KR20200031482A - A method of diagnosing cholangiocarcinoma - Google Patents

Abstract

The present invention relates to a method for diagnosing benign biliary diseases, pancreatic cancer and cholangiocarcinoma by measuring glycocholic acid (GCA) and taurochenodeoxycholic acid (TCDCA) from a cholangiocarcinoma patient. More specifically, as a result of measuring GCA and TCDCA from bile of benign biliary disease, pancreatic cancer and cholangiocarcinoma patients, the present invention confirmed that a cholangiocarcinoma patient has a high GCA and a low TCDCA, and GCA and TCDCA are a biomarker to identify a cholangiocarcinoma among benign biliary disease, pancreatic cancer and cholangiocarcinoma patients. In addition, the present invention relates to a method for diagnosing cholangiocarcinoma from patients with benign biliary diseases, pancreatic cancer and cholangiocarcinoma by using the same; and a screening method of a cholangiocarcinoma medicine. A composition for diagnosing cholangiocarcinoma may include a material for detecting GCA or TCDCA.

Description

Methods of diagnosing biliary cancer {A method of diagnosing cholangiocarcinoma}

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

Biliary tract cancer (Cholangiocarcinoma; CCA) is one of the malignant tumors of the bile duct epithelium. It is difficult to diagnose because of its location-specific location deep inside the body, and the initial symptoms are minimal. Therefore, early diagnosis of biliary tract cancer is difficult and, therefore, belongs to a carcinoma with a very high mortality rate. According to a recent statistical survey, the 5-year survival rate of biliary cancer is only 25%, and 10% or 0%, respectively, in patients with stage 3 or 4, respectively. In the United States, more than 7,000 biliary cancer patients have died each year since 2013, and the mortality rate has increased 36% between 1999 and 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 common biliary cancer biomarkers used in clinical practice. However, using the biomarker, the distinguishing power between the biliary cancer patient and the normal person is excellent, but it is difficult to distinguish biliary cancer, benign biliary disease, and pancreatic cancer. In order to solve the above problems, biomarker studies related to biliary cancer have been conducted. For example, MicroRNA-21 (miR-21) has been studied as a representative transcriptional biomarker of biliary cancer. MicroRNA-21 is highly sensitive and is associated with tumor growth in biliary cancer, but is tissue specific and increases in a variety of diseases. Among the proteomic biomarkers that distinguish biliary cancer are insulin-like growth factor-1 and elastase. However, these biomarkers are not specific for other diseases or have not yet been investigated in the mechanism of biliary cancer invention. Thus, there are few studies related to metabolic biomarkers of biliary cancer compared to transcriptional or proteomic biomarkers. The new metabolic biomarker is more powerful because it accurately represents the molecular phenotype than other markers. The present inventors discovered a specific metabolic biomarker related to a specific metabolic biomarker that occurs more or less specifically in the bile of patients with biliary cancer than patients with benign biliary disease and pancreatic cancer.

Unlike other biological fluids such as serum and urine, bile is located near the bile tumor, thereby increasing the probability of detecting bile duct specific biomarkers. Certain bile acids act as signaling molecules related to the invention of biliary cancer. Therefore, in order to discover potential biomarkers of biliary tract cancer, bile acids are currently being analyzed.

The rate of deoxycholic acid (DCA) in patients with biliary cancer was lower than bile 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 cancer than in patients with benign biliary disease or hepatocellular carcinoma (HCC). Patients with biliary cancer had higher levels of bile acid combined with taurine and glycine than patients diagnosed with benign biliary tract disease. However, this study failed to analyze the sample or type of bile acids and failed to interpret the role of bile acid biomarkers specific for the development of biliary cancer.

While studying biomarkers to distinguish patients with benign biliary tract disease, pancreatic cancer, and biliary cancer, the results of analyzing bile acids in patients with biliary cancer showed positive biliary tracts with different GCA (glycocholic acid) in bile cancer patients. The present invention was completed after confirming that it was higher than that of patients with diseases and pancreatic cancer and that TCDCA (Taurochenodeoxycholic acid) was low.

The present invention has been devised to solve the above problems, and an object of the present invention is to diagnose biliary cancer, which includes an agent that detects glycocholic acid (GCA; glycocholic acid) or taurochenodioxycholic acid (TCDCA; Taurochenodeoxycholic acid). It is to provide a composition.

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

Another object of the present invention is to provide a method for providing information of biliary 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 agents to be detected include substances used in antibody analysis, chemiluminescence analysis, or liquid chromatography mass spectrometry, but are not limited thereto.

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

The quantitative device may be a chromatography / mass spectrometer. The 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), but not limited to, any quantitative chromatography commonly used in the art may be used.

The present invention can also provide a method for providing information for diagnosing biliary cancer, which includes the following steps.

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

(b) The amount of glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA) in the patient is the amount of glycocholic acid (GCA) or taurochenodioxycholic acid (TCDCA) in the normal control. Step to compare with the amount of Taurochenodeoxycholic acid).

