WO2015182580A1 - Colorectal cancer metastasis detection method - Google Patents

Colorectal cancer metastasis detection method Download PDF

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WO2015182580A1
WO2015182580A1 PCT/JP2015/065029 JP2015065029W WO2015182580A1 WO 2015182580 A1 WO2015182580 A1 WO 2015182580A1 JP 2015065029 W JP2015065029 W JP 2015065029W WO 2015182580 A1 WO2015182580 A1 WO 2015182580A1
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colorectal cancer
metastasis
marker
ceacam1
subject
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PCT/JP2015/065029
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French (fr)
Japanese (ja)
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毅 朝長
秀明 久米
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国立研究開発法人 医薬基盤・健康・栄養研究所
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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
    • G01N27/622—Ion mobility spectrometry
    • G01N27/623—Ion mobility spectrometry combined with mass spectrometry
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

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  • the present invention relates to a method for detecting metastasis of colorectal cancer. More specifically, the present invention relates to a method for detecting colorectal cancer with metastasis by measuring the amount of a specific marker in a sample, and a kit used for the method.
  • Patent Document 1 RNA whose expression level is changed in the blood of patients with colorectal cancer is listed, and RNA derived from CD177 is disclosed as one of them.
  • Patent Document 2 reports that expression of CEACAM1 is reduced in the right and left tumors of the large intestine as a result of analyzing the expressed gene based on information such as the position of the sample derived from the large intestine tissue.
  • Non-patent document 1 reports the expression pattern of CEACAM1 gene and the progression of colorectal cancer.
  • An object of the present invention is to provide a method for detecting colon cancer metastasis simply and accurately, and a kit used in the method.
  • the present invention relates to the following [1] to [6].
  • [1] In a body fluid sample derived from a subject, the amount of at least one protein selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1 is measured, and the measured value is used as an index.
  • a method for detecting metastasis of colorectal cancer comprising a step of comparing the measured value and the reference value in the step, wherein the case where the measured value is recognized to be larger than the reference value is an indicator of the presence of metastasis of colorectal cancer .
  • Treatment evaluation method [4] A kit for detecting metastasis of colorectal cancer, comprising an antibody against one or more proteins selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1, or a fragment thereof.
  • [5] Use as a marker for colorectal cancer with metastasis of one or more proteins selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1.
  • Metastasis of colorectal cancer comprising detecting one or more proteins selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1 from a body fluid sample derived from a subject. A method of providing information on suspected colorectal cancer metastasis.
  • the presence or absence of colorectal cancer metastasis can be easily and accurately determined by the method or kit of the present invention.
  • the method or kit of the present invention makes it possible to specifically detect metastasis of colorectal cancer.
  • FIG. 1 is a diagram showing colorectal cancer marker candidates.
  • FIG. 2 shows the results of confirming the expression level of each marker in the extracted membrane vesicle fraction for each of the healthy group, the colon cancer patient group without metastasis, and the colon cancer patient group with metastasis.
  • FIG. 3 is a diagram showing the results of confirming the expression levels of markers derived from exosomes for individual specimens of a healthy group, a colon cancer patient group without metastasis, and a colon cancer patient group with metastasis. .
  • the method of the present invention is a method for detecting colorectal cancer metastasis in a subject, and may be affected by colorectal cancer with metastasis by measuring the amount of a specific marker in a body fluid sample. It is characterized by judging sex. Therefore, the present invention is also a method for providing information for detecting metastasis of colorectal cancer. Specifically, in a body fluid sample derived from a subject, a step of measuring the amount of a specific marker described later, and a measured value in the step are compared with a reference value, where the measured value in the subject is a reference value And when the subject is found to be larger than the colon cancer, the subject is judged to have colon cancer metastasized or suffering from metastasized colon cancer.
  • proteins to be measured in the present invention include CEACAM8, CEACAM1, CD177, and C5AR1.
  • CEACAM8 in the present specification refers to human-derived CEACAM8 (Carcinoembryonic antigen-related cell adhesion molecule 8) protein (GenBank accession No. P31997), which has the amino acid sequence shown in SEQ ID NO: 1. Although it is a 349-residue polypeptide, it may be a mutant or a fragment of a wild-type and / or mutant protein.
  • CEACAM1 in the present specification refers to human-derived CEACAM1 (Carcinoembryonic antigen-related cell adhesion molecule 1) protein (GenBank Accession No. P13688), which has the amino acid sequence shown in SEQ ID NO: 2. Although it is a polypeptide of 526 residues, it may be a mutant or a fragment of a wild-type and / or mutant protein.
  • CD177 refers to a human-derived CD177 protein (GenBank Accession No. Q8N6Q3), and the wild type is a 437-residue polypeptide having the amino acid sequence shown in SEQ ID NO: 3. This mutant or a fragment of a wild type and / or mutant type protein may be used.
  • C5AR1 refers to a human-derived C5AR1 protein (GenBank Accession No. P21730), which is a 350-residue polypeptide having the amino acid sequence shown in SEQ ID NO: 4 for the wild type. This mutant or a fragment of a wild type and / or mutant type protein may be used.
  • the present invention shows that there are more proteins in body fluid samples of metastatic colorectal cancer patients than in healthy individuals and colorectal cancer patients without metastasis.
  • the inventors have found the present invention for the first time and have completed the present invention (hereinafter, these four kinds of proteins may be collectively referred to as the marker of the present invention).
  • these four kinds of proteins may be collectively referred to as the marker of the present invention.
  • the marker of the present invention As described above, among CEACAM1 and CD177 among the markers of the present invention, there is a report on the relationship with colorectal cancer, but it can be detected with a sample derived from exosomes and the relationship with the presence or absence of metastasis. Until now, it has not been verified.
  • Patent Document 2 reports that the expression level of CEACAM1 decreases when suffering from colorectal cancer, and the tendency is different from the findings of the present inventors. Further, according to Non-Patent Document 1, it is reported that there is no significant difference in the expression pattern of CEACAM1 in each stage of colorectal cancer patients.
  • the method for identifying the marker of the present invention as a marker protein for colorectal cancer with metastasis will be described in detail in Examples below, but will be briefly described below.
  • a protein that produces a fluctuation signal is isolated and identified by a known proteomic technique. , Selected as a candidate protein.
  • these proteins were verified as follows. Specifically, the colon cancer patient group is divided into two groups according to the presence or absence of metastasis, and the body fluid samples of the healthy group, the colon cancer patient group without metastasis, and the colon cancer patient group with metastasis are obtained. As a result of comparison between groups of the amount of the candidate protein, the marker of the present invention was identified. In addition, the judgment of the presence or absence of metastasis in the colon cancer patient group was performed by image diagnosis.
  • colon cancer has metastasized or colon cancer metastasis means that primary cells of colon cancer migrate and reach other parts of the body to form cancer, or It means that it can be formed.
  • metalastatic colorectal cancer refers to primary colorectal cancer in which primary cells of colorectal cancer form or may have metastatic focus in other parts of the body. That's it. At this time, the degree of metastasis or the state of metastasis is not particularly limited.
  • Cold cancer without metastasis refers to colorectal cancer in which metastasis is not formed.
  • the method of the present invention specifically includes a protein amount measurement step (step A) and a colon cancer metastasis determination step (step B). Below, each process in this invention is demonstrated in detail.
  • Step A refers to a step of measuring the amount of one or more proteins selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1 in a body fluid sample derived from a subject.
  • body fluid sample used in the present specification, for example, blood, urine, saliva, milk, nasal discharge, cerebrospinal fluid and the like are exemplified, but blood is preferred.
  • blood can be whole blood, serum, and plasma.
  • the method for collecting and preparing the body fluid sample is not particularly limited, and can be performed according to a known method.
  • the marker of the present invention is also detected from an exosome or membrane vesicle fraction prepared from a body fluid sample, it can be used as a measurement target after preparing these. It is known that both the membrane vesicle fraction and the exosome can be obtained by aggregating a body fluid sample by ultracentrifugation or the like. In addition, since exosomes are vesicular granules and antigens that are specifically expressed (for example, CD9, CD63, CD147, EPCAM, etc.) are known, immunoprecipitation using an antibody that can specifically bind to these antigens. It is known that it can be captured (for example, International Publication No. 2013/099925).
  • the measurement of the marker of the present invention can be performed according to a known method. Specifically, for example, a mass spectrometric method and a method using an antibody capable of specifically recognizing a marker to be measured are preferable examples.
  • mass spectrometry examples include mass spectrometry using a MALDI (matrix-assisted laser desorption / ionization) mass spectrometer, an ESI (electrospray ionization) mass spectrometer, and the like. It may be a method to do. Of these, the LC-MS method using an ESI mass spectrometer is preferred.
  • MALDI matrix-assisted laser desorption / ionization
  • ESI electrospray ionization
  • Examples of methods using antibodies include Western blotting, radioimmunoassay (RIA), enzyme immunoassay (ELISA, EIA), luminescence immunoassay, and fluorescence immunoassay (exoscreen). Law).
  • the antibody used in such a method has the characteristic that the marker of the present invention can be detected or captured with good sensitivity and specificity.
  • Such an antibody can be prepared by a method well known to those skilled in the art.
  • a monoclonal antibody or a fragment thereof can be preferably used.
  • the monoclonal antibody or a fragment thereof can be used after labeling or solid-phase according to a generally known method.
  • the “monoclonal antibody fragment” means a fragment that is a part of the monoclonal antibody and has a specific binding property to the target protein in the same manner as the monoclonal antibody.
  • Specific examples include Fab, F (ab ′) 2 , Fab ′, single chain antibody (scFv), disulfide stabilized antibody (dsFv), dimerized V region fragment (Diabody), and CDR-containing peptides. be able to.
  • the marker amount can be measured.
