurochenodeoxycholic acid (TCDCA) is less than that of a patient with pancreatic cancer or benign biliary tract disease, it may be determined as biliary tract cancer.

The present invention can provide a method for screening a therapeutic agent for biliary tract cancer comprising the following steps.

(a) treating biliary tract cancer cells with a test substance; And

(b) measuring the amount of glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA) in the biliary tract cancer cells; And

(c) Selecting an experimental substance with reduced glycocholic acid (GCA) or increased taurochenodeoxycholic acid (TCDCA) compared to the control sample.

The present invention can provide a method for screening a therapeutic agent for biliary tract cancer comprising the following steps.

(a) treating biliary tract cancer cells with a test substance;

(b) measuring the expression level of an enzyme synthesizing glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA) or protein activity thereof in the biliary tract cancer cells; And

(c) the expression level of the enzyme synthesizing glycocholic acid (GCA) or the protein activity thereof is decreased compared to the control sample, or the expression level of the enzyme synthesizing taurochenodeoxycholic acid (TCDCA), or Selecting the test substance whose activity is increased.

As a result of comparing the bile acids of patients with biliary tract cancer, pancreatic cancer, and benign biliary tract cancer, the present inventors showed that the bile acids of biliary tract cancer patients had higher GCA (glycocholic acid) than other benign biliary disease patients and pancreatic cancer patients, and TCDCA (Taurochenodeoxycholic acid) was different than those of other benign biliary disease patients and patients with benign biliary tract cancer. It was confirmed that it was lower than that of patients with biliary tract disease and patients with pancreatic cancer, and eventually, GCA (glycocholic acid) and TCDCA (Taurochenodeoxycholic acid) have an effect that can distinguish patients with biliary tract cancer among pancreatic cancer, benign biliary tract patients, and biliary tract cancer patients.

1 is a total ion chromatogram separated from 15 kinds of bile acids, conditions of bile acids, and SRM chromatogram results.
2 is a result of comparing the concentration of total bile duct acid in patients with benign biliary tract disease, pancreatic cancer, and biliary tract cancer.
Figure 3 is a comparison result (A) of the composition ratio of bile acids in benign biliary tract disease of biliary tract cancer, a comparison result of the composition ratio of primary and secondary bile acids (B), non-conjugated damzic acid, taurine-binding bile acid and glycine-binding It is a comparison result (C) of bile acids.
4 is a result of comparing the composition ratio of GCA and TCDCA in patients with benign biliary tract disease, pancreatic cancer and biliary tract cancer.
5 is a result of comparing the relative gene expression of FXR, TGR5 and S1PR2 treated with GCA and TCDCA in biliary tract cancer cell lines.

Hereinafter, the present invention will be described in more detail.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1. Materials and reagents

Litocholic acid (LCA), ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), DCA, cholic acid (CA), GCA, taurodeoxycholic acid (TDCA) and Taurocholic acid (TCA) was purchased from Sigma-Aldrich. Glycolithocholic acid (GLCA), tauroursodeoxycholic acid (TUDCA) and taurochenodeoxycholic acid (TCDCA) were purchased from Carbosynth. Glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA) and taurolithocholic acid (TLCA) were purchased from Santa-Cruise. Glycoursodeoxycholic acid (GUDCA) was purchased from Achemblock. HPLC-grade water, methanol and acetonitrile were purchased from Deoksan.

Example 2. Patient and patient's bile juice collection

Bile samples were collected at Samsung Seoul Hospital. The research protocol was reviewed and approved by the Institutional Review Board of Samsung Medical Center. All experimental methods were performed in accordance with relevant guidelines and regulations. It consisted of 57 patients with benign biliary tract disease, 17 patients with pancreatic cancer, and 30 patients with biliary tract cancer. The bile samples were stored at -70 °C until the bile acid became.

