WO2023233945A1 - Biliary tract cancer testing method - Google Patents

Biliary tract cancer testing method Download PDF

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WO2023233945A1
WO2023233945A1 PCT/JP2023/017497 JP2023017497W WO2023233945A1 WO 2023233945 A1 WO2023233945 A1 WO 2023233945A1 JP 2023017497 W JP2023017497 W JP 2023017497W WO 2023233945 A1 WO2023233945 A1 WO 2023233945A1
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biliary tract
measured
tract cancer
mass
detection mode
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PCT/JP2023/017497
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French (fr)
Japanese (ja)
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隆士 石垣
眞由美 阿部
顕成 檜
広夫 内田
俊男 國料
隆史 水野
真輝 砂川
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株式会社日立ハイテク
国立大学法人東海国立大学機構
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Publication of WO2023233945A1 publication Critical patent/WO2023233945A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/493—Physical analysis of biological material of liquid biological material urine

Definitions

  • the present invention relates to a method, kit, and device for determining biliary tract cancer in a subject based on measured values of urinary tumor markers.
  • Biliary tract cancer is a relatively common cancer in Japan, and the 5-year survival rate, which is an indicator of prognosis, is low, so it is desirable to detect and treat it early.
  • Blood CEA, CA19-9, etc. are known as tumor markers for biliary tract cancer for early detection, but their tumor specificity is low and their accuracy is not high.
  • blood tests are invasive and are essentially limited to tests performed at medical institutions.
  • Patent Documents 1 to 5 are reports in Patent Documents 1 to 5 as tumor markers for cancer such as biliary tract cancer.
  • Patent Document 1 mainly describes genes and proteomics as tumor markers.
  • Patent Documents 2 to 5 describe metabolites as tumor markers for cancers such as colon cancer, but do not describe the urinary tumor marker disclosed herein.
  • An object of the present invention is to provide a means and method for non-invasively and easily determining biliary tract cancer, which has been desired in the past.
  • the present inventor identified a group of markers related to biliary tract cancer, and used these markers alone or in combination to determine biliary tract cancer and predict risk. discovered that biliary tract cancer monitoring can be performed easily and non-invasively.
  • the present invention relates to a method, device, and kit for determining biliary tract cancer and/or monitoring biliary tract cancer in a subject by measuring urinary metabolites that are urinary tumor markers.
  • Specific embodiments include:
  • a method for determining biliary tract cancer in a subject comprising: measuring a urinary tumor marker in a urine sample from a subject, the urinary tumor marker being measured in cholate, chenodeoxycholate sulfate, LC/MS negative ion detection mode as a mass-to-charge ratio of 259.028; compound (C10H12O6S), glycochenodeoxycholic acid 3-sulfate, isoleucylhydroxyproline, pro-hydroxy-pro, kynurenine, 4-methoxyphenol sulfate, 5-hydroxylysine, trans-4-hydroxyproline, glycylleucine , glycocholate, compound measured as mass-to-charge ratio 197.068 in LC/MS negative ion detection mode (C7H10N4O3), compound measured as mass-to-charge ratio 509.277 in LC/MS negative ion detection mode (C28H38N4O5), 3- Hydroxykynurenine, glycocheno
  • a device for determining biliary tract cancer A measurement unit for measuring a urinary tumor marker in a urine sample, wherein the urinary tumor marker is cholate, chenodeoxycholate sulfate, and a compound measured as a mass-to-charge ratio of 259.028 in LC/MS negative ion detection mode.
  • a method for evaluating the effectiveness of biliary tract cancer treatment comprising: measuring a urinary tumor marker in a urine sample from a patient with biliary tract cancer treated with an investigational therapeutic agent or therapy, the urinary tumor marker being cholate, chenodeoxycholate sulfate; , Compound measured as mass-to-charge ratio 259.028 in LC/MS negative ion detection mode (C10H12O6S), glycochenodeoxycholic acid 3-sulfate, isoleucylhydroxyproline, pro-hydroxy-pro, kynurenine, 4-methoxyphenol sulfate , 5-hydroxylysine, trans-4-hydroxyproline, glycylleucine, glycocholate, compound measured as mass-to-charge ratio 197.068 in LC/MS negative ion detection mode (C7H10N4O3), LC/MS negative ion detection mode (C28H38N4O5), 3-hydroxykynurenine, glycocheno
  • the present invention provides a method, device, and kit for determining biliary tract cancer in a minimally invasive, simple, and low-cost manner. Since the test uses urine, the collection method in clinical settings is also extremely simple, greatly improving convenience for medical professionals. Therefore, the present invention is useful in fields such as biliary tract cancer diagnosis, testing, therapeutic evaluation, and drug discovery.
  • RF random forest
  • For biliary tract cancer they are a graph (A) showing the calculation results of predicted values when 10 types of markers are applied to the cancer test model, and a graph (B) showing the AUC of the cancer test model.
  • For biliary tract cancer they are a graph (A) showing the calculation results of predicted values when 10 types of markers are applied to the cancer test model, and a graph (B) showing the AUC of the cancer test model.
  • biliary tract cancer For biliary tract cancer, they are a graph (A) showing the calculation results of predicted values when 20 types of markers are applied to the cancer test model, and a graph (B) showing the AUC of the cancer test model.
  • biliary tract cancer For biliary tract cancer, they are a graph (A) showing the calculation results of predicted values when 20 types of markers are applied to the cancer test model, and a graph (B) showing the AUC of the cancer test model.
  • these are a graph (A) showing the calculation results of predicted values when six types of markers that are considered important are applied to the cancer testing model, and a graph (B) showing the AUC of the cancer testing model.
  • a graph (A) showing the calculation results of predicted values when three types of markers considered to be important are applied to the cancer testing model, and a graph (B) showing the AUC of the cancer testing model.
  • these are a graph (A) showing the calculation results of predicted values when two types of markers considered to be important are applied to the cancer testing model, and a graph (B) showing the AUC of the cancer testing model.
  • An example of the configuration of a device to which the present invention is applied is shown. This is a graph showing the top 20 metabolites when urinary metabolites related to the difference between biliary tract cancer and healthy individuals are ranked by random forest (RF).
  • a graph (A) showing the calculation results of predicted values when five types of markers considered to be important are applied to the cancer testing model, and a graph (B) showing the AUC of the cancer testing model.
  • the methods, devices, and kits provided by the present invention utilize novel urinary tumor markers and marker groups associated with biliary tract cancer.
  • This urinary tumor marker is a metabolite whose urinary level differs depending on the presence or absence of biliary tract cancer, so it can be used to detect biliary tract cancer, predict the risk of biliary tract cancer, and determine the stage of biliary tract cancer. It is useful for determining the prognosis of biliary tract cancer, monitoring biliary tract cancer, and/or monitoring the therapeutic effect on biliary tract cancer.
  • the method for determining biliary tract cancer according to the present invention includes the steps of measuring a urinary tumor marker in a urine sample derived from a subject, and determining biliary tract cancer in the subject based on the measurement results.
  • Biliary tract cancer refers to cancer (malignant tumor) that occurs in the bile tract, including intrahepatic bile duct cancer, extrahepatic bile duct cancer (portal bile duct cancer, distal bile duct cancer), gallbladder cancer, It is classified as papillary cancer. Biliary tract cancers are classified into primary, metastatic, and recurrent types, and are classified into stages based on the degree of progression and spread. Necessary treatments (surgery, chemotherapy, radiotherapy, immunotherapy, etc.) also differ depending on whether the disease is primary, metastatic, or recurrent, and the stage.
  • a urinary tumor marker associated with biliary tract cancer is measured.
  • the "urinary metabolites” or “urinary tumor markers” to be measured in the present invention refer to the urinary metabolites listed in Table 1 below. Urinary metabolites are more convenient as tumor markers because they are less affected by enzymes and are structurally stable compared to substances in the blood. Furthermore, since urine is used as a specimen, it can be easily collected from a subject, making it extremely easy to use for cancer screening. Furthermore, a "marker group” is a combination of two or more urinary tumor markers.
  • Measure means determining the relative abundance or absolute concentration of a metabolite in a urine sample. Relative abundance is the ratio of the measured intensity of a metabolite of interest to an intentionally added standard substance.
  • the absolute concentration is defined as a calibration curve (relationship between the concentration of the metabolite and the measured intensity of the metabolite) created in advance using the same metabolite for the target metabolite, and calculated from the measured intensity. This is a method to calculate absolute concentration.
  • “measuring a urinary tumor marker” may mean measuring a metabolite that is a urinary tumor marker, or a derivative or a derivative thereof.
  • Derivative and “derivative” refer to a substance derived from a metabolite that is a urinary tumor marker and a substance derived from the metabolite, respectively. “Derivatives” and “derivatives” include, but are not limited to, fragments of metabolites, modified metabolites, and the like.
  • the main urinary tumor markers used in the present invention are summarized in Table 1 below.
  • the "metabolite” column shows the name of a metabolite whose structure was found as a result of a database search, or the symbol and estimated chemical formula if the structure is unknown.
  • the estimated chemical formula is estimated from the "measured mass” and “measurement mode” and a metabolite database such as the Human Metabolome Database (HMDB).
  • HMDB Human Metabolome Database
  • the CAS registration number in Table 1 is the de facto standard for chemical substance IDs and is a number for identifying chemical substances, and the HMDB ID is the ID of HMDB, an online database of small and medium molecule metabolites in the human body.
  • the "Measurement mass” column shows the mass-to-charge ratio when detected by the detection means described in the "Measurement mode” column.
  • “Neg”, “Pos Early”, and “Polar” in the “Measurement mode” column are “negative ion detection mode of liquid chromatograph mass spectrometer (LC/MS)” and “liquid chromatograph mass spectrometer (LC/MS)”, respectively.
  • LC/MS pos early is also simply referred to as "positive ion detection mode of liquid chromatograph mass spectrometer (LC/MS).”
  • the measured mass in Table 1 is basically the mass of the ionized metabolite, with one proton added or lost, and the mass varies by ⁇ 1 from the original metabolite mass. However, depending on the measurement conditions, multiple protons, sodium, etc. may be added or lost, and the measured mass will vary accordingly. Alternatively, a mass spectrum of fragment ions obtained by imparting energy to the metabolite and causing it to cleave may be measured.
  • cholate as shown in Table 1 is measured. That is, a compound measured as a mass of 407.280 in LC/MS negative ion detection mode is measured.
  • chenodeoxycholic acid sulfate (2) shown in Table 1 is measured. That is, a compound measured as a mass of 235.118 in LC/MS negative ion detection mode is measured.
  • X-17686 (C10H12O6S) shown in Table 1 is measured. That is, a compound (C10H12O6S) whose mass is measured as 259.028 in LC/MS negative ion detection mode is measured.
  • glycochenodeoxycholic acid 3-sulfate as shown in Table 1 is measured. That is, a compound measured as a mass of 263.628 in LC/MS negative ion detection mode is measured.
  • isoleucylhydroxyproline as shown in Table 1 is measured. That is, a compound measured as a mass of 245.150 in LC/MS positive ion detection mode is measured.
  • pro-hydroxy-pro as shown in Table 1 is measured. That is, a compound measured as a mass of 229.118 in LC/MS positive ion detection mode is measured.
  • kynurenine as shown in Table 1 is measured. That is, a compound measured as a mass of 209.092 in LC/MS positive ion detection mode is measured.
  • 4-methoxyphenol sulfate as shown in Table 1 is measured. That is, a compound measured as a mass of 203.002 in LC/MS negative ion detection mode is measured.
  • 5-hydroxylysine as shown in Table 1 is measured. That is, a compound measured as a mass of 163.108 in LC/MS positive ion detection mode is measured.
  • trans-4-hydroxyproline shown in Table 1 is measured. That is, a compound whose mass is measured as 130.051 in LC/MS polar detection mode is measured.
  • glycylleucine as shown in Table 1 is measured. That is, a compound measured as a mass of 189.123 in LC/MS positive ion detection mode is measured.
  • the glycocholates shown in Table 1 are measured. That is, a compound measured as a mass of 464.302 in LC/MS negative ion detection mode is measured.
  • X-13728 (C7H10N4O3) shown in Table 1 is measured. That is, a compound (C7H10N4O3) whose mass is measured as 197.068 in LC/MS negative ion detection mode is measured.
  • X-21851 (C28H38N4O5) shown in Table 1 is measured. That is, a compound (C28H38N4O5) measured as a mass of 509.277 in LC/MS negative ion detection mode is measured.
  • 3-hydroxykynurenine as shown in Table 1 is measured. That is, a compound measured as a mass of 225.087 in LC/MS positive ion detection mode is measured.
  • the glycochenodeoxycholates shown in Table 1 are measured. That is, a compound measured as a mass of 448.307 in LC/MS negative ion detection mode is measured.
  • isoleucylglycine shown in Table 1 is measured. That is, a compound measured as a mass of 187.116 in LC/MS negative ion detection mode is measured.
  • the phenylalanyl hydroxyproline shown in Table 1 is measured. That is, a compound measured as a mass of 279.134 in LC/MS positive ion detection mode is measured.
  • 4-hydroxyphenylpyruvate as shown in Table 1 is measured. That is, a compound measured as having a mass of 179.035 in LC/MS negative ion detection mode is measured.
  • lactate as shown in Table 1 is measured. That is, a compound whose mass is measured as 89.024 in LC/MS polar detection mode is measured.
  • cyclo(pro-hydroxypro) as shown in Table 1 is measured. That is, a compound measured as a mass of 211.108 in LC/MS positive ion detection mode is measured.
  • tryptophan as shown in Table 1 is measured. That is, a compound measured as a mass of 205.097 in LC/MS positive ion detection mode is measured.
  • N6-acetyl lysine as shown in Table 1 is measured. That is, a compound measured as a mass of 187.109 in LC/MS polar detection mode is measured.
  • gamma-glutamylphenylalanine as shown in Table 1 is measured. That is, a compound measured as a mass of 295.129 in LC/MS positive ion detection mode is measured.
  • leucyl hydroxyproline as shown in Table 1 is measured. That is, a compound measured as a mass of 245.150 in LC/MS positive ion detection mode is measured.
  • X-18887 (C14H23N3O6) shown in Table 1 is measured. That is, a compound (C14H23N3O6) whose mass is measured as 328.152 in LC/MS negative ion detection mode is measured.
  • X-24475 (C6H11NO3) shown in Table 1 is measured. That is, a compound (C6H11NO3) whose mass is measured as 146.081 in LC/MS positive ion detection mode is measured.
  • the androsterone glucuronides shown in Table 1 are measured. That is, a compound measured as a mass of 465.249 in LC/MS negative ion detection mode is measured.
  • the 3-hydroxyanthranilates shown in Table 1 are measured. That is, a compound measured as a mass of 154.050 in LC/MS positive ion detection mode is measured.
  • 11 beta-hydroxyandrosterone glucuronide as shown in Table 1 is measured. That is, a compound measured as a mass of 481.244 in LC/MS negative ion detection mode is measured.
  • cystathionine as shown in Table 1 is measured. That is, a compound measured as a mass of 221.060 in LC/MS positive ion detection mode is measured.
  • carnosine as shown in Table 1 is measured. That is, a compound measured as a mass of 227.114 in LC/MS positive ion detection mode is measured.
  • proline as shown in Table 1 is measured. That is, a compound measured as a mass of 116.071 in LC/MS positive ion detection mode is measured.
  • anserine as shown in Table 1 is measured. That is, a compound measured as a mass of 239.115 in LC/MS negative ion detection mode is measured.
  • arabitol/xylitol as shown in Table 1 is measured. That is, a compound whose mass is measured as 151.061 in LC/MS polar detection mode is measured.
  • 3-hydroxy-2-ethylpropionate as shown in Table 1 is measured. That is, a compound whose mass is measured as 117.056 in LC/MS polar detection mode is measured.
  • the 2R,3R-dihydroxybutyrate shown in Table 1 is measured. That is, a compound whose mass is measured as 119.035 in LC/MS polar detection mode is measured.
  • gamma-glutamyltyrosine as shown in Table 1 is measured. That is, a compound measured as a mass of 311.124 in LC/MS negative ion detection mode is measured.
  • 1-methyladenine as shown in Table 1 is measured. That is, a compound measured as having a mass of 150.077 in LC/MS positive ion detection mode is measured.
  • the dihydroorotonic acid shown in Table 1 is measured. That is, a compound whose mass is measured as 157.026 in LC/MS polar detection mode is measured.
  • alanine as shown in Table 1 is measured. That is, a compound measured as having a mass of 90.055 in LC/MS positive ion detection mode is measured.
  • 17 alpha-hydroxypregnenolone glucuronide as shown in Table 1 is measured. That is, a compound measured as a mass of 509.276 in LC/MS negative ion detection mode is measured.
  • the mass spectrometer used to analyze the metabolites shown in Table 1 has a very high resolution, so it is possible to measure mass to about five decimal places. However, taking into account measurement errors, Table 1 is It is written in 3 digits after the decimal point. Furthermore, when using a mass spectrometer with low resolution, an integer mass or a mass of one or two digits after the decimal point is measured.
  • At least one of the urinary tumor markers shown in Table 1 can be used to determine biliary tract cancer and monitor the effects of treatment.
  • the present invention by using at least two or three or more urinary tumor markers in combination, more accurate and precise determination and monitoring of the effectiveness of treatment are possible.
  • at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, At least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or more can be combined.
  • the combination of markers is not particularly limited.
  • at least three urinary tumor markers are measured.
  • the urinary tumor markers shown in Table 1 can be used alone to determine biliary tract cancer.
  • the marker includes at least glycochenodeoxycholic acid 3-sulfate, or glycocholate, or 4-hydroxyphenylpyruvate.
  • Yobs is the measured value
  • Ycalc is the calculated value by OPLS
  • Ypred is the predicted value when cross-validated, represents the average value.
  • Cross-validation refers to a method in which data is divided, a part of it is analyzed first, and the remaining part is used to test the analysis to verify and confirm the validity of the analysis itself. According to this, it can be said that the closer the value of the accuracy variable R2Y is to 1, the higher the accuracy of the model is, and the closer the Q2 value of the predictor variable is to 1, the higher the predictiveness of the model is. It is thought that by using a combination with high values of the accuracy variable and the predictive variable for biliary tract cancer determination, more accurate determination will be possible.
  • the combination of urinary tumor markers is determined based on the type, sex, and age of the subject, the determination of biliary tract cancer, and the follow-up of subjects at high risk (e.g., based on family history) or subjects with no abnormalities (monitoring of biliary tract cancer). ), or can be selected as appropriate depending on the purpose including treatment monitoring.
  • the partial least squares method which is a type of multivariate analysis, particularly OPLS-DA can be used.
  • multivariate analysis it may be difficult to understand the characteristics of the data if multidimensional data is used as is, so it may be necessary to reduce it to 2D or 3D data for visual visualization.
  • analysis methods known in the art such as principal component analysis.
  • Urine sample refers to urine collected from a subject and a sample obtained by processing the urine (for example, urine to which a preservative such as toluene, xylene, or hydrochloric acid has been added).
  • the target is human urine. Since metabolic activity in the human body is assumed to differ depending on race, etc., monogoroid people, including Japanese, who are the subjects of analysis of the present invention, are preferred, but not limited thereto. For example, it may be during mass screening such as health checkups or cancer tests, during additional examinations after such mass screening, or before and after surgery at a hospital, or during treatment such as chemotherapy or radiation. Good too. Furthermore, even in the same individual, the concentration of metabolites in urine easily varies depending on the timing of urine collection, water intake, etc.
  • the amount of creatinine in the same urine or the osmolality (osmolality) of the same urine is measured and the amount of each metabolite is divided. Standardize.
  • the urinary metabolite amount hereinafter basically means a standardized amount.
  • Measuring a urinary tumor marker means measuring its amount or concentration in a urine sample, preferably semi-quantitatively or quantitatively, and the amount may be an absolute amount or a relative amount. There may be. Measurements can be made directly or indirectly. Direct measurement involves determining the amount or concentration of a urinary metabolite based on a signal that directly correlates with the number of molecules present in the sample. Such signals are based, for example, on certain physical or chemical properties of the urinary metabolites. Indirect measurements are measurements of signals obtained from secondary components (ie components other than urinary metabolites), such as ligands, labels or enzymatic reaction products.
  • secondary components ie components other than urinary metabolites
  • a urinary tumor marker that is, a urinary metabolite is measured, but the measurement method is not particularly limited, and any method or means known in the art can be used.
  • measurement of urinary tumor markers can be performed by means of measuring physical or chemical properties specific to urinary metabolites, such as precise molecular weight or NMR spectra.
  • means for measuring metabolites in urine include analysis devices such as a mass spectrometer, an NMR analyzer, a two-dimensional electrophoresis device, a chromatograph, and a liquid chromatography mass spectrometer (LC/MS). Although these analyzers may be used alone to measure a urinary tumor marker, a plurality of analyzers may be used to measure a urinary tumor marker.
  • a reagent for detecting a metabolite to be measured such as an immunoreaction reagent or an enzyme reaction reagent, is available, such a reagent can be used to measure the metabolite in urine.
  • urinary tumor marker contained in the urine sample collected from the subject and determine biliary tract cancer in the subject based on the results. Additionally, urinary tumor markers may be measured in urine samples taken from the subject at multiple time points.
  • the presence and progression of biliary tract cancer can be determined at an early stage, which is useful for detailed examinations and determining treatment strategies. If it becomes possible to diagnose biliary tract cancer with a simple test, it can be expected to not only provide treatment but also prevent the risk of invasion caused by the test. Patients will be able to receive early treatment for biliary tract cancer, and those at high risk will be able to be monitored for the development of biliary tract cancer. Furthermore, the effect of biliary tract cancer treatment can be monitored, and it becomes possible to consider stopping, continuing, or changing the treatment depending on the treatment effect.
  • the method for determining biliary tract cancer of the present invention can be carried out easily and conveniently by using a kit and/or device equipped with a means for measuring a urinary tumor marker, which is a urinary metabolite.
  • the kit for determining biliary tract cancer according to the present invention includes at least a means for measuring at least one (preferably at least three) of the urinary tumor markers shown in Table 1 above.
  • kits of the present invention are a mass spectrometry reagent set, which includes, for example, an isotope labeling reagent, a fractionation minicolumn, a buffer solution, and the like.
  • a kit is an immunoreaction reagent set, which includes, for example, a substrate on which a primary antibody is immobilized, a secondary antibody, and the like.
  • an enzyme reaction reagent set is composed of, for example, an enzyme, a buffer solution, and the like.
  • the kit of the present invention may include an instruction manual describing the procedure and protocol for implementing the method of the present invention, a table showing reference values or reference ranges used in determining biliary tract cancer, and the like.
  • kit of the present invention may be provided individually or within a single container.
  • the kit of the invention contains all of the components necessary to carry out the method of the invention, eg as components in adjusted concentrations, ready for immediate use.
  • the biliary tract cancer determination device includes the following means: a measurement unit that measures at least one (preferably at least three) of the urinary tumor markers shown in Table 1 above in the urine sample; a comparison unit that compares the measurement value of the urinary tumor marker measured by the measurement unit with a reference value or a previous measurement value; A determination unit that determines biliary tract cancer from the comparison results obtained by the comparison unit.
  • the biliary tract cancer determination device includes the following means: a measurement unit that measures at least one (preferably at least three) of the urinary tumor markers shown in Table 1 above in the urine sample; From the explanatory variables measured by the above measurement unit (the amount or concentration of urinary tumor markers, or the observed ion intensity ratio of urinary tumor markers that increases or decreases in biliary tract cancer patients, for example, compared to benign or no abnormalities) The calculated value of the objective variable (biliary tract cancer, abnormal a comparison unit that compares it with a reference value that is an index (indicating whether there is no A determination unit that determines biliary tract cancer from the comparison results obtained by the comparison unit.
  • the apparatus of the present invention is preferably a system in which the measuring section, the comparing section, and the determining section described above are operably connected to each other so that the method of the present invention can be carried out.
  • One embodiment of the device of the invention is shown in FIG.
  • the measurement unit includes a means for measuring a urinary tumor marker in a urine sample as described above, such as a mass spectrometer, an NMR analyzer, a two-dimensional electrophoresis device, a chromatograph, a liquid chromatography mass Equipped with analysis equipment such as analysis (LC/MS) equipment.
  • a means for measuring a urinary tumor marker in a urine sample as described above such as a mass spectrometer, an NMR analyzer, a two-dimensional electrophoresis device, a chromatograph, a liquid chromatography mass Equipped with analysis equipment such as analysis (LC/MS) equipment.