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

The biological sample may be bile.

When the amount of glycocholic acid (GCA) is greater than the sample of the normal control group, it can be determined to be biliary cancer.

When the amount of taurochenodeoxycholic acid (TCDCA; Taurochenodeoxycholic acid) is less than that of a sample of a normal control patient, it can be determined to be biliary cancer.

The present invention can also provide information for diagnosing biliary cancer, which includes the following steps.

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

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

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

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

(e) The amount of glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA) in patients with suspected biliary cancer is glycocholic acid (GCA) in a sample of patients with pancreatic cancer or benign biliary disease. ) Or comparing with the amount of Taurochenodeoxycholic acid (TCDCA).

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

When 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 cancer.

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

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

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

(c) selecting a test substance in which glycocholic acid (GCA) is reduced or taurochenodeoxycholic acid (TCDCA) is increased compared to a control sample.

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

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

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

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

As a result of comparing the bile acids of the patients with biliary cancer, pancreatic cancer and benign biliary cancer, the present inventors found that glycic acid (GCA) of bile acids in patients with biliary cancer is higher than that of patients with other benign biliary diseases and pancreatic cancer, and that TCDCA (Taurochenodeoxycholic acid) is different. It was confirmed that it is lower than that of patients with biliary tract disease and pancreatic cancer, and eventually, GCA (glycocholic acid) and TCDCA (Taurochenodeoxycholic acid) have an effect of distinguishing biliary cancer patients among patients with pancreatic cancer, benign biliary tract and biliary cancer.

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

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 only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1. Materials and reagents

Lithocholic acid (LCA), ursodeoxycholic acid (UDCA), kenodioxycholic 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 Cruz. Glycoursodeoxycholic acid (GUDCA) was purchased from Achemblock. HPLC-grade water, methanol and acetonitrile were purchased from Deoksan.

Example 2. Collection of patient and patient's bile fluid

Bile samples were collected at Samsung Seoul Hospital. The research protocol was reviewed and approved by an institutional reviewer at Samsung Medical Center. All experimental methods were performed according to the relevant guidelines and regulations. It consisted of 57 benign biliary diseases, 17 patients with pancreatic cancer and 30 patients with biliary cancer. The bile samples were stored at -70 ° C until they became bile acids.

Example 3. Preparation of bile acids

After thawing 30 μL of the bile sample on ice, bile acid was extracted with 400 μL of chloroform and 200 μL of methanol. After the mixture was well mixed, 120 μL of distilled water was added to the mixture. The mixture was vortexed and centrifuged for 5 minutes, then 100 μl of the supernatant was transferred to a 1.5 ml micro tube and dried in a centrifugal vacuum concentrator. After drying was completed, bile acids were dissolved in 100 μL of 50% methanol before transfer to an HPLC autosampler vial for LC-MS / MS analysis.

Example 4. LC-MS / MS analysis

The bile acid (10 μL) resuspended 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 an analytical column at a flow rate of 400 μL / min. The LC gradient method was set up 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 included 100% water and 0.1% (v / v) formic acid, 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. A reverse phase LC method was developed based on SRM conditions to isolate 15 important bile acids in human bile (FIG. 1). Using the optimized LC-MS / MS method, 15 important bile acids were quantified in the bile of patients with benign biliary disease, pancreatic cancer, and biliary cancer. The average concentration of bile acids was highest in patients with pancreatic cancer (FIG. 2). In addition, the average concentration of bile acids in the biliary benign disease was twice that of the biliary cancer patients. In patients with biliary cancer, when the concentration of bile bile acid is low, bile duct obstruction and inflammation may result in a decrease in bile acid excretion and bile acid excretion. Bile acids can be classified into synthetic (primary and secondary) and conjugating molecules (glycine, taurine and non-conjugated). The average ratio of secondary bile acids was found to be nearly twice that of patients with biliary benign disease compared to pancreatic cancer and biliary cancer (Fig. 3). In patients with biliary tract cancer, the average percentage of total non-conjugated bile acids was 10 times lower than that of patients with biliary positivity. The unbound bile acids inhibit the growth of biliary cancer cell lines. Conjugated bile acids activate epidermal growth factor receptors and increase the expression of the cold element cyclooxygenase-2 present in many cancers. It also promotes biliary cancer cell growth and contributes to the activation of NF-kB, which blocks cell death. It promotes the growth and invasiveness of biliary cancer through activation of S1PR2 rather than non-conjugated bile acids. Therefore, the increased proportion of bile acids encountered in biliary cancer pills is related to the pathogenesis of biliary cancer. Quantitative analysis of 15 bile acids in patients with biliary benign disease, pancreatic cancer, and biliary cancer (Table 1) .The average composition of GCA was compared with biliary benign disease (22.3%, p <0.0001) and pancreatic cancer (19.9%, p <0.0001). By biliary cancer (35.6%) was the highest (Fig. 4). In contrast to GCA, the mean composition of TCDCA was significantly lower in patients with biliary cancer (7.31%) than in patients with biliary-positive 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 wetted 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. Cultured in an environment of 5% CO ₂ . GCA and TCDCA were dissolved in 100 μL of DMSO (Sigma Aldrich, MO, USA), respectively, 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. Cells were incubated for 48 hours after treatment with each bile acid.