  • the following process B is performed using the obtained measured value.
  • Step B is a step of determining (or evaluating) colon cancer metastasis in vitro based on the amount of the marker obtained in Step A.
  • the determination method when the amount of the marker of the subject is statistically significant compared to the reference value, the colorectal cancer has metastasized or is suffering from colorectal cancer with metastasis.
  • the method of determining (or evaluating) is mentioned.
  • “determining” refers to evaluating metastasis of colorectal cancer or morbidity of colorectal cancer with metastasis based on the measurement result obtained by the detection method of the present invention. It is intended not to include judgment by a physician.
  • the “reference value” can be, for example, a measurement value of the marker amount in a healthy person.
  • the amount of marker contained in a sample (positive sample) confirmed to be derived from a plurality of colorectal cancer patients with metastases and a plurality of healthy individuals (or from colorectal cancer patients without metastases) is measured and compared, and based on the result, a value that can distinguish the positive sample and the negative sample with the highest probability is obtained.
  • Healthy person means an individual who is not affected by at least colon cancer (with or without metastasis), preferably a healthy individual. Further, the healthy person needs to be the same species as the subject. For example, when the subject to be detected is a human (subject), the healthy person must also be a human (hereinafter referred to as “healthy person” in this specification).
  • the physical condition of a healthy person is preferably the same as or close to that of the subject. For example, in the case of a human, the physical condition corresponds to race, sex, age, height, weight, and the like.
  • the body fluid sample derived from a healthy person is preferably the same type of sample as the body fluid sample derived from the subject.
  • the body fluid sample derived from the healthy person is also preferably blood.
  • the amount of the marker in the body fluid sample of the healthy person is preferably measured by the same method as the method for measuring the amount of the marker in the body fluid sample of the subject described in the above step.
  • the amount of the marker in the bodily fluid sample of the healthy person can be newly measured every time the amount of the marker in the bodily fluid sample of the subject is measured, but the amount of the marker measured in advance can also be used.
  • the amount of a marker in various physical conditions of a healthy person is measured in advance and the value is input to a computer and stored in a database, the physical condition of the subject can be input to the computer, This is convenient because the amount of marker of a healthy person who has the optimal physical condition for comparison with the subject can be used immediately.
  • “Statistically significant” includes, for example, a case where the risk value (significance level) of the obtained value is less than 5%, 1%, or 0.1%. Therefore, “statistically significant” for the measured value means that there is a significant difference between the two when the quantitative difference between the markers obtained from the subject and the healthy subject is statistically processed, And it means that the amount of the marker of the subject is relatively large compared to that of the healthy subject. For example, the amount of the marker in the body fluid sample corresponds to a case where the subject is twice or more, preferably 3 times or more, more preferably 4 times or more, most preferably 5 times or more that of a healthy person. If the quantitative difference is 3 times or more, the reliability is high, and it can be said that there is a significant statistical amount.
  • the test method for statistical processing is not particularly limited as long as a known test method capable of determining the presence or absence of significance is appropriately used. For example, Student's t test or multiple comparison test can be used.
  • the subject is evaluated as having colon cancer metastasized or suffering from colon cancer with metastasis.
  • the stage of colorectal cancer that is the subject of the present invention is not particularly limited, and ranges from early stage cancer to end stage cancer.
  • the colorectal cancer metastasis detection method of the present invention includes a mode in which a marker in a body fluid sample is measured using a mass spectrometer and a mode in which an antibody is used to measure immunologically. According to the method of the present invention, it is possible not only to determine whether or not a subject suffers from colorectal cancer with metastasis, or whether or not colorectal cancer metastasis has occurred. Enables identification of healthy individuals or colorectal cancer patients with metastasis from those with no metastasis.
  • the marker amount of the healthy subject is set to the marker amount before treatment of the subject, and the marker amount after treatment is set to If the amount of marker after treatment is shown to be reduced by comparing with the amount of marker in the subject, it is judged that the treatment is likely to be effective in the treatment of metastatic colorectal cancer be able to. Therefore, the present invention also measures the marker amount of the present invention before and after receiving cancer treatment, and determines that the treatment is effective when the value after treatment is smaller than that before treatment. It is possible to provide an evaluation method characterized by The evaluation of the treatment of “colorectal cancer with metastasis” herein includes evaluating the treatment of cancer at the metastasis destination in addition to the treatment of colorectal cancer that is the primary lesion.
  • a kit for detecting colon cancer metastasis (colorectal cancer with metastasis) (hereinafter, also referred to as “colorectal cancer metastasis detection kit”) is provided.
  • Colorectal cancer metastasis detection kit is used to evaluate the presence or absence of metastatic colorectal cancer, the degree of morbidity or the improvement or degree of improvement, or the presence or absence of colorectal cancer metastasis, It is used directly or indirectly to screen candidate substances useful for the prevention, amelioration or treatment of colorectal cancer metastasis.
  • the kit of this embodiment is a marker of the present invention whose expression varies in a body fluid sample, preferably blood, serum, plasma, membrane vesicle fraction, or exosome, in relation to the onset of colorectal cancer.
  • a body fluid sample preferably blood, serum, plasma, membrane vesicle fraction, or exosome.
  • an antibody or a fragment thereof or a chemically modified derivative thereof is included.
  • These antibodies may be bound to a solid phase carrier.
  • Others include, for example, labeled secondary antibodies, as well as substrates necessary for label detection, carriers, washing buffers, sample diluents, enzyme substrates, reaction stop solutions, purified proteins as standards, instructions for use, etc. You may go out.
  • the antibody or fragment thereof that can recognize the marker of the present invention is as described above.
  • kits can be used as long as they use an antibody (for example, Western blot method, ELISA method, exoscreen method, etc.) when measuring the protein amount of the marker of the present invention in a body fluid sample.
  • an antibody for example, Western blot method, ELISA method, exoscreen method, etc.
  • the body fluid sample is an exosome
  • a protein not derived from the exosome may be simultaneously detected by the antibody.
  • kit of the present invention for example, when the amount of protein of the marker of the present invention present in the blood sample of a healthy subject and a subject is measured, and there is a significant difference between the expression levels of both, A determination and / or diagnosis of cancer metastasis can be made.
  • Example 1 Selection of biomarker candidate membrane protein using tissue
  • a membrane fraction prepared from a benign tumor of the large intestine and a colon cancer patient (including “no metastasis” and “with metastasis”) according to a conventional method is subjected to the isobaric tags for relative and absolute quantification method (hereinafter referred to as “iTRAQ (registered trademark)”).
  • iTRAQ relative and absolute quantification method
  • Proteome analysis by the “Applied Biosystems” method). Proteins were identified from the fluctuation signals seen here, and proteome analysis was performed by the Selected Reaction Monitoring method (hereinafter referred to as “SRM method”) with higher quantitative accuracy for further verification (results not shown).
  • SRM method Selected Reaction Monitoring method
  • SI peptide a stable isotope-labeled peptide identified by the proteome analysis by the iTRAQ method is mixed and used as an internal standard. The quantitative accuracy was improved.
  • Example 2 (Confirmation of candidate protein expression in membrane vesicle fraction in mixed blood) Next, a total of 3 specimens: a sample mixed with the serum of 4 healthy subjects, a sample mixed with the serum of 4 colorectal cancer patients without metastasis, and a sample mixed with the serum of 4 colorectal cancer patients with metastasis A membrane vesicle fraction was prepared for the specimen, and the expression level of the candidate protein selected in Example 1 was examined. The presence or absence of colorectal cancer metastasis was confirmed by pathological findings and laparoscopic findings.
  • the membrane vesicle fraction was prepared by the following method. Specifically, 550 ⁇ L of PBS was added to 100 ⁇ L of serum, centrifuged at 2,000 ⁇ g and 4 ° C. for 30 minutes, and then filtered using a 0.22 ⁇ m spin column. Then, after ultracentrifugation at 100,000 ⁇ g and 4 ° C. for 90 minutes, the precipitate was washed with PBS, and ultracentrifugated again at 100,000 ⁇ g and 4 ° C for 90 minutes. Further, the precipitate was washed with PBS containing 50 mM DTT, and the precipitate after ultracentrifugation at 100,000 ⁇ g and 4 ° C. for 90 minutes was obtained as a membrane vesicle fraction.
  • the obtained membrane vesicle fraction was solubilized with Phase Transfer Surfactant solution (hereinafter referred to as “PTS solution”) containing surfactants (deoxycholic acid and lauroyl sarcosine acid), digested with trypsin, and then added with ethyl acetate.
  • PTS solution Phase Transfer Surfactant solution
  • surfactants deoxycholic acid and lauroyl sarcosine acid
  • the TSQ ⁇ vantage mass spectrometer manufactured by Thermo Fisher Scientific Co., Ltd.
  • SRM method proteome analysis is performed. It was.
  • Example 3 (Confirmation of candidate protein expression in membrane vesicle fraction in blood in individual specimen)
  • the candidate proteins extracted in Example 2 were verified and further refined using individual serum samples.
  • the test used sera from 20 healthy individuals, 18 patients with stage 1 or stage 2 colorectal cancer who had no metastasis, and 19 patients with stage 4 colorectal cancer with metastasis.
  • a membrane vesicle fraction was prepared in the same manner as in Example 2, and the membrane fraction corresponding to 40 ⁇ L of serum was solubilized with a PTS solution containing a surfactant (deoxycholic acid and lauroyl sarcosine acid). After digestion with trypsin, ethyl acetate was added to remove the surfactant.
  • the SI peptide was added here, and the proteome analysis by SRM method was performed.
  • the SI peptide used here was a stable isotope-labeled peptide (purchased from Greiner) of a peptide sequence (trypsin digested fragment) specific to each protein based on amino acid sequence information.