Example 3. Preparation of bile acids

After thawing 30 μL of a bile sample on ice, the bile acid was extracted with 400 μL of chloroform and 200 μL of methanol. After mixing the mixture well, 120 μL of distilled water was added to the mixture. After vortexing the mixture and centrifugation for 5 minutes, 100 µl of the supernatant was transferred to a 1.5 ml microtube, and dried in a centrifugal vacuum concentrator. After drying was completed, bile acid was dissolved in 100 μL of 50% methanol before transferring to an HPLC autosampler vial for LC-MS/MS analysis.

Example 4. LC-MS / MS analysis

Bile acid (10 μL) re-suspended in methanol was injected into a Unison UK-C18 column (3 μm, ID 3 mm×100 mm). LC-MS/MS analysis was performed using a system consisting of Accela LC (ThermoFisher, MA, USA) and TSQ Quantum access max (ThermoFisher, MA, USA). Bile acids were separated in the analytical column at a flow rate of 400 μL/min. The LC slope method was set as follows: t=0 min, 35% B; t=2 min, 35% B; t=20 min, 50% B; t=30 min, 80% B; t=33 min, 80% B; t=35 min, 35% B; Solvent A contained 100% water and 0.1% (v/v) formic acid, and solvent B was 100% acetonitrile and 0.1% (v/v) formic acid. The mass spectrometer was operated in negative ion mode. Electric spray ionization spray voltage was used at 3 kV and the capillary temperature was 300°C. In order to separate 15 important bile acids in human bile, a reversed-phase LC method was developed based on SRM conditions (FIG. 1). An optimized LC-MS/MS method was used to quantify 15 important bile acids in bile of patients with benign biliary disease, pancreatic cancer and biliary tract cancer. The average concentration of bile acids was highest in pancreatic cancer patients (Fig. 2). In addition, the average concentration of total bile acids in benign biliary tract disease was twice that of the group of patients with biliary tract cancer. In patients with biliary tract cancer, when the concentration of bile bile acids is low, bile duct obstruction and inflammation may result in decreased bile acid excretion due to bile duct obstruction. Bile acids can be classified into synthetic (primary and secondary) and conjugated molecules (glycine, taurine and non-conjugated). The average ratio of secondary bile acids was found to be close to twice that in patients with benign biliary tract disease compared to pancreatic cancer and biliary tract cancer (Fig. 3). The average ratio of total unconjugated bile acids in patients with biliary tract cancer was 10 times lower than in patients with positive biliary tract. Unbound bile acids inhibit the growth of biliary cancer cell lines. Conjugated bile acids activate epidermal growth factor receptors and increase the expression of cyclooxygenase-2, a cold factor present in many cancers. It also contributes to the activation of NF-kB, which promotes the growth of biliary tract cancer cells and prevents cell death. It promotes the growth and invasiveness of biliary tract cancer through activation of S1PR2 rather than non-conjugated bile acids. Therefore, the increased proportion of bile acids encountered in biliary tract cancer pills is related to the pathogenesis of biliary tract cancer. 15 bile acids were quantitatively analyzed in patients with benign biliary tract disease, pancreatic cancer and biliary tract cancer (Table 1). The average composition of GCA was compared with benign biliary tract disease (22.3%, p <0.0001) and pancreatic cancer (19.9%, p<0.0001). Therefore, it was highest in biliary tract cancer (35.6%) (Fig. 4). In contrast to GCA, the average composition of TCDCA was significantly lower in patients with biliary tract cancer (7.31%) than those with benign biliary tract disease (12.45%, p = 0.001) and pancreatic cancer (13.82%, p = 0.002).

Example 5. Cell culture

The CCA cell line named SNU-245 was purchased from Korean Cell Line Bank37. Cells were moistened in RPMI-1640 medium containing 10% fetal bovine serum (Biowest, Nuaille, France) and 1 mg/mL gentamicin (Sigma Aldrich, MO, USA) for 5 days (Biowest, Nuaille, France) 37° C. in an incubator. It was cultured in an environment of 5% CO ₂ . GCA and TCDCA were each dissolved in 100 μL of DMSO (Sigma Aldrich, MO, USA) and added to the medium to a final concentration of 1.6 μmol/mL. As a control, 100 μL of DMSO was added to the medium without bile acid. After treatment with each bile acid, the cells were incubated for 48 hours.