  • LC/MS analysis
  • the measurement unit includes a data analysis unit consisting of a computer and software that processes the measurement values obtained from the above-mentioned analysis device or the like.
  • the data analysis unit calculates the amount or concentration of the urinary tumor marker contained in the urine sample by referring to data such as a calibration curve based on the measurement values obtained from the above-mentioned analyzer or the like.
  • the data analysis unit analyzes the explanatory variables measured by the measurement unit (the amount or concentration of urinary tumor markers, or whether they increase or decrease in patients with biliary tract cancer relative to benign or no abnormality, for example).
  • the calculated value of the objective variable (whether there is biliary tract cancer or no abnormality) is calculated based on the cancer test model obtained by multivariate analysis from the observed ion intensity ratio of the urinary tumor marker. Calculate the index (indicating whether The data analysis section can include, for example, a signal display section, a unit for analyzing measured values, a computer unit, etc.
  • the comparison section reads out a reference value regarding the amount or concentration of the urinary tumor marker from a storage device (database), etc., and compares the measurement value of the urinary tumor marker measured by the measurement section with the reference value.
  • the comparison section reads the reference value of the target variable from a storage device (database), etc., and compares the calculated value of the target variable obtained by the measurement section with the reference value.
  • the comparison section selects and reads an appropriate reference value according to the type of urinary tumor marker.
  • the comparison section reads the previous measurement value from a storage device (database), etc., and compares it with the measurement value of the urinary tumor marker measured by the measurement section.
  • the determination section determines whether the measurement value of the urinary tumor marker is compared with the reference value in the comparison section, or based on the result of comparing the measurement value of the urinary tumor marker at a plurality of time points in the comparison section. , determine biliary tract cancer.
  • the determination section is based on the results of comparing the calculated value of the objective variable and the reference value in the comparing section, or the results of comparing the calculated values of the objective variable at multiple points in time in the comparing section. Based on this, biliary tract cancer is determined.
  • the determination unit acquires information indicating the presence of biliary tract cancer in the subject, the stage of biliary tract cancer, and whether there is any abnormality.
  • Preferred devices are those that can be used without the knowledge of a specialized clinician, such as electronic devices that simply require the addition of a sample.
  • the determination unit determines that the subject may have biliary tract cancer when the urinary tumor marker is equal to or higher than the reference value or the previous measurement value. For example, if the urinary tumor marker is lower than the reference value or the previous measurement value, the determination unit determines that there is a possibility that the subject has no abnormality.
  • the device of the present invention may further include a data storage section, a data output/display section, and the like.
  • determination of biliary tract cancer means not only detecting biliary tract cancer in a subject, but also predicting the risk of biliary tract cancer in a subject, to determine the stage of biliary tract cancer, to determine the prognosis of biliary tract cancer in a subject, to monitor biliary tract cancer in a subject, to monitor the effect of treatment for biliary tract cancer existing in a subject, and to monitor biliary tract cancer in a subject. This meaning includes assisting in diagnosis. Furthermore, in the present invention, “determination” includes continuous monitoring of biliary tract cancer that has already been detected or diagnosed, and confirmation of the detection or diagnosis of biliary tract cancer that has already been performed.
  • determination by the biliary tract cancer determination method, determination kit, and determination device according to the present invention is intended to be able to determine a statistically significant proportion of subjects. Therefore, “determination” by the biliary tract cancer determination method, determination kit, and determination device according to the present invention includes cases where correct results are not always obtained for all (ie, 100%) of the subjects. Statistically significant proportions can be determined using a variety of well-known statistical evaluation tools, such as determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney test, and the like. A preferred confidence interval is at least 90%. The p-value is preferably 0.1, 0.01, 0.05, 0.005 or 0.0001. More preferably, at least 60%, at least 80%, or at least 90% of the subjects can be appropriately determined by the biliary tract cancer determination method, determination kit, and determination device according to the present invention.
  • a urinary tumor marker is measured in a subject's urine sample and the measurement is compared to a baseline or previous measurement.
  • each urinary tumor marker may be compared with its reference value or previous measurement value, or the calculated value of the target variable obtained by multivariate analysis may be calculated. , it may be compared with a reference value or a previous measurement value.
  • the standard is the amount or concentration of a urinary tumor marker related to biliary tract cancer, or the range of the amount or concentration, or the amount or concentration of a urinary tumor marker that is an indicator of no abnormality, or the amount or concentration of the urinary tumor marker. range of concentrations.
  • the calculated value of the objective variable that identifies biliary tract cancer/no abnormality becomes the reference value.
  • the reference value can be derived from healthy subjects (population) or low-risk biliary tract cancer subjects (population).
  • the reference value may be derived from patients (patient populations) who have biliary tract cancer (e.g., a certain stage of biliary tract cancer) or who have biliary tract cancer with a particular prognosis. .
  • the reference value applied to an individual subject may vary depending on various physiological parameters such as the species, age, and sex of the subject animal.
  • the correlation between the amount or concentration of a urinary tumor marker and the presence of biliary tract cancer or a specific prognosis is recorded as a database.
  • the measured value of the urinary tumor marker in the urine sample can then be compared with the reference value in the database.
  • Such a database is useful as an indicator of the presence or absence of biliary tract cancer (or a specific stage of biliary tract cancer), or a reference value or reference range that is an indicator of prognosis.
  • the amount or concentration of the urinary tumor markers shown in Table 1 differs depending on the presence or absence of biliary tract cancer, and the amount or concentration changes depending on the presence of biliary tract cancer and before or after the start of treatment.
  • the markers shown in Table 1 have increased amounts or concentrations in patients with biliary tract cancer compared to subjects without biliary tract cancer. Therefore, if the marker shown in Table 1 is higher than the reference value derived from the normal population (subjects without biliary tract cancer) or equal to or higher than the reference value derived from the patient population with biliary tract cancer.
  • the subject may have or is at high risk of biliary tract cancer.
  • the threshold value of the predicted value is not limited to 0 and may be varied.
  • the magnitude of the predicted value may be quantitatively associated with the risk (probability) of cancer without clearly defining the threshold value.
  • a urine sample is collected from the subject at multiple time points, a urinary tumor marker is determined in the urine sample at each time point, and the urinary tumor marker measurements are compared at each time point. do. More specifically, the amount or concentration of the urinary tumor marker at the first time point (a) is compared with the amount or concentration of the urinary tumor marker at the second time point (b).
  • the calculated value of one component at the first time point and the calculated value at the second time point are compared. Measurements are taken at least 2, 3, 4, 5, 10, 15, 20, 30 or more times over time, e.g. 1 day, 2 days, 5 days, 1 week.
  • the urinary tumor marker used in the present invention can be used to monitor the effect of treatment (therapeutic agent or treatment method) on biliary tract cancer in a subject.
  • a urine sample is collected from a patient with biliary tract cancer, and urinary tumor markers in the urine sample are measured before receiving treatment with a therapeutic agent or treatment method.
  • a urine sample is collected at an appropriate time and urinary tumor markers are measured in the urine sample. For example, immediately after treatment, 30 minutes, 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours (1 day), 2-10 days, 10-20 Collect urine samples after 1 day, 20 to 30 days, and 1 month to 6 months. Measurement of urinary tumor markers in urine samples can be performed in the same manner as described above. By measuring urinary tumor markers before and after treatment, it becomes possible to monitor the effectiveness of treatment with the therapeutic agent or method. Based on the results of monitoring, it helps to consider stopping, continuing, or changing treatment.
  • the method for determining biliary tract cancer may be performed in combination with other conventionally known methods for diagnosing biliary tract cancer.
  • Such known methods for diagnosing biliary tract cancer include blood tests (measurement of blood cancer markers, liver function tests, etc.), image tests (such as abdominal ultrasound, computed tomography (CT), MRI), Positron CT (PET), etc.), endoscopy, and pathological examinations such as biopsy or cytology.
  • the doctor can diagnose the subject's biliary tract cancer and take appropriate treatment. That is, the present invention also relates to a method for determining and treating biliary tract cancer in a subject. For example, if biliary tract cancer is determined in a subject according to the method of the present invention and it is evaluated that the subject is likely to have biliary tract cancer, the subject is treated for biliary tract cancer or Take measures to prevent progression. In addition, if it is assessed that the stage of biliary tract cancer in the subject is advanced or that the prognosis of biliary tract cancer is likely to be poor, treatment may be continued or, if necessary, a change in treatment method may be considered. do.
  • urinary tumor markers may be measured over time to avoid excessive testing and treatment. May be monitored for biliary tract cancer. Further, if it is evaluated that there is a high possibility that biliary tract cancer exists in the subject, other biliary tract cancer diagnostic methods such as those described above are performed to confirm the presence of biliary tract cancer. Furthermore, based on the evaluation results before and after the treatment, the effectiveness of the treatment is monitored and a decision is made to stop, continue, or change the treatment. Furthermore, if it is determined that there is no abnormality, urinary tumor markers can be measured over time to monitor the progress.
  • biliary tract cancer For biliary tract cancer, surgery (surgical resection), chemotherapy, radiation therapy, immunotherapy, proton beam therapy, heavy particle beam therapy, etc. can be performed alone or in appropriate combinations. Treatment for biliary tract cancer can be selected appropriately by those skilled in the art, taking into consideration the type, stage, malignancy, gender, age and condition of biliary tract cancer, responsiveness to treatment, genetic polymorphisms (SNPs), etc. can do.
  • SNPs genetic polymorphisms
  • the testing center provides guidance on cancer testing in response to requests from test subjects.
  • the test subject may select the number of biomarkers for the test.
  • the number of biomarkers includes 1 to 3 types of urinary tumor markers. This can also be used as a pan-cancer test (analyzing various cancers at once) in combination with other biomarkers.
  • the testing center hands the test subject the test kit necessary for urine collection. Send by mail, etc. as necessary. After receiving the test kit, the person to be tested hands or sends the specimen to the test center.
  • samples are frozen and stored at approximately -80°C for subsequent testing as needed.
  • storage at -80°C is not the only option, but frozen storage at approximately -5°C and refrigerated storage at approximately 5°C are recommended. , room temperature storage, etc.
  • the testing center performs a primary test and sends the test results to the person being tested.
  • the person to be tested may apply for a secondary test depending on the content, or may receive a more detailed diagnosis. This makes it possible to confirm the suspicion of biliary tract cancer in the primary examination and furthermore to identify the stage of biliary tract cancer.
  • the urinary tumor marker used in the present invention can be used to evaluate the effectiveness of biliary tract cancer treatment (therapeutic drug or treatment method) or to screen therapeutic drug candidates for biliary tract cancer.
  • a method for evaluating the effectiveness of biliary tract cancer treatment or a method for screening therapeutic drug candidates for biliary tract cancer includes: (a) measuring a urinary tumor marker in a urine sample from an animal with biliary tract cancer treated with the investigational therapeutic agent or therapy; (b) It includes the step of evaluating the effectiveness of the test drug or treatment method for biliary tract cancer based on the measurement results in (a).
  • a urine sample is collected from a patient with biliary tract cancer or a human without biliary tract cancer, and urinary tumor markers in the urine sample are measured.
  • a urine sample is collected from a person with biliary tract cancer and urinary tumor markers are measured in the urine sample prior to treatment with the test therapeutic agent or therapy.
  • urine samples are collected at appropriate times and urinary tumor markers are measured in the urine samples. For example, immediately after treatment, 30 minutes, 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours (1 day), 2-10 days, 10-20 Collect urine samples after 1 day, 20 to 30 days, and 1 month to 6 months. Measurement of urinary tumor markers in urine samples and determination of biliary tract cancer can be performed in the same manner as described above.
  • test therapeutic drug or treatment method to be evaluated or screened is not particularly limited.
  • the test therapeutic agent or therapy may include any material agent, specifically a naturally occurring molecule, such as an amino acid, a peptide, an oligopeptide, a polypeptide, a protein, a nucleic acid, a lipid, a carbohydrate (such as a sugar), steroids, glycopeptides, glycoproteins, proteoglycans, etc.; synthetic analogs or derivatives of naturally occurring molecules, such as peptidomimetics, nucleic acid molecules (aptamers, antisense nucleic acids, double-stranded RNA (RNAi), etc.); naturally occurring and mixtures thereof.
  • the therapeutic agent or treatment method may be a single substance, a complex composed of multiple substances, food, diet, or the like.
  • the test therapeutic agent or treatment method may include radiation, ultraviolet light, etc. in addition to the above-mentioned physical factors.
  • test therapeutic drug or treatment method can be examined under several conditions. Such conditions include the time or duration, amount (large or small), number of times, etc., of treatment with the test therapeutic agent or treatment method. For example, multiple doses can be established by preparing a dilution series of the test therapeutic agent. Furthermore, when examining additive effects, synergistic effects, etc. of multiple test therapeutic agents or treatments, the therapeutic agents or treatments may be used in combination.
  • the fact that the measured value after treatment is lower than the measured value before treatment means that the test drug or treatment is effective for the disappearance of biliary tract cancer, Indicates that the drug shrinks cancer, improves symptoms caused by biliary tract cancer, halts the progression of biliary tract cancer, or is effective.
  • the measured value after treatment is higher than the measured value before treatment or is not significantly different from the measured value before treatment, indicating that the test therapeutic agent or treatment method is not effective in treating biliary tract cancer.
  • Example 1 Comprehensive analysis of urinary metabolites related to biliary tract cancer At Nagoya University Hospital, permission was obtained from 27 patients with biliary tract cancer before tumor resection, 2 weeks after tumor resection, and 4 weeks after tumor resection.
  • Urine samples were collected at the end of the week (62 samples in total).
  • Patient information includes sample ID, sample collection date, age, gender, pre- and postoperative status, osmolarity, prognosis, diagnosis name, surgical method, histopathological type, pathological margin, pathological lymph node metastasis, stage, intraoperative blood transfusion, and medical history. Information regarding medical history, other marker measurements, etc. was recorded.
  • the specific breakdown of urine samples is as follows.
  • LC/MS liquid chromatography mass spectrometer
  • metabolites are identified using a database, that is, peak annotation is performed. If a metabolite not registered in the database is detected, structure estimation may be performed using tandem mass spectrometry (MS/MS), which actively generates fragment ions from target ions. Although it is not always possible to clearly estimate the structure using the MS/MS method, it is much easier to estimate the structure than isolating the target component and analyzing it using a nuclear magnetic resonance apparatus (NMR). Once candidate substances have been narrowed down using the MS/MS method, they are actually synthesized and their mass spectra and MS/MS spectra are compared to confirm the estimated structure.
  • MS/MS tandem mass spectrometry
  • PCA principal component analysis
  • Example 2 ROC curves were determined for the top 20 RF markers identified in Example 1 before and after surgery (with or without tumor), and AUC was determined. The results are shown in the table below.
  • the AUC value represents the discrimination ability for biliary tract cancer when the indicated marker is used alone. The closer the AUC value is to 1, the higher the discrimination ability is, and generally, an AUC value of 0.7 or higher can be considered a good model or a good discrimination ability.
  • Example 3 Construction of a cancer test model that combines multiple markers A cancer test model that combines multiple markers identified in Example 1 was constructed.
  • explanatory variables representing the fit to the training data used for model construction
  • predictor variables representing the predictive performance of the model (leave-one-out cross-validation). Verification
  • a cancer testing model was determined using the following indicators using the OPLS discriminant analysis method.
  • Yobs is the measured value
  • Ycalc is the calculated value by OPLS
  • Ypred is the predicted value when cross-validated, represents the average value.
  • Cross-validation refers to a method in which data is divided, a part of it is analyzed first, and the remaining part is used to test the analysis to verify and confirm the validity of the analysis itself.
  • the explanatory variable R2Y value indicates the fit to the training data used for model construction, and the closer it is to 1, the higher the model accuracy
  • the predictor variable Q2 value indicates the predictive performance of the model (leave-one-out cross -validation (leave-one-out cross-validation), the closer it is to 1, the more predictive the model is.
  • the cancer test model is, for example, when five types of markers are used, the concentration of each marker in the urine sample or the intensity of ions corresponding to each marker (actually, the mass chromatogram obtained by LC/MS measurement).
  • the prediction formula ⁇ x (marker 1 intensity) + ⁇ x (marker 2 intensity) + ⁇ x (marker 3 intensity) + ⁇ x (marker 4 intensity) + ⁇ x (marker 5 intensity) + ⁇ ( ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ are constants) are used to determine the risk of cancer. Specifically, the higher the predicted value, the higher the risk of cancer, and the lower the predicted value, the lower the risk of cancer. When determining whether cancer or normality is true or false, the threshold value of the predicted value is appropriately set.
  • the cancer testing model was evaluated using the OPLS discriminant analysis method for the top 10 or top 20 RF markers identified in Example 1. Specifically, we constructed cancer testing models for the following six areas. In Figures 2 to 8, white bar graphs indicate urine samples collected before biliary tract cancer resection, black bar graphs indicate urine samples collected 2 weeks after surgery, and diagonal bar graphs indicate urine samples collected 4 weeks after surgery. Shows the urine specimen collected in .
  • a model was constructed using the top 10 RF markers preoperatively versus 2 weeks postoperatively, and applied to specimens 4 weeks postoperatively. The results are shown in Figure 2, A (predicted value) and B (AUC). The diagonally lined bar graph is the test sample.
  • a model was constructed using the top 20 RF markers before surgery versus 2 weeks after surgery, and applied to specimens 4 weeks after surgery. The results are shown in Figure 4, A (predicted value) and B (AUC). The diagonally lined bar graph is the test sample.
  • a model was constructed using the top 20 RF markers before surgery versus 4 weeks after surgery, and applied to specimens 2 weeks after surgery. The results are shown in Figure 5, A (predicted value) and B (AUC). The black bar graph is the test sample.
  • markers considered to be particularly important were used to construct a model preoperatively versus postoperatively (both 2 weeks and 4 weeks). These six markers are metabolites that have a high ability to distinguish between biliary tract cancer and healthy individuals, as described below (Example 4).
  • glycochenodeoxycholate 3-sulfate (Table 3: 4th place, Table 6: 1st place), glycocholate (Table 3: 12th place, Table 6: 2nd place), 4-hydroxyphenylpyruvate (Table 3: 19th place, Table 6 : 6th place), glycochenodeoxycholate (Table 3: 16th place, Table 6: 9th place), trans-4-hydroxyproline (Table 3: 10th place, Table 6: 12th place), kynurenine (Table 3: 7th place, Table 6: 18th place).
  • A predicted value
  • B AUC
  • the black bars are postoperative specimens (both 2 and 4 weeks).
  • R2Y 0.447
  • predictive variable Q2 0.359
  • RF markers three markers considered to be particularly important were used to construct a model preoperatively versus postoperatively (both 2 weeks and 4 weeks). These three markers are metabolites that have a high ability to distinguish between biliary tract cancer and healthy individuals, as will be described later (Example 4). Specifically, glycochenodeoxycholate 3-sulfate (Table 3: 4th place, Table 6: 1st place), glycocholate (Table 3: 12th place, Table 6: 2nd place), 4-hydroxyphenylpyruvate (Table 3: 19th place, Table 6 :6th place). The results are shown in A (predicted value) and B (AUC) in FIG. Here, the black bars are postoperative specimens (both 2 and 4 weeks).
  • markers considered to be particularly important were used to build a model preoperatively versus postoperatively (both 2 weeks and 4 weeks). These two markers are metabolites that have a high ability to distinguish between biliary tract cancer and healthy individuals, as described below (Example 4). Specifically, they are glycochenodeoxycholate 3-sulfate (Table 3: 4th place, Table 6: 1st place) and glycocholate (Table 3: 12th place, Table 6: 2nd place). The results are shown in Figure 8, A (predicted value) and B (AUC). Here, the black bars are postoperative specimens (both 2 and 4 weeks).
  • Example 4 Comprehensive analysis of urinary metabolites by comparison of biliary tract cancer patients and healthy subjects With permission from 25 biliary tract cancer patients, urine samples were collected before tumor resection. In addition, as a control group, urine samples were collected from 25 people who were found to be healthy through medical examinations, with their permission. The specific breakdown of urine samples is as follows.
  • LC/MS liquid chromatography mass spectrometer
  • metabolites are identified using a database, that is, peak annotation is performed. If a metabolite not registered in the database is detected, structure estimation may be performed using tandem mass spectrometry (MS/MS), which actively generates fragment ions from target ions. Although it is not always possible to clearly estimate the structure using the MS/MS method, it is much easier to estimate the structure than isolating the target component and analyzing it using a nuclear magnetic resonance apparatus (NMR). Once candidate substances have been narrowed down using the MS/MS method, they are actually synthesized and their mass spectra and MS/MS spectra are compared to confirm the estimated structure.
  • MS/MS tandem mass spectrometry
  • RF random forest analysis
  • Example 5 The cancer test model was evaluated using the OPLS discriminant analysis method for the top 20, top 10, or top 5 RF markers identified in Example 4. Specifically, we constructed cancer testing models for the following three areas. In Figures 11 to 13, white bar graphs indicate urine samples from biliary tract cancer patients (before tumor resection), and black bar graphs indicate urine samples from healthy individuals.

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Abstract

The present invention provides a means and a method for determining biliary tract cancer in a non-invasive and simple manner. Specifically, the present invention relates to a method, a device, and a kit for determining biliary tract cancer in a subject by measuring a tumor marker in urine from a urine sample derived from a subject.

Description

胆道がん検査方法Biliary tract cancer testing method
　本発明は、尿中腫瘍マーカの測定値に基づいて、対象の胆道がんを判定するための方法、キット及び装置に関する。 The present invention relates to a method, kit, and device for determining biliary tract cancer in a subject based on measured values of urinary tumor markers.
　胆道がんは、日本において比較的症例が多いがんであり、予後の指標となる5年生存率も低いことから、早期に発見して治療を行うことが望ましい。早期発見のための胆道がんの腫瘍マーカとして、血液中のCEA、CA19-9等が知られているが、腫瘍特異性は低く、精度も高くない。また、血液検査は侵襲を伴い、実質的に医療機関で行われる検査に限定される。 Biliary tract cancer is a relatively common cancer in Japan, and the 5-year survival rate, which is an indicator of prognosis, is low, so it is desirable to detect and treat it early. Blood CEA, CA19-9, etc. are known as tumor markers for biliary tract cancer for early detection, but their tumor specificity is low and their accuracy is not high. Furthermore, blood tests are invasive and are essentially limited to tests performed at medical institutions.
　胆道がんなどのがんの腫瘍マーカとして、例えば特許文献１～５の報告がある。特許文献１では、腫瘍マーカとして主に遺伝子及びプロテオミクスが記載されている。特許文献２～５には、大腸がんなどのがんの腫瘍マーカとして代謝物が記載されているが、本明細書に開示する尿中腫瘍マーカは記載されていない。 For example, there are reports in Patent Documents 1 to 5 as tumor markers for cancer such as biliary tract cancer. Patent Document 1 mainly describes genes and proteomics as tumor markers. Patent Documents 2 to 5 describe metabolites as tumor markers for cancers such as colon cancer, but do not describe the urinary tumor marker disclosed herein.
US特許出願公開2012/053073A1US Patent Application Publication 2012/053073A1 US特許出願公開2015/044716A1US Patent Application Publication 2015/044716A1 US特許出願公開2004/121375A1US Patent Application Publication 2004/121375A1 US特許出願公開2019/094205A1US Patent Application Publication 2019/094205A1 US特許出願公開2018/299448A1US Patent Application Publication 2018/299448A1
　本発明は、従来から望まれている、非侵襲的かつ簡便に胆道がんを判定するための手段及び方法を提供することを目的とする。 An object of the present invention is to provide a means and method for non-invasively and easily determining biliary tract cancer, which has been desired in the past.
　本発明者は、がん尿中腫瘍マーカを探索する過程で、胆道がんに関連するマーカ群を特定し、これらのマーカを単独で又は組み合わせて使用することで胆道がんの判定やリスク予測、胆道がんのモニタリングを簡便かつ無侵襲に行うことができることを見出した。 In the process of searching for cancer urinary tumor markers, the present inventor identified a group of markers related to biliary tract cancer, and used these markers alone or in combination to determine biliary tract cancer and predict risk. discovered that biliary tract cancer monitoring can be performed easily and non-invasively.