  • candidates that did not show the same change as the result of the mixed sample in individual samples or candidates detected only in some samples were excluded. The results are shown in FIG. The significant difference test in the figure was performed according to the Mann-Whitney U test test.
  • CEACAM1, CEACAM8, CD177, and C5AR1 were significantly increased in colorectal cancer patients with metastasis compared to healthy subjects. This shows that it is possible to detect colon cancer with metastasis by measuring CEACAM1, CEACAM8, CD177, and C5AR1 present in the membrane vesicle fraction in blood. Furthermore, CEACAM1, CEACAM8, CD177, and C5AR1 show a tendency to increase in patients with metastatic colorectal cancer compared to those with no metastasis, and among them CEACAM1, CEACAM8, and CD177 There was a significant increase.
  • Example 4 (Confirmation of candidate protein expression in exosomes) CD63 was employed as an exosome marker protein, and exosomes were prepared from serum by immunoprecipitation using an anti-CD63 antibody and examined for the expression of candidate proteins. Sera from two healthy subjects, two Stage 1 or Stage 2 colorectal cancer patients with no metastasis, and two Stage 4 colorectal cancer patients with metastasis were provided for the study. As the anti-CD63 antibody, Anti CD63 for Exosome Isolation (Cosmo Bio) was used.
  • a serum membrane vesicle fraction using an antibody was prepared as follows. After adding 100 ⁇ L of PBS to 100 ⁇ L of serum, a resin having an anti-CD63 antibody covalently bonded to the magnetic beads was added and allowed to react overnight at 4 ° C. Exosomes were prepared by removing non-specific binders by washing several times with PBS.
  • the obtained exosome was solubilized with a PTS solution containing surfactants (deoxycholic acid and lauroyl sarcosine acid), digested with trypsin, and then ethyl acetate was added to remove the surfactant.
  • the CEACAM1, CEACAM8, CD177, and C5AR1 SI peptides used in Example 3 were added as internal standard peptides to the trypsin-digested sample, and then measured by the SRM method using a TSQ vantage mass spectrometer and subjected to proteomic analysis. At this time, the membrane vesicle fraction prepared by ultracentrifugation was also measured at the same time and used as a comparative control. The results are shown in FIG.
  • CD63-IP-SRM is a sample obtained by treating exosomes immunoprecipitated with an anti-CD63 antibody by the SRM method, and Centrifugation-SRM indicates each sample treated by the SRM method by ultracentrifugation.
  • CEACAM1, CEACAM8, CD177, and C5AR1 were also expressed in exosomes, and their amounts showed the same tendency as those of membrane vesicle fractions prepared by ultracentrifugation. .
  • the method of the present invention allows the sample provider to determine whether or not there is a high possibility of colorectal cancer with metastasis or whether or not colorectal cancer has metastasized. This is useful because the sample provider can take measures to prevent the progression of colorectal cancer.
  • Sequence number 1 of a sequence table is an amino acid sequence of CEACAM8 protein.
  • Sequence number 2 of a sequence table is an amino acid sequence of CEACAM1 protein.
  • Sequence number 3 of a sequence table is an amino acid sequence of CD177 protein.
  • Sequence number 4 of a sequence table is an amino acid sequence of C5AR1 protein.

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Abstract

A colorectal cancer metastasis detection method characterized by measuring the amount of at least one type of protein selected from a group comprising CEACAM8, CEACAM1, CD177, and C5AR1 and using this measurement value as an indicator, in a body fluid sample obtained from a subject. As a result of this method, a determination can be made as to whether or not there is a high likelihood of a sample provider having metastatic colorectal cancer or whether or not colorectal cancer has metastasized. This method is useful because, as a result, the sample provider can take measures to prevent progress of the colorectal cancer.

Description

大腸がんの転移検出方法Colorectal cancer metastasis detection method
　本発明は、大腸がんの転移検出方法に関する。さらに詳しくは、サンプル中の特定のマーカーの量を測定することで、転移のある大腸がんを検出する方法、及び該方法に用いるキットに関する。 The present invention relates to a method for detecting metastasis of colorectal cancer. More specifically, the present invention relates to a method for detecting colorectal cancer with metastasis by measuring the amount of a specific marker in a sample, and a kit used for the method.
　大腸がん患者の３０～４０％においては再発や転移が見られる。大腸がんの転移としては、肝臓への転移によるものが最も多い。大腸がんのスクリーニングには便潜血検査が主に使われている。この検査が陽性であった場合、その多くは内視鏡検査を行い、組織を採取して確定診断を行う。一方で、そのような組織診断は作業が煩雑であることから簡易な検査方法として、あるいは、切除術を受けた大腸がん患者の術後管理においては組織採取ができないことから、生体試料を用いたがんマーカー測定を行うことが主流となっている。 Relapse or metastasis is seen in 30-40% of colorectal cancer patients. The most common colorectal cancer metastasis is due to metastasis to the liver. The fecal occult blood test is mainly used for screening for colorectal cancer. If this test is positive, many perform endoscopy, collect tissue, and make a definitive diagnosis. On the other hand, such a tissue diagnosis is a complicated examination because it is complicated, or a tissue sample cannot be collected in the postoperative management of colorectal cancer patients who have undergone resection. It is mainstream to measure cancer markers.
　特許文献１では、大腸がんの患者の血液で発現量が変化したＲＮＡが挙げられており、そのうちの一つとしてＣＤ１７７に由来のＲＮＡが開示されている。特許文献２では、大腸組織由来のサンプルの位置などの情報を基に発現遺伝子を解析した結果、ＣＥＡＣＡＭ１が大腸の右側腫瘍及び左側腫瘍でその発現が低下していることが報告されている。 In Patent Document 1, RNA whose expression level is changed in the blood of patients with colorectal cancer is listed, and RNA derived from CD177 is disclosed as one of them. Patent Document 2 reports that expression of CEACAM1 is reduced in the right and left tumors of the large intestine as a result of analyzing the expressed gene based on information such as the position of the sample derived from the large intestine tissue.
　また、非特許文献１には、ＣＥＡＣＡＭ１の遺伝子の発現パターンと大腸がんの進行について報告されている。 Non-patent document 1 reports the expression pattern of CEACAM1 gene and the progression of colorectal cancer.
ＷＯ２０１２／１５６５２９号公報WO2012 / 156529 特開２００７－２２４００９号公報JP 2007-224209 A
　大腸がんの転移症例に対して行われる化学療法や分子標的治療法は、その抑制効果、或いは延命効果が必ずしも十分とは言えないのが現状である。また、手術後の患者には転移の有無に関わらず抗がん剤投与をするなど、患者に経済的・精神的な負担を強いている問題がある。一方、大腸がんの転移に対して特異性を有するマーカーは臨床において使用されていない。そこで、転移のある大腸がんを事前に予測できるようなマーカーが求められている。 The current situation is that chemotherapy and molecular target therapy for metastatic cases of colorectal cancer are not necessarily sufficient for their inhibitory effect or life-prolonging effect. In addition, there are problems that impose an economic and mental burden on patients, such as administration of anticancer drugs to patients after surgery regardless of the presence or absence of metastasis. On the other hand, a marker having specificity for colorectal cancer metastasis has not been used clinically. Therefore, there is a need for a marker that can predict colorectal cancer with metastasis in advance.
　本発明の課題は、大腸がんの転移を簡便にかつ精度よく検出するための方法、及び該方法に用いるキットを提供することにある。 An object of the present invention is to provide a method for detecting colon cancer metastasis simply and accurately, and a kit used in the method.
　本発明は、以下の〔１〕～〔６〕に関する。
〔１〕　被験者に由来する体液試料において、ＣＥＡＣＡＭ８、ＣＥＡＣＡＭ１、ＣＤ１７７、及びＣ５ＡＲ１からなる群から選択される少なくとも１種のタンパク質の量を測定し、その測定値を指標にすることを特徴とする、大腸がんの転移検出方法。
〔２〕　ＣＥＡＣＡＭ８、ＣＥＡＣＡＭ１、ＣＤ１７７、及びＣ５ＡＲ１からなる群から選択される少なくとも１種のタンパク質の量を測定する工程と、
前記工程における測定値と基準値とを対比する工程
とを含み、前記測定値が基準値よりも大きいと認められる場合が大腸がんの転移の存在の指標となる、大腸がんの転移検出方法。
〔３〕　治療後の被験者に由来する体液試料中のＣＥＡＣＡＭ８、ＣＥＡＣＡＭ１、ＣＤ１７７、及びＣ５ＡＲ１からなる群から選択される少なくとも１種のタンパク質の量を測定する工程と、
前記工程における測定値と、治療前の測定値とを対比する工程と、
を含み、治療後における測定値が治療前における測定値より小さいと認められる場合に、前記治療が転移のある大腸がんの治療効果を有すると評価する工程を含む、転移のある大腸がんの治療の評価方法。
〔４〕　ＣＥＡＣＡＭ８、ＣＥＡＣＡＭ１、ＣＤ１７７、及びＣ５ＡＲ１からなる群から選択される１つ又は２つ以上のタンパク質に対する抗体又はその断片を含有してなる、大腸がんの転移を検出するためのキット。
〔５〕　ＣＥＡＣＡＭ８、ＣＥＡＣＡＭ１、ＣＤ１７７、及びＣ５ＡＲ１からなる群から選択される１つ又は２つ以上のタンパク質の転移のある大腸がんのマーカーとしての使用。
〔６〕　ＣＥＡＣＡＭ８、ＣＥＡＣＡＭ１、ＣＤ１７７、及びＣ５ＡＲ１からなる群から選択される１つ又は２つ以上のタンパク質を、被験者に由来する体液試料中から検出することを特徴とする、大腸がんの転移又は大腸がんの転移の疑いの情報を提供する方法。 The present invention relates to the following [1] to [6].