Example 6. RNA preparation and qRT-PCR

Cells were harvested using trypsin (Biowest, Nuaille, France). Total RNA was isolated from the collected cells using the QIAGEN RNA mini kit (QIAGEN, Hilden, Germany). RNA concentration and purity were confirmed by spectrophotometer (ThermoFisher, MA, USA) and gel electrophoresis, respectively. Reverse transcription was performed with cDNA using M-MLV reverse transcriptase (Promega, WI, USA) and oligo(dT)primer (ThermoFisher, MA, USA). PCR primer sequences for FXR, S1PR2, TGR5 and β-actin were used previously reported primers, and β-actin was used as an internal control for RT-PCR. RT-PCR analysis was performed using LightCycler 480 II (Roche, Basel, Swiss) under the following conditions: denaturation at 95°C for 20 seconds, annealing at 60°C for 20 seconds, extension at 72°C for 20 seconds. Bile acids bind to bile acid receptors in bile duct cells that lead to several biological phenotypes. In particular, quantitative changes in bile acid receptors such as FXR, TGR5, and S1PR2 affect the development and activity of biliary cancer cells. Therefore, in order to verify the phenotypic effect of GCR and TCDCA, changes in the expression of FXR, TGR5 and S1PR2 genes by qRT-PCR were quantified (FIG. 5). Since TCDCA was selected as a negative biomarker for biliary tract cancer, it was expected that gene expression related to biliary tract cancer pathogenesis would decrease compared to GCA. FXR is a bile acid receptor mainly present in the liver and intestine and is a gene that mediates the pathogenesis of various biliary tract diseases, as well as bile acid transport and metabolism. Several studies have reported that low expression of FXR is associated with various biliary tract diseases caused by abnormal bile acid transport. In transcriptomic analysis, the expression of FXR gene in GCA-treated (0.17 fold less control) cell line was lower than that of control. Decreased FXR gene expression has been reported to cause human biliary cancer, suggesting that GCA is closely correlated with tumor differentiation. TGR5 gene expression was 8.55 times higher in GCA-treated biliary tract cancer cell lines than in control (p <0.0001) or TCDCA treated cells (p <0.0001). TGR5 is a G protein-coupled receptor present in cholangiocytes, is activated in response to bile acids and regulates the proliferation of bile duct cells. Overexpression of TGR5 in biliary tract cancer induces resistance to cell death and contributes to the pathogenesis of tumors. As the expression of TGR5 increased in the biliary tract cancer cell line treated with GCA, it was confirmed that GCA may have an effect on the development of biliary tract cancer. In GCA-treated biliary tract cancer cell lines, S1PR2 gene expression was 3.4 times and 3.9 times higher than that of TCDCA-treated cells and (p = 0.0006) control (p = 0.0008), respectively. S1PR2 is also a G protein-coupled receptor activated by sphingosine monophosphate, thus causing cell proliferation by ERK1/2 and protein kinase B in bile duct cells. GCA and TCDCA were identified as biomarkers capable of discriminating between biliary tract cancer, benign biliary tract disease and pancreatic cancer.

Claims (11)

delete delete delete delete (a) measuring the amount of glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA) from the biological sample isolated from the patient; And
(b) The amount of glycocholic acid (GCA; glycocholic acid) or taurochenodeoxycholic acid (TCDCA; Taurochenodeoxycholic acid) in the patient was adjusted to the glycocholic acid (GCA; glycocholic acid) or taurokenodioxycholic acid (TCDCA) of the normal control group. Comparing with the amount of Taurochenodeoxycholic acid);
Including,
A method of providing information for diagnosing biliary tract cancer when the amount of the taurochenodeoxycholic acid (TCDCA) is less than that of a normal control patient sample.
The method of claim 5, wherein the amount of glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA) is measured by chromatography/mass spectrometry, and provides information for diagnosing biliary tract cancer. .
The method of claim 5, wherein the biological sample is bile.
The method of claim 5, wherein when the amount of the glycocholic acid (GCA) is greater than that of the normal control sample, it is determined that it is biliary tract cancer.
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