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 of FXR, S1PR2, TGR5 and β-actin used previously reported primers, and β-actin was used as internal control of 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, and extension at 72 ° C for 20 seconds. Bile acids bind to the bile acid receptors of bile duct cells resulting in several biological phenotypes. In particular, quantitative changes of 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 FXR, TGR5 and S1PR2 gene expression by qRT-PCR were quantified (FIG. 5). Since TCDCA was selected as a negative biomarker of biliary cancer, it was expected that gene expression related to biliary etiology would decrease compared to GCA. FXR is a bile acid receptor primarily present in the liver and intestine and is a gene that mediates the pathogenesis of various biliary diseases as well as bile acid transport and metabolism. Several studies have reported that low expression of FXR is associated with various biliary diseases caused by abnormal bile acid transport. In the transcriptomic analysis, the expression of the FXR gene in the GCA-treated (0.17 fold less control) cell line was lower than the control. Decreasing FXR gene expression has been reported to cause human biliary cancer, suggesting that GCA is closely related to tumor differentiation. TGR5 gene expression was 8.55 times higher in GCA-treated biliary cancer cell lines than control (p <0.0001) or TCDCA treated cells (p <0.0001). TGR5 is a G protein binding receptor present in cholangiocytes and is activated in response to bile acids and regulates the proliferation of bile duct cells. Overexpression of TGR5 in biliary cancer induces resistance to cell death and contributes to the pathogenesis of tumors. As the expression of TGR5 increased in the biliary cancer cell line treated with GCA, it was confirmed that GCA may influence the development of biliary cancer. S1PR2 gene expression in GCA-treated biliary cancer cell lines was 3.4 and 3.9 times higher, respectively, than TCDCA-treated cells and (p = 0.0006) control (p = 0.0008). S1PR2 is also a G protein-coupled receptor activated by sphingosine monophosphate, causing cell proliferation by ERK1 / 2 and protein kinase B in bile duct cells. GCA and TCDCA were identified as biomarkers capable of distinguishing biliary cancer, biliary benign disease, and pancreatic cancer.

Claims (11)

A composition for diagnosing biliary cancer, comprising an agent that detects glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA).
The composition for diagnosing biliary cancer according to claim 1, wherein the agent to be detected is an agent used in antibody analysis, chemiluminescence analysis, or liquid chromatography mass spectrometry.
A kit for diagnosing biliary cancer, comprising a quantitation device for glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA).
The kit for diagnosing biliary cancer according to claim 3, wherein the quantitative device is a chromatography / mass spectrometer.
(a) measuring the amount of glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA; Taurochenodeoxycholic acid) from a biological sample isolated from the patient; And
(b) The amount of glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA) of the patient is the amount of glycocholic acid (GCA) or taurochenodioxycholic acid (TCDCA) in the normal control. Comparing with the amount of Taurochenodeoxycholic acid);
Method for providing information for diagnosing biliary cancer, including.
The method of claim 5, wherein the amount of the glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA) is measured by chromatography / mass spectrometry. .
The method of claim 5, wherein the biological sample is bile.
According to claim 5, If the amount of glycocholic acid (GCA; glycocholic acid) is greater than the sample of the normal control, it is determined that the biliary cancer, information providing method for diagnosing biliary cancer.
According to claim 5, If the amount of taurochenodeoxycholic acid (TCDCA; Taurochenodeoxycholic acid) is less than the sample of the normal control patient, the method of providing information for diagnosing biliary cancer.
(a) treating the test substance with biliary cancer cells; And
(b) measuring the amount of glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA; Taurochenodeoxycholic acid) in the biliary cancer cells; And
(c) selecting a test substance in which glycocholic acid (GCA) is reduced or taurochenodeoxycholic acid (TCDCA) is increased as compared to a control sample;
Method of screening for a treatment for biliary tract cancer comprising a.
(a) treating the test substance with biliary cancer cells; And
(b) measuring the expression level or protein activity of an enzyme synthesizing glycocholic acid (GCA) or taurochenodeoxycholic acid (TCDCA) in the biliary cancer cells; And
(c) the expression level of an enzyme synthesizing glycocholic acid (GCA) or its protein activity is reduced, or the expression level of an enzyme synthesizing taurochenodeoxycholic acid (TCDCA) compared to a control sample, or Selecting a test substance whose activity is increased;
Method of screening for a treatment for biliary tract cancer comprising a.

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