[1] In a body fluid sample derived from a subject, the amount of at least one protein selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1 is measured, and the measured value is used as an index. A method for detecting metastasis of colorectal cancer.
[2] measuring the amount of at least one protein selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1,
A method for detecting metastasis of colorectal cancer, comprising a step of comparing the measured value and the reference value in the step, wherein the case where the measured value is recognized to be larger than the reference value is an indicator of the presence of metastasis of colorectal cancer .
[3] measuring the amount of at least one protein selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1 in a body fluid sample derived from a subject after treatment;
Comparing the measured value in the step with the measured value before treatment;
Including a step of evaluating that the treatment has a therapeutic effect on metastatic colorectal cancer when the measured value after treatment is recognized to be smaller than the measured value before treatment. Treatment evaluation method.
[4] A kit for detecting metastasis of colorectal cancer, comprising an antibody against one or more proteins selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1, or a fragment thereof.
[5] Use as a marker for colorectal cancer with metastasis of one or more proteins selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1.
[6] Metastasis of colorectal cancer, comprising detecting one or more proteins selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1 from a body fluid sample derived from a subject. A method of providing information on suspected colorectal cancer metastasis.
　本発明の方法もしくはキットにより、大腸がんの転移の有無を簡便にかつ精度よく判断することができる。また、本発明の方法もしくはキットにより、大腸がんの転移を特異的に検出することが可能になる。 The presence or absence of colorectal cancer metastasis can be easily and accurately determined by the method or kit of the present invention. In addition, the method or kit of the present invention makes it possible to specifically detect metastasis of colorectal cancer.
図１は、大腸がんのマーカー候補を示す図である。FIG. 1 is a diagram showing colorectal cancer marker candidates. 図２は、健常者群、転移のない大腸がん患者群、及び転移のある大腸がん患者群それぞれの個体について、抽出された膜小胞画分における各マーカーの発現量を確認した結果を示す図である。FIG. 2 shows the results of confirming the expression level of each marker in the extracted membrane vesicle fraction for each of the healthy group, the colon cancer patient group without metastasis, and the colon cancer patient group with metastasis. FIG. 図３は、健常者群、転移のない大腸がん患者群、及び転移のある大腸がん患者群それぞれの個体検体について、エクソソームに由来する各マーカーの発現量を確認した結果を示す図である。FIG. 3 is a diagram showing the results of confirming the expression levels of markers derived from exosomes for individual specimens of a healthy group, a colon cancer patient group without metastasis, and a colon cancer patient group with metastasis. .
　本発明の方法は、被験者における大腸がんの転移を検出するための方法であって、体液試料中の特定のマーカーの量を測定することで、転移のある大腸がんに罹患している可能性を判断することを特徴とする。従って、本発明は、大腸がんの転移検出用の情報を提供する方法でもある。具体的には、被験者に由来する体液試料において、後述の特定のマーカーの量を測定する工程と、前記工程における測定値を基準値と対比して、ここで、前記被験者における測定値が基準値よりも大きいと認められる場合に、被験者が大腸がんが転移している、もしくは転移のある大腸がんに罹患していると判断する工程を含む。 The method of the present invention is a method for detecting colorectal cancer metastasis in a subject, and may be affected by colorectal cancer with metastasis by measuring the amount of a specific marker in a body fluid sample. It is characterized by judging sex. Therefore, the present invention is also a method for providing information for detecting metastasis of colorectal cancer. Specifically, in a body fluid sample derived from a subject, a step of measuring the amount of a specific marker described later, and a measured value in the step are compared with a reference value, where the measured value in the subject is a reference value And when the subject is found to be larger than the colon cancer, the subject is judged to have colon cancer metastasized or suffering from metastasized colon cancer.
　本発明における測定対象のタンパク質としては、ＣＥＡＣＡＭ８、ＣＥＡＣＡＭ１、ＣＤ１７７、及びＣ５ＡＲ１が挙げられる。 Examples of proteins to be measured in the present invention include CEACAM8, CEACAM1, CD177, and C5AR1.
　本明細書におけるＣＥＡＣＡＭ８とは、ヒト由来のＣＥＡＣＡＭ８（Ｃａｒｃｉｎｏｅｍｂｒｙｏｎｉｃ　ａｎｔｉｇｅｎ－ｒｅｌａｔｅｄ　ｃｅｌｌ　ａｄｈｅｓｉｏｎ　ｍｏｌｅｃｕｌｅ　８）タンパク質（ＧｅｎＢａｎｋ　アクセッションＮｏ．Ｐ３１９９７）のことであり、野生型については配列番号１に示されるアミノ酸配列を有する３４９残基のポリペプチドであるが、この変異体又は、野生型及び／又は変異型のタンパク質の断片であってもよい。 CEACAM8 in the present specification refers to human-derived CEACAM8 (Carcinoembryonic antigen-related cell adhesion molecule 8) protein (GenBank accession No. P31997), which has the amino acid sequence shown in SEQ ID NO: 1. Although it is a 349-residue polypeptide, it may be a mutant or a fragment of a wild-type and / or mutant protein.
　本明細書におけるＣＥＡＣＡＭ１とは、ヒト由来のＣＥＡＣＡＭ１（Ｃａｒｃｉｎｏｅｍｂｒｙｏｎｉｃ　ａｎｔｉｇｅｎ－ｒｅｌａｔｅｄ　ｃｅｌｌ　ａｄｈｅｓｉｏｎ　ｍｏｌｅｃｕｌｅ　１）タンパク質（ＧｅｎＢａｎｋ　アクセッションＮｏ．Ｐ１３６８８）のことであり、野生型については配列番号２に示されるアミノ酸配列を有する５２６残基のポリペプチドであるが、この変異体又は、野生型及び／又は変異型のタンパク質の断片であってもよい。 CEACAM1 in the present specification refers to human-derived CEACAM1 (Carcinoembryonic antigen-related cell adhesion molecule 1) protein (GenBank Accession No. P13688), which has the amino acid sequence shown in SEQ ID NO: 2. Although it is a polypeptide of 526 residues, it may be a mutant or a fragment of a wild-type and / or mutant protein.
　本明細書におけるＣＤ１７７とは、ヒト由来のＣＤ１７７タンパク質（ＧｅｎＢａｎｋ　アクセッションＮｏ．Ｑ８Ｎ６Ｑ３）のことであり、野生型については配列番号３に示されるアミノ酸配列を有する４３７残基のポリペプチドであるが、この変異体又は、野生型及び／又は変異型のタンパク質の断片であってもよい。 In this specification, CD177 refers to a human-derived CD177 protein (GenBank Accession No. Q8N6Q3), and the wild type is a 437-residue polypeptide having the amino acid sequence shown in SEQ ID NO: 3. This mutant or a fragment of a wild type and / or mutant type protein may be used.
　本明細書におけるＣ５ＡＲ１とは、ヒト由来のＣ５ＡＲ１タンパク質（ＧｅｎＢａｎｋ　アクセッションＮｏ．Ｐ２１７３０）のことであり、野生型については配列番号４に示されるアミノ酸配列を有する３５０残基のポリペプチドであるが、この変異体又は、野生型及び／又は変異型のタンパク質の断片であってもよい。 In this specification, C5AR1 refers to a human-derived C5AR1 protein (GenBank Accession No. P21730), which is a 350-residue polypeptide having the amino acid sequence shown in SEQ ID NO: 4 for the wild type. This mutant or a fragment of a wild type and / or mutant type protein may be used.
　これらのタンパク質はいずれのタンパク質もその存在自体は公知であったが、転移のある大腸がん患者の体液試料中において、健常者や転移のない大腸がん患者よりも多く存在することを本発明者らが初めて見出して、本発明を完成するに至った（以降、これら４種類のタンパク質をまとめて、本発明のマーカーと記載することもある）。なお、前述のとおり本発明のマーカーのなかでも、ＣＥＡＣＡＭ１やＣＤ１７７については、大腸がんとの関係についての報告があるが、エクソソーム由来の試料で検出可能であることや、転移の有無との関連までは検証されていない。また、特許文献２においては、大腸がんに罹患している場合にはＣＥＡＣＡＭ１の発現量が低下することが報告されており、本発明者らの発見とはその傾向は異なっている。さらに、非特許文献１によれば、大腸がん患者の各ステージにおけるＣＥＡＣＡＭ１の発現パターンには有意差がないと報告されている。 The presence of any of these proteins is known per se, but the present invention shows that there are more proteins in body fluid samples of metastatic colorectal cancer patients than in healthy individuals and colorectal cancer patients without metastasis. The inventors have found the present invention for the first time and have completed the present invention (hereinafter, these four kinds of proteins may be collectively referred to as the marker of the present invention). As described above, among CEACAM1 and CD177 among the markers of the present invention, there is a report on the relationship with colorectal cancer, but it can be detected with a sample derived from exosomes and the relationship with the presence or absence of metastasis. Until now, it has not been verified. Patent Document 2 reports that the expression level of CEACAM1 decreases when suffering from colorectal cancer, and the tendency is different from the findings of the present inventors. Further, according to Non-Patent Document 1, it is reported that there is no significant difference in the expression pattern of CEACAM1 in each stage of colorectal cancer patients.
