JP2011106994A - Biomarker on colon cancer,and method of measuring biomarker on colon cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biomarker for diagnosing colon cancer, and a method of measuring the same. <P>SOLUTION: Diagnosis can be conducted on colon cancer by measuring compounds in collected blood, the compounds being selected from among the group, including indole-3-acetic acid, hippuric acid, citric acid, 3-indoxyl sulfate, thioproline, urocanic acid, acetoacetic acid, 6-hydroxy hexanoic acid, decanoic acid, cis-aconitic acid, lauric acid, O-acetylcarnitine, cystine, ribavirin, 3-phenylpropionic acid, hydroxyproline and pipecolinic acid. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a biomarker for diagnosing colorectal cancer.

  Colorectal cancer is one of the most frequently occurring cancers. In Europe and the United States, the number of cancers observed at the anatomical site is the second highest after lung cancer. In the United States, about 130,000 cases develop annually, and the annual number of deaths of about 57,000 cases is reported (for example, refer nonpatent literature 1).

  A fecal occult blood test (FBO) is usually used for screening whether or not the patient suffers from colorectal cancer, and diagnosis is performed by colonoscopy. In the case of early colorectal cancer, the possibility of cure is high. For example, the 10-year survival rate is about 90% when the cancer is confined to the mucous membrane (early colorectal cancer) and penetrates through the intestinal wall. When lymph node metastasis is positive (non-remote metastasis advanced colorectal cancer), about 30 to 50%, and when metastasized to other organs (distant metastasis advanced colorectal cancer) is less than 20% (for example, non-patent literature) 1).

  The fecal occult blood test is widely used as a test that can be received with less burden on the subject, and although it has shown a certain effect as a screening method for people with a possibility of colorectal cancer from a healthy population, it is low in accuracy and positive It is a general perception that a negative cannot be established. On the other hand, colonoscopy with an endoscope requires dietary restriction and laxative pretreatment, and further inserts an endoscope into the intestine, which places a great physical and mental burden on the subject.

Directed by Masanori Fukushima, “Merck Manual 18th Edition, Japanese Edition”, Nikkei Business Publications, December 25, 2006

  Then, an object of this invention is to provide the biomarker for diagnosing colon cancer, and its measuring method.

  That is, the present invention comprises the following items.

(1) A biomarker for diagnosing colorectal cancer, which is indole-3-acetic acid, hippuric acid, citric acid, 3-indoxyl sulfate, thioproline, urocanic acid, acetoacetic acid, 6-hydroxyhexanoic acid, decanoic acid A biomarker selected from the group consisting of cis-aconitic acid, lauric acid, O-acetylcarnitine, cystine, ribavirin, 3-phenylpropionic acid, hydroxyproline, and pipecolic acid.
(2) Indole-3-acetic acid, hippuric acid, citric acid, 3-indoxyl sulfate, thioproline, urocanic acid, acetoacetic acid, 6-hydroxyhexanoic acid, decanoic acid, cis-aconitic acid, laurin in the collected blood A measurement method comprising a step of measuring the content of one or more compounds selected from the group consisting of an acid, O-acetylcarnitine, cystine, ribavirin, 3-phenylpropionic acid, hydroxyproline, and pipecolic acid.
(3) measuring the content of indole-3-acetic acid in the collected blood;
Measuring the content of thioproline and / or urocanic acid in the blood, and the measuring method according to (2).
(4) The measurement according to (2), comprising a step of measuring the content of acetoacetic acid in the collected blood and a step of measuring the content of hippuric acid in the blood Method.
(5) a step of measuring the content of citrate in the collected blood, and one or more compounds selected from the group consisting of 3-phenylpropionic acid, 3-indoxyl sulfate, and hydroxyproline in the blood And a step of measuring the content of. The measurement method according to (2), comprising the step of:
(6) a step of measuring the content of 3-indoxyl sulfate in the collected blood, and 4-acetylbutyric acid, heptanoic acid, 6-hydroxyhexanoic acid, uric acid, decanoic acid, cis-aconitic acid in the blood Measuring the content of one or more compounds selected from the group consisting of: undecanoic acid, lauric acid, 2'-deoxyuridine, urocanic acid, O-acetylcarnitine, cystine, ribavirin, and 1-methyladenosine The measuring method according to (2).
(7) The measurement according to (2), comprising a step of measuring the content of urocanic acid in the collected blood and a step of measuring the content of pipecolic acid in the blood Method.
(8) The method according to claim 2, comprising a step of measuring the content of cis-aconitic acid in the collected blood and a step of measuring the content of hydroxyproline in the blood. Measuring method.
(9) A method for determining the effect of a drug on colorectal cancer, the measurement method according to any one of (2) to (8) before and after the drug is administered to a patient suffering from colorectal cancer Carrying out
Comparing the compound content obtained before and after administering the drug.
(10) A method for determining the effect of a drug on colorectal cancer, wherein the drug is administered to a patient suffering from colorectal cancer, and then the measurement according to any of (2) to (8) above A method comprising a step of performing the method, and a step of comparing the content of the compound with a threshold value of the content of the compound in blood for distinguishing a colorectal cancer patient from a healthy person.
(11) The content of one or more compounds selected from the group consisting of indole-3-acetic acid, hippuric acid, citric acid, and 3-indoxyl sulfate in the blood in order to discriminate between colorectal cancer patients and healthy individuals A method for determining the threshold value of the compound, comprising measuring the content of the compound contained in blood collected from a plurality of patients diagnosed with colorectal cancer and a plurality of healthy individuals.
(12) A method for screening a therapeutic agent for a colon cancer patient, the step of administering a candidate substance of the therapeutic agent to the model animal for colon cancer, and the step of collecting blood before and after administering the candidate substance Measuring the content of one or more compounds selected from the group consisting of indole-3-acetic acid, hippuric acid, citric acid, and 3-indoxyl sulfate in the blood, and administering the candidate substance Comparing the content of the compound in the blood before and after.
(13) A content of one or more compounds selected from the group consisting of indole-3-acetic acid, hippuric acid, citric acid, and 3-indoxyl sulfate in the blood for determining the degree of progression of colorectal cancer A method for determining thresholds collected from multiple patients diagnosed with early colorectal cancer, multiple patients diagnosed with non-remotely advanced colorectal cancer, and multiple patients diagnosed with distantly transferred colorectal cancer Measuring the content of the compound contained in blood.