　すなわち、本発明は、尿中腫瘍マーカである尿中代謝物を測定することにより、対象において、胆道がんを判定する及び／又は胆道がんについてモニタリングするための方法、装置及びキットに関する。具体的な実施形態として以下を包含する： That is, the present invention relates to a method, device, and kit for determining biliary tract cancer and/or monitoring biliary tract cancer in a subject by measuring urinary metabolites that are urinary tumor markers. Specific embodiments include:
［１］対象における胆道がんを判定する方法であって、
　対象由来の尿サンプル中の尿中腫瘍マーカを測定するステップであって、該尿中腫瘍マーカが、コール酸塩、ケノデオキシコール酸硫酸塩、LC/MSネガティブイオン検出モードで質量電荷比259.028として計測される化合物（C10H12O6S）、グリコケノデオキシコール酸3-硫酸塩、イソロイシルヒドロキシプロリン、pro-ヒドロキシ-pro、キヌレニン、4-メトキシフェノール硫酸塩、5-ヒドロキシリシン、trans-4-ヒドロキシプロリン、グリシルロイシン、グリココール酸塩、LC/MSネガティブイオン検出モードで質量電荷比197.068として計測される化合物（C7H10N4O3）、LC/MSネガティブイオン検出モードで質量電荷比509.277として計測される化合物（C28H38N4O5）、3-ヒドロキシキヌレニン、グリコケノデオキシコール酸塩、イソロイシルグリシン、フェニルアラニルヒドロキシプロリン、4-ヒドロキシフェニルピルビン酸塩、乳酸塩、シクロ(pro-ヒドロキシpro)、トリプトファン、N6-アセチルリシン、ガンマ-グルタミルフェニルアラニン、ロイシルヒドロキシプロリン、LC/MSネガティブイオン検出モードで質量電荷比328.152として計測される化合物（C14H23N3O6）、LC/MSポジティブイオン検出モードで質量電荷比146.081として計測される化合物（C6H11NO3）、アンドロステロングルクロニド、3-ヒドロキシアントラニレート、11ベータ-ヒドロキシアンドロステロングルクロニド、シスタチオニン、カルノシン、プロリン、アンセリン、アラビトール/キシリトール、3-ヒドロキシ-2-エチルプロピオネート、2R,3R-ジヒドロキシブチレート、ガンマ-グルタミルチロシン、1-メチルアデニン、ジヒドロオロト酸、アラニン、及び17アルファ-ヒドロキシプレグネノロングルクロニドから選択される少なくとも1種の尿中腫瘍マーカを含む、上記ステップ；
　上記測定結果に基づいて対象における胆道がんを判定するステップ
を含む、方法。 [1] A method for determining biliary tract cancer in a subject, comprising:
measuring a urinary tumor marker in a urine sample from a subject, the urinary tumor marker being measured in cholate, chenodeoxycholate sulfate, LC/MS negative ion detection mode as a mass-to-charge ratio of 259.028; compound (C10H12O6S), glycochenodeoxycholic acid 3-sulfate, isoleucylhydroxyproline, pro-hydroxy-pro, kynurenine, 4-methoxyphenol sulfate, 5-hydroxylysine, trans-4-hydroxyproline, glycylleucine , glycocholate, compound measured as mass-to-charge ratio 197.068 in LC/MS negative ion detection mode (C7H10N4O3), compound measured as mass-to-charge ratio 509.277 in LC/MS negative ion detection mode (C28H38N4O5), 3- Hydroxykynurenine, glycochenodeoxycholate, isoleucylglycine, phenylalanylhydroxyproline, 4-hydroxyphenylpyruvate, lactate, cyclo(pro-hydroxypro), tryptophan, N6-acetyllysine, gamma-glutamylphenylalanine, Leucyl hydroxyproline, a compound measured as a mass-to-charge ratio of 328.152 in LC/MS negative ion detection mode (C14H23N3O6), a compound measured as a mass-to-charge ratio of 146.081 in LC/MS positive ion detection mode (C6H11NO3), androsterone glucuronide , 3-hydroxyantranilate, 11beta-hydroxyandrosterone glucuronide, cystathionine, carnosine, proline, anserine, arabitol/xylitol, 3-hydroxy-2-ethylpropionate, 2R,3R-dihydroxybutyrate, gamma-glutamyl said step comprising at least one urinary tumor marker selected from tyrosine, 1-methyladenine, dihydroorotate, alanine, and 17 alpha-hydroxypregnenolone glucuronide;
A method comprising determining biliary tract cancer in a subject based on the measurement results.
［２］胆道がんの判定装置であって、
　尿サンプル中の尿中腫瘍マーカを測定する測定部であって、該尿中腫瘍マーカが、コール酸塩、ケノデオキシコール酸硫酸塩、LC/MSネガティブイオン検出モードで質量電荷比259.028として計測される化合物（C10H12O6S）、グリコケノデオキシコール酸3-硫酸塩、イソロイシルヒドロキシプロリン、pro-ヒドロキシ-pro、キヌレニン、4-メトキシフェノール硫酸塩、5-ヒドロキシリシン、trans-4-ヒドロキシプロリン、グリシルロイシン、グリココール酸塩、LC/MSネガティブイオン検出モードで質量電荷比197.068として計測される化合物（C7H10N4O3）、LC/MSネガティブイオン検出モードで質量電荷比509.277として計測される化合物（C28H38N4O5）、3-ヒドロキシキヌレニン、グリコケノデオキシコール酸塩、イソロイシルグリシン、フェニルアラニルヒドロキシプロリン、4-ヒドロキシフェニルピルビン酸塩、乳酸塩、シクロ(pro-ヒドロキシpro)、トリプトファン、N6-アセチルリシン、ガンマ-グルタミルフェニルアラニン、ロイシルヒドロキシプロリン、LC/MSネガティブイオン検出モードで質量電荷比328.152として計測される化合物（C14H23N3O6）、LC/MSポジティブイオン検出モードで質量電荷比146.081として計測される化合物（C6H11NO3）、アンドロステロングルクロニド、3-ヒドロキシアントラニレート、11ベータ-ヒドロキシアンドロステロングルクロニド、シスタチオニン、カルノシン、プロリン、アンセリン、アラビトール/キシリトール、3-ヒドロキシ-2-エチルプロピオネート、2R,3R-ジヒドロキシブチレート、ガンマ-グルタミルチロシン、1-メチルアデニン、ジヒドロオロト酸、アラニン、及び17アルファ-ヒドロキシプレグネノロングルクロニドから選択される少なくとも1種の尿中腫瘍マーカを含む、測定部と、
　上記測定部で測定した尿中腫瘍マーカの測定値を基準値又は前回の測定値と比較する比較部と、
　上記比較部で得られた比較結果から胆道がんを判定する判定部と
を備えることを特徴とする装置。 [2] A device for determining biliary tract cancer,
A measurement unit for measuring a urinary tumor marker in a urine sample, wherein the urinary tumor marker is cholate, chenodeoxycholate sulfate, and a compound measured as a mass-to-charge ratio of 259.028 in LC/MS negative ion detection mode. (C10H12O6S), glycochenodeoxycholic acid 3-sulfate, isoleucylhydroxyproline, pro-hydroxy-pro, kynurenine, 4-methoxyphenol sulfate, 5-hydroxylysine, trans-4-hydroxyproline, glycylleucine, glycosyl Cholate, Compound measured as mass to charge ratio 197.068 in LC/MS negative ion detection mode (C7H10N4O3), Compound measured as mass to charge ratio 509.277 in LC/MS negative ion detection mode (C28H38N4O5), 3-Hydroxykynurenine , glycochenodeoxycholate, isoleucylglycine, phenylalanyl hydroxyproline, 4-hydroxyphenylpyruvate, lactate, cyclo(pro-hydroxypro), tryptophan, N6-acetyllysine, gamma-glutamylphenylalanine, leucyl Hydroxyproline, compound measured as mass-to-charge ratio 328.152 in LC/MS negative ion detection mode (C14H23N3O6), compound measured as mass-to-charge ratio 146.081 in LC/MS positive ion detection mode (C6H11NO3), androsterone glucuronide, 3 -Hydroxyantranilate, 11beta-hydroxyandrosterone glucuronide, cystathionine, carnosine, proline, anserine, arabitol/xylitol, 3-hydroxy-2-ethylpropionate, 2R,3R-dihydroxybutyrate, gamma-glutamyltyrosine, a measurement section comprising at least one urinary tumor marker selected from 1-methyladenine, dihydroorotate, alanine, and 17 alpha-hydroxypregnenolone glucuronide;
a comparison unit that compares the measurement value of the urinary tumor marker measured by the measurement unit with a reference value or a previous measurement value;
An apparatus comprising: a determination section that determines biliary tract cancer from the comparison results obtained by the comparison section.
［３］胆道がんの判定用キットであって、
　コール酸塩、ケノデオキシコール酸硫酸塩、LC/MSネガティブイオン検出モードで質量電荷比259.028として計測される化合物（C10H12O6S）、グリコケノデオキシコール酸3-硫酸塩、イソロイシルヒドロキシプロリン、pro-ヒドロキシ-pro、キヌレニン、4-メトキシフェノール硫酸塩、5-ヒドロキシリシン、trans-4-ヒドロキシプロリン、グリシルロイシン、グリココール酸塩、LC/MSネガティブイオン検出モードで質量電荷比197.068として計測される化合物（C7H10N4O3）、LC/MSネガティブイオン検出モードで質量電荷比509.277として計測される化合物（C28H38N4O5）、3-ヒドロキシキヌレニン、グリコケノデオキシコール酸塩、イソロイシルグリシン、フェニルアラニルヒドロキシプロリン、4-ヒドロキシフェニルピルビン酸塩、乳酸塩、シクロ(pro-ヒドロキシpro)、トリプトファン、N6-アセチルリシン、ガンマ-グルタミルフェニルアラニン、ロイシルヒドロキシプロリン、LC/MSネガティブイオン検出モードで質量電荷比328.152として計測される化合物（C14H23N3O6）、LC/MSポジティブイオン検出モードで質量電荷比146.081として計測される化合物（C6H11NO3）、アンドロステロングルクロニド、3-ヒドロキシアントラニレート、11ベータ-ヒドロキシアンドロステロングルクロニド、シスタチオニン、カルノシン、プロリン、アンセリン、アラビトール/キシリトール、3-ヒドロキシ-2-エチルプロピオネート、2R,3R-ジヒドロキシブチレート、ガンマ-グルタミルチロシン、1-メチルアデニン、ジヒドロオロト酸、アラニン、及び17アルファ-ヒドロキシプレグネノロングルクロニドから選択される少なくとも1種の尿中腫瘍マーカを測定するための手段を含むことを特徴とするキット。 [3] A kit for determining biliary tract cancer,
Cholate, chenodeoxycholic acid sulfate, compound measured as mass-to-charge ratio 259.028 in LC/MS negative ion detection mode (C10H12O6S), glycochenodeoxycholic acid 3-sulfate, isoleucylhydroxyproline, pro-hydroxy-pro, Kynurenine, 4-methoxyphenol sulfate, 5-hydroxylysine, trans-4-hydroxyproline, glycylleucine, glycocholate, compound measured as mass-to-charge ratio 197.068 in LC/MS negative ion detection mode (C7H10N4O3) , Compound measured as mass-to-charge ratio 509.277 in LC/MS negative ion detection mode (C28H38N4O5), 3-hydroxykynurenine, glycochenodeoxycholate, isoleucylglycine, phenylalanylhydroxyproline, 4-hydroxyphenylpyruvate , lactate, cyclo(pro-hydroxypro), tryptophan, N6-acetyllysine, gamma-glutamylphenylalanine, leucylhydroxyproline, compound measured as mass-to-charge ratio 328.152 in LC/MS negative ion detection mode (C14H23N3O6), Compound (C6H11NO3) measured as mass-to-charge ratio 146.081 in LC/MS positive ion detection mode, androsterone glucuronide, 3-hydroxyanthranilate, 11beta-hydroxyandrosterone glucuronide, cystathionine, carnosine, proline, anserine, arabitol/ At least one member selected from xylitol, 3-hydroxy-2-ethylpropionate, 2R,3R-dihydroxybutyrate, gamma-glutamyltyrosine, 1-methyladenine, dihydroorotate, alanine, and 17 alpha-hydroxypregnenolone glucuronide A kit comprising a means for measuring a urinary tumor marker.
［４］胆道がんの治療の有効性の評価方法であって、
　被験治療薬又は治療法による処置を受けた胆道がんを有する患者からの尿サンプルにおいて、尿中腫瘍マーカを測定するステップであって、該尿中腫瘍マーカが、コール酸塩、ケノデオキシコール酸硫酸塩、LC/MSネガティブイオン検出モードで質量電荷比259.028として計測される化合物（C10H12O6S）、グリコケノデオキシコール酸3-硫酸塩、イソロイシルヒドロキシプロリン、pro-ヒドロキシ-pro、キヌレニン、4-メトキシフェノール硫酸塩、5-ヒドロキシリシン、trans-4-ヒドロキシプロリン、グリシルロイシン、グリココール酸塩、LC/MSネガティブイオン検出モードで質量電荷比197.068として計測される化合物（C7H10N4O3）、LC/MSネガティブイオン検出モードで質量電荷比509.277として計測される化合物（C28H38N4O5）、3-ヒドロキシキヌレニン、グリコケノデオキシコール酸塩、イソロイシルグリシン、フェニルアラニルヒドロキシプロリン、4-ヒドロキシフェニルピルビン酸塩、乳酸塩、シクロ(pro-ヒドロキシpro)、トリプトファン、N6-アセチルリシン、ガンマ-グルタミルフェニルアラニン、ロイシルヒドロキシプロリン、LC/MSネガティブイオン検出モードで質量電荷比328.152として計測される化合物（C14H23N3O6）、LC/MSポジティブイオン検出モードで質量電荷比146.081として計測される化合物（C6H11NO3）、アンドロステロングルクロニド、3-ヒドロキシアントラニレート、11ベータ-ヒドロキシアンドロステロングルクロニド、シスタチオニン、カルノシン、プロリン、アンセリン、アラビトール/キシリトール、3-ヒドロキシ-2-エチルプロピオネート、2R,3R-ジヒドロキシブチレート、ガンマ-グルタミルチロシン、1-メチルアデニン、ジヒドロオロト酸、アラニン、及び17アルファ-ヒドロキシプレグネノロングルクロニドから選択される少なくとも1種の尿中腫瘍マーカを含む、上記ステップ、
　上記測定結果に基づいて胆道がんに対する被験治療薬又は治療法の有効性を評価するステップ
を含む方法。 [4] A method for evaluating the effectiveness of biliary tract cancer treatment, comprising:
measuring a urinary tumor marker in a urine sample from a patient with biliary tract cancer treated with an investigational therapeutic agent or therapy, the urinary tumor marker being cholate, chenodeoxycholate sulfate; , Compound measured as mass-to-charge ratio 259.028 in LC/MS negative ion detection mode (C10H12O6S), glycochenodeoxycholic acid 3-sulfate, isoleucylhydroxyproline, pro-hydroxy-pro, kynurenine, 4-methoxyphenol sulfate , 5-hydroxylysine, trans-4-hydroxyproline, glycylleucine, glycocholate, compound measured as mass-to-charge ratio 197.068 in LC/MS negative ion detection mode (C7H10N4O3), LC/MS negative ion detection mode (C28H38N4O5), 3-hydroxykynurenine, glycochenodeoxycholate, isoleucylglycine, phenylalanylhydroxyproline, 4-hydroxyphenylpyruvate, lactate, cyclo(pro- Hydroxy pro), tryptophan, N6-acetyl lysine, gamma-glutamylphenylalanine, leucyl hydroxyproline, compound measured as mass-to-charge ratio 328.152 in LC/MS negative ion detection mode (C14H23N3O6), in LC/MS positive ion detection mode Compound (C6H11NO3) measured as mass-to-charge ratio 146.081, androsterone glucuronide, 3-hydroxyanthranilate, 11beta-hydroxyandrosterone glucuronide, cystathionine, carnosine, proline, anserine, arabitol/xylitol, 3-hydroxy-2- comprising at least one urinary tumor marker selected from ethyl propionate, 2R,3R-dihydroxybutyrate, gamma-glutamyltyrosine, 1-methyladenine, dihydroorotate, alanine, and 17 alpha-hydroxypregnenolone glucuronide; The above steps,
A method comprising the step of evaluating the effectiveness of a test therapeutic agent or treatment method for biliary tract cancer based on the measurement results.
　本明細書は、本願の優先権の基礎となる2022年6月3日出願の日本国特許出願番号2022-090965号の開示内容を包含する。 This specification includes the disclosure content of Japanese Patent Application No. 2022-090965 filed on June 3, 2022, which is the basis of the priority of this application.
　本発明により、低侵襲性で、簡便かつ低コストに胆道がんを判定するための方法、装置及びキットが提供される。尿による検査のため、臨床現場における採取法も非常に簡便になり、医療従事者の利便性も大きく向上する。したがって、本発明は、胆道がんの診断、検査、治療評価、創薬などの分野に有用である。 The present invention provides a method, device, and kit for determining biliary tract cancer in a minimally invasive, simple, and low-cost manner. Since the test uses urine, the collection method in clinical settings is also extremely simple, greatly improving convenience for medical professionals. Therefore, the present invention is useful in fields such as biliary tract cancer diagnosis, testing, therapeutic evaluation, and drug discovery.
胆道がんに関連する尿中代謝物をランダムフォレスト（RF）によりランク付けした場合の上位30の代謝物を示すグラフである。This is a graph showing the top 30 metabolites when urinary metabolites related to biliary tract cancer are ranked by random forest (RF). 胆道がんについて、10種のマーカをがん検査モデルに適用した場合の予測値の計算結果を示すグラフ（Ａ）、及びがん検査モデルのAUCを示すグラフ（Ｂ）である。For biliary tract cancer, they are a graph (A) showing the calculation results of predicted values when 10 types of markers are applied to the cancer test model, and a graph (B) showing the AUC of the cancer test model. 胆道がんについて、10種のマーカをがん検査モデルに適用した場合の予測値の計算結果を示すグラフ（Ａ）、及びがん検査モデルのAUCを示すグラフ（Ｂ）である。For biliary tract cancer, they are a graph (A) showing the calculation results of predicted values when 10 types of markers are applied to the cancer test model, and a graph (B) showing the AUC of the cancer test model. 胆道がんについて、20種のマーカをがん検査モデルに適用した場合の予測値の計算結果を示すグラフ（Ａ）、及びがん検査モデルのAUCを示すグラフ（Ｂ）である。For biliary tract cancer, they are a graph (A) showing the calculation results of predicted values when 20 types of markers are applied to the cancer test model, and a graph (B) showing the AUC of the cancer test model. 胆道がんについて、20種のマーカをがん検査モデルに適用した場合の予測値の計算結果を示すグラフ（Ａ）、及びがん検査モデルのAUCを示すグラフ（Ｂ）である。For biliary tract cancer, they are a graph (A) showing the calculation results of predicted values when 20 types of markers are applied to the cancer test model, and a graph (B) showing the AUC of the cancer test model. 胆道がんについて、重要と考えられる6種のマーカをがん検査モデルに適用した場合の予測値の計算結果を示すグラフ（Ａ）、及びがん検査モデルのAUCを示すグラフ（Ｂ）である。For biliary tract cancer, these are a graph (A) showing the calculation results of predicted values when six types of markers that are considered important are applied to the cancer testing model, and a graph (B) showing the AUC of the cancer testing model. . 胆道がんについて、重要と考えられる3種のマーカをがん検査モデルに適用した場合の予測値の計算結果を示すグラフ（Ａ）、及びがん検査モデルのAUCを示すグラフ（Ｂ）である。For biliary tract cancer, a graph (A) showing the calculation results of predicted values when three types of markers considered to be important are applied to the cancer testing model, and a graph (B) showing the AUC of the cancer testing model. . 胆道がんについて、重要と考えられる2種のマーカをがん検査モデルに適用した場合の予測値の計算結果を示すグラフ（Ａ）、及びがん検査モデルのAUCを示すグラフ（Ｂ）である。For biliary tract cancer, these are a graph (A) showing the calculation results of predicted values when two types of markers considered to be important are applied to the cancer testing model, and a graph (B) showing the AUC of the cancer testing model. . 本発明を適用した装置の構成例を示す。An example of the configuration of a device to which the present invention is applied is shown. 胆道がんと健常者の差に関連する尿中代謝物をランダムフォレスト（RF）によりランク付けした場合の上位20の代謝物を示すグラフである。This is a graph showing the top 20 metabolites when urinary metabolites related to the difference between biliary tract cancer and healthy individuals are ranked by random forest (RF). 胆道がんについて、重要と考えられる20種のマーカをがん検査モデルに適用した場合の予測値の計算結果を示すグラフ（Ａ）、及びがん検査モデルのAUCを示すグラフ（Ｂ）である。For biliary tract cancer, these are a graph (A) showing the calculation results of predicted values when 20 types of markers considered to be important are applied to the cancer testing model, and a graph (B) showing the AUC of the cancer testing model. . 胆道がんについて、重要と考えられる10種のマーカをがん検査モデルに適用した場合の予測値の計算結果を示すグラフ（Ａ）、及びがん検査モデルのAUCを示すグラフ（Ｂ）である。For biliary tract cancer, a graph (A) showing the calculation results of predicted values when 10 types of markers considered to be important are applied to the cancer testing model, and a graph (B) showing the AUC of the cancer testing model. . 胆道がんについて、重要と考えられる5種のマーカをがん検査モデルに適用した場合の予測値の計算結果を示すグラフ（Ａ）、及びがん検査モデルのAUCを示すグラフ（Ｂ）である。For biliary tract cancer, these are a graph (A) showing the calculation results of predicted values when five types of markers considered to be important are applied to the cancer testing model, and a graph (B) showing the AUC of the cancer testing model. .
　本発明が提供する方法、装置及びキットでは、胆道がんに関連する新規な尿中腫瘍マーカ及びマーカ群を利用する。この尿中腫瘍マーカは、胆道がんの有無に関連してその尿中レベルに差異がある代謝物であるため、胆道がんの検出、胆道がんのリスク予測、胆道がんのステージ判定、胆道がんの予後判定、胆道がんについてのモニタリング及び／又は胆道がんに対する治療効果のモニタリングなどに有用である。 The methods, devices, and kits provided by the present invention utilize novel urinary tumor markers and marker groups associated with biliary tract cancer. This urinary tumor marker is a metabolite whose urinary level differs depending on the presence or absence of biliary tract cancer, so it can be used to detect biliary tract cancer, predict the risk of biliary tract cancer, and determine the stage of biliary tract cancer. It is useful for determining the prognosis of biliary tract cancer, monitoring biliary tract cancer, and/or monitoring the therapeutic effect on biliary tract cancer.
　本発明に係る胆道がんの判定方法は、対象由来の尿サンプル中の尿中腫瘍マーカを測定するステップと、その測定結果に基づいて対象における胆道がんを判定するステップを含む。 The method for determining biliary tract cancer according to the present invention includes the steps of measuring a urinary tumor marker in a urine sample derived from a subject, and determining biliary tract cancer in the subject based on the measurement results.
　本発明では、胆道がんを判定する。胆道がんとは、胆道に発生するがん（悪性腫瘍）をいい、肝内胆管がん、肝外胆管がん（肝門部領域胆管がん、遠位胆管がん）、胆嚢がん、及び乳頭部がんに分類される。胆道がんには、原発性、転移性、再発性のものがあり、またその進行度と広がりの程度からステージに分類されている。この原発性、転移性又は再発性の違いや、ステージの違いに応じて、必要な処置（外科手術、化学療法、放射線療法、免疫療法など）も異なる。 In the present invention, biliary tract cancer is determined. Biliary tract cancer refers to cancer (malignant tumor) that occurs in the bile tract, including intrahepatic bile duct cancer, extrahepatic bile duct cancer (portal bile duct cancer, distal bile duct cancer), gallbladder cancer, It is classified as papillary cancer. Biliary tract cancers are classified into primary, metastatic, and recurrent types, and are classified into stages based on the degree of progression and spread. Necessary treatments (surgery, chemotherapy, radiotherapy, immunotherapy, etc.) also differ depending on whether the disease is primary, metastatic, or recurrent, and the stage.