　本発明のマーカーを、転移のある大腸がんのマーカータンパク質として同定した方法は、後述の実施例にて詳述するが、以下に簡単に説明する。先ず、大腸の良性腫瘍と大腸がんの患者群（転移の有無については区別しない）の腫瘍組織から調製した膜画分を用いて、公知のプロテオミクス手法により変動シグナルを生じるタンパク質を分離・同定し、候補タンパク質として選択した。次に、これらのタンパク質については以下の検証を行った。具体的には、大腸がんの患者群を転移の有無により２群に分けて、健常者群、転移のない大腸がん患者群、転移のある大腸がん患者群の各群の体液試料を用いて、前記候補タンパク質の量の群間比較を行った結果、本発明のマーカーを同定した。なお、大腸がんの患者群における転移の有無の判断は、画像診断により行った。 The method for identifying the marker of the present invention as a marker protein for colorectal cancer with metastasis will be described in detail in Examples below, but will be briefly described below. First, using a membrane fraction prepared from a tumor tissue of a benign tumor of the large intestine and a colon cancer patient group (with or without metastasis), a protein that produces a fluctuation signal is isolated and identified by a known proteomic technique. , Selected as a candidate protein. Next, these proteins were verified as follows. Specifically, the colon cancer patient group is divided into two groups according to the presence or absence of metastasis, and the body fluid samples of the healthy group, the colon cancer patient group without metastasis, and the colon cancer patient group with metastasis are obtained. As a result of comparison between groups of the amount of the candidate protein, the marker of the present invention was identified. In addition, the judgment of the presence or absence of metastasis in the colon cancer patient group was performed by image diagnosis.
　本明細書において、「大腸がんが転移している又は大腸がんの転移」とは、大腸がんの原発細胞が移動し、身体の他の部分に到達してがんを形成する、あるいは形成し得る状態にあることを意味する。また、本明細書において「転移のある大腸がん」とは、大腸がんの原発細胞が身体の他の部分において転移巣を形成している、あるいはその可能性がある原発性の大腸がんのことである。このとき、転移の程度又は転移巣の状態は特に限定されない。また「転移のない大腸がん」とは、転移巣の形成が見られない大腸がんをさす。 In the present specification, “colon cancer has metastasized or colon cancer metastasis” means that primary cells of colon cancer migrate and reach other parts of the body to form cancer, or It means that it can be formed. Further, in the present specification, “metastatic colorectal cancer” refers to primary colorectal cancer in which primary cells of colorectal cancer form or may have metastatic focus in other parts of the body. That's it. At this time, the degree of metastasis or the state of metastasis is not particularly limited. “Colon cancer without metastasis” refers to colorectal cancer in which metastasis is not formed.
　本発明の方法には、具体的には、タンパク質量の測定工程（工程Ａ）及び大腸がんの転移決定工程（工程Ｂ）が含まれる。以下に、本発明における各工程について詳細に説明する。 The method of the present invention specifically includes a protein amount measurement step (step A) and a colon cancer metastasis determination step (step B). Below, each process in this invention is demonstrated in detail.
　工程Ａとは、被験者に由来する体液試料において、ＣＥＡＣＡＭ８、ＣＥＡＣＡＭ１、ＣＤ１７７、及びＣ５ＡＲ１からなる群から選択される１つ又は２つ以上のタンパク質の量を測定する工程をさす。 Step A refers to a step of measuring the amount of one or more proteins selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1 in a body fluid sample derived from a subject.
　本明細書で用いられる「体液試料」としては、例えば、血液、尿、唾液、乳汁、鼻汁、脳脊髄液などが例示されるが、好ましくは、血液である。本明細書中「血液」としては、全血、血清、及び血漿を用いることができる。なお、体液試料の採取方法や調製方法は特に限定はなく、公知の方法に従って行うことができる。 As the “body fluid sample” used in the present specification, for example, blood, urine, saliva, milk, nasal discharge, cerebrospinal fluid and the like are exemplified, but blood is preferred. In the present specification, “blood” can be whole blood, serum, and plasma. The method for collecting and preparing the body fluid sample is not particularly limited, and can be performed according to a known method.
　本発明のマーカーは、体液試料から調製されるエクソソームあるいは膜小胞画分からも検出されることから、これらを調製してから測定対象として用いることができる。膜小胞画分及びエクソソームのいずれも体液試料を超遠心法などにより凝集することで得られることが知られている。また、エクソソームは小胞顆粒であり、特異的に発現している抗原（例えば、ＣＤ９、ＣＤ６３、ＣＤ１４７、ＥＰＣＡＭなど）が知られているので、これらに特異的に結合できる抗体を用いて免疫沈降させることで捕捉できることが知られている（例えば、国際公報２０１３／０９９９２５号）。 Since the marker of the present invention is also detected from an exosome or membrane vesicle fraction prepared from a body fluid sample, it can be used as a measurement target after preparing these. It is known that both the membrane vesicle fraction and the exosome can be obtained by aggregating a body fluid sample by ultracentrifugation or the like. In addition, since exosomes are vesicular granules and antigens that are specifically expressed (for example, CD9, CD63, CD147, EPCAM, etc.) are known, immunoprecipitation using an antibody that can specifically bind to these antigens. It is known that it can be captured (for example, International Publication No. 2013/099925).
　本発明のマーカーの測定は、公知の方法に従って行うことができる。具体的には、例えば、質量分析方法や測定対象のマーカーを特異的に認識できる抗体を用いる方法が好適例として挙げられる。 The measurement of the marker of the present invention can be performed according to a known method. Specifically, for example, a mass spectrometric method and a method using an antibody capable of specifically recognizing a marker to be measured are preferable examples.
　質量分析法としては、MALDI（マトリックス支援レーザ脱離イオン化法）型質量分析装置、ESI（エレクトロスプレイイオン化法）型質量分析装置等を用いる質量分析方法が挙げられ、タンパク質の一部のペプチドから定量する方法であってもよい。なかでも、ESI型質量分析装置を用いるLC-MS法が好ましい。 Examples of the mass spectrometry include mass spectrometry using a MALDI (matrix-assisted laser desorption / ionization) mass spectrometer, an ESI (electrospray ionization) mass spectrometer, and the like. It may be a method to do. Of these, the LC-MS method using an ESI mass spectrometer is preferred.
　抗体を用いる方法（免疫学的測定法）としては、例えば、ウェスタンブロット法、放射免疫測定法（RIA）、酵素免疫測定法（ELISA、EIA）、発光免疫測定法、蛍光免疫測定法（エクソスクリーン法等）が挙げられる。かかる方法において用いる抗体は、本発明のマーカーを良好な感度及び特異性で検出又は捕捉することができるという特徴を有する。このような抗体は、当業者に周知の方法により作製することができ、例えば、モノクローナル抗体又はその断片を好適に用いることができる。また、前記モノクローナル抗体又はその断片は、通常公知の方法に従って、標識化や固相化して用いることができる。なお、本明細書において、「モノクローナル抗体の断片」としては、前記のモノクローナル抗体の一部であって、当該モノクローナル抗体と同様に目的のタンパク質に対して特異的な結合性を有する断片を意味する。具体的には、Ｆａｂ、Ｆ（ａｂ’）２、Ｆａｂ’、一本鎖抗体（ｓｃＦｖ）、ジスルフィド安定化抗体（ｄｓＦｖ）、２量化体Ｖ領域断片（Ｄｉａｂｏｄｙ）、ＣＤＲを含むペプチド等を挙げることができる。 Examples of methods using antibodies (immunological assay) include Western blotting, radioimmunoassay (RIA), enzyme immunoassay (ELISA, EIA), luminescence immunoassay, and fluorescence immunoassay (exoscreen). Law). The antibody used in such a method has the characteristic that the marker of the present invention can be detected or captured with good sensitivity and specificity. Such an antibody can be prepared by a method well known to those skilled in the art. For example, a monoclonal antibody or a fragment thereof can be preferably used. In addition, the monoclonal antibody or a fragment thereof can be used after labeling or solid-phase according to a generally known method. In the present specification, the “monoclonal antibody fragment” means a fragment that is a part of the monoclonal antibody and has a specific binding property to the target protein in the same manner as the monoclonal antibody. . Specific examples include Fab, F (ab ′) 2 , Fab ′, single chain antibody (scFv), disulfide stabilized antibody (dsFv), dimerized V region fragment (Diabody), and CDR-containing peptides. be able to.
　かくして、マーカー量を測定することができる。得られた測定値を用いて、以下の工程Ｂを行う。 Thus, the marker amount can be measured. The following process B is performed using the obtained measured value.
　工程Ｂとは、工程Ａで得られたマーカーの量に基づいてインビトロで大腸がんの転移を決定（又は評価）する工程である。決定方法の一例として、被験者のマーカーの量が基準値と比較して統計学的に有意に多いときに、大腸がんが転移している、もしくは転移のある大腸がんに罹患していると決定（又は評価）する方法が挙げられる。本明細書中「決定」とは、本発明の検出方法において得られた測定結果に基づいて、大腸がんの転移、もしくは転移のある大腸がんの罹患を評価することを指し、評価には、医師による判定を含まないことを意図している。 Step B is a step of determining (or evaluating) colon cancer metastasis in vitro based on the amount of the marker obtained in Step A. As an example of the determination method, when the amount of the marker of the subject is statistically significant compared to the reference value, the colorectal cancer has metastasized or is suffering from colorectal cancer with metastasis. The method of determining (or evaluating) is mentioned. In the present specification, “determining” refers to evaluating metastasis of colorectal cancer or morbidity of colorectal cancer with metastasis based on the measurement result obtained by the detection method of the present invention. It is intended not to include judgment by a physician.
　「基準値」とは、例えば、健常者におけるマーカー量の測定値とすることができる。好ましくは、複数の転移のある大腸がん患者由来であることが確認された試料（陽性試料）に含まれるマーカー量と、複数の健常者由来（もしくは転移のない大腸がん患者由来）であることが確認された試料（陰性試料）に含まれるマーカー量とを測定して対比し、その結果に基づき最も高確率に陽性試料と陰性試料とを区別できる値とする。 The “reference value” can be, for example, a measurement value of the marker amount in a healthy person. Preferably, the amount of marker contained in a sample (positive sample) confirmed to be derived from a plurality of colorectal cancer patients with metastases and a plurality of healthy individuals (or from colorectal cancer patients without metastases) The amount of marker contained in the confirmed sample (negative sample) is measured and compared, and based on the result, a value that can distinguish the positive sample and the negative sample with the highest probability is obtained.