  The present invention can provide a biomarker for colorectal cancer and a measurement method thereof.

  Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to examples. The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== Biomarkers for colorectal cancer diagnosis ==
In this specification, a biomarker for diagnosing colorectal cancer (also referred to herein as a diagnostic marker) is a diagnostic marker (affected marker) that can identify a colorectal cancer patient with high probability, and a healthy person is highly Includes diagnostic markers (healthy markers) that can be eliminated with probability.

  Here, the colorectal cancer in this specification refers to early colorectal cancer in which cancer is confined to the mucosa, invasion through the intestinal wall, non-distantly metastatic advanced colorectal cancer positive for lymph node metastasis, and other organs Although all the advanced colorectal cancers of distant metastasis that have metastasized are included, the term “colorectal cancer” includes all colorectal cancers regardless of the degree of progression.

  The diagnostic markers according to the present invention include indole-3-acetic acid, hippuric acid, citric acid, and 3-indoxyl sulfate, thioproline, urocanic acid, acetoacetic acid, 6-hydroxyhexanoic acid, decanoic acid, cis-aconitic acid, laurin Content of a diagnostic marker in blood collected from a vertebrate to be diagnosed that is an acid, O-acetylcarnitine, cystine, ribavirin, 3-phenylpropionic acid, hydroxyproline, and pipecolic acid (herein, the amount of marker It is possible to diagnose whether or not the individual suffers from colorectal cancer.

== Measurement method of diagnostic marker ==
The content of the compound that is a colorectal cancer diagnostic marker contained in the collected blood can be measured by a known method. For example, a method of measuring mass after isolating a target compound such as high performance liquid chromatography-mass spectrometer (LC-MS) or gas chromatography-mass spectrometer (GC-MS) can be mentioned. However, it is not limited to these. As other methods, a measurement method using NMR analysis, a measurement method using acid-alkali neutralization titration, a measurement method using an amino acid analyzer, a measurement method using an enzymatic method, a measurement method using a nucleic acid aptamer / peptide aptamer, Measurement methods using colorimetric determination, measurement methods using only high-performance liquid chromatography, measurement methods using only gas chromatography, measurement methods using only a mass spectrometer, and the like can be mentioned. It can be measured before or after the compound to be isolated is isolated.

  The animal from which blood is collected is preferably a vertebrate in which at least one type of diagnostic marker according to the present invention is present in the blood, more preferably a mammal, rodents such as mice and rats, Although it is more preferable that it is human, it is not particularly limited.

  When using high performance liquid chromatography, it is preferable to use a column (for example, an ion exchange column) capable of simultaneous analysis of ionic metabolites.