　一態様において、尿中腫瘍マーカを測定するステップでは、胆道がんに関連する尿中腫瘍マーカを測定する。本発明において測定する対象となる「尿中代謝物」又は「尿中腫瘍マーカ」は、以下の表1に列挙される尿中代謝物を意味する。尿中代謝物は、血中の物質と比較すると酵素の影響を受けにくく構造的に安定しているため、腫瘍マーカとしての利便性が高い。その上、尿を検体に用いるため対象から容易に採取でき、がんのスクリーニング用途としても非常に利用しやすい。また「マーカ群」とは、2以上の尿中腫瘍マーカからなる組み合わせである。 In one embodiment, in the step of measuring a urinary tumor marker, a urinary tumor marker associated with biliary tract cancer is measured. The "urinary metabolites" or "urinary tumor markers" to be measured in the present invention refer to the urinary metabolites listed in Table 1 below. Urinary metabolites are more convenient as tumor markers because they are less affected by enzymes and are structurally stable compared to substances in the blood. Furthermore, since urine is used as a specimen, it can be easily collected from a subject, making it extremely easy to use for cancer screening. Furthermore, a "marker group" is a combination of two or more urinary tumor markers.
　「測定する」とは、代謝物の尿サンプル中の相対存在量又は絶対濃度を求めることを意味する。相対存在量とは、意図的に添加した標準物質に対して、目的とする代謝物の測定強度の比である。一方、絶対濃度とは、目的とする代謝物に対して、あらかじめ同じ代謝物を用いて検量線（代謝物の濃度と代謝物の測定強度との関係）を作成し、測定された強度からその絶対濃度を算出する方法である。また本発明では、「尿中腫瘍マーカを測定する」とは、尿中腫瘍マーカである代謝物を測定してもよいし、又はその派生物若しくは誘導体を測定してもよい。「派生物」及び「誘導体」とは、尿中腫瘍マーカである代謝物から派生する物質及び当該代謝物に由来する物質をそれぞれ意味する。「派生物」及び「誘導体」には、例えば、代謝物の断片、修飾された代謝物などが含まれるが、これに限定されるものではない。 "Measure" means determining the relative abundance or absolute concentration of a metabolite in a urine sample. Relative abundance is the ratio of the measured intensity of a metabolite of interest to an intentionally added standard substance. On the other hand, the absolute concentration is defined as a calibration curve (relationship between the concentration of the metabolite and the measured intensity of the metabolite) created in advance using the same metabolite for the target metabolite, and calculated from the measured intensity. This is a method to calculate absolute concentration. Furthermore, in the present invention, "measuring a urinary tumor marker" may mean measuring a metabolite that is a urinary tumor marker, or a derivative or a derivative thereof. "Derivative" and "derivative" refer to a substance derived from a metabolite that is a urinary tumor marker and a substance derived from the metabolite, respectively. "Derivatives" and "derivatives" include, but are not limited to, fragments of metabolites, modified metabolites, and the like.
　本発明において使用する主な尿中腫瘍マーカを以下の表1にまとめる。表中、「代謝物」の欄では、データベースによる検索の結果、構造がわかった代謝物の名称、又は構造がわからない場合には記号及び推定化学式を示す。推定化学式は、「測定質量」及び「測定モード」とHuman Metabolome Database (HMDB)などの代謝物データベースから推定したものである。表１のCAS登録番号は、化学物質IDのデファクトスタンダードであり、化学物質を特定するための番号であり、HMDB IDは、人体中小分子代謝物のオンラインデータベースHMDBのIDである。表1において、「測定質量」の欄には、「測定モード」の欄に記載された検出手段により検出した場合の質量電荷比を示す。「測定モード」の欄における「Neg」、「Pos Early」、及び「Polar」とは、それぞれ「液体クロマトグラフ質量分析装置（LC/MS）のネガティブイオン検出モード」、「液体クロマトグラフ質量分析装置（LC/MS）のポジティブイオン検出モードのうち親水性化合物向けに最適化したモード」、及び「親水性相互作用液体クロマトグラフィ（HILIC）カラムを用いた液体クロマトグラフ質量分析装置（LC/MS）のネガティブイオン検出モードであり、極性化合物向けに最適化した検出モード」を表している。本明細書中では、「LC/MS pos early」を単に「液体クロマトグラフ質量分析装置（LC/MS）のポジティブイオン検出モード」ともいう。表1の測定質量は、基本的に、イオン化された代謝物の質量であり、プロトンが一つ付加あるいは一つ失われ、元の代謝物の質量から±1変動している。ただし、測定条件によっては、複数のプロトンやナトリウムなどが付加あるいは失われる場合があり、その分、測定質量が変動する。また、代謝物にエネルギーを与えて開裂させたフラグメントイオンの質量スペクトルを測定してもよい。 The main urinary tumor markers used in the present invention are summarized in Table 1 below. In the table, the "metabolite" column shows the name of a metabolite whose structure was found as a result of a database search, or the symbol and estimated chemical formula if the structure is unknown. The estimated chemical formula is estimated from the "measured mass" and "measurement mode" and a metabolite database such as the Human Metabolome Database (HMDB). The CAS registration number in Table 1 is the de facto standard for chemical substance IDs and is a number for identifying chemical substances, and the HMDB ID is the ID of HMDB, an online database of small and medium molecule metabolites in the human body. In Table 1, the "Measurement mass" column shows the mass-to-charge ratio when detected by the detection means described in the "Measurement mode" column. "Neg", "Pos Early", and "Polar" in the "Measurement mode" column are "negative ion detection mode of liquid chromatograph mass spectrometer (LC/MS)" and "liquid chromatograph mass spectrometer (LC/MS)", respectively. (LC/MS) positive ion detection mode optimized for hydrophilic compounds” and “Liquid chromatography mass spectrometry (LC/MS) using a hydrophilic interaction liquid chromatography (HILIC) column”. This is a negative ion detection mode, which is a detection mode optimized for polar compounds. In this specification, "LC/MS pos early" is also simply referred to as "positive ion detection mode of liquid chromatograph mass spectrometer (LC/MS)." The measured mass in Table 1 is basically the mass of the ionized metabolite, with one proton added or lost, and the mass varies by ±1 from the original metabolite mass. However, depending on the measurement conditions, multiple protons, sodium, etc. may be added or lost, and the measured mass will vary accordingly. Alternatively, a mass spectrum of fragment ions obtained by imparting energy to the metabolite and causing it to cleave may be measured.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-I000002
Figure JPOXMLDOC01-appb-I000003
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-I000002
Figure JPOXMLDOC01-appb-I000003
　一実施形態において、表1に示すコール酸塩を測定する。すなわち、LC/MSネガティブイオン検出モードで質量407.280として計測される化合物を測定する。 In one embodiment, cholate as shown in Table 1 is measured. That is, a compound measured as a mass of 407.280 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示すケノデオキシコール酸硫酸塩(2)を測定する。すなわち、LC/MSネガティブイオン検出モードで質量235.118として計測される化合物を測定する。 In one embodiment, chenodeoxycholic acid sulfate (2) shown in Table 1 is measured. That is, a compound measured as a mass of 235.118 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示すX-17686（C10H12O6S）を測定する。すなわち、LC/MSネガティブイオン検出モードで質量259.028として計測される化合物（C10H12O6S）を測定する。 In one embodiment, X-17686 (C10H12O6S) shown in Table 1 is measured. That is, a compound (C10H12O6S) whose mass is measured as 259.028 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示すグリコケノデオキシコール酸3-硫酸塩を測定する。すなわち、LC/MSネガティブイオン検出モードで質量263.628として計測される化合物を測定する。 In one embodiment, glycochenodeoxycholic acid 3-sulfate as shown in Table 1 is measured. That is, a compound measured as a mass of 263.628 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示すイソロイシルヒドロキシプロリンを測定する。すなわち、LC/MSポジティブイオン検出モードで質量245.150として計測される化合物を測定する。 In one embodiment, isoleucylhydroxyproline as shown in Table 1 is measured. That is, a compound measured as a mass of 245.150 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示すpro-ヒドロキシ-proを測定する。すなわち、LC/MSポジティブイオン検出モードで質量229.118として計測される化合物を測定する。 In one embodiment, pro-hydroxy-pro as shown in Table 1 is measured. That is, a compound measured as a mass of 229.118 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示すキヌレニンを測定する。すなわち、LC/MSポジティブイオン検出モードで質量209.092として計測される化合物を測定する。 In one embodiment, kynurenine as shown in Table 1 is measured. That is, a compound measured as a mass of 209.092 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示す4-メトキシフェノール硫酸塩を測定する。すなわち、LC/MSネガティブイオン検出モードで質量203.002として計測される化合物を測定する。 In one embodiment, 4-methoxyphenol sulfate as shown in Table 1 is measured. That is, a compound measured as a mass of 203.002 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示す5-ヒドロキシリシンを測定する。すなわち、LC/MSポジティブイオン検出モードで質量163.108として計測される化合物を測定する。 In one embodiment, 5-hydroxylysine as shown in Table 1 is measured. That is, a compound measured as a mass of 163.108 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示すtrans-4-ヒドロキシプロリンを測定する。すなわち、LC/MS極性検出モードで質量130.051として計測される化合物を測定する。 In one embodiment, trans-4-hydroxyproline shown in Table 1 is measured. That is, a compound whose mass is measured as 130.051 in LC/MS polar detection mode is measured.
　一実施形態において、表1に示すグリシルロイシンを測定する。すなわち、LC/MSポジティブイオン検出モードで質量189.123として計測される化合物を測定する。 In one embodiment, glycylleucine as shown in Table 1 is measured. That is, a compound measured as a mass of 189.123 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示すグリココール酸塩を測定する。すなわち、LC/MSネガティブイオン検出モードで質量464.302として計測される化合物を測定する。 In one embodiment, the glycocholates shown in Table 1 are measured. That is, a compound measured as a mass of 464.302 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示すX-13728（C7H10N4O3）を測定する。すなわち、LC/MSネガティブイオン検出モードで質量197.068として計測される化合物（C7H10N4O3）を測定する。 In one embodiment, X-13728 (C7H10N4O3) shown in Table 1 is measured. That is, a compound (C7H10N4O3) whose mass is measured as 197.068 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示すX-21851（C28H38N4O5）を測定する。すなわち、LC/MSネガティブイオン検出モードで質量509.277として計測される化合物（C28H38N4O5）を測定する。 In one embodiment, X-21851 (C28H38N4O5) shown in Table 1 is measured. That is, a compound (C28H38N4O5) measured as a mass of 509.277 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示す3-ヒドロキシキヌレニンを測定する。すなわち、LC/MSポジティブイオン検出モードで質量225.087として計測される化合物を測定する。 In one embodiment, 3-hydroxykynurenine as shown in Table 1 is measured. That is, a compound measured as a mass of 225.087 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示すグリコケノデオキシコール酸塩を測定する。すなわち、LC/MSネガティブイオン検出モードで質量448.307として計測される化合物を測定する。 In one embodiment, the glycochenodeoxycholates shown in Table 1 are measured. That is, a compound measured as a mass of 448.307 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示すイソロイシルグリシンを測定する。すなわち、LC/MSネガティブイオン検出モードで質量187.116として計測される化合物を測定する。 In one embodiment, isoleucylglycine shown in Table 1 is measured. That is, a compound measured as a mass of 187.116 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示すフェニルアラニルヒドロキシプロリンを測定する。すなわち、LC/MSポジティブイオン検出モードで質量279.134として計測される化合物を測定する。 In one embodiment, the phenylalanyl hydroxyproline shown in Table 1 is measured. That is, a compound measured as a mass of 279.134 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示す4-ヒドロキシフェニルピルビン酸塩を測定する。すなわち、LC/MSネガティブイオン検出モードで質量179.035として計測される化合物を測定する。 In one embodiment, 4-hydroxyphenylpyruvate as shown in Table 1 is measured. That is, a compound measured as having a mass of 179.035 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示す乳酸塩を測定する。すなわち、LC/MS極性検出モードで質量89.024として計測される化合物を測定する。 In one embodiment, lactate as shown in Table 1 is measured. That is, a compound whose mass is measured as 89.024 in LC/MS polar detection mode is measured.
　一実施形態において、表1に示すシクロ(pro-ヒドロキシpro)を測定する。すなわち、LC/MSポジティブイオン検出モードで質量211.108として計測される化合物を測定する。 In one embodiment, cyclo(pro-hydroxypro) as shown in Table 1 is measured. That is, a compound measured as a mass of 211.108 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示すトリプトファンを測定する。すなわち、LC/MSポジティブイオン検出モードで質量205.097として計測される化合物を測定する。 In one embodiment, tryptophan as shown in Table 1 is measured. That is, a compound measured as a mass of 205.097 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示すN6-アセチルリシンを測定する。すなわち、LC/MS極性検出モードで質量187.109として計測される化合物を測定する。 In one embodiment, N6-acetyl lysine as shown in Table 1 is measured. That is, a compound measured as a mass of 187.109 in LC/MS polar detection mode is measured.
　一実施形態において、表1に示すガンマ-グルタミルフェニルアラニンを測定する。すなわち、LC/MSポジティブイオン検出モードで質量295.129として計測される化合物を測定する。 In one embodiment, gamma-glutamylphenylalanine as shown in Table 1 is measured. That is, a compound measured as a mass of 295.129 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示すロイシルヒドロキシプロリンを測定する。すなわち、LC/MSポジティブイオン検出モードで質量245.150として計測される化合物を測定する。 In one embodiment, leucyl hydroxyproline as shown in Table 1 is measured. That is, a compound measured as a mass of 245.150 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示すX-18887（C14H23N3O6）を測定する。すなわち、LC/MSネガティブイオン検出モードで質量328.152として計測される化合物（C14H23N3O6）を測定する。 In one embodiment, X-18887 (C14H23N3O6) shown in Table 1 is measured. That is, a compound (C14H23N3O6) whose mass is measured as 328.152 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示すX-24475（C6H11NO3）を測定する。すなわち、LC/MSポジティブイオン検出モードで質量146.081として計測される化合物（C6H11NO3）を測定する。 In one embodiment, X-24475 (C6H11NO3) shown in Table 1 is measured. That is, a compound (C6H11NO3) whose mass is measured as 146.081 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示すアンドロステロングルクロニドを測定する。すなわち、LC/MSネガティブイオン検出モードで質量465.249として計測される化合物を測定する。 In one embodiment, the androsterone glucuronides shown in Table 1 are measured. That is, a compound measured as a mass of 465.249 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示す3-ヒドロキシアントラニレートを測定する。すなわち、LC/MSポジティブイオン検出モードで質量154.050として計測される化合物を測定する。 In one embodiment, the 3-hydroxyanthranilates shown in Table 1 are measured. That is, a compound measured as a mass of 154.050 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示す11ベータ-ヒドロキシアンドロステロングルクロニドを測定する。すなわち、LC/MSネガティブイオン検出モードで質量481.244として計測される化合物を測定する。 In one embodiment, 11 beta-hydroxyandrosterone glucuronide as shown in Table 1 is measured. That is, a compound measured as a mass of 481.244 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示すシスタチオニンを測定する。すなわち、LC/MSポジティブイオン検出モードで質量221.060として計測される化合物を測定する。 In one embodiment, cystathionine as shown in Table 1 is measured. That is, a compound measured as a mass of 221.060 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示すカルノシンを測定する。すなわち、LC/MSポジティブイオン検出モードで質量227.114として計測される化合物を測定する。 In one embodiment, carnosine as shown in Table 1 is measured. That is, a compound measured as a mass of 227.114 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示すプロリンを測定する。すなわち、LC/MSポジティブイオン検出モードで質量116.071として計測される化合物を測定する。 In one embodiment, proline as shown in Table 1 is measured. That is, a compound measured as a mass of 116.071 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示すアンセリンを測定する。すなわち、LC/MSネガティブイオン検出モードで質量239.115として計測される化合物を測定する。 In one embodiment, anserine as shown in Table 1 is measured. That is, a compound measured as a mass of 239.115 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示すアラビトール/キシリトールを測定する。すなわち、LC/MS極性検出モードで質量151.061として計測される化合物を測定する。 In one embodiment, arabitol/xylitol as shown in Table 1 is measured. That is, a compound whose mass is measured as 151.061 in LC/MS polar detection mode is measured.
　一実施形態において、表1に示す3-ヒドロキシ-2-エチルプロピオネートを測定する。すなわち、LC/MS極性検出モードで質量117.056として計測される化合物を測定する。 In one embodiment, 3-hydroxy-2-ethylpropionate as shown in Table 1 is measured. That is, a compound whose mass is measured as 117.056 in LC/MS polar detection mode is measured.
　一実施形態において、表1に示す2R,3R-ジヒドロキシブチレートを測定する。すなわち、LC/MS極性検出モードで質量119.035として計測される化合物を測定する。 In one embodiment, the 2R,3R-dihydroxybutyrate shown in Table 1 is measured. That is, a compound whose mass is measured as 119.035 in LC/MS polar detection mode is measured.
　一実施形態において、表1に示すガンマ-グルタミルチロシンを測定する。すなわち、LC/MSネガティブイオン検出モードで質量311.124として計測される化合物を測定する。 In one embodiment, gamma-glutamyltyrosine as shown in Table 1 is measured. That is, a compound measured as a mass of 311.124 in LC/MS negative ion detection mode is measured.
　一実施形態において、表1に示す1-メチルアデニンを測定する。すなわち、LC/MSポジティブイオン検出モードで質量150.077として計測される化合物を測定する。 In one embodiment, 1-methyladenine as shown in Table 1 is measured. That is, a compound measured as having a mass of 150.077 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示すジヒドロオロト酸を測定する。すなわち、LC/MS極性検出モードで質量157.026として計測される化合物を測定する。 In one embodiment, the dihydroorotonic acid shown in Table 1 is measured. That is, a compound whose mass is measured as 157.026 in LC/MS polar detection mode is measured.
　一実施形態において、表1に示すアラニンを測定する。すなわち、LC/MSポジティブイオン検出モードで質量90.055として計測される化合物を測定する。 In one embodiment, alanine as shown in Table 1 is measured. That is, a compound measured as having a mass of 90.055 in LC/MS positive ion detection mode is measured.
　一実施形態において、表1に示す17アルファ-ヒドロキシプレグネノロングルクロニドを測定する。すなわち、LC/MSネガティブイオン検出モードで質量509.276として計測される化合物を測定する。 In one embodiment, 17 alpha-hydroxypregnenolone glucuronide as shown in Table 1 is measured. That is, a compound measured as a mass of 509.276 in LC/MS negative ion detection mode is measured.
　なお、表1に示した代謝物の解析に使用した質量分析計は非常に高分解能であるため、質量が小数点以下5桁程度まで測定可能であるが、測定誤差等を考慮して表１は小数点以下3桁で記載している。また、分解能の低い質量分析計を使用する場合には、整数質量又は小数点以下1桁、2桁程度の質量を測定することになる。 The mass spectrometer used to analyze the metabolites shown in Table 1 has a very high resolution, so it is possible to measure mass to about five decimal places. However, taking into account measurement errors, Table 1 is It is written in 3 digits after the decimal point. Furthermore, when using a mass spectrometer with low resolution, an integer mass or a mass of one or two digits after the decimal point is measured.
　本発明の一実施形態では、表1に示した尿中腫瘍マーカのうち少なくとも1つのマーカを使用して、胆道がんの判定や治療の効果のモニタリングを行うことができる。 In one embodiment of the present invention, at least one of the urinary tumor markers shown in Table 1 can be used to determine biliary tract cancer and monitor the effects of treatment.
　また本発明では、尿中腫瘍マーカを少なくとも2つ、又は3つ以上組み合わせて使用することによって、より正確かつ高精度な判定や、治療の効果のモニタリングが可能となる。例えば、少なくとも2個、少なくとも3個、少なくとも4個、少なくとも5個、少なくとも6個、少なくとも7個、少なくとも8個、少なくとも9個、少なくとも10個、少なくとも11個、少なくとも12個、少なくとも13個、少なくとも14個、少なくとも15個、少なくとも16個、少なくとも17個、少なくとも18個、少なくとも19個、少なくとも20個、又はそれ以上を組み合わせることができる。マーカの組み合わせは特に限定されるものではない。例えば、一実施形態において、尿中腫瘍マーカの少なくとも3種を測定する。 Furthermore, in the present invention, by using at least two or three or more urinary tumor markers in combination, more accurate and precise determination and monitoring of the effectiveness of treatment are possible. For example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, At least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or more can be combined. The combination of markers is not particularly limited. For example, in one embodiment, at least three urinary tumor markers are measured.
　表1に示した尿中腫瘍マーカは、いずれも単独で使用して胆道がんを判定できるものである。一実施形態では、マーカとして、少なくともグリコケノデオキシコール酸3-硫酸塩、あるいはグリココール酸塩、あるいは4-ヒドロキシフェニルピルビン酸塩を含める。 All of the urinary tumor markers shown in Table 1 can be used alone to determine biliary tract cancer. In one embodiment, the marker includes at least glycochenodeoxycholic acid 3-sulfate, or glycocholate, or 4-hydroxyphenylpyruvate.
　なお、単独の尿中腫瘍マーカの場合は個別に比較・解析していけばよいが、2個以上の複数の尿中腫瘍マーカを検討する場合には、その組み合わせが多様となるため、比較・解析が非常に煩雑である。そこで、2個以上の組み合わせについて、どのような組み合わせが良いかを判断する基準として、以下に示す精度変数R2Yと予測変数Q2という評価変数を用いることができる。 In addition, in the case of a single urinary tumor marker, it is sufficient to compare and analyze them individually, but when considering two or more urinary tumor markers, the combinations will be diverse, so it is necessary to compare and analyze them individually. Analysis is extremely complicated. Therefore, evaluation variables called accuracy variable R2Y and predictor variable Q2 shown below can be used as criteria for determining which combination of two or more is good.
Figure JPOXMLDOC01-appb-M000004
Figure JPOXMLDOC01-appb-M000004
　ここで、Yobsは実測値、YcalcはOPLSによる計算値、Ypredは交差検証を行った際の予測値、
Figure JPOXMLDOC01-appb-I000005
は平均値を表す。交差検証とは、データを分割し、その一部をまず解析して残る部分でその解析のテストを行い、解析自身の妥当性の検証・確認に当てる手法を示す。これによれば、精度変数R2Yの値が1に近いほどモデルの精度は高く、予測変数のQ2値が1に近いほどモデルの予測性は高いといえる。この精度変数及び予測変数の値の高い組み合わせを胆道がんの判定に使用することで、より高精度な判定が可能になると考えられる。 Here, Yobs is the measured value, Ycalc is the calculated value by OPLS, Ypred is the predicted value when cross-validated,
Figure JPOXMLDOC01-appb-I000005
represents the average value. Cross-validation refers to a method in which data is divided, a part of it is analyzed first, and the remaining part is used to test the analysis to verify and confirm the validity of the analysis itself. According to this, it can be said that the closer the value of the accuracy variable R2Y is to 1, the higher the accuracy of the model is, and the closer the Q2 value of the predictor variable is to 1, the higher the predictiveness of the model is. It is thought that by using a combination with high values of the accuracy variable and the predictive variable for biliary tract cancer determination, more accurate determination will be possible.
　なお、尿中腫瘍マーカの組み合わせは、対象の種類、性別、年齢や、胆道がんの判定、リスクが高い対象（例えば家族歴などに基づく）若しくは異常なし対象の経過観察（胆道がんのモニタリング）、又は治療のモニタリングを含む目的などに応じて、適宜選択することができる。 The combination of urinary tumor markers is determined based on the type, sex, and age of the subject, the determination of biliary tract cancer, and the follow-up of subjects at high risk (e.g., based on family history) or subjects with no abnormalities (monitoring of biliary tract cancer). ), or can be selected as appropriate depending on the purpose including treatment monitoring.
　尿中腫瘍マーカの識別の方法の一例として、多変量解析の一種である部分最小二乗法、特にOPLS-DAを用いることができる。2群間で変動する代謝物を複数組み合わせて多変量解析を行う場合、多次元データをそのまま用いるとデータが持つ特徴がわかりにくい場合があるため、2次元又は3次元データに縮約して視覚化することが好ましい。多変量解析としては、主成分分析などの当技術分野で公知の解析方法を使用することも可能である。 As an example of a method for identifying urinary tumor markers, the partial least squares method, which is a type of multivariate analysis, particularly OPLS-DA can be used. When performing multivariate analysis by combining multiple metabolites that vary between two groups, it may be difficult to understand the characteristics of the data if multidimensional data is used as is, so it may be necessary to reduce it to 2D or 3D data for visual visualization. It is preferable to As the multivariate analysis, it is also possible to use analysis methods known in the art, such as principal component analysis.
　尿サンプルとは、対象から採取した尿、及び当該尿を処理して得られるサンプル（例えば、トルエン、キシレン、塩酸などの保存料を添加した尿）を意味する。 Urine sample refers to urine collected from a subject and a sample obtained by processing the urine (for example, urine to which a preservative such as toluene, xylene, or hydrochloric acid has been added).