　「健常者」とは、少なくとも大腸がん（転移の有無をとわない）に罹患していない個体、好ましくは健康な個体をいう。さらに、健常者は、被験者と同一の生物種であることを要する。例えば、検出に供する被験者がヒト（被検者）の場合には、健常者もヒト（本明細書では、以降「健常者」とする）でなければばらない。健常者の身体的条件は、被験者のそれと同一又は近似することが好ましい。身体的条件とは、例えば、ヒトの場合であれば、人種、性別、年齢、身長、体重等が該当する。 “Healthy person” means an individual who is not affected by at least colon cancer (with or without metastasis), preferably a healthy individual. Further, the healthy person needs to be the same species as the subject. For example, when the subject to be detected is a human (subject), the healthy person must also be a human (hereinafter referred to as “healthy person” in this specification). The physical condition of a healthy person is preferably the same as or close to that of the subject. For example, in the case of a human, the physical condition corresponds to race, sex, age, height, weight, and the like.
　健常者由来の体液試料としては、被験者由来の体液試料と同種の試料であることが好ましく、例えば、被験者由来の体液試料が血液である場合、健常者由来の体液試料も血液が好ましい。 The body fluid sample derived from a healthy person is preferably the same type of sample as the body fluid sample derived from the subject. For example, when the body fluid sample derived from the subject is blood, the body fluid sample derived from the healthy person is also preferably blood.
　健常者の体液試料におけるマーカーの量は、前記工程で説明をした被験者の体液試料におけるマーカーの量の測定方法と同様の方法で測定することが好ましい。健常者の体液試料におけるマーカーの量は、被験者の体液試料におけるマーカーの量を測定する都度、新たに測定することもできるが、予め測定しておいたマーカーの量を利用することもできる。特に、健常者の様々な身体的条件におけるマーカーの量を予め測定しておき、その値をコンピューターに入力してデータベース化しておけば、被験者の身体的条件を当該コンピューターに入力することで、その被験者との比較に最適な身体的条件を有する健常者のマーカーの量を即座に利用できるので便利である。 The amount of the marker in the body fluid sample of the healthy person is preferably measured by the same method as the method for measuring the amount of the marker in the body fluid sample of the subject described in the above step. The amount of the marker in the bodily fluid sample of the healthy person can be newly measured every time the amount of the marker in the bodily fluid sample of the subject is measured, but the amount of the marker measured in advance can also be used. In particular, if the amount of a marker in various physical conditions of a healthy person is measured in advance and the value is input to a computer and stored in a database, the physical condition of the subject can be input to the computer, This is convenient because the amount of marker of a healthy person who has the optimal physical condition for comparison with the subject can be used immediately.
　「統計学的に有意」とは、例えば、得られた値の危険率（有意水準）が５％、１％又は０．１％より小さい場合が挙げられる。それ故、測定値について「統計学的に有意に大きい」とは、被験者と健常者のそれぞれから得られたマーカーの量的差異を統計学的に処理したときに両者間に有意差があり、かつ被験者の前記マーカーの量が健常者のそれと比較して相対的に多いことをいう。例えば、体液試料中のマーカーの量に関して、被験者が健常者の２倍以上、好ましくは３倍以上、より好ましくは４倍以上、最も好ましくは５倍以上多い場合が該当する。量的差異が３倍以上であれば信頼度は高く、統計学的にも有意に多いといえる。統計学的処理の検定方法は、有意性の有無を判断可能な公知の検定方法を適宜使用すればよく、特に限定しない。例えば、スチューデントｔ検定法、多重比較検定法を用いることができる。 “Statistically significant” includes, for example, a case where the risk value (significance level) of the obtained value is less than 5%, 1%, or 0.1%. Therefore, “statistically significant” for the measured value means that there is a significant difference between the two when the quantitative difference between the markers obtained from the subject and the healthy subject is statistically processed, And it means that the amount of the marker of the subject is relatively large compared to that of the healthy subject. For example, the amount of the marker in the body fluid sample corresponds to a case where the subject is twice or more, preferably 3 times or more, more preferably 4 times or more, most preferably 5 times or more that of a healthy person. If the quantitative difference is 3 times or more, the reliability is high, and it can be said that there is a significant statistical amount. The test method for statistical processing is not particularly limited as long as a known test method capable of determining the presence or absence of significance is appropriately used. For example, Student's t test or multiple comparison test can be used.
　被験者の体液中のマーカーの量が基準値よりも統計学的に有意に多い場合、その被験者は大腸がんが転移している、もしくは転移のある大腸がんに罹患していると評価する。本発明において対象となる大腸がんの病期は、特に限定はなく、早期癌から末期癌に及ぶ。 If the amount of marker in the body fluid of the subject is statistically significantly higher than the reference value, the subject is evaluated as having colon cancer metastasized or suffering from colon cancer with metastasis. The stage of colorectal cancer that is the subject of the present invention is not particularly limited, and ranges from early stage cancer to end stage cancer.
　このように、本発明の大腸がんの転移検出方法は、体液試料中のマーカーを、質量分析装置を用いて測定する態様及び抗体を用いて免疫学的に測定する態様を含む。本発明の方法によって、被験者が転移のある大腸がんに罹患しているか否か、あるいは、大腸がんの転移の有無を決定又は評価することができるだけでなく、転移のある大腸がん患者と健常者の識別、あるいは、転移のある大腸がん患者と転移のない大腸がん患者の識別を可能にする。 Thus, the colorectal cancer metastasis detection method of the present invention includes a mode in which a marker in a body fluid sample is measured using a mass spectrometer and a mode in which an antibody is used to measure immunologically. According to the method of the present invention, it is possible not only to determine whether or not a subject suffers from colorectal cancer with metastasis, or whether or not colorectal cancer metastasis has occurred. Enables identification of healthy individuals or colorectal cancer patients with metastasis from those with no metastasis.
　また、前記解析において、例えば、被験者が転移のある大腸がん患者であると判断された場合においては、健常者のマーカー量を被験者の治療前のマーカー量に設定し、治療後のマーカー量を被験者のマーカー量として対比することで、治療後のマーカー量が減少していることが示された場合は、当該治療が転移のある大腸がんの治療に有効である可能性が高いと判断することができる。従って、本発明はまた、がん治療を受ける前と受けた後において、本発明のマーカー量を測定し、治療後の値が治療前のそれより小さい場合に、当該治療が効果を有すると判断することを特徴とする評価方法を提供することができる。ここでの「転移のある大腸がん」の治療の評価とは、原発巣である大腸がんの治療に加えて、転移先のがんの治療を評価することも含む。 In the above analysis, for example, when it is determined that the subject is a colorectal cancer patient with metastasis, the marker amount of the healthy subject is set to the marker amount before treatment of the subject, and the marker amount after treatment is set to If the amount of marker after treatment is shown to be reduced by comparing with the amount of marker in the subject, it is judged that the treatment is likely to be effective in the treatment of metastatic colorectal cancer be able to. Therefore, the present invention also measures the marker amount of the present invention before and after receiving cancer treatment, and determines that the treatment is effective when the value after treatment is smaller than that before treatment. It is possible to provide an evaluation method characterized by The evaluation of the treatment of “colorectal cancer with metastasis” herein includes evaluating the treatment of cancer at the metastasis destination in addition to the treatment of colorectal cancer that is the primary lesion.
　本発明の別の態様では、大腸がんの転移（転移のある大腸がん）を検出するためのキット（以下、「大腸がん転移検出用キット」ともいう）が提供される。「大腸がん転移検出用キット」とは、転移のある大腸がんの罹患の有無、罹患の程度若しくは改善の有無や改善の程度、あるいは大腸がんの転移の有無を評価するために、また大腸がんの転移の予防、改善又は治療に有用な候補物質をスクリーニングするために、直接又は間接的に利用されるものをいう。 In another embodiment of the present invention, a kit for detecting colon cancer metastasis (colorectal cancer with metastasis) (hereinafter, also referred to as “colorectal cancer metastasis detection kit”) is provided. “Colorectal cancer metastasis detection kit” is used to evaluate the presence or absence of metastatic colorectal cancer, the degree of morbidity or the improvement or degree of improvement, or the presence or absence of colorectal cancer metastasis, It is used directly or indirectly to screen candidate substances useful for the prevention, amelioration or treatment of colorectal cancer metastasis.
　本態様のキットは、その構成物として、大腸がんの罹患に関連して体液試料中、好ましくは、血液、血清、血漿、膜小胞画分、あるいはエクソソームにおいて発現が変動する本発明のマーカーを特異的に認識し、また結合可能な物質が包含される。具体的には、例えば、抗体若しくはその断片又はそれらの化学修飾誘導体が含まれる。これらの抗体は、固相担体に結合されていてもよい。その他、例えば、標識二次抗体、さらには標識の検出に必要な基質、担体、洗浄バッファー、試料希釈液、酵素基質、反応停止液、精製された標準物質としてのタンパク質、使用説明書等を含んでいてもよい。なお、本発明のマーカーを認識できる抗体又はその断片としては、前述の通りである。 The kit of this embodiment is a marker of the present invention whose expression varies in a body fluid sample, preferably blood, serum, plasma, membrane vesicle fraction, or exosome, in relation to the onset of colorectal cancer. Are specifically recognized and bindable. Specifically, for example, an antibody or a fragment thereof or a chemically modified derivative thereof is included. These antibodies may be bound to a solid phase carrier. Others include, for example, labeled secondary antibodies, as well as substrates necessary for label detection, carriers, washing buffers, sample diluents, enzyme substrates, reaction stop solutions, purified proteins as standards, instructions for use, etc. You may go out. The antibody or fragment thereof that can recognize the marker of the present invention is as described above.