  From the viewpoint that all types of marker amounts contained in blood can be measured simultaneously, it is preferable to measure using a capillary electrophoresis-time-of-flight mass spectrometer (CE-TOFMS).

  When measuring the amount of marker contained in the collected blood, it is preferable to pre-treat the blood prior to the measurement. For example, it is preferable to separate serum or plasma from blood by standing or centrifuging, and use the removed serum or plasma for measurement.

  In measurement using CE-TOFMS, it is preferable to further carry out the following pretreatment on the serum or plasma thus obtained.

  Serum or plasma is mixed with an alcohol solvent such as methanol or ethanol to stop the reaction of enzymes contained in the serum or plasma. The kind of alcohol solvent to be used is preferably methanol, and the purity of the alcohol solvent is preferably for LC / MS.

  Next, an organic solvent containing water-soluble substances such as phospholipids is obtained by adding and mixing an organic solvent and water to serum or plasma in which the enzymatic reaction has been stopped, and mixing the resulting mixture. Remove the phase.

  The type of the organic solvent to be used is not particularly limited as long as it is an organic solvent that can be phase-separated from water, but dichloromethane, chloroform, dichloroethane, and the like are preferable, and chloroform is particularly preferable. Further, the purity of the organic solvent to be used is preferably special grade or analytical. The purity of the water used is preferably distilled water, deionized water, or Milli-Q water, particularly Milli-Q water.

  The method of mixing serum, organic solvent, and water that has stopped the enzyme reaction is preferably a method using a magnetic stirrer or vortex, etc., considering that mixing can be performed efficiently even with a small amount of sample. The method is particularly preferred.

  The method of phase-separating the obtained liquid mixture is preferably stationary or centrifugation, and centrifugation is particularly preferred.

  It is preferable to remove proteins from the obtained aqueous phase. The method for removing the protein is not particularly limited, but for example, ultrafiltration is preferable.

  After removing the protein, it is preferable to distill off the alcohol solvent contained in the aqueous phase. The method for distilling off the solvent is not particularly limited, such as natural drying, reduced pressure drying, or reduced pressure centrifugal drying, but reduced pressure centrifugal drying is preferred in view of the fact that it can be carried out easily in a short time.

  Thus, it is preferable to prepare an aqueous solution from which the insoluble matter has been removed from the collected blood and perform CE-TOFMS measurement using the aqueous solution from which the insoluble matter has been removed.

  In measuring the amount of marker contained in the collected blood, those skilled in the art can appropriately select and set the measurement conditions according to the type of the compound to be measured.

  For example, when measuring using CE-TOFMS, it can set and measure as follows.

  First, it is preferable that the sample used for the measurement is an aqueous solution in which insoluble matters are removed from the collected blood according to the above method.

  Moreover, the sample used for the measurement may contain an internal standard substance that serves as a measurement standard for the electrophoresis time and content of the compound that is a diagnostic marker. The internal standard substance is not particularly limited as long as it does not affect the efficiency of electrophoresis and mass spectrometry of the compound that is a diagnostic marker. For example, methionine sulfone or 10-camphorsulfonic acid (CSA) It is preferable that

  The capillary for capillary electrophoresis is preferably a fused silica capillary. In addition, the inner diameter of the capillary is preferably 100 μm or less, and particularly preferably 50 μm, in view of the improvement in resolution. The total length of the capillary is preferably 50 cm to 150 cm, and particularly preferably 100 cm.

  The method for identifying the fraction containing the compound that is the target diagnostic marker in each fraction obtained by the capillary electrophoresis is not particularly limited. For example, using a preparation of the target compound in advance, Examples thereof include a method of measuring the electrophoresis time of the compound and a method of using a relative time with the electrophoresis time of the internal standard substance.

  Next, the content of the compound having m / z of the target compound in the fraction identified as containing the compound as the target diagnostic marker is measured as a peak area.

  This measured peak area can be corrected by taking a ratio with the peak area of the internal standard substance. In addition, by creating a calibration curve using a sample of the target compound, the absolute concentration, that is, the content of the compound that is the target disease marker contained in the collected blood can be obtained from the measured peak area. it can.

== How to use diagnostic markers ==
Examples of the method of using the diagnostic marker include the following modes.

<Diagnosis method for colorectal cancer>
First, colorectal cancer can be diagnosed by measuring the content of a diagnostic marker in blood using blood obtained by collecting blood from vertebrates. Here, the content of the diagnostic marker in blood is preferably the absolute concentration of the diagnostic marker, but is not limited as long as the absolute concentration of the diagnostic marker is correlated with the absolute concentration of the diagnostic marker, and the relative concentration is not limited. Alternatively, the weight per unit volume, raw data measured to know the absolute concentration (for example, a value obtained by standardizing the peak area of a graph obtained by CE-TOFMS measurement), or the like may be used.