　また対象はヒト尿である。ヒト体内における代謝活動は、人種等によって異なることが想定されるため、本発明の解析対象である日本人を含むモノゴロイド人が好ましいが、その限りではない。例えば、健康診断やがん検査によるマススクリーニング時であってもよいし、そのようなマススクリーニング後の追加検査時、あるいは、病院等での手術前後や化学療法や放射線等の治療中であってもよい。また、尿は、同一個人であっても、採取のタイミングや水分の摂取などによって尿中代謝物の濃度は容易に変動する。この随時尿における尿中代謝物の含有量を標準化するため、一般的には、同尿中のクレアチニン量、あるいは、同尿の浸透圧（オスモラリティ）を測定し、各代謝物量を割ることで規格化する。以降での尿中代謝物量とは、基本的に、規格化された量を意味する。 The target is human urine. Since metabolic activity in the human body is assumed to differ depending on race, etc., monogoroid people, including Japanese, who are the subjects of analysis of the present invention, are preferred, but not limited thereto. For example, it may be during mass screening such as health checkups or cancer tests, during additional examinations after such mass screening, or before and after surgery at a hospital, or during treatment such as chemotherapy or radiation. Good too. Furthermore, even in the same individual, the concentration of metabolites in urine easily varies depending on the timing of urine collection, water intake, etc. In order to standardize the content of urinary metabolites in random urine, generally, the amount of creatinine in the same urine or the osmolality (osmolality) of the same urine is measured and the amount of each metabolite is divided. Standardize. The urinary metabolite amount hereinafter basically means a standardized amount.
　尿中腫瘍マーカの測定は、尿サンプル中のその量又は濃度を、好ましくは半定量的又は定量的に測定することを意味し、その量は、絶対量であってもよいし又は相対量であってもよい。測定は、直接的又は間接的に行うことができる。直接的な測定は、サンプル中に存在する尿中代謝物の分子数と直接相関するシグナルに基づいて、その量又は濃度を測定することを含む。そのようなシグナルは、例えば尿中代謝物の特定の物理的又は化学的な特性に基づいている。間接的な測定は、二次成分（すなわち尿中代謝物以外の成分）、例えばリガンド、標識又は酵素反応生成物から得られるシグナルの測定である。 Measuring a urinary tumor marker means measuring its amount or concentration in a urine sample, preferably semi-quantitatively or quantitatively, and the amount may be an absolute amount or a relative amount. There may be. Measurements can be made directly or indirectly. Direct measurement involves determining the amount or concentration of a urinary metabolite based on a signal that directly correlates with the number of molecules present in the sample. Such signals are based, for example, on certain physical or chemical properties of the urinary metabolites. Indirect measurements are measurements of signals obtained from secondary components (ie components other than urinary metabolites), such as ligands, labels or enzymatic reaction products.
　本発明の一実施形態では、尿中腫瘍マーカ、すなわち尿中代謝物を測定するが、その測定方法は、当技術分野で公知の方法又は手段を用いることができ、特に限定されるものではない。例えば、尿中腫瘍マーカの測定は、尿中代謝物に特有の物理的又は化学的特性を測定するための手段、例えば正確な分子量又はNMRスペクトル等を測定するための手段によって行うことができる。尿中代謝物を測定するための手段としては、質量分析計、NMR分析計、二次元電気泳動装置、クロマトグラフ、液体クロマトグラフィ質量分析装置（LC/MS）等の分析装置が挙げられる。これら分析装置を単独で使用して尿中腫瘍マーカを測定してもよいが、複数の分析装置により尿中腫瘍マーカを測定してもよい。 In one embodiment of the present invention, a urinary tumor marker, that is, a urinary metabolite is measured, but the measurement method is not particularly limited, and any method or means known in the art can be used. . For example, measurement of urinary tumor markers can be performed by means of measuring physical or chemical properties specific to urinary metabolites, such as precise molecular weight or NMR spectra. Examples of means for measuring metabolites in urine include analysis devices such as a mass spectrometer, an NMR analyzer, a two-dimensional electrophoresis device, a chromatograph, and a liquid chromatography mass spectrometer (LC/MS). Although these analyzers may be used alone to measure a urinary tumor marker, a plurality of analyzers may be used to measure a urinary tumor marker.
　あるいは、測定対象の代謝物を検出するための試薬、例えば免疫反応試薬、酵素反応試薬などが利用できる場合には、そのような試薬を利用して尿中の代謝物を測定することができる。 Alternatively, if a reagent for detecting a metabolite to be measured, such as an immunoreaction reagent or an enzyme reaction reagent, is available, such a reagent can be used to measure the metabolite in urine.
　表1に示された尿中代謝物は、LC/MSにより見出されたものであるため、LC/MSを使用すればこれらの尿中代謝物を測定することができる。 Since the urinary metabolites shown in Table 1 were found by LC/MS, these urinary metabolites can be measured using LC/MS.
　以上のようにして、対象から採取した尿サンプルに含まれる尿中腫瘍マーカを測定し、その結果に基づいて対象における胆道がんを判定することが可能である。さらに、対象から複数の時点に採取した尿サンプルにおいて尿中腫瘍マーカを測定してもよい。 As described above, it is possible to measure the urinary tumor marker contained in the urine sample collected from the subject and determine biliary tract cancer in the subject based on the results. Additionally, urinary tumor markers may be measured in urine samples taken from the subject at multiple time points.
　本発明の胆道がんの判定方法によって、胆道がんの存在や進行を早期に判定することができ、詳細な検査や治療方針の決定に役立つ。簡便な検査で胆道がんか否かの診断が可能となれば、治療はもちろん検査による侵襲リスクを防ぐことが期待できる。対象は、胆道がんの治療を早期に受けたり、リスクが高い場合には胆道がんの発症について経過観察することが可能となる。また、胆道がんの治療の効果についてモニターすることができ、治療の効果に応じて治療の停止、継続又は変更を検討することが可能となる。さらに、尿サンプルを利用することから低侵襲性であり、心理的障壁が非常に低く、簡便かつ低コストに胆道がんを判定できる。そのため、早期発見のためのスクリーニング用途として非常に適しており、また尿は繰り返し採取が可能であることから、日常的なモニタリングにも適している。 By the method for determining biliary tract cancer of the present invention, the presence and progression of biliary tract cancer can be determined at an early stage, which is useful for detailed examinations and determining treatment strategies. If it becomes possible to diagnose biliary tract cancer with a simple test, it can be expected to not only provide treatment but also prevent the risk of invasion caused by the test. Patients will be able to receive early treatment for biliary tract cancer, and those at high risk will be able to be monitored for the development of biliary tract cancer. Furthermore, the effect of biliary tract cancer treatment can be monitored, and it becomes possible to consider stopping, continuing, or changing the treatment depending on the treatment effect. Furthermore, since it uses a urine sample, it is minimally invasive, has very low psychological barriers, and can easily and inexpensively determine biliary tract cancer. Therefore, it is very suitable for screening purposes for early detection, and since urine can be collected repeatedly, it is also suitable for daily monitoring.
　本発明の胆道がんの判定方法は、尿中代謝物である尿中腫瘍マーカを測定するための手段を備えたキット及び／又は装置を用いることによって、容易かつ簡便に行うことができる。 The method for determining biliary tract cancer of the present invention can be carried out easily and conveniently by using a kit and/or device equipped with a means for measuring a urinary tumor marker, which is a urinary metabolite.
　本発明に係る胆道がんの判定用キットは、少なくとも、上記表1に示される尿中腫瘍マーカのうち少なくとも1つ（好ましくは少なくとも3種）を測定するための手段を含む。 The kit for determining biliary tract cancer according to the present invention includes at least a means for measuring at least one (preferably at least three) of the urinary tumor markers shown in Table 1 above.
　本発明のキットの一例は、質量分析用試薬セットであり、例えば同位体標識試薬、分画用ミニカラム、緩衝液等により構成される。キットの別の例は、免疫反応用試薬セットであり、例えば、一次抗体を固定した基板、二次抗体等により構成される。また別の例として、酵素反応用試薬セットは、例えば、酵素、緩衝液等により構成される。本発明のキットは、本発明の方法を実施するための手順及びプロトコールを記載した説明書、胆道がんの判定において使用する基準値又は基準範囲を示した表などを含んでもよい。 An example of the kit of the present invention is a mass spectrometry reagent set, which includes, for example, an isotope labeling reagent, a fractionation minicolumn, a buffer solution, and the like. Another example of a kit is an immunoreaction reagent set, which includes, for example, a substrate on which a primary antibody is immobilized, a secondary antibody, and the like. As another example, an enzyme reaction reagent set is composed of, for example, an enzyme, a buffer solution, and the like. The kit of the present invention may include an instruction manual describing the procedure and protocol for implementing the method of the present invention, a table showing reference values or reference ranges used in determining biliary tract cancer, and the like.
　本発明のキットに含まれる構成要素は、個別に提供されてもよいし、又は単一の容器内に提供されてもよい。好ましくは、本発明のキットは、本発明の方法を実施するために必要な構成要素の全てを、即時に使用することができるように、例えば調整された濃度の構成要素として含む。 The components included in the kit of the present invention may be provided individually or within a single container. Preferably, the kit of the invention contains all of the components necessary to carry out the method of the invention, eg as components in adjusted concentrations, ready for immediate use.
　本発明に係る胆道がんの判定装置は、以下の手段を備える：
　尿サンプル中の、上記表1に示される尿中腫瘍マーカのうち少なくとも1つ（好ましくは少なくとも3種）を測定する測定部と、
　上記測定部で測定した尿中腫瘍マーカの測定値を基準値又は前回の測定値と比較する比較部と、
　上記比較部で得られた比較結果から胆道がんを判定する判定部。 The biliary tract cancer determination device according to the present invention includes the following means:
a measurement unit that measures at least one (preferably at least three) of the urinary tumor markers shown in Table 1 above in the urine sample;
a comparison unit that compares the measurement value of the urinary tumor marker measured by the measurement unit with a reference value or a previous measurement value;
A determination unit that determines biliary tract cancer from the comparison results obtained by the comparison unit.
　また、本発明に係る胆道がんの判定装置は、多変量解析を用いる場合、以下の手段を備える：
　尿サンプル中の、上記表1に示される尿中腫瘍マーカのうち少なくとも1つ（好ましくは少なくとも3種）を測定する測定部と、
　上記測定部で測定した説明変数（尿中腫瘍マーカの量若しくは濃度、又は例えば良性若しくは異常なしに対して胆道がん患者で増減している尿中腫瘍マーカの観測されたイオンの強度比）から多変量解析して得られた計算値を、蓄積されたデータに基づいて多変量解析して得られたがん検査モデルに基づいて計算された目的変数の計算値（胆道がんであるか、異常なしであるかを示す指標）である基準値又は前回までの目的変数の計算値と比較する比較部と、
　上記比較部で得られた比較結果から胆道がんを判定する判定部。 Furthermore, when using multivariate analysis, the biliary tract cancer determination device according to the present invention includes the following means:
a measurement unit that measures at least one (preferably at least three) of the urinary tumor markers shown in Table 1 above in the urine sample;
From the explanatory variables measured by the above measurement unit (the amount or concentration of urinary tumor markers, or the observed ion intensity ratio of urinary tumor markers that increases or decreases in biliary tract cancer patients, for example, compared to benign or no abnormalities) The calculated value of the objective variable (biliary tract cancer, abnormal a comparison unit that compares it with a reference value that is an index (indicating whether there is no
A determination unit that determines biliary tract cancer from the comparison results obtained by the comparison unit.
　本発明の装置は、好ましくは、本発明の方法を実施することができるように、上記の測定部、比較部及び判定部が互いに動作可能なように連結されたシステムである。本発明の装置の一実施形態を図６に示す。 The apparatus of the present invention is preferably a system in which the measuring section, the comparing section, and the determining section described above are operably connected to each other so that the method of the present invention can be carried out. One embodiment of the device of the invention is shown in FIG.
　ここで、測定部は、上述のように、尿サンプル中の尿中腫瘍マーカを測定するための手段を含み、例えば質量分析計、NMR分析計、二次元電気泳動装置、クロマトグラフ、液体クロマトグラフィ質量分析（LC/MS）装置等の分析装置を備えている。 Here, the measurement unit includes a means for measuring a urinary tumor marker in a urine sample as described above, such as a mass spectrometer, an NMR analyzer, a two-dimensional electrophoresis device, a chromatograph, a liquid chromatography mass Equipped with analysis equipment such as analysis (LC/MS) equipment.
　測定部は、上述したような分析装置等から得られた測定値を処理するソフトウエアと計算機よりなるデータ解析部を備えている。データ解析部は、上述したような分析装置等から得られた測定値に基づいて検量線等のデータを参照することで、尿サンプルに含まれる尿中腫瘍マーカの量若しくは濃度を算出する。一方、データ解析部は、多変量解析を用いる場合には、上記測定部で測定した説明変数（尿中腫瘍マーカの量若しくは濃度、又は例えば良性若しくは異常なしに対して胆道がん患者で増減している尿中腫瘍マーカの観測されたイオンの強度比）から多変量解析して得られたがん検査モデルに基づいて計算された目的変数の計算値（胆道がんあるか、異常なしであるかを示す指標）を算出する。データ解析部は、例えば、シグナル表示部分、測定値を分析するユニット、コンピュータユニット等を含むことができる。 The measurement unit includes a data analysis unit consisting of a computer and software that processes the measurement values obtained from the above-mentioned analysis device or the like. The data analysis unit calculates the amount or concentration of the urinary tumor marker contained in the urine sample by referring to data such as a calibration curve based on the measurement values obtained from the above-mentioned analyzer or the like. On the other hand, when using multivariate analysis, the data analysis unit analyzes the explanatory variables measured by the measurement unit (the amount or concentration of urinary tumor markers, or whether they increase or decrease in patients with biliary tract cancer relative to benign or no abnormality, for example). The calculated value of the objective variable (whether there is biliary tract cancer or no abnormality) is calculated based on the cancer test model obtained by multivariate analysis from the observed ion intensity ratio of the urinary tumor marker. Calculate the index (indicating whether The data analysis section can include, for example, a signal display section, a unit for analyzing measured values, a computer unit, etc.
　また、比較部は、尿中腫瘍マーカの量若しくは濃度に関する基準値を記憶装置（データベース）等から読み出し、上記測定部で測定した尿中腫瘍マーカの測定値と基準値とを比較する。一方、比較部は、多変量解析を用いる場合には、目的変数の基準値を記憶装置（データベース）等から読み出し、上記測定部で得られた目的変数の計算値と基準値とを比較する。このとき、比較部は、尿中腫瘍マーカの種類に応じて適切な基準値を選択して読み出す。あるいは、同一対象における経時的モニタリングの場合には、比較部は、前回の測定値を記憶装置（データベース）等から読み出し、測定部で測定した尿中腫瘍マーカの測定値と比較する。 Further, the comparison section reads out a reference value regarding the amount or concentration of the urinary tumor marker from a storage device (database), etc., and compares the measurement value of the urinary tumor marker measured by the measurement section with the reference value. On the other hand, when using multivariate analysis, the comparison section reads the reference value of the target variable from a storage device (database), etc., and compares the calculated value of the target variable obtained by the measurement section with the reference value. At this time, the comparison section selects and reads an appropriate reference value according to the type of urinary tumor marker. Alternatively, in the case of monitoring over time for the same subject, the comparison section reads the previous measurement value from a storage device (database), etc., and compares it with the measurement value of the urinary tumor marker measured by the measurement section.
　さらに、判定部は、比較部において尿中腫瘍マーカの測定値と基準値とを比較した結果に基づいて、あるいは比較部において複数の時点における尿中腫瘍マーカの測定値を比較した結果に基づいて、胆道がんを判定する。一方、判定部は、多変量解析を用いる場合、比較部において目的変数の計算値と基準値を比較した結果に基づいて、あるいは比較部において複数の時点における目的変数の計算値を比較した結果に基づいて、胆道がんを判定する。ここで、判定部は、対象における胆道がんの存在や胆道がんのステージ、異常なし等を示す情報を取得する。好ましい装置は、専門の臨床医の知識がなくても使用することができるものであり、例えば、単にサンプルを付加すればよい電子的装置がある。 Further, the determination section determines whether the measurement value of the urinary tumor marker is compared with the reference value in the comparison section, or based on the result of comparing the measurement value of the urinary tumor marker at a plurality of time points in the comparison section. , determine biliary tract cancer. On the other hand, when using multivariate analysis, the determination section is based on the results of comparing the calculated value of the objective variable and the reference value in the comparing section, or the results of comparing the calculated values of the objective variable at multiple points in time in the comparing section. Based on this, biliary tract cancer is determined. Here, the determination unit acquires information indicating the presence of biliary tract cancer in the subject, the stage of biliary tract cancer, and whether there is any abnormality. Preferred devices are those that can be used without the knowledge of a specialized clinician, such as electronic devices that simply require the addition of a sample.
　例えば、判定部は、尿中腫瘍マーカが基準値又は前回の測定値以上である場合、対象が胆道がんを有する可能性があると判定する。また例えば、判定部は、尿中腫瘍マーカが基準値又は前回の測定値よりも低い場合、対象が異常なしの可能性があると判定する。 For example, the determination unit determines that the subject may have biliary tract cancer when the urinary tumor marker is equal to or higher than the reference value or the previous measurement value. For example, if the urinary tumor marker is lower than the reference value or the previous measurement value, the determination unit determines that there is a possibility that the subject has no abnormality.
　本発明の装置は、データ保存部、データ出力・表示部などをさらに備えるものであってもよい。 The device of the present invention may further include a data storage section, a data output/display section, and the like.
　本明細書中、「胆道がんの判定（の補助）」とは、対象における胆道がんを検出することだけではなく、対象における胆道がんのリスクを予測すること、対象における胆道がんのステージを判定すること、対象における胆道がんの予後を判定すること、対象において胆道がんについてモニタリングすること、対象に存在する胆道がんに対する治療の効果をモニタリングすること、さらには胆道がんの診断を補助することを含む意味である。また本発明において「判定」は、既に検出又は診断された胆道がんの継続的なモニタリング、及び既に行った胆道がんの検出又は診断の確認も包含する。 In this specification, "determination of biliary tract cancer (assistance)" means not only detecting biliary tract cancer in a subject, but also predicting the risk of biliary tract cancer in a subject, to determine the stage of biliary tract cancer, to determine the prognosis of biliary tract cancer in a subject, to monitor biliary tract cancer in a subject, to monitor the effect of treatment for biliary tract cancer existing in a subject, and to monitor biliary tract cancer in a subject. This meaning includes assisting in diagnosis. Furthermore, in the present invention, "determination" includes continuous monitoring of biliary tract cancer that has already been detected or diagnosed, and confirmation of the detection or diagnosis of biliary tract cancer that has already been performed.
　なお、本発明に係る胆道がんの判定方法、判定用キット及び判定装置による「判定」は、統計学的に有意な割合の対象を判定できることを意図している。よって本発明に係る胆道がんの判定方法、判定用キット及び判定装置による「判定」には、対象の全て（すなわち100％）について必ず正しい結果が得られない場合も含まれる。統計的に有意な割合は、様々な周知の統計評価ツール、例えば信頼区間の決定、p値の決定、スチューデントのt検定、マン・ホイットニー検定等を用いて決定することができる。好ましい信頼区間は、少なくとも90％である。p値は、好ましくは、0.1、0.01、0.05、0.005又は0.0001である。より好ましくは、対象の少なくとも60％、少なくとも80％又は少なくとも90％を、本発明に係る胆道がんの判定方法、判定用キット及び判定装置によって適切に判定することができる。 Note that "determination" by the biliary tract cancer determination method, determination kit, and determination device according to the present invention is intended to be able to determine a statistically significant proportion of subjects. Therefore, "determination" by the biliary tract cancer determination method, determination kit, and determination device according to the present invention includes cases where correct results are not always obtained for all (ie, 100%) of the subjects. Statistically significant proportions can be determined using a variety of well-known statistical evaluation tools, such as determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney test, and the like. A preferred confidence interval is at least 90%. The p-value is preferably 0.1, 0.01, 0.05, 0.005 or 0.0001. More preferably, at least 60%, at least 80%, or at least 90% of the subjects can be appropriately determined by the biliary tract cancer determination method, determination kit, and determination device according to the present invention.
　胆道がんの判定の具体例は次のとおりである。一実施形態において、対象の尿サンプル中の尿中腫瘍マーカを測定し、その測定値を基準値又は前回の測定値と比較する。複数の尿中腫瘍マーカを測定する場合には、それぞれの尿中腫瘍マーカをそれぞれの基準値又は前回の測定値と比較してもよいし、多変量解析により得られる目的変数の計算値を求め、それを基準値又は前回の測定値と比較してもよい。 Specific examples of biliary tract cancer determination are as follows. In one embodiment, a urinary tumor marker is measured in a subject's urine sample and the measurement is compared to a baseline or previous measurement. When measuring multiple urinary tumor markers, each urinary tumor marker may be compared with its reference value or previous measurement value, or the calculated value of the target variable obtained by multivariate analysis may be calculated. , it may be compared with a reference value or a previous measurement value.
　基準（基準値）は、胆道がんに関連する尿中腫瘍マーカの量若しくは濃度、又はその量若しくは濃度の範囲、あるいは異常なしの指標となる尿中腫瘍マーカの量若しくは濃度、又はその量若しくは濃度の範囲である。一方、多変量解析を用いる場合、胆道がん/異常なしを識別する目的変数の計算値が基準値となる。例えば、基準値は、健常者（集団）又は胆道がんの低リスク者（集団）に由来するものとすることができる。あるいは、基準値は、胆道がん（例えば、特定のステージの胆道がん）に罹患している又は特定の予後を示す胆道がんを有する患者（患者集団）に由来するものとすることができる。個々の対象に適用する基準値は、対象動物の種類、年齢、性別などの様々な生理学的パラメータに応じて変化しうる。 The standard (reference value) is the amount or concentration of a urinary tumor marker related to biliary tract cancer, or the range of the amount or concentration, or the amount or concentration of a urinary tumor marker that is an indicator of no abnormality, or the amount or concentration of the urinary tumor marker. range of concentrations. On the other hand, when using multivariate analysis, the calculated value of the objective variable that identifies biliary tract cancer/no abnormality becomes the reference value. For example, the reference value can be derived from healthy subjects (population) or low-risk biliary tract cancer subjects (population). Alternatively, the reference value may be derived from patients (patient populations) who have biliary tract cancer (e.g., a certain stage of biliary tract cancer) or who have biliary tract cancer with a particular prognosis. . The reference value applied to an individual subject may vary depending on various physiological parameters such as the species, age, and sex of the subject animal.
　好ましくは、尿中腫瘍マーカの量又は濃度と、胆道がんの存在若しくは特定の予後との相関をデータベースとして記録する。そして、測定された尿サンプル中の尿中腫瘍マーカの測定値を、データベース中の基準値と比較することができる。このようなデータベースは、胆道がんの有無（若しくは特定のステージの胆道がん）の指標、又は予後の指標となる基準値又は基準範囲として有用である。 Preferably, the correlation between the amount or concentration of a urinary tumor marker and the presence of biliary tract cancer or a specific prognosis is recorded as a database. The measured value of the urinary tumor marker in the urine sample can then be compared with the reference value in the database. Such a database is useful as an indicator of the presence or absence of biliary tract cancer (or a specific stage of biliary tract cancer), or a reference value or reference range that is an indicator of prognosis.
　表1に示した尿中腫瘍マーカは、胆道がんの有無においてその量又は濃度に差があり、胆道がんの存在により、また治療の開始前又は開始後に、その量又は濃度が変化する。具体的には、表1に示すマーカは、胆道がんのない対象と比較して胆道がんを有する患者においてその量又は濃度が上昇する。したがって、表1に示すマーカが、異常なし集団（胆道がんを有しない対象）に由来する基準値よりも高い場合又は胆道がんを有する患者集団に由来する基準値と同等若しくはそれより高い場合には、対象が胆道がんを有する可能性がある又はそのリスクが高いといえる。 The amount or concentration of the urinary tumor markers shown in Table 1 differs depending on the presence or absence of biliary tract cancer, and the amount or concentration changes depending on the presence of biliary tract cancer and before or after the start of treatment. Specifically, the markers shown in Table 1 have increased amounts or concentrations in patients with biliary tract cancer compared to subjects without biliary tract cancer. Therefore, if the marker shown in Table 1 is higher than the reference value derived from the normal population (subjects without biliary tract cancer) or equal to or higher than the reference value derived from the patient population with biliary tract cancer. The subject may have or is at high risk of biliary tract cancer.