　これらのキットは、体液試料中の本発明のマーカーのタンパク質量を測定する際に抗体を用いる方法（例えば、ウェスタンブロット法、ＥＬＩＳＡ法、エクソスクリーン法等）であれば、用いることができる。なお、体液試料がエクソソームである場合、本発明のマーカーのタンパク質が定量されるのであれば、エクソソームに由来しないタンパク質も前記抗体により同時に検出されることがあってもよい。 These kits can be used as long as they use an antibody (for example, Western blot method, ELISA method, exoscreen method, etc.) when measuring the protein amount of the marker of the present invention in a body fluid sample. When the body fluid sample is an exosome, if the protein of the marker of the present invention is quantified, a protein not derived from the exosome may be simultaneously detected by the antibody.
　本発明のキットを用いて、例えば、健常者と被験者の血液サンプル中に存在する本発明のマーカーのタンパク質量を測定して、両者の発現量に有意差が生じた場合には、被験者における大腸がんの転移の決定及び／又は診断を行うことができる。 Using the kit of the present invention, for example, when the amount of protein of the marker of the present invention present in the blood sample of a healthy subject and a subject is measured, and there is a significant difference between the expression levels of both, A determination and / or diagnosis of cancer metastasis can be made.
　以下、本発明を実施例に基づいて説明するが、実施例は本発明をより良く理解するために例示するものであって、本発明の範囲がこれらの実施例に限定されることを意図するものではない。 Hereinafter, the present invention will be described based on examples. However, the examples are provided for better understanding of the present invention, and the scope of the present invention is intended to be limited to these examples. It is not a thing.
実施例１（組織を用いたバイオマーカー候補膜タンパク質の選定）
　大腸の良性腫瘍と大腸がん患者組織（「転移なし」及び「転移あり」を含む）から、常法に従って調製した膜画分についてｉｓｏｂａｒｉｃ　Ｔａｇｓ　ｆｏｒ　Ｒｅｌａｔｉｖｅ　ａｎｄ　Ａｂｓｏｌｕｔｅ　Ｑｕａｎｔｉｔａｔｉｏｎ法（以下、「iTRAQ（登録商標、Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）法」という）によるプロテオーム解析を行った。ここで見られた変動シグナルからタンパク質を同定し、さらなる検証のため、より定量精度の高いＳｅｌｅｃｔｅｄ　Ｒｅａｃｔｉｏｎ　Ｍｏｎｉｔｏｒｉｎｇ法（以下、「SRM法」という）によるプロテオーム解析を行った（結果は表示せず）。なお、このSRM法を行うに当たっては、iTRAQ法によるプロテオーム解析で同定された配列の安定同位体標識ペプチド（グライナー社から購入、以下、「SIペプチド」という）を混和させ、内部標準として用いることによって定量精度を向上させた。 Example 1 (Selection of biomarker candidate membrane protein using tissue)
A membrane fraction prepared from a benign tumor of the large intestine and a colon cancer patient (including “no metastasis” and “with metastasis”) according to a conventional method is subjected to the isobaric tags for relative and absolute quantification method (hereinafter referred to as “iTRAQ (registered trademark)”). Proteome analysis by the “Applied Biosystems” method). Proteins were identified from the fluctuation signals seen here, and proteome analysis was performed by the Selected Reaction Monitoring method (hereinafter referred to as “SRM method”) with higher quantitative accuracy for further verification (results not shown). In performing this SRM method, a stable isotope-labeled peptide (purchased from Greiner, hereinafter referred to as “SI peptide”) identified by the proteome analysis by the iTRAQ method is mixed and used as an internal standard. The quantitative accuracy was improved.
実施例２（混合血液中の膜小胞画分における候補タンパク質発現の確認）
　続いて、健常者4名の血清を混合した検体、転移のない大腸がん患者4名の血清を混合した検体、及び転移のある大腸がん患者4名の血清を混合した検体、の計3検体について膜小胞画分を調製し、実施例１において選択された候補タンパク質の発現量を調べた。なお、大腸がんの転移の有無は、病理所見及び開腹所見によって確認した。 Example 2 (Confirmation of candidate protein expression in membrane vesicle fraction in mixed blood)
Next, a total of 3 specimens: a sample mixed with the serum of 4 healthy subjects, a sample mixed with the serum of 4 colorectal cancer patients without metastasis, and a sample mixed with the serum of 4 colorectal cancer patients with metastasis A membrane vesicle fraction was prepared for the specimen, and the expression level of the candidate protein selected in Example 1 was examined. The presence or absence of colorectal cancer metastasis was confirmed by pathological findings and laparoscopic findings.
　膜小胞画分の調製は、次の方法で行った。具体的には、血清100μLにPBS 550μLを加え、2,000×g、4℃で30分間遠心後、0.22μmのspinカラムを用いてフィルター濾過した。その後、100,000×g、4℃で90分間超遠心後、沈殿物をPBSで洗浄し、再び100,000×g、4℃で90分間超遠心を行った。さらに、その沈殿物を50mM DTTを含むPBSで洗浄し、100,000×g、4℃で90分間超遠心後の沈殿物を膜小胞画分として得た。 The membrane vesicle fraction was prepared by the following method. Specifically, 550 μL of PBS was added to 100 μL of serum, centrifuged at 2,000 × g and 4 ° C. for 30 minutes, and then filtered using a 0.22 μm spin column. Then, after ultracentrifugation at 100,000 × g and 4 ° C. for 90 minutes, the precipitate was washed with PBS, and ultracentrifugated again at 100,000 × g and 4 ° C for 90 minutes. Further, the precipitate was washed with PBS containing 50 mM DTT, and the precipitate after ultracentrifugation at 100,000 × g and 4 ° C. for 90 minutes was obtained as a membrane vesicle fraction.
　得られた膜小胞画分は、界面活性剤（デオキシコール酸及びラウロイルサルコシン酸）を含むＰｈａｓｅ　Ｔｒａｎｓｆｅｒ　Ｓｕｒｆａｃｔａｎｔ溶液（以下、「PTS溶液」という）で可溶化し、トリプシン消化後、酢酸エチルを加えて酸性条件にすること（相間移動溶解法（PTS法）という）で質量分析の際に悪影響となる界面活性剤を除いた。トリプシン消化したサンプルに、実施例１で用いたSIペプチドを内部標準ペプチドとして添加後、TSQ vantage質量分析計（サーモフィッシャーサイエンティフィック社製）を用いて、SRM法で測定し、プロテオーム解析を行った。 The obtained membrane vesicle fraction was solubilized with Phase Transfer Surfactant solution (hereinafter referred to as “PTS solution”) containing surfactants (deoxycholic acid and lauroyl sarcosine acid), digested with trypsin, and then added with ethyl acetate. In this case, the surfactants, which are adversely affected during mass spectrometry, were removed by using acidic conditions (referred to as phase transfer dissolution method (PTS method)). After adding the SI peptide used in Example 1 as an internal standard peptide to the trypsin-digested sample, the TSQ で vantage mass spectrometer (manufactured by Thermo Fisher Scientific Co., Ltd.) is used for measurement by SRM method, and proteome analysis is performed. It was.
　その結果、健常者及び転移のない大腸がん患者に比べて、転移のある大腸がん患者で増加したタンパク質として9種類、健常者に比べてがん患者（転移あり、転移なしを含む）で増加したタンパク質として1種類、健常者に比べてがん患者（転移あり、転移なしを含む）で減少したタンパク質として1種類を抽出することができた（図１）。 As a result, 9 types of proteins increased in colorectal cancer patients with metastasis compared to healthy subjects and colorectal cancer patients without metastasis, and cancer patients (including metastasis, including no metastasis) compared to healthy individuals It was possible to extract one type of increased protein and one type of protein decreased in cancer patients (including metastasis and no metastasis) compared to healthy subjects (Fig. 1).
実施例３（個別検体での血液中の膜小胞画分における候補タンパク質発現の確認）
　実施例２で抽出された候補タンパク質について、個別の血清サンプルを用いて検証と、さらなる絞り込みを行った。試験には、健常者20名、転移の認められないStage1又はStage2の大腸がん患者18名、及び転移のあるStage4の大腸がん患者19名の血清を用いた Example 3 (Confirmation of candidate protein expression in membrane vesicle fraction in blood in individual specimen)
The candidate proteins extracted in Example 2 were verified and further refined using individual serum samples. The test used sera from 20 healthy individuals, 18 patients with stage 1 or stage 2 colorectal cancer who had no metastasis, and 19 patients with stage 4 colorectal cancer with metastasis.
　具体的には、実施例２と同様にして膜小胞画分を調製し、血清40μLに相当する膜画分を界面活性剤（デオキシコール酸及びラウロイルサルコシン酸）を含むPTS溶液で可溶化し、トリプシン消化後、酢酸エチルを加えて界面活性剤を除いた。ここに、SIペプチドを添加してSRM法によるプロテオーム解析を行った。なお、ここで用いたSIペプチドは、アミノ酸配列情報をもとに、それぞれのタンパク質に特異的なペプチド配列（トリプシン消化断片）の安定同位体標識ペプチド（グライナー社から購入）を用いた。また、個別検体では混合検体の結果と同様の変化を示さなかった候補や、一部検体でのみ検出された候補を除外した。結果を図２に示す。図中の有意差検定は、Mann-Whitney U test検定に従って行った。 Specifically, a membrane vesicle fraction was prepared in the same manner as in Example 2, and the membrane fraction corresponding to 40 μL of serum was solubilized with a PTS solution containing a surfactant (deoxycholic acid and lauroyl sarcosine acid). After digestion with trypsin, ethyl acetate was added to remove the surfactant. The SI peptide was added here, and the proteome analysis by SRM method was performed. The SI peptide used here was a stable isotope-labeled peptide (purchased from Greiner) of a peptide sequence (trypsin digested fragment) specific to each protein based on amino acid sequence information. In addition, candidates that did not show the same change as the result of the mixed sample in individual samples or candidates detected only in some samples were excluded. The results are shown in FIG. The significant difference test in the figure was performed according to the Mann-Whitney U test test.