  When making a diagnosis, a vertebrate that has been previously diagnosed as having colon cancer (also referred to herein as a diseased vertebrate), and a vertebrate that has been diagnosed as not having colon cancer (this book) The range of the marker amount in the blood of the vertebrate blood to be diagnosed falls within the range of the marker amount in the blood of the healthy vertebrate. If this is the case, the vertebrate is more likely not to have colorectal cancer, and if it falls within the range of marker levels in the blood of the diseased vertebrate, it is likely that the vertebrate is affected with colorectal cancer.

  In addition, as the range of marker amount in the blood of healthy vertebrates, the range of marker amount in the blood at normal time of the vertebrate individual to be diagnosed is measured in advance, and the marker amount at the time of diagnosis is You may compare with the range of marker amount.

  Here, the range of the marker amount in the blood of a healthy vertebrate may be a range from a value obtained by subtracting the standard deviation to an average value of a plurality of measurement values for each animal to a value obtained by adding the standard deviation to the average value. The range may be a range from the lower limit value to the upper limit value of the average value, and is not particularly limited, but the most preferable value or range may be selected when diagnosing a disease by a diagnostic marker.

  Or you may determine the threshold value of the marker amount in the blood for discriminating a diseased vertebrate and a healthy vertebrate. For example, the amount of markers in the blood of a plurality of diseased vertebrates and a plurality of healthy vertebrates is measured in advance, and the threshold value is determined. The method for determining the threshold value may be in accordance with a standard method of a person skilled in the art, and is not particularly limited, but the diseased vertebrate is included in the first predetermined ratio below the threshold (or above the threshold), and the healthy vertebrate is above the threshold ( (Or less than the threshold) may be determined so that the second predetermined ratio is included. As a specific method, for example, the statistical software JMP of SAS may be used to obtain a value that provides the best chi-square value for discrimination.

  As the diagnostic marker, the first predetermined ratio and the second predetermined ratio are preferably higher, preferably 50% or more, more preferably 70% or more, still more preferably 90% or more, and most preferably 100%. If both values are set high, the specificity and sensitivity become high. However, if both the first and second predetermined ratios cannot be increased, the threshold value may be determined so that either the specificity or the sensitivity is increased. Higher specificity and sensitivity are preferable, preferably 50% or more, more preferably 70% or more, still more preferably 90% or more, and most preferably 100%. The specificity is (number of healthy vertebrates above or below threshold) × 100 / (total number of healthy vertebrates), and sensitivity is (number of diseased vertebrates below or above threshold) × 100 / (disease) The total number of vertebrates. Diagnostic markers with high specificity have a high probability of suffering from colorectal cancer when the amount of the marker is less than or greater than the threshold, and diagnostic markers for identifying colorectal cancer (in this specification, these diagnostic markers are referred to as It is also preferably used as a disease marker). A highly sensitive diagnostic marker has a high probability of not suffering from colorectal cancer when the amount of the marker is greater than or less than a threshold, and a diagnostic marker that excludes healthy individuals from colorectal cancer patients (herein, these The diagnostic marker is also preferably used as a healthy marker.

  The diagnostic marker according to the present invention can be used as a disease marker or a healthy marker, but when diagnosing colorectal cancer in humans, for example, a diagnostic marker having high sensitivity and specificity, such as indole-3-acetic acid, It is effective as both a disease marker and a healthy marker. Therefore, when the plasma concentration of the subject is measured and the threshold value for distinguishing between healthy and affected is within the affected range, the possibility of colorectal cancer is high, or when the sample is within the healthy range It can be diagnosed that the nature is high. In addition, a diagnostic marker with particularly high sensitivity such as citric acid and 3-indoxyl sulfate is preferably used as a healthy marker, and the concentration of the subject in plasma is measured to determine whether the patient is healthy from the threshold for distinguishing between healthy and diseased. If it is in the range, it can be diagnosed that the possibility of being healthy is high. On the other hand, a marker with particularly high specificity such as hippuric acid is preferably used as a disease marker, and when the plasma concentration of a subject is measured and within a diseased range with respect to a threshold value for distinguishing between healthy and diseased Can be diagnosed as having a high possibility of having colorectal cancer. However, the measurement value, threshold value, sensitivity, and specificity are likely to change if the instrument, measurement method, population, and calculation method for measuring the marker amount are different. Which marker is suitable as a disease marker or a healthy marker may vary depending on the animal species, population, measurement method, and calculation method to be diagnosed.