　基準との比較では、予測値を求めて判定を行うことも可能である。尿中腫瘍マーカによるがん検査では、複数の尿中腫瘍マーカを用いてがんのリスクを評価することが多いため、そのような場合に予測値を求めることが望ましい場合がある。例えば、尿中腫瘍マーカを3種選択した場合には、以下の予測式で与えられた予測値よりがんのリスクを評価する。なお、式中の強度とは、各質量に対応するマスクロマトグラムの面積値となる。
［数２］
　予測値＝α×(尿中腫瘍マーカ１の強度)＋β×(尿中腫瘍マーカ２の強度)
　　　　　　＋γ×(尿中腫瘍マーカ３の強度)＋δ
　　　　　　　（式中、α、β、γ、δは定数である） In comparison with a standard, it is also possible to determine a predicted value. Cancer tests using urinary tumor markers often use multiple urinary tumor markers to evaluate cancer risk, so it may be desirable to obtain predictive values in such cases. For example, when three types of urinary tumor markers are selected, the cancer risk is evaluated based on the predicted value given by the following prediction formula. Note that the intensity in the formula is the area value of the mass chromatogram corresponding to each mass.
[Number 2]
Predicted value = α × (intensity of urinary tumor marker 1) + β × (intensity of urinary tumor marker 2)
+γ×(intensity of urinary tumor marker 3)+δ
(In the formula, α, β, γ, and δ are constants)
　上記がん検査モデルでは、例えば、予測値が0以上であればがんのリスクが高く、0より小さければがんのリスクは小さいと判断する。ただし、予測値のしきい値は0に限らず可変させてもよい。また、しきい値を明確に定めず、予測値の大小を、がんのリスク（確率）と定量的に対応させてもよい。 In the above cancer testing model, for example, if the predicted value is 0 or more, it is determined that the risk of cancer is high, and if it is smaller than 0, the risk of cancer is determined to be low. However, the threshold value of the predicted value is not limited to 0 and may be varied. Alternatively, the magnitude of the predicted value may be quantitatively associated with the risk (probability) of cancer without clearly defining the threshold value.
　例えば、予測式は以下のように表される：
［数３］
　予測式＝0.3336×(尿中腫瘍マーカ１の強度)＋0.2408×(尿中腫瘍マーカ２の強度)＋0.4132×(尿中腫瘍マーカ３の強度)＋0.1234 For example, the prediction formula is expressed as:
[Number 3]
Prediction formula = 0.3336 x (intensity of urinary tumor marker 1) + 0.2408 x (intensity of urinary tumor marker 2) + 0.4132 x (intensity of urinary tumor marker 3) + 0.1234
　別の実施形態では、対象から複数の時点で尿サンプルを採取し、それぞれの測定時点における尿サンプルに含まれる尿中腫瘍マーカを測定し、尿中腫瘍マーカの測定値をそれぞれの測定時点で比較する。より具体的には、第1の時点における尿中腫瘍マーカの量又は濃度（a）と第2の時点における尿中腫瘍マーカの量又は濃度（b）とを比較する。多変量解析を行った場合には、例えば、1つの成分の第1の時点における計算値と第2の時点における計算値とを比較する。測定は、経時的に少なくとも2回、3回、4回、5回、10回、15回、20回、30回、又はそれ以上の回で、例えば1日、2日、5日、1週間、2週間、3週間、1ヶ月、2ヶ月、3ヶ月、半年、1年、2年、3年、5年、又はそれ以上の期間を空けて、行うことができる。この比較によって、経時的なモニタリングを行うことができ、胆道がんの進行、胆道がんの転移若しくは再発、良性腫瘍の悪性化、異常なしからの胆道がんの発症などを評価することができる。 In another embodiment, a urine sample is collected from the subject at multiple time points, a urinary tumor marker is determined in the urine sample at each time point, and the urinary tumor marker measurements are compared at each time point. do. More specifically, the amount or concentration of the urinary tumor marker at the first time point (a) is compared with the amount or concentration of the urinary tumor marker at the second time point (b). When multivariate analysis is performed, for example, the calculated value of one component at the first time point and the calculated value at the second time point are compared. Measurements are taken at least 2, 3, 4, 5, 10, 15, 20, 30 or more times over time, e.g. 1 day, 2 days, 5 days, 1 week. , 2 weeks, 3 weeks, 1 month, 2 months, 3 months, half a year, 1 year, 2 years, 3 years, 5 years, or more. Through this comparison, it is possible to perform monitoring over time, and it is possible to evaluate the progression of biliary tract cancer, metastasis or recurrence of biliary tract cancer, malignant transformation of benign tumors, and the development of biliary tract cancer in the absence of abnormalities. .
　また別の実施形態では、本発明に使用される尿中腫瘍マーカを利用して、対象における胆道がんに対する治療（治療薬又は治療法）の効果をモニタリングすることができる。具体的には、
（a）治療薬又は治療法による処置を受ける前に、胆道がんを有する患者からの尿サンプルにおいて尿中腫瘍マーカを測定するステップ、
（b）治療薬又は治療法による処置を受けた後に、胆道がんを有する患者からの尿サンプルにおいて尿中腫瘍マーカを測定するステップ、
（c）必要に応じて、ステップ（b）を繰り返すステップ、
（d）（a）～（c）の測定結果に基づいて胆道がんに対する治療薬又は治療法の効果をモニタリングするステップ
を含む。 In another embodiment, the urinary tumor marker used in the present invention can be used to monitor the effect of treatment (therapeutic agent or treatment method) on biliary tract cancer in a subject. in particular,
(a) measuring a urinary tumor marker in a urine sample from a patient with biliary tract cancer prior to receiving treatment with a therapeutic agent or therapy;
(b) measuring a urinary tumor marker in a urine sample from a patient with biliary tract cancer after receiving treatment with a therapeutic agent or therapy;
(c) repeating step (b) as necessary;
(d) It includes the step of monitoring the effect of a therapeutic agent or treatment method for biliary tract cancer based on the measurement results of (a) to (c).
　上記方法では、治療薬又は治療法による処置を受ける前に、胆道がんを有する患者から尿サンプルを採取し、尿サンプル中の尿中腫瘍マーカを測定する。胆道がんを有する患者に治療薬又は治療法による処置が行われた後、適当な時期に尿サンプルを採取して、尿サンプル中の尿中腫瘍マーカを測定する。例えば、処置の直後、30分後、1時間後、3時間後、5時間後、10時間後、15時間後、20時間後、24時間（1日）後、2～10日後、10～20日後、20～30日後、1ヶ月～6ヵ月後に尿サンプルを採取する。尿サンプル中の尿中腫瘍マーカの測定については前記と同様に行うことができる。治療前後に尿中腫瘍マーカを測定することによって、その治療薬又は治療法による処置の効果をモニタリングすることが可能となる。モニタリングの結果に基づいて、治療の停止、継続又は変更を検討する一助となる。 In the above method, a urine sample is collected from a patient with biliary tract cancer, and urinary tumor markers in the urine sample are measured before receiving treatment with a therapeutic agent or treatment method. After a patient with biliary tract cancer is treated with a therapeutic agent or therapy, a urine sample is collected at an appropriate time and urinary tumor markers are measured in the urine sample. For example, immediately after treatment, 30 minutes, 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours (1 day), 2-10 days, 10-20 Collect urine samples after 1 day, 20 to 30 days, and 1 month to 6 months. Measurement of urinary tumor markers in urine samples can be performed in the same manner as described above. By measuring urinary tumor markers before and after treatment, it becomes possible to monitor the effectiveness of treatment with the therapeutic agent or method. Based on the results of monitoring, it helps to consider stopping, continuing, or changing treatment.
　さらに胆道がんの判定方法は、他の従来公知の胆道がんの診断方法と組み合わせて行ってもよい。そのような公知の胆道がんの診断方法としては、血液検査（血中がんマーカの測定、肝機能検査など）、画像検査（例えば腹部超音波検査、コンピュータ断層撮影（CT）、MRI検査、ポジトロンCT（PET）等）、内視鏡検査、生検若しくは細胞診による病理検査などが挙げられる。 Furthermore, the method for determining biliary tract cancer may be performed in combination with other conventionally known methods for diagnosing biliary tract cancer. Such known methods for diagnosing biliary tract cancer include blood tests (measurement of blood cancer markers, liver function tests, etc.), image tests (such as abdominal ultrasound, computed tomography (CT), MRI), Positron CT (PET), etc.), endoscopy, and pathological examinations such as biopsy or cytology.
　上述の判定結果に基づいて、医師は、対象の胆道がんについて診断を行い、適切な処置を行うことができる。すなわち本発明は、対象において胆道がんを判定し、治療する方法にも関する。例えば、本発明に係る方法に従って対象における胆道がんを判定し、対象が胆道がんを有している可能性が高いと評価された場合、対象において胆道がんを治療する又は胆道がんの進行を予防する処置を行う。また、対象における胆道がんのステージが進行している又は胆道がんの予後が悪い可能性が高いと評価された場合には、治療を継続したり、必要であれば治療法の変更を検討する。あるいは、対象がリスクは高いがその時点で胆道がんを発症していないと評価された場合には、過度の検査及び治療を回避するため、尿中腫瘍マーカの測定を経時的に行って、胆道がんについてモニタリングしてもよい。また、対象において胆道がんが存在する可能性が高いと評価された場合には、上述したような他の胆道がんの診断方法を行って、胆道がんの存在を確定する。さらに、治療前後での評価結果に基づいて、治療の効果をモニタリングし、治療の停止、継続又は変更を決定する。また、異常なしと判定された場合には、尿中腫瘍マーカの測定を経時的に行って、経過観察することができる。 Based on the above-mentioned determination results, the doctor can diagnose the subject's biliary tract cancer and take appropriate treatment. That is, the present invention also relates to a method for determining and treating biliary tract cancer in a subject. For example, if biliary tract cancer is determined in a subject according to the method of the present invention and it is evaluated that the subject is likely to have biliary tract cancer, the subject is treated for biliary tract cancer or Take measures to prevent progression. In addition, if it is assessed that the stage of biliary tract cancer in the subject is advanced or that the prognosis of biliary tract cancer is likely to be poor, treatment may be continued or, if necessary, a change in treatment method may be considered. do. Alternatively, if the subject is assessed to be at high risk but not developing biliary tract cancer at that time, urinary tumor markers may be measured over time to avoid excessive testing and treatment. May be monitored for biliary tract cancer. Further, if it is evaluated that there is a high possibility that biliary tract cancer exists in the subject, other biliary tract cancer diagnostic methods such as those described above are performed to confirm the presence of biliary tract cancer. Furthermore, based on the evaluation results before and after the treatment, the effectiveness of the treatment is monitored and a decision is made to stop, continue, or change the treatment. Furthermore, if it is determined that there is no abnormality, urinary tumor markers can be measured over time to monitor the progress.
　胆道がんは、外科手術（外科的切除）、化学療法、放射線療法、免疫療法、陽子線治療、重粒子線治療などを、単独で又は適宜組み合わせて行うことができる。胆道がんの治療は、胆道がんの種類、ステージ、悪性度、性別、年齢及び状態、治療に対する応答性、保有する遺伝子多型（SNP）などを考慮して、当業者であれば適宜選択することができる。 For biliary tract cancer, surgery (surgical resection), chemotherapy, radiation therapy, immunotherapy, proton beam therapy, heavy particle beam therapy, etc. can be performed alone or in appropriate combinations. Treatment for biliary tract cancer can be selected appropriately by those skilled in the art, taking into consideration the type, stage, malignancy, gender, age and condition of biliary tract cancer, responsiveness to treatment, genetic polymorphisms (SNPs), etc. can do.
　本発明を適用した例として、検査センタにおけるがん検査について説明する。検査センタでは、被検査者からの請求等に応じてがん検査の案内を行う。被検査者は一次検査の申し込みに際し、検査のバイオマーカ数の選択を行ってもよい。例えば、バイオマーカ数としては1～3種類の尿中腫瘍マーカが挙げられる。これは、他のバイオマーカと組み合わせて、全がん検査（いろいろながんを一度に解析する）として利用することもできる。 As an example to which the present invention is applied, cancer testing at a testing center will be described. The testing center provides guidance on cancer testing in response to requests from test subjects. When applying for the primary test, the test subject may select the number of biomarkers for the test. For example, the number of biomarkers includes 1 to 3 types of urinary tumor markers. This can also be used as a pan-cancer test (analyzing various cancers at once) in combination with other biomarkers.
　続いて、検査センタは尿採取に必要な検査キットを被検査者に渡す。必要に応じて郵送などにより送付する。被検査者は検査キットを受け取った後、検査センタに検体を渡す、又は送付等行う。検査センタでは、検体を続く検査のため必要に応じて、約－80℃に冷凍保存しておく。ただし、対象の尿中代謝物が温度や経過日数に対して安定であることがわかっていれば、-80℃の冷凍保管に限らず、約-5℃の冷凍保管、約5℃の冷蔵保管、室温保管等であってもよい。検査センタでは一次検査を行い、検査結果を被検査者に送る。 Next, the testing center hands the test subject the test kit necessary for urine collection. Send by mail, etc. as necessary. After receiving the test kit, the person to be tested hands or sends the specimen to the test center. At the testing center, samples are frozen and stored at approximately -80°C for subsequent testing as needed. However, if the target urinary metabolites are known to be stable over temperature and the number of days that have elapsed, storage at -80°C is not the only option, but frozen storage at approximately -5°C and refrigerated storage at approximately 5°C are recommended. , room temperature storage, etc. The testing center performs a primary test and sends the test results to the person being tested.
　被検査者は、一次検査の結果を受け取り、内容に応じて二次検査の申し込みを行ってもよいし、より詳細な診断を受けてもよい。これにより、一次検査での胆道がんの疑いを確証したり、さらには胆道がんのステージを特定することが可能となる。 After receiving the results of the primary test, the person to be tested may apply for a secondary test depending on the content, or may receive a more detailed diagnosis. This makes it possible to confirm the suspicion of biliary tract cancer in the primary examination and furthermore to identify the stage of biliary tract cancer.
　また、本発明に使用される尿中腫瘍マーカを利用して、胆道がんの治療（治療薬又は治療法）の有効性を評価する、あるいは胆道がんの治療薬候補をスクリーニングすることができる。具体的には、胆道がんの治療の有効性を評価する方法、又は胆道がんの治療薬候補をスクリーニング方法は、
（a）被験治療薬又は治療法による処置を受けた胆道がんを有する動物からの尿サンプルにおいて、尿中腫瘍マーカを測定するステップ、
（b）（a）の測定結果に基づいて胆道がんに対する被験治療薬又は治療法の有効性を評価するステップ
を含む。 Furthermore, the urinary tumor marker used in the present invention can be used to evaluate the effectiveness of biliary tract cancer treatment (therapeutic drug or treatment method) or to screen therapeutic drug candidates for biliary tract cancer. . Specifically, a method for evaluating the effectiveness of biliary tract cancer treatment or a method for screening therapeutic drug candidates for biliary tract cancer includes:
(a) measuring a urinary tumor marker in a urine sample from an animal with biliary tract cancer treated with the investigational therapeutic agent or therapy;
(b) It includes the step of evaluating the effectiveness of the test drug or treatment method for biliary tract cancer based on the measurement results in (a).
　本発明の方法では、胆道がんを有する患者あるいは胆道がんを有しないヒトから尿サンプルを採取し、尿サンプル中の尿中腫瘍マーカを測定する。好ましくは、被験治療薬又は治療法による処置を行う前に、胆道がんを有するヒトから尿サンプルを採取し、尿サンプル中の尿中腫瘍マーカを測定する。胆道がんを有する動物に被験治療薬又は治療法による処置が行われた後、適当な時期に尿サンプルを採取して、尿サンプル中の尿中腫瘍マーカを測定する。例えば、処置の直後、30分後、1時間後、3時間後、5時間後、10時間後、15時間後、20時間後、24時間（1日）後、2～10日後、10～20日後、20～30日後、1ヶ月～6ヵ月後に尿サンプルを採取する。尿サンプル中の尿中腫瘍マーカの測定、胆道がんの判定については前記と同様に行うことができる。 In the method of the present invention, a urine sample is collected from a patient with biliary tract cancer or a human without biliary tract cancer, and urinary tumor markers in the urine sample are measured. Preferably, a urine sample is collected from a person with biliary tract cancer and urinary tumor markers are measured in the urine sample prior to treatment with the test therapeutic agent or therapy. After animals with biliary tract cancer are treated with the test therapeutic agent or therapy, urine samples are collected at appropriate times and urinary tumor markers are measured in the urine samples. For example, immediately after treatment, 30 minutes, 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours (1 day), 2-10 days, 10-20 Collect urine samples after 1 day, 20 to 30 days, and 1 month to 6 months. Measurement of urinary tumor markers in urine samples and determination of biliary tract cancer can be performed in the same manner as described above.
　評価又はスクリーニングの対象となる被験治療薬又は治療法の種類は特に限定されるものではない。例えば、被験治療薬又は治療法は、任意の物質的因子、具体的には、天然に生じる分子、例えば、アミノ酸、ペプチド、オリゴペプチド、ポリペプチド、タンパク質、核酸、脂質、炭水化物（糖等）、ステロイド、グリコペプチド、糖タンパク質、プロテオグリカンなど；天然に生じる分子の合成アナログ又は誘導体、例えば、ペプチド擬態物、核酸分子（アプタマー、アンチセンス核酸、二本鎖RNA（RNAi）等）など；天然に生じない分子、例えば低分子有機化合物（無機及び有機化合物ライブラリー、又はコンビナトリアルライブラリー等）など；並びにそれらの混合物を挙げることができる。また治療薬又は治療法は、単一物質であってもよいし、複数の物質から構成される複合体や、食品及び食餌等であってもよい。さらに、被験治療薬又は治療法は、上記のような物質的因子に加えて、放射線、紫外線などであってもよい。 The type of test therapeutic drug or treatment method to be evaluated or screened is not particularly limited. For example, the test therapeutic agent or therapy may include any material agent, specifically a naturally occurring molecule, such as an amino acid, a peptide, an oligopeptide, a polypeptide, a protein, a nucleic acid, a lipid, a carbohydrate (such as a sugar), steroids, glycopeptides, glycoproteins, proteoglycans, etc.; synthetic analogs or derivatives of naturally occurring molecules, such as peptidomimetics, nucleic acid molecules (aptamers, antisense nucleic acids, double-stranded RNA (RNAi), etc.); naturally occurring and mixtures thereof. Furthermore, the therapeutic agent or treatment method may be a single substance, a complex composed of multiple substances, food, diet, or the like. Furthermore, the test therapeutic agent or treatment method may include radiation, ultraviolet light, etc. in addition to the above-mentioned physical factors.
　また、被験治療薬又は治療法の有効性は、いくつかの条件で検討することも可能である。そのような条件としては、被験治療薬又は治療法で処置する時間又は期間、量（大小）、回数などが挙げられる。例えば、被験治療薬の希釈系列を調製するなどして複数の用量を設定することができる。さらに、複数の被験治療薬又は治療法の相加作用、相乗作用などを検討する場合には、治療薬又は治療法を組み合わせて用いてもよい。 Additionally, the effectiveness of a test therapeutic drug or treatment method can be examined under several conditions. Such conditions include the time or duration, amount (large or small), number of times, etc., of treatment with the test therapeutic agent or treatment method. For example, multiple doses can be established by preparing a dilution series of the test therapeutic agent. Furthermore, when examining additive effects, synergistic effects, etc. of multiple test therapeutic agents or treatments, the therapeutic agents or treatments may be used in combination.
　被験治療薬又は治療法の処置後の採取した尿サンプル中の尿中腫瘍マーカを測定し、処置前の量又は濃度と比較することによって、被験治療薬又は治療法が、胆道がんの消失、胆道がんの縮小、胆道がんによる症状の改善、胆道がんの進行の停止又は減速化に有効であるか否かを評価することができる。 By measuring urinary tumor markers in urine samples collected after treatment with the investigational therapeutic agent or therapy and comparing them with the amount or concentration before treatment, it is possible to determine whether the investigational therapeutic agent or therapy eliminates biliary tract cancer, It can be evaluated whether it is effective in shrinking biliary tract cancer, improving symptoms caused by biliary tract cancer, and stopping or slowing down the progression of biliary tract cancer.
　例えば、表1に示したマーカの場合には、胆道がん患者において、処置後の測定値が処置前の測定値より低いことは、被験治療薬又は治療法が、胆道がんの消失、胆道がんの縮小、胆道がんによる症状の改善、胆道がんの進行の停止又は有効であることを示す。一方、処置後の測定値が処置前の測定値より高い又は処置前の測定値と有意差がないことは、被験治療薬又は治療法が胆道がんの治療に有効でないことを示す。 For example, in the case of the markers shown in Table 1, in patients with biliary tract cancer, the fact that the measured value after treatment is lower than the measured value before treatment means that the test drug or treatment is effective for the disappearance of biliary tract cancer, Indicates that the drug shrinks cancer, improves symptoms caused by biliary tract cancer, halts the progression of biliary tract cancer, or is effective. On the other hand, the measured value after treatment is higher than the measured value before treatment or is not significantly different from the measured value before treatment, indicating that the test therapeutic agent or treatment method is not effective in treating biliary tract cancer.
　以上から、本発明に係る胆道がんの治療の有効性の評価方法により、胆道がんを治療若しくは予防するための治療薬若しくは治療法を見出し、さらには治療薬又は治療法の有効性を確認することができる。 From the above, by using the method for evaluating the effectiveness of biliary tract cancer treatment according to the present invention, a therapeutic agent or method for treating or preventing biliary tract cancer was discovered, and the effectiveness of the therapeutic agent or treatment method was confirmed. can do.
　以下に実施例を例示し、本発明を具体的に説明するが、この実施例は単に本発明の説明のために提供するものであり、本出願において開示する発明の範囲を限定したり制限したりするものではない。 The present invention will be specifically explained below by way of examples, but these examples are provided merely to explain the present invention, and do not limit or restrict the scope of the invention disclosed in this application. It's not something you can do.
［実施例１］胆道がんと関連する尿中代謝物の網羅的解析
　名古屋大学病院において、胆道がん患者27名より許可を得て、腫瘍切除前と、腫瘍切除後2週間経過時及び4週間経過時に尿検体の採取を行った（合計62検体）。患者情報として、サンプルID、検体採取日、年齢、性別、術前術後、オスモラリティ、予後、診断名、術式、病理組織型、病理断端、病理リンパ節転移、ステージ、術中輸血、既往歴、他のマーカ測定値などについての情報を記録した。尿検体の具体的な内訳は以下の通りである。 [Example 1] Comprehensive analysis of urinary metabolites related to biliary tract cancer At Nagoya University Hospital, permission was obtained from 27 patients with biliary tract cancer before tumor resection, 2 weeks after tumor resection, and 4 weeks after tumor resection. Urine samples were collected at the end of the week (62 samples in total). Patient information includes sample ID, sample collection date, age, gender, pre- and postoperative status, osmolarity, prognosis, diagnosis name, surgical method, histopathological type, pathological margin, pathological lymph node metastasis, stage, intraoperative blood transfusion, and medical history. Information regarding medical history, other marker measurements, etc. was recorded. The specific breakdown of urine samples is as follows.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　患者の尿中代謝物（メタボローム）について、溶液中の混合成分を高感度に分析するのに好適な液体クロマトグラフィ質量分析装置（LC/MS）を用いて網羅的に解析を行った。できるだけ多くの代謝物を検出するため、イオン化には正負のエレクトロスプレーイオン化法を用いるとともに、混合成分の分離には、逆相クロマトグラフィ、親水性相互作用クロマトグラフィなど、複数の分離モードを使用した。 A comprehensive analysis of the patient's urinary metabolites (metabolome) was conducted using a liquid chromatography mass spectrometer (LC/MS) suitable for highly sensitive analysis of mixed components in solutions. In order to detect as many metabolites as possible, positive and negative electrospray ionization was used for ionization, and multiple separation modes were used to separate the mixed components, including reversed-phase chromatography and hydrophilic interaction chromatography.