  By using the diagnostic markers according to the present invention in combination, it is possible to increase diagnostic accuracy. For example, if two types of diagnostic markers are used and only one of them is a concentration that indicates the possibility of colon cancer, it may be diagnosed that there is a possibility of colon cancer, but both indicate the possibility of colon cancer. If it is a concentration, the possibility of colorectal cancer is higher than when one type of diagnostic marker is used. Preferred is a case where a healthy marker is used in combination with a disease marker. For example, for a certain animal individual, if it is diagnosed with the healthy marker and the concentration indicates “not healthy”, the same individual is further used. If it is diagnosed with a disease marker and shows that it is “affected”, it is determined that there is a high possibility of colorectal cancer. If it is not a concentration that indicates “affected”, it may be healthy. Judgment is high. The diagnostic accuracy at this time is higher than when only the disease marker is used. If the concentration indicates that the healthy marker is “healthy”, it is diagnosed that there is a high possibility of being healthy. Is more likely. If the concentration of the affected marker indicates “affected”, it is preferable to perform further diagnosis by determining the distance from the threshold of the concentrations of both diagnostic markers and the determination of the third biomarker.

  Alternatively, as a method of using a combination of the diagnostic markers according to the present invention, the measurement values of a plurality of markers are used to determine the threshold value for distinguishing between healthy subjects and patients suffering from colorectal cancer, or the degree of progression of colorectal cancer A threshold value may be calculated. In this case, for example, from statistical data of the peak area obtained by measuring each marker measurement in accordance with the above “diagnostic marker measurement method”, the statistical software JMP ver. 7.0.1 can be used to calculate the threshold by finding the value that gives the best chi-square value for discrimination and dividing that best value in one marker by that best value in the other marker . By using such a threshold value, colorectal cancer can be diagnosed more accurately than when a threshold value obtained from the measurement value of a single marker is used.

  In particular, when the diagnostic marker according to the present invention is used for screening from a healthy population or for diagnosing a population suspected of being affected by screening, it is highly accurate even with patients with early colorectal cancer. It is preferable to select a marker or a combination of markers that can be discriminated.

  The diagnosis of colorectal cancer using the diagnostic marker according to the present invention may be performed in combination with a conventional fecal occult blood test, enema examination, endoscopy, biopsy, CT scan, MRI, ultrasonic examination and the like.

  As described above, by using the diagnostic marker according to the present invention, it becomes possible to diagnose colorectal cancer without using a special device and without using a test method that burdens the patient.

<Method for determining the progression of colorectal cancer>
Since the blood content of the diagnostic marker according to the present invention increases or decreases in proportion to the degree of progression of colorectal cancer, it can be used as a diagnostic marker for determining the degree of progression of colorectal cancer.

  Specifically, by measuring in advance the amount of markers in the blood of a plurality of healthy vertebrates and a plurality of vertebrates suffering from early colon cancer, non-distant metastasis progression colon cancer or distant metastasis progression colon cancer, A range of marker amount in the blood corresponding to the degree of progression of colorectal cancer may be set, and the degree of progression of colorectal cancer may be diagnosed according to which range the amount of marker in the subject falls.

  Alternatively, in the same manner as the threshold value of the marker amount in the blood for determining the diseased vertebrate and the healthy vertebrate is determined as described above, for example, a plurality of healthy vertebrates and early colon cancer, Threshold level of marker amount in blood to measure the amount of marker in blood of multiple vertebrates suffering from distant metastasis advanced colorectal cancer or distant metastasis advanced colorectal cancer, and to determine the progression of colorectal cancer in diseased vertebrates May be determined.

<Drug determination>
In general, a therapeutic agent for a disease may vary in its effect from individual to individual. Therefore, it is very beneficial to examine the efficacy of a therapeutic drug in each individual, and the efficacy of the therapeutic drug can be easily examined using the diagnostic marker of the present invention. For example, before and after administering a therapeutic agent for colorectal cancer, blood is collected from a diseased vertebrate, the content of the diagnostic marker contained in the collected blood is measured, and the diagnostic marker in the blood before and after the therapeutic agent is administered Compare the contents of. If the content of the diagnostic marker is close to the normal range after administration of the therapeutic agent, it can be determined that the therapeutic agent is effective. Thus, by using the diagnostic marker of the present invention, it can be easily determined whether or not there is an effect of a therapeutic agent.

  In addition, the content of a diagnostic marker contained in blood after the administration of a therapeutic agent for colorectal cancer is measured, and this value is used as a threshold value for distinguishing between a colorectal cancer patient and a healthy person, or the degree of progression of colorectal cancer. It is possible to determine whether or not the therapeutic agent is effective in improving the cancer of the patient.

<Screening method for drugs effective for colorectal cancer>
Using experimental animals with colorectal cancer, compounds that are effective in treating colorectal cancer can be identified. For example, a compound that is a candidate for a therapeutic agent for colorectal cancer is administered to a model animal for colorectal cancer, blood is collected before and after administration, the content of a diagnostic marker contained in the blood is measured, and before and after administration of the compound Compare the content of diagnostic markers in the blood. If the content of the diagnostic marker is close to the normal range after administration of the compound, it can be determined that the administered compound is effective in treating colorectal cancer. Thus, by using the diagnostic marker of the present invention, it is possible to easily screen for compounds that are effective in treating diseases.

<Screening method for drugs most effective for diseased animals>
In the case of drugs having different drug effects depending on the individual, it is possible to try a plurality of drugs on an individual having colorectal cancer, diagnose with a diagnostic marker, and screen for the most effective drug for that individual.

[Example 1] Collecting samples from patients with colorectal cancer and healthy individuals In social insurance Kyoto Hospital, Nishijin Hospital, or Kyoto Prefectural University of Medicine Hospital, early colon cancer (8 persons) by endoscopy and histopathological examination, For patients diagnosed with non-distant metastatic advanced colorectal cancer (9 patients) or distant metastatic advanced colorectal cancer (8 patients), with the approval of the Ethics Committee, Requested research cooperation. Early colorectal cancer is a benign tumor that has become malignant, and invasion is limited to the submucosa. Non-distant metastatic colorectal cancer is invaded to the muscle lamina or lymph nodes. Distant metastatic colorectal cancer is an advanced cancer that has been confirmed to metastasize to other organs.

  Healthy individuals were convened by advertisement and selected from those who requested research cooperation (21 people).

  Blood was collected from the above patients and healthy subjects for measurement of diagnostic markers, and plasma was separated within 2 hours and stored frozen until measurement.

Table 1 shows the age and sex of each patient and each healthy person, the degree of progression of colorectal cancer by histopathological diagnosis, and the like.

Table 2 shows the average age and the number of men and women in each progression of colorectal cancer.

[Example 2] Preparation of plasma sample In order to measure the amount of each diagnostic marker compound contained in blood with a capillary electrophoresis-time-of-flight mass spectrometer (CE-TOFMS), CE- Plasma samples for TOFMS measurement were prepared.

  100 μL of plasma prepared from the blood of the patient of Example 1 (early colorectal cancer, non-distant metastasis advanced colorectal cancer, distant metastasis advanced colorectal cancer), and healthy individuals was placed in a centrifuge tube. Methanol (Wako Pure Chemicals, LC / MS) containing 10 μM methionine sulfone and 10 μM 10-camphorsulfonic acid (H3304-1002, sold by Human Metabolome Technologies, Inc.) as an internal standard solution Add 9 mL, add 1 mL of chloroform (Wako Pure Chemicals, reagent grade) and 400 μL of Milli-Q water, mix well by vortex for 30 seconds, and centrifuge (4 ° C., 2,300 × g, 5 minutes) )did. The aqueous phase was transferred to an ultrafiltration filter (Millipore Ultrafree-MCPBCC centrifugal filter unit 5 kDa), and centrifuged (4 ° C., 9,100 × g, 2 to 4 hours) until the solution in the filter cup almost disappeared. . The filter cup was removed and the filtrate was centrifuged and dried under reduced pressure. A sample for CE-TOFMS measurement was prepared by re-dissolving the dried product in 50 μL of Milli-Q water added with an internal standard.

[Example 3] CE-TOFMS measurement method and analysis method of results CE-TOFMS measurement and subsequent data analysis were performed using an Agilent CE-TOF-MSD system (Agilent Technologies) using a fused silica capillary as a capillary. I went.