　得られたマススペクトルから、データベースを用いて代謝物の同定、すなわちピークアノテーションを行う。データベースに登録されていない代謝物が検出された場合、ターゲットイオンからフラグメントイオンを積極的に生成させるタンデム質量分析法（MS/MS）による構造推定を行う場合がある。MS/MS法では必ずしも明確に構造が推定できない場合もあるが、目的成分を単離して核磁気共鳴装置（NMR）による解析を行うよりはるかに構造推定を簡便に行うことができる。MS/MS法により候補物質が絞れた場合には、それらを実際に合成してマススペクトル、MS/MSスペクトルを比較することで推定された構造を確かなものにする。 From the obtained mass spectrum, metabolites are identified using a database, that is, peak annotation is performed. If a metabolite not registered in the database is detected, structure estimation may be performed using tandem mass spectrometry (MS/MS), which actively generates fragment ions from target ions. Although it is not always possible to clearly estimate the structure using the MS/MS method, it is much easier to estimate the structure than isolating the target component and analyzing it using a nuclear magnetic resonance apparatus (NMR). Once candidate substances have been narrowed down using the MS/MS method, they are actually synthesized and their mass spectra and MS/MS spectra are compared to confirm the estimated structure.
　以上の解析から、尿中代謝物1524種の物質が検出され、うち1027種が構造既知物質として同定された。同定されていない未知構造化合物についても、測定で得られる質量電荷比（m/z）やMSスペクトル、MS/MSスペクトル、保持時間、分離モードの情報があるため、IDを付与し、化学式の同定などを実施した。 From the above analysis, 1524 types of urinary metabolites were detected, of which 1027 types were identified as substances with known structures. Even for compounds with unknown structures that have not been identified, since we have information on the mass-to-charge ratio (m/z), MS spectrum, MS/MS spectrum, retention time, and separation mode obtained through measurement, we can assign an ID and identify the chemical formula. etc. were carried out.
　以下の解析を行うためのデータ前処理として、各代謝物の測定値毎に対し、Osmolalityで規格化した。更に、中央値で規格化（median = 1）し、欠損値は最小値を代入した。最後に、対数変換を行った。一部の解析では、欠損率が大きいマーカを除外した（例えば、術前の27例で欠損率50％以上のマーカを除外した）。マーカ探索では、薬物や食品等の外因性代謝物を除外した。OPLS-DAでは、各変数の重みを統一するため、平均0、標準偏差1とする標準化（Auto scaling）を行った。 As data preprocessing for the following analysis, each measured value of each metabolite was normalized by Osmolality. Furthermore, the values were normalized using the median value (median = 1), and missing values were substituted with the minimum value. Finally, logarithmic transformation was performed. In some analyses, markers with a high missing rate were excluded (eg, markers with a missing rate of 50% or more in 27 cases preoperatively were excluded). In the marker search, exogenous metabolites such as drugs and foods were excluded. In OPLS-DA, in order to unify the weight of each variable, standardization (auto scaling) was performed to set the mean to 0 and standard deviation to 1.
　最初に、全代謝物を用いて主成分解析（Principal Component Analysis: PCA）を行った。その結果、切除術前と術後でマーカ主成分値が区分された。術後2週間と術後4週間でも区分が観測され、術後4週間の方が術前に近かった。これは、術後2週間では、手術による過渡的な影響や、治癒における代謝活性が反映されていると推測できる。一方、男女、疾患部位、予後生死、等では、明確な区分はなかった。 First, principal component analysis (PCA) was performed using all metabolites. As a result, the marker principal component values were differentiated before and after resection. Segregation was observed at 2 weeks and 4 weeks after surgery, and 4 weeks after surgery was closer to preoperative values. It can be inferred that this reflects the transient effects of surgery and metabolic activity during healing in the 2 weeks after surgery. On the other hand, there were no clear divisions in terms of gender, site of disease, prognosis of life or death, etc.
　続いて、全代謝物を用いて、各グループ間で対応ありの有意差検定（Matched pair t-tests）を行った。その結果、切除術前と術後2週間で、494の代謝物に有意差があった（p<＝0.05）。切除術前と術後4週間では、113の代謝物に有意差があった。PCAと同様に、術後2週間では、手術の影響あるいはその回復に関連する生化学的変化の代謝物も含めて抽出していると推測した。なお、術前に対し、術後2週間と術後4週間の双方で有意差ありと評価されたのは85代謝物であった。 Subsequently, matched pair t-tests were performed between each group using all metabolites. As a result, there were significant differences in 494 metabolites between before and 2 weeks after resection (p<=0.05). There were significant differences in 113 metabolites before and 4 weeks after resection. Similar to PCA, we assumed that metabolites of biochemical changes related to the effects of surgery or recovery were also extracted during the 2 weeks after surgery. In addition, 85 metabolites were evaluated as having significant differences both 2 weeks and 4 weeks after surgery compared to before surgery.
　次いで、術前と術後4週間で有意差のある113代謝物のうち、外因性及び欠損率の高いものを除外した86代謝物を用い、（i）術前 対 術後2週間、（ii）術前 対 術後4週間、（iii）術前 対 術後2週間及び術後4週間について、ランダムフォレスト（RF）解析を行った。N数が最大になり、かつ腫瘍なし（術後2週間及び術後4週間）に対するものとなるため、（iii）術前 対 術後2週間及び術後4週間の結果をマーカ重要度とした。RFを3回行った平均順位で並べた上位30マーカを以下の表3及び図1のグラフに示す。 Next, of the 113 metabolites that had significant differences between preoperatively and 4 weeks postoperatively, 86 metabolites were excluded that were exogenous and had a high deficiency rate, and were used to determine whether (i) preoperatively vs. 2 weeks postoperatively, (ii) Random Forest (RF) analysis was performed for preoperative vs. 4 weeks postoperatively, (iii) preoperative vs. 2 weeks postoperatively, and 4 weeks postoperatively. Since the N number is the largest and is for no tumor (2 weeks postoperatively and 4 weeks postoperatively), (iii) the results before surgery versus 2 weeks postoperatively and 4 weeks postoperatively were set as marker importance. . The top 30 markers arranged by average ranking after performing RF three times are shown in Table 3 below and the graph in Figure 1.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-I000008
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-I000008
　上述のようにして、胆道がん（腫瘍有無）に関連するマーカ30種を同定した。上記マーカは、胆道がんの有無によりその存在量に変化が生じるため、上記マーカを測定することにより、胆道がんの有無の判定や手術後のモニタリングを行うことが可能となる。 As described above, 30 markers related to biliary tract cancer (presence or absence of tumor) were identified. The abundance of the above marker changes depending on the presence or absence of biliary tract cancer, so by measuring the above marker, it becomes possible to determine the presence or absence of biliary tract cancer and to perform post-surgery monitoring.
［実施例２］
　実施例１で同定したRFの上位20マーカについて手術前後（腫瘍有無）に対するROC曲線を求め、AUCを求めた。その結果を以下の表に示す。AUCの値は、示したマーカを単独で使用した場合の胆道がんについての判別能力を表す。AUC値は1に近いほど判別能力が高いことを示し、一般的に0.7以上で良好なモデル、あるいは、良好な判別能力とみなせる。 [Example 2]
ROC curves were determined for the top 20 RF markers identified in Example 1 before and after surgery (with or without tumor), and AUC was determined. The results are shown in the table below. The AUC value represents the discrimination ability for biliary tract cancer when the indicated marker is used alone. The closer the AUC value is to 1, the higher the discrimination ability is, and generally, an AUC value of 0.7 or higher can be considered a good model or a good discrimination ability.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　上記表4に示されるマーカは、単独で使用した場合でも、胆道がんの有無について高い判別能力で評価することが可能であることがわかった。 It was found that the markers shown in Table 4 above can evaluate the presence or absence of biliary tract cancer with high discrimination ability even when used alone.
［実施例３］複数マーカを組み合わせたがん検査モデルの構築
　実施例１で同定されたマーカを複数組み合わせた場合のがん検査モデルを構築した。組み合わせたマーカの妥当性を評価するために、説明変数（モデル構築に用いた学習データに対する当てはまりを表わす）と予測変数（モデルの予測性能を示す(leave-one-out cross-validation：一個抜き交差検証)）による評価を行った。これらの変数は、値が1に近いほど良いモデルであることを示しており、どのモデルの妥当性が高いかを検証する上で非常に有効な指標となる。具体的には、OPLS判別分析法により、以下のような指標を用いてがん検査モデルを求めた。 [Example 3] Construction of a cancer test model that combines multiple markers A cancer test model that combines multiple markers identified in Example 1 was constructed. In order to evaluate the validity of the combined markers, we use explanatory variables (representing the fit to the training data used for model construction) and predictor variables (representing the predictive performance of the model (leave-one-out cross-validation). Verification) The closer the value of these variables is to 1, the better the model, and is a very effective indicator for verifying which model has higher validity. Specifically, a cancer testing model was determined using the following indicators using the OPLS discriminant analysis method.
Figure JPOXMLDOC01-appb-M000010
Figure JPOXMLDOC01-appb-M000010
　ここで、Yobsは実測値、YcalcはOPLSによる計算値、Ypredは交差検証を行った際の予測値、
Figure JPOXMLDOC01-appb-I000011
は平均値を表す。交差検証とは、データを分割し、その一部をまず解析して残る部分でその解析のテストを行い、解析自身の妥当性の検証・確認に当てる手法を示す。これによれば、説明変数R2Y値はモデル構築に用いた学習データに対する当てはまりを表わし、1に近いほどモデルの精度は高く、予測変数Q2値はモデルの予測性能を示し（leave-one-out cross-validation 一個抜き交差検証）、1に近いほどモデルの予測性は高いといえる。 Here, Yobs is the measured value, Ycalc is the calculated value by OPLS, Ypred is the predicted value when cross-validated,
Figure JPOXMLDOC01-appb-I000011
represents the average value. Cross-validation refers to a method in which data is divided, a part of it is analyzed first, and the remaining part is used to test the analysis to verify and confirm the validity of the analysis itself. According to this, the explanatory variable R2Y value indicates the fit to the training data used for model construction, and the closer it is to 1, the higher the model accuracy, and the predictor variable Q2 value indicates the predictive performance of the model (leave-one-out cross -validation (leave-one-out cross-validation), the closer it is to 1, the more predictive the model is.
　複数のマーカの組み合わせについて、がん検査モデルを構築する。ここでがん検査モデルとは、例えば、マーカを5種用いる場合、尿検体中の各マーカ濃度あるいは各マーカに相当するイオンの強度（実際にはLC/MS測定で得られたマスクロマトグラムの面積）を用いて、予測式＝α×（マーカ1強度）＋β×（マーカ2強度）＋γ×（マーカ3強度）＋δ×（マーカ4強度）＋ε×（マーカ5強度）＋ζ（α、β、γ、δ、ε、ζは定数）で計算される予測値を用いて、がんのリスクを判定するというものである。具体的には、予測値が高いほどがんのリスクが高いと判断し、予測値が低いほどがんのリスクが低いと判断する。がんか健常かのどちらかを正誤で判別する場合は、予測値の閾値を適切に設定する。 Build a cancer testing model for the combination of multiple markers. Here, the cancer test model is, for example, when five types of markers are used, the concentration of each marker in the urine sample or the intensity of ions corresponding to each marker (actually, the mass chromatogram obtained by LC/MS measurement). Using the prediction formula = α x (marker 1 intensity) + β x (marker 2 intensity) + γ x (marker 3 intensity) + δ x (marker 4 intensity) + ε x (marker 5 intensity) + ζ (α, β, γ, δ, ε, and ζ are constants) are used to determine the risk of cancer. Specifically, the higher the predicted value, the higher the risk of cancer, and the lower the predicted value, the lower the risk of cancer. When determining whether cancer or normality is true or false, the threshold value of the predicted value is appropriately set.
　実施例１で同定したRFの上位10マーカ又は上位20マーカについて、OPLS判別分析法によるがん検査モデルの評価を行った。具体的には、以下の6つについてがん検査モデルを構築した。図２～８において、白色の棒グラフは、胆道がんの切除術前に採取した尿検体を示し、黒色の棒グラフは術後2週間に採取した尿検体を示し、斜線の棒グラフは術後4週間に採取した尿検体を示す。 The cancer testing model was evaluated using the OPLS discriminant analysis method for the top 10 or top 20 RF markers identified in Example 1. Specifically, we constructed cancer testing models for the following six areas. In Figures 2 to 8, white bar graphs indicate urine samples collected before biliary tract cancer resection, black bar graphs indicate urine samples collected 2 weeks after surgery, and diagonal bar graphs indicate urine samples collected 4 weeks after surgery. Shows the urine specimen collected in .
（１）RFの上位10マーカにより、術前 対 術後2週間でモデルを構築し、術後4週間の検体に適用した。その結果を図2のA（予測値）及びB（AUC）に示す。斜線の棒グラフがテスト検体である。本がん検査モデルは、説明変数R2Y：0.706、予測変数Q2：0.632、AUC値（N＝62）：0.929であった。図からわかるように、複数のマーカの組み合わせを用いることでより高精度な識別が可能となる。また、トレーニングデータとテストデータで同様の結果が得られており、モデルの汎用性も高い。 (1) A model was constructed using the top 10 RF markers preoperatively versus 2 weeks postoperatively, and applied to specimens 4 weeks postoperatively. The results are shown in Figure 2, A (predicted value) and B (AUC). The diagonally lined bar graph is the test sample. This cancer testing model had explanatory variable R2Y: 0.706, predictive variable Q2: 0.632, and AUC value (N = 62): 0.929. As can be seen from the figure, more accurate identification is possible by using a combination of multiple markers. In addition, similar results were obtained for training data and test data, and the model is highly versatile.
（２）RFの上位10マーカにより、術前 対 術後4週間でモデルを構築し、術後2週間の検体に適用した。その結果を図3のA（予測値）及びB（AUC）に示す。黒色の棒グラフがテスト検体である。本がん検査モデルは、説明変数R2Y：0.694、予測変数Q2：0.537、AUC値（N＝62）：0.930であった。図からわかるように、複数のマーカの組み合わせを用いることでより高精度な識別が可能となる。また、トレーニングデータとテストデータで同様の結果が得られており、モデルの汎用性も高い。 (2) A model was constructed using the top 10 RF markers before surgery versus 4 weeks after surgery, and was applied to specimens 2 weeks after surgery. The results are shown in Figure 3, A (predicted value) and B (AUC). The black bar graph is the test sample. This cancer testing model had explanatory variable R2Y: 0.694, predictive variable Q2: 0.537, and AUC value (N = 62): 0.930. As can be seen from the figure, more accurate identification is possible by using a combination of multiple markers. In addition, similar results were obtained for training data and test data, and the model is highly versatile.
（３）RFの上位20マーカにより、術前 対 術後2週間でモデルを構築し、術後4週間の検体に適用した。その結果を図4のA（予測値）及びB（AUC）に示す。斜線の棒グラフがテスト検体である。本がん検査モデルは、説明変数R2Y：0.773、予測変数Q2：0.689、AUC値（N＝62）：0.91であった。図からわかるように、複数のマーカの組み合わせを用いることでより高精度な識別が可能となる。また、トレーニングデータとテストデータで同様の結果が得られており、モデルの汎用性も高い。 (3) A model was constructed using the top 20 RF markers before surgery versus 2 weeks after surgery, and applied to specimens 4 weeks after surgery. The results are shown in Figure 4, A (predicted value) and B (AUC). The diagonally lined bar graph is the test sample. This cancer testing model had explanatory variable R2Y: 0.773, predictive variable Q2: 0.689, and AUC value (N = 62): 0.91. As can be seen from the figure, more accurate identification is possible by using a combination of multiple markers. In addition, similar results were obtained for training data and test data, and the model is highly versatile.
（４）RFの上位20マーカにより、術前 対 術後4週間でモデルを構築し、術後2週間の検体に適用した。その結果を図5のA（予測値）及びB（AUC）に示す。黒色の棒グラフがテスト検体である。本がん検査モデルは、説明変数R2Y：0.755、予測変数Q2：0.561、AUC値（N＝62）：0.939であった。図からわかるように、複数のマーカの組み合わせを用いることでより高精度な識別が可能となる。また、トレーニングデータとテストデータで同様の結果が得られており、モデルの汎用性も高い。 (4) A model was constructed using the top 20 RF markers before surgery versus 4 weeks after surgery, and applied to specimens 2 weeks after surgery. The results are shown in Figure 5, A (predicted value) and B (AUC). The black bar graph is the test sample. This cancer testing model had explanatory variable R2Y: 0.755, predictive variable Q2: 0.561, and AUC value (N = 62): 0.939. As can be seen from the figure, more accurate identification is possible by using a combination of multiple markers. In addition, similar results were obtained for training data and test data, and the model is highly versatile.
（５）RFの上位20マーカのうち、特に重要と思われる6マーカを用いて、術前　対　術後（2週間と4週間の双方）でモデルを構築した。この6マーカは、後述するように胆道がんと健常者を判別する能力も高い代謝物である（実施例４）。具体的には、glycochenodeoxycholate 3-sulfate（表3：4位、表6：1位）、glycocholate（表3：12位、表6：2位）、4-hydroxyphenylpyruvate（表3：19位、表6：6位）、glycochenodeoxycholate（表3：16位、表6：9位）、trans-4-hydroxyproline（表3：10位、表6：12位）、kynurenine（表3：7位、表6：18位）、の6つである。その結果を図6のA（予測値）及びB（AUC）に示す。ここでは、黒色の棒グラフが術後検体（2週間と4週間の双方）である。本がん検査モデルは、説明変数R2Y：0.447、予測変数Q2：0.359、AUC値（N＝62）：0.903であった。図からわかるように、複数のマーカの組み合わせを用いることでより高精度な識別が可能となる。 (5) Among the top 20 RF markers, 6 markers considered to be particularly important were used to construct a model preoperatively versus postoperatively (both 2 weeks and 4 weeks). These six markers are metabolites that have a high ability to distinguish between biliary tract cancer and healthy individuals, as described below (Example 4). Specifically, glycochenodeoxycholate 3-sulfate (Table 3: 4th place, Table 6: 1st place), glycocholate (Table 3: 12th place, Table 6: 2nd place), 4-hydroxyphenylpyruvate (Table 3: 19th place, Table 6 : 6th place), glycochenodeoxycholate (Table 3: 16th place, Table 6: 9th place), trans-4-hydroxyproline (Table 3: 10th place, Table 6: 12th place), kynurenine (Table 3: 7th place, Table 6: 18th place). The results are shown in Figure 6, A (predicted value) and B (AUC). Here, the black bars are postoperative specimens (both 2 and 4 weeks). This cancer testing model had explanatory variable R2Y: 0.447, predictive variable Q2: 0.359, and AUC value (N = 62): 0.903. As can be seen from the figure, more accurate identification is possible by using a combination of multiple markers.
（６）RFの上位20マーカのうち、特に重要と思われる3マーカを用いて、術前　対　術後（2週間と4週間の双方）でモデルを構築した。この3マーカは、後述するように胆道がんと健常者を判別する能力も高い代謝物である（実施例４）。具体的には、glycochenodeoxycholate 3-sulfate（表3：4位、表6：1位）、glycocholate（表3：12位、表6：2位）、4-hydroxyphenylpyruvate（表3：19位、表6：6位）、の3つである。その結果を図７のA（予測値）及びB（AUC）に示す。ここでは、黒色の棒グラフが術後検体（2週間と4週間の双方）である。本がん検査モデルは、説明変数R2Y：0.412、予測変数Q2：0.350、AUC値（N＝62）：0.883であった。図からわかるように、複数のマーカの組み合わせを用いることでより高精度な識別が可能となる。 (6) Among the top 20 RF markers, three markers considered to be particularly important were used to construct a model preoperatively versus postoperatively (both 2 weeks and 4 weeks). These three markers are metabolites that have a high ability to distinguish between biliary tract cancer and healthy individuals, as will be described later (Example 4). Specifically, glycochenodeoxycholate 3-sulfate (Table 3: 4th place, Table 6: 1st place), glycocholate (Table 3: 12th place, Table 6: 2nd place), 4-hydroxyphenylpyruvate (Table 3: 19th place, Table 6 :6th place). The results are shown in A (predicted value) and B (AUC) in FIG. Here, the black bars are postoperative specimens (both 2 and 4 weeks). This cancer testing model had explanatory variable R2Y: 0.412, predictive variable Q2: 0.350, and AUC value (N = 62): 0.883. As can be seen from the figure, more accurate identification is possible by using a combination of multiple markers.
（７）RFの上位20マーカのうち、特に重要と思われる2マーカを用いて、術前　対　術後（2週間と4週間の双方）でモデルを構築した。この2マーカは、後述するように胆道がんと健常者を判別する能力も高い代謝物である（実施例４）。具体的には、glycochenodeoxycholate 3-sulfate（表3：4位、表6：1位）、glycocholate（表3：12位、表6：2位）、の2つである。その結果を図8のA（予測値）及びB（AUC）に示す。ここでは、黒色の棒グラフが術後検体（2週間と4週間の双方）である。本がん検査モデルは、説明変数R2Y：0.306、予測変数Q2：0.241、AUC値（N＝62）：0.809であった。図からわかるように、複数のマーカの組み合わせを用いることでより高精度な識別が可能となる。 (7) Among the top 20 markers of RF, two markers considered to be particularly important were used to build a model preoperatively versus postoperatively (both 2 weeks and 4 weeks). These two markers are metabolites that have a high ability to distinguish between biliary tract cancer and healthy individuals, as described below (Example 4). Specifically, they are glycochenodeoxycholate 3-sulfate (Table 3: 4th place, Table 6: 1st place) and glycocholate (Table 3: 12th place, Table 6: 2nd place). The results are shown in Figure 8, A (predicted value) and B (AUC). Here, the black bars are postoperative specimens (both 2 and 4 weeks). This cancer testing model had explanatory variable R2Y: 0.306, predictive variable Q2: 0.241, and AUC value (N = 62): 0.809. As can be seen from the figure, more accurate identification is possible by using a combination of multiple markers.
［実施例４］胆道がん患者と健常者の比較による尿中代謝物の網羅的解析
　胆道がん患者25名より許可を得て、腫瘍切除前に尿検体の採取を行った。また、対照群として、健診により健常とされた25名より許可を得て、尿検体の採取を行った。尿検体の具体的な内訳は以下の通りである。 [Example 4] Comprehensive analysis of urinary metabolites by comparison of biliary tract cancer patients and healthy subjects With permission from 25 biliary tract cancer patients, urine samples were collected before tumor resection. In addition, as a control group, urine samples were collected from 25 people who were found to be healthy through medical examinations, with their permission. The specific breakdown of urine samples is as follows.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
　被験者の尿中代謝物（メタボローム）について、溶液中の混合成分を高感度に分析するのに好適な液体クロマトグラフィ質量分析装置（LC/MS）を用いて網羅的に解析を行った。できるだけ多くの代謝物を検出するため、イオン化には正負のエレクトロスプレーイオン化法を用いるとともに、混合成分の分離には、逆相クロマトグラフィ、親水性相互作用クロマトグラフィなど、複数の分離モードを使用した。 A comprehensive analysis of the subjects' urinary metabolites (metabolome) was conducted using a liquid chromatography mass spectrometer (LC/MS) suitable for highly sensitive analysis of mixed components in solutions. In order to detect as many metabolites as possible, positive and negative electrospray ionization was used for ionization, and multiple separation modes were used to separate the mixed components, including reversed-phase chromatography and hydrophilic interaction chromatography.
　得られたマススペクトルから、データベースを用いて代謝物の同定、すなわちピークアノテーションを行う。データベースに登録されていない代謝物が検出された場合、ターゲットイオンからフラグメントイオンを積極的に生成させるタンデム質量分析法（MS/MS）による構造推定を行う場合がある。MS/MS法では必ずしも明確に構造が推定できない場合もあるが、目的成分を単離して核磁気共鳴装置（NMR）による解析を行うよりはるかに構造推定を簡便に行うことができる。MS/MS法により候補物質が絞れた場合には、それらを実際に合成してマススペクトル、MS/MSスペクトルを比較することで推定された構造を確かなものにする。 From the obtained mass spectrum, metabolites are identified using a database, that is, peak annotation is performed. If a metabolite not registered in the database is detected, structure estimation may be performed using tandem mass spectrometry (MS/MS), which actively generates fragment ions from target ions. Although it is not always possible to clearly estimate the structure using the MS/MS method, it is much easier to estimate the structure than isolating the target component and analyzing it using a nuclear magnetic resonance apparatus (NMR). Once candidate substances have been narrowed down using the MS/MS method, they are actually synthesized and their mass spectra and MS/MS spectra are compared to confirm the estimated structure.
　以上の解析から、尿中代謝物1574種の物質が検出され、うち1070種が構造既知物質として同定された。 From the above analysis, 1574 types of urinary metabolites were detected, of which 1070 types were identified as substances with known structures.