The measurement conditions for the CE-TOFMS measurement are as follows.
(A) Cation mode
Run buffer: Cation Buffer Solution (p / n: H3301-1001)
Rinse buffer: Cation Buffer Solution (p / n: H3301-1001)
Sample injection: Pressure injection 50 mbar, 10 sec
CE voltage: Positive, 30 kV
MS ionization: ESI Positive
MS capillary voltage: 4,000 V
MS scan range: m / z 50-1,000
Sheath liquid: HMT Sheath Liquid (p / n: H3301-1020)

(B) Anion mode
Run buffer: Anion Buffer Solution (p / n: H3302-1021)
Rinse buffer: Anion Buffer Solution (p / n: H3302-1022)
Sample injection: Pressure injection 50 mbar, 25 sec
CE voltage: Positive, 30 kV
MS ionization: ESI Negative
MS capillary voltage: 3,500 V
MS scan range: m / z 50-1,000
Sheath liquid: HMT Sheath Liquid (p / n: H3301-1020)

For the peaks detected by CE-TOFMS, mass-to-charge ratio (m / z), migration time (Migration time: MT) and area value were obtained as peak information. The obtained peak area value was converted into a relative area value using the following formula 1.

[Example 4] Measurement of plasma concentration of diagnostic marker by CE-TOFMS and analysis of results Plasma-derived CE-TOFMS prepared from colorectal cancer patients described in Example 1 and healthy subjects and prepared according to Example 2 The measurement sample was subjected to CE-TOFMS measurement under the conditions shown in Example 3. By this measurement, data on the amount of each compound contained in each sample was obtained as a peak area.

From this peak area data, statistical software JMP ver. 7.0.1 is used to obtain a value that provides the best chi-square value for discrimination, and as a diagnostic marker, early colorectal cancer and healthy person, non-distant metastatic advanced colorectal cancer and healthy person, distant metastatic advanced colorectal cancer and The thresholds were set such that a healthy person, advanced colorectal cancer (including non-metastasis and metastasis) and a healthy person could be distinguished. The results are shown in Table 3.

Table 4 shows the number of people whose measured content of each marker is greater than or less than the threshold value.

  In the case of a diagnostic marker whose blood content is high in cancer patients and low in healthy individuals, healthy individuals whose values are higher than the threshold are false positives. On the other hand, a cancer patient showing a value lower than the threshold is false negative. Further, in the case of a diagnostic marker whose blood content is low in cancer patients and high in healthy individuals, healthy individuals who show lower values than the threshold are false positives. On the other hand, cancer patients showing a higher value than the threshold are false negatives (Table 3).

Table 5 shows the sensitivity and specificity of each diagnostic marker.

  When discriminating between early colorectal cancer patients and healthy individuals, citric acid and 3-indoxyl sulfate have a specificity of 50% or less, but the sensitivity is high and the ability to exclude healthy individuals from the cancer patient group is high. It is preferably used as a healthy marker. On the other hand, hippuric acid is low in sensitivity, but has high specificity and high ability to supplement affected individuals, so it is preferably used as a disease marker. In addition, a compound such as indole-3-acetic acid having both sensitivity and specificity of 60% or more can distinguish an affected person from a healthy person with high accuracy, and can be used alone for diagnosis. . Indole-3-acetic acid, hippuric acid, citric acid, and 3-indoxyl sulfate have high sensitivity and specificity when distinguishing patients with advanced colorectal cancer from healthy individuals. It is also possible to use it alone for diagnosis.

  However, when diagnosing the possibility of illness using a diagnostic marker such as a cancer dock or the like for a subject who does not know whether or not he / she is afflicted with cancer, he / she is afflicted with early colorectal cancer An accuracy that can distinguish a patient from a healthy person is required.

  With respect to the above compounds, all the contents in the blood can be measured simultaneously only by measuring the blood by CE-TOFMS, so it is extremely easy to make a diagnosis by combining the results of a plurality of compounds.

[Example 5] Diagnosis using a plurality of biomarkers The diagnosis accuracy can be increased by using a plurality of diagnosis markers among the above-mentioned compounds. In particular, a combination of a disease marker and a healthy marker, a combination of a diagnostic marker and a disease marker effective for both, a combination of a diagnostic marker and a healthy marker effective for both, and a combination of markers effective for both are considered effective. Specific combinations of diagnostic markers are exemplified in Table 6.

  When diagnosing colorectal cancer using a plurality of biomarkers, the statistical software JMP ver. 7.0.1 was used to obtain a value that would provide the best chi-square value for discrimination. Then, a ratio calculated by dividing the best value of the chi-square value of discrimination in the marker 2 by the best value of chi-square value of the discrimination in the marker 1 was used as the threshold of the marker combination.

  As shown in Table 6, in any combination of markers, the sensitivity and specificity were the same or improved compared to when each marker was used alone.

  Therefore, by using the markers according to the present invention in combination, it is possible to diagnose colorectal cancer with higher accuracy.

Example 6 Biomarker for Determining Progression of Colorectal Cancer Blood-derived CE-TOFMS measurement sample collected from the colorectal cancer patient described in Example 1 and healthy subjects and prepared according to Example 2 Was subjected to CE-TOFMS measurement under the conditions shown in Example 3.

Table 7 shows the average value of each marker concentration in blood in healthy subjects and patients suffering from various stages of colorectal cancer.

  Indole-3-acetic acid, hippuric acid, citric acid, and 3-indoxyl sulfate tend to increase or decrease the marker concentration in the order of healthy subject, early cancer, non-distant metastasis advanced colon cancer, and distant metastasis progression colon cancer. is there. Therefore, these markers can be used as biomarkers for determining the degree of progression of colorectal cancer.

  In addition, other marker concentrations shown in Table 7 are also observed between healthy subjects and colon cancer patients, between early cancers and advanced colorectal cancer patients (including non-remote metastases and distant metastases), and non-remote metastasis advanced cancers. There is a tendency to increase or decrease, either between patients and patients with distant metastatic cancer. Therefore, these markers can also be used as biomarkers for determining the degree of progression of colorectal cancer.

Claims (9)

A biomarker for diagnosing colorectal cancer,
Indole-3-acetic acid, hippuric acid, citric acid, 3-indoxyl sulfate, thioproline, urocanic acid, acetoacetic acid, 6-hydroxyhexanoic acid, decanoic acid, cis-aconitic acid, lauric acid, O-acetylcarnitine, cystine, A biomarker selected from the group consisting of ribavirin, 3-phenylpropionic acid, hydroxyproline, and pipecolic acid.   Indole-3-acetic acid, hippuric acid, citric acid, 3-indoxyl sulfate, thioproline, urocanic acid, acetoacetic acid, 6-hydroxyhexanoic acid, decanoic acid, cis-aconitic acid, lauric acid, O in the collected blood -Measuring method including the process of measuring content of 1 or more compounds selected from the group which consists of acetylcarnitine, cystine, ribavirin, 3-phenylpropionic acid, hydroxyproline, and pipecolic acid. Measuring the content of indole-3-acetic acid in the collected blood;
And measuring the content of thioproline and / or urocanic acid in the blood. Measuring the content of 3-indoxyl sulfate in the collected blood;
4-acetylbutyric acid, heptanoic acid, 6-hydroxyhexanoic acid, uric acid, decanoic acid, cis-aconitic acid, undecanoic acid, lauric acid, 2'-deoxyuridine, urocanic acid, O-acetylcarnitine, cystine in the blood, The measuring method according to claim 2, comprising a step of measuring the content of one or more compounds selected from the group consisting of ribavirin and 1-methyladenosine. A method for determining the effect of a drug on colorectal cancer,
Performing the measurement method according to any one of claims 2 to 4, before and after administering the drug to a patient suffering from colorectal cancer;
Comparing the compound content obtained before and after administering the drug. A method for determining the effect of a drug on colorectal cancer,
Performing the measurement method according to any one of claims 2 to 4 after administering the drug to the patient suffering from colorectal cancer;
A method comprising a step of comparing a content threshold value of the compound in blood and the content of the compound for distinguishing between a colorectal cancer patient and a healthy person.   In order to discriminate between a colorectal cancer patient and a healthy person, a threshold value for the content of one or more compounds selected from the group consisting of indole-3-acetic acid, hippuric acid, citric acid, and 3-indoxyl sulfate in blood A method for determining, comprising measuring a content of the compound contained in blood collected from a plurality of patients diagnosed with colorectal cancer and a plurality of healthy individuals.   A method for screening a therapeutic agent for a colorectal cancer patient, the step of administering a candidate substance of a therapeutic agent to the model animal of colorectal cancer, the step of collecting blood before and after administering the candidate substance, Measuring the content of one or more compounds selected from the group consisting of indole-3-acetic acid, hippuric acid, citric acid, and 3-indoxyl sulfate in the blood, and before and after administering the candidate substance, Comparing the content of the compound in the blood.   Determination of a threshold value for the content of one or more compounds selected from the group consisting of indole-3-acetic acid, hippuric acid, citric acid, and 3-indoxyl sulfate in the blood for determining the progression of colorectal cancer Included in blood collected from multiple patients diagnosed with early colorectal cancer, multiple patients diagnosed with non-remotely advanced colorectal cancer, and multiple patients diagnosed with distantly transferred colorectal cancer Measuring the content of the compound.