　以下の解析を行うためのデータ前処理として、各代謝物の測定値毎に対し、Osmolalityで規格化した。更に、中央値で規格化（median = 1）し、欠損値は最小値を代入した。最後に、対数変換を行った。一部の解析では、欠損率が大きいマーカを除外した。マーカ探索では、薬物や食品等の外因性代謝物を除外した。OPLS-DAでは、各変数の重みを統一するため、平均0、標準偏差1とする標準化（Auto scaling）を行った。 As data preprocessing for the following analysis, each measured value of each metabolite was normalized by Osmolality. Furthermore, the values were normalized using the median value (median = 1), and missing values were substituted with the minimum value. Finally, logarithmic transformation was performed. In some analyses, markers with high missingness rates were excluded. In the marker search, exogenous metabolites such as drugs and foods were excluded. In OPLS-DA, in order to unify the weight of each variable, standardization (auto scaling) was performed to set the mean to 0 and standard deviation to 1.
　全代謝物を用いて、グループ間で有意差検定を行った。ここでは、対応無しのノンパラメトリックに適用できるウィルコクソンの順位和検定を用いた。その結果、がん患者と健常者で、外因性代謝物を除いた293の代謝物に有意差があった（p<＝0.05）。実施例１でランダムフォレスト解析を実施した86代謝物と、ここでの293代謝物との間に共通するものは、32代謝物であった。 A significant difference test was performed between groups using all metabolites. Here, the Wilcoxon rank sum test, which can be applied to unpaired nonparametric tests, was used. As a result, there were significant differences in 293 metabolites, excluding exogenous metabolites, between cancer patients and healthy subjects (p<=0.05). There were 32 metabolites in common between the 86 metabolites subjected to random forest analysis in Example 1 and the 293 metabolites here.
　次いで、この胆道がんの腫瘍切除で有意差があり、かつ、がん患者と健常者で有意差がある32代謝物を用い、がん患者　対　健常者の判別について、ランダムフォレスト解析（RF）を行った。RFを3回行った平均順位で並べた上位20マーカを以下の表6及び図10のグラフに示す。 Next, random forest analysis (RF) was performed to discriminate between cancer patients and healthy individuals using 32 metabolites that showed significant differences in tumor resection of biliary tract cancer and between cancer patients and healthy individuals. I did it. The top 20 markers arranged by average ranking after performing RF three times are shown in Table 6 below and the graph in FIG. 10.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
　上述のようにして、胆道がんの腫瘍有無と関連し（実施例１～３）、かつ、健常者と差があるマーカ20種を同定した。上記マーカを測定することにより、胆道がんの早期発見やスクリーニングを行うことが可能となる。 As described above, 20 markers that are associated with the presence or absence of biliary tract cancer (Examples 1 to 3) and that are different from healthy subjects were identified. By measuring the above markers, it becomes possible to perform early detection and screening of biliary tract cancer.
［実施例５］
　実施例４で同定したRFの上位20マーカ、上位10マーカ、あるいは上位5マーカについて、OPLS判別分析法によるがん検査モデルの評価を行った。具体的には、以下の3つについてがん検査モデルを構築した。図11～13において、白色の棒グラフは、胆道がん患者（腫瘍切除前）の尿検体を示し、黒色の棒グラフは健常者の尿検体を示す。 [Example 5]
The cancer test model was evaluated using the OPLS discriminant analysis method for the top 20, top 10, or top 5 RF markers identified in Example 4. Specifically, we constructed cancer testing models for the following three areas. In Figures 11 to 13, white bar graphs indicate urine samples from biliary tract cancer patients (before tumor resection), and black bar graphs indicate urine samples from healthy individuals.
（１）RFの上位20マーカにより、がん患者　対　健常者でモデルを構築した。その結果を図11のA（予測値）及びB（AUC）に示す。本がん検査モデルは、説明変数R2Y：0.598、予測変数Q2：0.405、AUC値（N＝50）：0.934であった。図からわかるように、複数のマーカの組み合わせを用いることで高精度な識別が可能となる。 (1) Using the top 20 RF markers, a model was constructed for cancer patients versus healthy individuals. The results are shown in Figure 11, A (predicted value) and B (AUC). This cancer testing model had explanatory variable R2Y: 0.598, predictive variable Q2: 0.405, and AUC value (N = 50): 0.934. As can be seen from the figure, highly accurate identification is possible by using a combination of multiple markers.
（２）RFの上位10マーカにより、がん患者　対　健常者でモデルを構築した。その結果を図12のA（予測値）及びB（AUC）に示す。本がん検査モデルは、説明変数R2Y：0.471、予測変数Q2：0.333、AUC値（N＝50）：0.941であった。図からわかるように、複数のマーカの組み合わせを用いることで高精度な識別が可能となる。 (2) A model was constructed for cancer patients versus healthy subjects using the top 10 RF markers. The results are shown in Figure 12, A (predicted value) and B (AUC). This cancer testing model had explanatory variable R2Y: 0.471, predictive variable Q2: 0.333, and AUC value (N = 50): 0.941. As can be seen from the figure, highly accurate identification is possible by using a combination of multiple markers.
（３）RFの上位5マーカにより、がん患者　対　健常者でモデルを構築した。その結果を図13のA（予測値）及びB（AUC）に示す。本がん検査モデルは、説明変数R2Y：0.346、予測変数Q2：0.254、AUC値（N＝50）：0.899であった。マーカ数が少なくなることにより、説明変数や予測変数の数値が低くなるが、AUC値としては高い値を維持できている。また、マーカ数が少ないほど、1検体測定に対する検査時間や試薬、データ解析におけるコストを低減することが可能となる。 (3) A model was constructed for cancer patients versus healthy subjects using the top five RF markers. The results are shown in Figure 13, A (predicted value) and B (AUC). This cancer testing model had explanatory variable R2Y: 0.346, predictive variable Q2: 0.254, and AUC value (N = 50): 0.899. As the number of markers decreases, the numerical values of explanatory variables and predictor variables decrease, but the AUC value remains high. Furthermore, the fewer the number of markers, the lower the cost of testing time, reagents, and data analysis for measuring one sample.
　本明細書で引用した全ての刊行物、特許及び特許出願をそのまま参考として本明細書にとり入れるものとする。 All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

Claims (13)

  1. 　対象における胆道がんを判定する方法であって、
    　対象由来の尿サンプル中の尿中腫瘍マーカを測定するステップであって、該尿中腫瘍マーカが、コール酸塩、ケノデオキシコール酸硫酸塩、LC/MSネガティブイオン検出モードで質量259.028として計測される化合物（C10H12O6S）、グリコケノデオキシコール酸3-硫酸塩、イソロイシルヒドロキシプロリン、pro-ヒドロキシ-pro、キヌレニン、4-メトキシフェノール硫酸塩、5-ヒドロキシリシン、trans-4-ヒドロキシプロリン、グリシルロイシン、グリココール酸塩、LC/MSネガティブイオン検出モードで質量197.068として計測される化合物（C7H10N4O3）、LC/MSネガティブイオン検出モードで質量509.277として計測される化合物（C28H38N4O5）、3-ヒドロキシキヌレニン、グリコケノデオキシコール酸塩、イソロイシルグリシン、フェニルアラニルヒドロキシプロリン、4-ヒドロキシフェニルピルビン酸塩、乳酸塩、シクロ(pro-ヒドロキシpro)、トリプトファン、N6-アセチルリシン、ガンマ-グルタミルフェニルアラニン、ロイシルヒドロキシプロリン、LC/MSネガティブイオン検出モードで質量328.152として計測される化合物（C14H23N3O6）、LC/MSポジティブイオン検出モードで質量146.081として計測される化合物（C6H11NO3）、アンドロステロングルクロニド、3-ヒドロキシアントラニレート、11ベータ-ヒドロキシアンドロステロングルクロニド、シスタチオニン、カルノシン、プロリン、アンセリン、アラビトール/キシリトール、3-ヒドロキシ-2-エチルプロピオネート、2R,3R-ジヒドロキシブチレート、ガンマ-グルタミルチロシン、1-メチルアデニン、ジヒドロオロト酸、アラニン、及び17アルファ-ヒドロキシプレグネノロングルクロニドから選択される少なくとも1種の尿中腫瘍マーカを含む、上記ステップ；
    　上記測定結果に基づいて対象における胆道がんを判定するステップ
    を含む、方法。     A method for determining biliary tract cancer in a subject, the method comprising:
    measuring a urinary tumor marker in a urine sample from a subject, the urinary tumor marker being cholate, chenodeoxycholate sulfate, a compound measured as mass 259.028 in LC/MS negative ion detection mode; (C10H12O6S), glycochenodeoxycholic acid 3-sulfate, isoleucylhydroxyproline, pro-hydroxy-pro, kynurenine, 4-methoxyphenol sulfate, 5-hydroxylysine, trans-4-hydroxyproline, glycylleucine, glycosyl Cholate, Compound measured as mass 197.068 in LC/MS negative ion detection mode (C7H10N4O3), Compound measured as mass 509.277 in LC/MS negative ion detection mode (C28H38N4O5), 3-Hydroxykynurenine, Glycochenodeoxycholic acid salt, isoleucylglycine, phenylalanylhydroxyproline, 4-hydroxyphenylpyruvate, lactate, cyclo(pro-hydroxypro), tryptophan, N6-acetyllysine, gamma-glutamylphenylalanine, leucylhydroxyproline, LC /Compound measured as mass 328.152 in MS negative ion detection mode (C14H23N3O6), compound measured as mass 146.081 in LC/MS positive ion detection mode (C6H11NO3), androsterone glucuronide, 3-hydroxyanthranilate, 11beta -Hydroxyandrosterone glucuronide, cystathionine, carnosine, proline, anserine, arabitol/xylitol, 3-hydroxy-2-ethylpropionate, 2R,3R-dihydroxybutyrate, gamma-glutamyltyrosine, 1-methyladenine, dihydroorotate, said step comprising at least one urinary tumor marker selected from alanine and 17 alpha-hydroxypregnenolone glucuronide;
    A method comprising determining biliary tract cancer in a subject based on the measurement results.
  2. 　前記尿中腫瘍マーカの少なくとも3種を測定する、請求項１に記載の方法。 The method according to claim 1, wherein at least three of the urinary tumor markers are measured.
  3. 　前記尿中腫瘍マーカが基準値以上である場合、対象が胆道がんを有する可能性があることを示す、請求項１に記載の方法。 The method according to claim 1, wherein when the urinary tumor marker is equal to or higher than a reference value, it indicates that the subject may have biliary tract cancer.
  4. 　前記胆道がんの判定が、対象における胆道がんの検出、対象における胆道がんのリスク予測、対象における胆道がんのステージ判定、対象における胆道がんの予後判定、対象における胆道がんについてのモニタリング又は対象に存在する胆道がんに対する治療の効果のモニタリングである、請求項１に記載の方法。 The determination of biliary tract cancer includes detecting biliary tract cancer in the subject, predicting the risk of biliary tract cancer in the subject, determining the stage of biliary tract cancer in the subject, determining the prognosis of biliary tract cancer in the subject, and determining the prognosis of biliary tract cancer in the subject. 2. The method of claim 1, wherein the method is monitoring or monitoring the effect of a treatment on biliary tract cancer present in the subject.
  5. 　前記胆道がんが、肝内胆管がん、肝門部領域胆管がん、遠位胆管がん、胆嚢がん、及び乳頭部がんからなる群より選択されるものである、請求項１に記載の方法。 Claim 1, wherein the biliary tract cancer is selected from the group consisting of intrahepatic bile duct cancer, hilar region bile duct cancer, distal bile duct cancer, gallbladder cancer, and papillary cancer. Method described.
  6. 　前記尿中腫瘍マーカの測定を液体クロマトグラフィ質量分析法（LC/MS）により行う、請求項１に記載の方法。 The method according to claim 1, wherein the measurement of the urinary tumor marker is performed by liquid chromatography mass spectrometry (LC/MS).
  7. 　胆道がんの判定装置であって、
    　尿サンプル中の尿中腫瘍マーカを測定する測定部であって、該尿中腫瘍マーカが、コール酸塩、ケノデオキシコール酸硫酸塩、LC/MSネガティブイオン検出モードで質量259.028として計測される化合物（C10H12O6S）、グリコケノデオキシコール酸3-硫酸塩、イソロイシルヒドロキシプロリン、pro-ヒドロキシ-pro、キヌレニン、4-メトキシフェノール硫酸塩、5-ヒドロキシリシン、trans-4-ヒドロキシプロリン、グリシルロイシン、グリココール酸塩、LC/MSネガティブイオン検出モードで質量197.068として計測される化合物（C7H10N4O3）、LC/MSネガティブイオン検出モードで質量509.277として計測される化合物（C28H38N4O5）、3-ヒドロキシキヌレニン、グリコケノデオキシコール酸塩、イソロイシルグリシン、フェニルアラニルヒドロキシプロリン、4-ヒドロキシフェニルピルビン酸塩、乳酸塩、シクロ(pro-ヒドロキシpro)、トリプトファン、N6-アセチルリシン、ガンマ-グルタミルフェニルアラニン、ロイシルヒドロキシプロリン、LC/MSネガティブイオン検出モードで質量328.152として計測される化合物（C14H23N3O6）、LC/MSポジティブイオン検出モードで質量146.081として計測される化合物（C6H11NO3）、アンドロステロングルクロニド、3-ヒドロキシアントラニレート、11ベータ-ヒドロキシアンドロステロングルクロニド、シスタチオニン、カルノシン、プロリン、アンセリン、アラビトール/キシリトール、3-ヒドロキシ-2-エチルプロピオネート、2R,3R-ジヒドロキシブチレート、ガンマ-グルタミルチロシン、1-メチルアデニン、ジヒドロオロト酸、アラニン、及び17アルファ-ヒドロキシプレグネノロングルクロニドから選択される少なくとも1種の尿中腫瘍マーカを含む、測定部と、
    　上記測定部で測定した尿中腫瘍マーカの測定値を基準値又は前回の測定値と比較する比較部と、
    　上記比較部で得られた比較結果から胆道がんを判定する判定部と
    を備えることを特徴とする装置。     A device for determining biliary tract cancer,
    A measurement unit for measuring a urinary tumor marker in a urine sample, wherein the urinary tumor marker is cholate, chenodeoxycholate sulfate, a compound (C10H12O6S) whose mass is measured as 259.028 in LC/MS negative ion detection mode. ), glycochenodeoxycholic acid 3-sulfate, isoleucylhydroxyproline, pro-hydroxy-pro, kynurenine, 4-methoxyphenol sulfate, 5-hydroxylysine, trans-4-hydroxyproline, glycylleucine, glycocholic acid Salt, compound measured as mass 197.068 in LC/MS negative ion detection mode (C7H10N4O3), compound measured as mass 509.277 in LC/MS negative ion detection mode (C28H38N4O5), 3-hydroxykynurenine, glycochenodeoxycholate, Isoleucylglycine, phenylalanylhydroxyproline, 4-hydroxyphenylpyruvate, lactate, cyclo(pro-hydroxypro), tryptophan, N6-acetyllysine, gamma-glutamylphenylalanine, leucylhydroxyproline, LC/MS Compound measured as mass 328.152 in negative ion detection mode (C14H23N3O6), compound measured as mass 146.081 in LC/MS positive ion detection mode (C6H11NO3), androsterone glucuronide, 3-hydroxyanthranilate, 11beta-hydroxy Androsterone glucuronide, cystathionine, carnosine, proline, anserine, arabitol/xylitol, 3-hydroxy-2-ethylpropionate, 2R,3R-dihydroxybutyrate, gamma-glutamyltyrosine, 1-methyladenine, dihydroorotate, alanine, and a measurement unit comprising at least one urinary tumor marker selected from 17 alpha-hydroxypregnenolone glucuronide;
    a comparison unit that compares the measurement value of the urinary tumor marker measured by the measurement unit with a reference value or a previous measurement value;
    An apparatus comprising: a determination section that determines biliary tract cancer from the comparison results obtained by the comparison section.
  8. 　前記尿中腫瘍マーカが基準値又は前回の測定値以上である場合、前記判定部は、対象が胆道がんを有する可能性があると判定する、請求項７に記載の装置。 The device according to claim 7, wherein when the urinary tumor marker is equal to or higher than a reference value or a previous measurement value, the determination unit determines that the subject may have biliary tract cancer.
  9. 　前記測定部は、前記尿中腫瘍マーカの少なくとも3種を測定する、請求項７に記載の装置。 The device according to claim 7, wherein the measurement unit measures at least three types of the urinary tumor markers.
  10. 　前記測定部が液体クロマトグラフィ質量分析（LC/MS）装置を備える、請求項７に記載の装置。 The apparatus according to claim 7, wherein the measurement section includes a liquid chromatography mass spectrometry (LC/MS) device.
  11. 　胆道がんの判定用キットであって、
    　コール酸塩、ケノデオキシコール酸硫酸塩、LC/MSネガティブイオン検出モードで質量259.028として計測される化合物（C10H12O6S）、グリコケノデオキシコール酸3-硫酸塩、イソロイシルヒドロキシプロリン、pro-ヒドロキシ-pro、キヌレニン、4-メトキシフェノール硫酸塩、5-ヒドロキシリシン、trans-4-ヒドロキシプロリン、グリシルロイシン、グリココール酸塩、LC/MSネガティブイオン検出モードで質量197.068として計測される化合物（C7H10N4O3）、LC/MSネガティブイオン検出モードで質量509.277として計測される化合物（C28H38N4O5）、3-ヒドロキシキヌレニン、グリコケノデオキシコール酸塩、イソロイシルグリシン、フェニルアラニルヒドロキシプロリン、4-ヒドロキシフェニルピルビン酸塩、乳酸塩、シクロ(pro-ヒドロキシpro)、トリプトファン、N6-アセチルリシン、ガンマ-グルタミルフェニルアラニン、ロイシルヒドロキシプロリン、LC/MSネガティブイオン検出モードで質量328.152として計測される化合物（C14H23N3O6）、LC/MSポジティブイオン検出モードで質量146.081として計測される化合物（C6H11NO3）、アンドロステロングルクロニド、3-ヒドロキシアントラニレート、11ベータ-ヒドロキシアンドロステロングルクロニド、シスタチオニン、カルノシン、プロリン、アンセリン、アラビトール/キシリトール、3-ヒドロキシ-2-エチルプロピオネート、2R,3R-ジヒドロキシブチレート、ガンマ-グルタミルチロシン、1-メチルアデニン、ジヒドロオロト酸、アラニン、及び17アルファ-ヒドロキシプレグネノロングルクロニドから選択される少なくとも1種の尿中腫瘍マーカを測定するための手段を含むことを特徴とするキット。     A kit for determining biliary tract cancer,
    Cholate, chenodeoxycholic acid sulfate, compound measured as mass 259.028 in LC/MS negative ion detection mode (C10H12O6S), glycochenodeoxycholic acid 3-sulfate, isoleucylhydroxyproline, pro-hydroxy-pro, kynurenine, 4-Methoxyphenol sulfate, 5-hydroxylysine, trans-4-hydroxyproline, glycylleucine, glycocholate, compound measured as mass 197.068 in LC/MS negative ion detection mode (C7H10N4O3), LC/MS Compound (C28H38N4O5) measured as mass 509.277 in negative ion detection mode, 3-hydroxykynurenine, glycochenodeoxycholate, isoleucylglycine, phenylalanylhydroxyproline, 4-hydroxyphenylpyruvate, lactate, cyclo( pro-hydroxypro), tryptophan, N6-acetyllysine, gamma-glutamylphenylalanine, leucylhydroxyproline, compound (C14H23N3O6) measured as mass 328.152 in LC/MS negative ion detection mode, in LC/MS positive ion detection mode Compound measured as mass 146.081 (C6H11NO3), androsterone glucuronide, 3-hydroxyanthranilate, 11beta-hydroxyandrosterone glucuronide, cystathionine, carnosine, proline, anserine, arabitol/xylitol, 3-hydroxy-2-ethylpro for measuring at least one urinary tumor marker selected from pionate, 2R,3R-dihydroxybutyrate, gamma-glutamyltyrosine, 1-methyladenine, dihydroorotate, alanine, and 17 alpha-hydroxypregnenolone glucuronide; A kit comprising the means.
  12. 　質量分析用試薬セットである、請求項１１に記載のキット。 The kit according to claim 11, which is a reagent set for mass spectrometry.
  13. 　胆道がんの治療の有効性の評価方法であって、
    　被験治療薬又は治療法による処置を受けた胆道がんを有する動物からの尿サンプルにおいて、尿中腫瘍マーカを測定するステップであって、該尿中腫瘍マーカが、コール酸塩、ケノデオキシコール酸硫酸塩、LC/MSネガティブイオン検出モードで質量259.028として計測される化合物（C10H12O6S）、グリコケノデオキシコール酸3-硫酸塩、イソロイシルヒドロキシプロリン、pro-ヒドロキシ-pro、キヌレニン、4-メトキシフェノール硫酸塩、5-ヒドロキシリシン、trans-4-ヒドロキシプロリン、グリシルロイシン、グリココール酸塩、LC/MSネガティブイオン検出モードで質量197.068として計測される化合物（C7H10N4O3）、LC/MSネガティブイオン検出モードで質量509.277として計測される化合物（C28H38N4O5）、3-ヒドロキシキヌレニン、グリコケノデオキシコール酸塩、イソロイシルグリシン、フェニルアラニルヒドロキシプロリン、4-ヒドロキシフェニルピルビン酸塩、乳酸塩、シクロ(pro-ヒドロキシpro)、トリプトファン、N6-アセチルリシン、ガンマ-グルタミルフェニルアラニン、ロイシルヒドロキシプロリン、LC/MSネガティブイオン検出モードで質量328.152として計測される化合物（C14H23N3O6）、LC/MSポジティブイオン検出モードで質量146.081として計測される化合物（C6H11NO3）、アンドロステロングルクロニド、3-ヒドロキシアントラニレート、11ベータ-ヒドロキシアンドロステロングルクロニド、シスタチオニン、カルノシン、プロリン、アンセリン、アラビトール/キシリトール、3-ヒドロキシ-2-エチルプロピオネート、2R,3R-ジヒドロキシブチレート、ガンマ-グルタミルチロシン、1-メチルアデニン、ジヒドロオロト酸、アラニン、及び17アルファ-ヒドロキシプレグネノロングルクロニドから選択される少なくとも1種の尿中腫瘍マーカを含む、上記ステップ、
    　上記測定結果に基づいて胆道がんに対する被験治療薬又は治療法の有効性を評価するステップ
    を含む方法。     A method for evaluating the effectiveness of biliary tract cancer treatment, the method comprising:
    measuring a urinary tumor marker in a urine sample from an animal with biliary tract cancer treated with a test therapeutic agent or therapy, the urinary tumor marker being cholate, chenodeoxycholate sulfate; , compound measured as mass 259.028 in LC/MS negative ion detection mode (C10H12O6S), glycochenodeoxycholic acid 3-sulfate, isoleucylhydroxyproline, pro-hydroxy-pro, kynurenine, 4-methoxyphenol sulfate, 5 -Hydroxylysine, trans-4-hydroxyproline, glycylleucine, glycocholate, compound measured as mass 197.068 in LC/MS negative ion detection mode (C7H10N4O3), as mass 509.277 in LC/MS negative ion detection mode Compounds to be measured (C28H38N4O5), 3-hydroxykynurenine, glycochenodeoxycholate, isoleucylglycine, phenylalanylhydroxyproline, 4-hydroxyphenylpyruvate, lactate, cyclo(pro-hydroxypro), tryptophan, N6-acetyllysine, gamma-glutamylphenylalanine, leucyl hydroxyproline, compound measured as mass 328.152 in LC/MS negative ion detection mode (C14H23N3O6), compound measured as mass 146.081 in LC/MS positive ion detection mode ( C6H11NO3), androsterone glucuronide, 3-hydroxyanthranilate, 11beta-hydroxyandrosterone glucuronide, cystathionine, carnosine, proline, anserine, arabitol/xylitol, 3-hydroxy-2-ethylpropionate, 2R,3R-dihydroxy said step, comprising at least one urinary tumor marker selected from butyrate, gamma-glutamyltyrosine, 1-methyladenine, dihydroorotate, alanine, and 17 alpha-hydroxypregnenolone glucuronide;
    A method comprising the step of evaluating the effectiveness of a test therapeutic agent or treatment method for biliary tract cancer based on the measurement results.
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JP2019105456A (en) * 2017-12-08 2019-06-27 株式会社日立製作所 Biomarker Search Method for Urinary Metabolites
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