　図２より、ＣＥＡＣＡＭ１、ＣＥＡＣＡＭ８、ＣＤ１７７、及びＣ５ＡＲ１が、健常者に比べて、転移のある大腸がん患者で有意な増加が見られた。これは、血液中の膜小胞画分中に存在するＣＥＡＣＡＭ１、ＣＥＡＣＡＭ８、ＣＤ１７７、及びＣ５ＡＲ１を測定することによって、転移のある大腸がんを検出することが可能であることを示している。さらに、ＣＥＡＣＡＭ１、ＣＥＡＣＡＭ８、ＣＤ１７７、及びＣ５ＡＲ１は、転移の認められない大腸がん患者に比べても、転移のある大腸がん患者で増加する傾向を示し、なかでも、ＣＥＡＣＡＭ１、ＣＥＡＣＡＭ８、及びＣＤ１７７が有意な増加を示した。このことから、血液中の膜小胞画分中に存在するＣＥＡＣＡＭ１、ＣＥＡＣＡＭ８、ＣＤ１７７、及びＣ５ＡＲ１の量を測定することによって、転移のある大腸がんと転移のない大腸がんを区別して、転移のある大腸がんを検出できることが示唆される。 2. From FIG. 2, CEACAM1, CEACAM8, CD177, and C5AR1 were significantly increased in colorectal cancer patients with metastasis compared to healthy subjects. This shows that it is possible to detect colon cancer with metastasis by measuring CEACAM1, CEACAM8, CD177, and C5AR1 present in the membrane vesicle fraction in blood. Furthermore, CEACAM1, CEACAM8, CD177, and C5AR1 show a tendency to increase in patients with metastatic colorectal cancer compared to those with no metastasis, and among them CEACAM1, CEACAM8, and CD177 There was a significant increase. From this, by measuring the amount of CEACAM1, CEACAM8, CD177, and C5AR1 present in the membrane vesicle fraction in blood, colon cancer with metastasis and colon cancer without metastasis can be distinguished from each other. It is suggested that colorectal cancer can be detected.
実施例４（エクソソームにおけるにおける候補タンパク質発現の確認）
　エクソソームのマーカータンパク質としてＣＤ６３を採用し、抗ＣＤ６３抗体を用いた免疫沈降法により血清からエクソソームを調製し、それに候補タンパク質が発現していないかを調べた。試験には、健常者2名、転移の認められないStage1又はStage2の大腸がん患者2名、及び転移のあるStage4の大腸がん患者2名の血清を供した。なお、抗ＣＤ６３抗体としては、Ａｎｔｉ　ＣＤ６３　ｆｏｒ　Ｅｘｏｓｏｍｅ　Ｉｓｏｌａｔｉｏｎ　（コスモバイオ社）を用いた。 Example 4 (Confirmation of candidate protein expression in exosomes)
CD63 was employed as an exosome marker protein, and exosomes were prepared from serum by immunoprecipitation using an anti-CD63 antibody and examined for the expression of candidate proteins. Sera from two healthy subjects, two Stage 1 or Stage 2 colorectal cancer patients with no metastasis, and two Stage 4 colorectal cancer patients with metastasis were provided for the study. As the anti-CD63 antibody, Anti CD63 for Exosome Isolation (Cosmo Bio) was used.
　先ず、抗体を用いた血清膜小胞画分の調製は次のとおりに行った。血清100μLにPBS 100μLを加えた後、磁気ビーズに抗ＣＤ６３抗体を共有結合させたレジンを加え、一晩4℃で反応させた。PBSで数回洗浄することで非特異的な結合物を除き、エクソソームを調製した。 First, a serum membrane vesicle fraction using an antibody was prepared as follows. After adding 100 μL of PBS to 100 μL of serum, a resin having an anti-CD63 antibody covalently bonded to the magnetic beads was added and allowed to react overnight at 4 ° C. Exosomes were prepared by removing non-specific binders by washing several times with PBS.
　次に、得られたエクソソームを界面活性剤（デオキシコール酸及びラウロイルサルコシン酸）を含むPTS溶液で可溶化し、トリプシン消化後、酢酸エチルを加えて界面活性剤を除いた。トリプシン消化したサンプルに、実施例３で用いたＣＥＡＣＡＭ１、ＣＥＡＣＡＭ８、ＣＤ１７７、及びＣ５ＡＲ１のSIペプチドを内部標準ペプチドとして添加後、TSQ vantage質量分析計を用いてSRM法で測定、プロテオーム解析を行った。この時、超遠心法により調製した膜小胞画分も同時に測定を行い、比較対照とした。結果を図３に示す。なお、図３中、CD63-IP-SRMは、抗ＣＤ６３抗体で免疫沈降したエクソソームをSRM法により処理したサンプルであり、Centrifugation-SRMは、超遠心によるSRM法により処理した各サンプルを示す。 Next, the obtained exosome was solubilized with a PTS solution containing surfactants (deoxycholic acid and lauroyl sarcosine acid), digested with trypsin, and then ethyl acetate was added to remove the surfactant. The CEACAM1, CEACAM8, CD177, and C5AR1 SI peptides used in Example 3 were added as internal standard peptides to the trypsin-digested sample, and then measured by the SRM method using a TSQ vantage mass spectrometer and subjected to proteomic analysis. At this time, the membrane vesicle fraction prepared by ultracentrifugation was also measured at the same time and used as a comparative control. The results are shown in FIG. In FIG. 3, CD63-IP-SRM is a sample obtained by treating exosomes immunoprecipitated with an anti-CD63 antibody by the SRM method, and Centrifugation-SRM indicates each sample treated by the SRM method by ultracentrifugation.
　図３より、エクソソームにおいてもＣＥＡＣＡＭ１、ＣＥＡＣＡＭ８、ＣＤ１７７、及びＣ５ＡＲ１は発現しており、それらの量は、超遠心法で調製した膜小胞画分のそれらの量と同じような傾向を示していた。健常者と転移の認められる大腸がん患者との間で明確な差があり、同様に、転移の認められない大腸がん患者と転移の認められる大腸がん患者との間でも明確な差が認められることが示された。このことは、血清エクソソーム上のこれらのタンパク質を定量することによって、転移のある大腸がんを検出できることを示している。 From FIG. 3, CEACAM1, CEACAM8, CD177, and C5AR1 were also expressed in exosomes, and their amounts showed the same tendency as those of membrane vesicle fractions prepared by ultracentrifugation. . There is a clear difference between healthy and metastatic colorectal cancer patients, as well as a clear difference between colorectal cancer patients with no metastasis and colorectal cancer patients with metastasis. It was shown to be accepted. This indicates that metastatic colorectal cancer can be detected by quantifying these proteins on serum exosomes.
　本発明の方法により、サンプル提供者が転移のある大腸がんの可能性が高いか否か、あるいは大腸がんが転移しているか否かを判定することができる。これにより、サンプル提供者は大腸がんの進行を阻止する手段を講じることができるため、有用である。 The method of the present invention allows the sample provider to determine whether or not there is a high possibility of colorectal cancer with metastasis or whether or not colorectal cancer has metastasized. This is useful because the sample provider can take measures to prevent the progression of colorectal cancer.
配列表の配列番号１は、ＣＥＡＣＡＭ８タンパク質のアミノ酸配列である。
配列表の配列番号２は、ＣＥＡＣＡＭ１タンパク質のアミノ酸配列である。
配列表の配列番号３は、ＣＤ１７７タンパク質のアミノ酸配列である。
配列表の配列番号４は、Ｃ５ＡＲ１タンパク質のアミノ酸配列である。 Sequence number 1 of a sequence table is an amino acid sequence of CEACAM8 protein.
Sequence number 2 of a sequence table is an amino acid sequence of CEACAM1 protein.
Sequence number 3 of a sequence table is an amino acid sequence of CD177 protein.
Sequence number 4 of a sequence table is an amino acid sequence of C5AR1 protein.

Claims (12)

  1. 　被験者に由来する体液試料において、ＣＥＡＣＡＭ８、ＣＥＡＣＡＭ１、ＣＤ１７７、及びＣ５ＡＲ１からなる群から選択される少なくとも１種のタンパク質の量を測定し、その測定値を指標にすることを特徴とする、大腸がんの転移検出方法。 A colorectal cancer characterized in that in a body fluid sample derived from a subject, the amount of at least one protein selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1 is measured, and the measured value is used as an index. Metastasis detection method.
  2. 　前記測定値を基準値と対比して、その値が基準値よりも大きいと認められる場合を大腸がんの転移の存在の指標とする請求項１記載の方法。 The method according to claim 1, wherein the measured value is compared with a reference value, and a case where the measured value is recognized to be larger than the reference value is used as an indicator of the presence of colorectal cancer metastasis.
  3. 　前記体液試料が血液試料である、請求項１又は２記載の方法。 The method according to claim 1 or 2, wherein the body fluid sample is a blood sample.
  4. 　前記体液試料が膜小胞画分である、請求項１又は２記載の方法。 The method according to claim 1 or 2, wherein the body fluid sample is a membrane vesicle fraction.
  5. 　前記タンパク質がエクソソームに由来するものである、請求項１～４いずれか記載の方法。 The method according to any one of claims 1 to 4, wherein the protein is derived from exosomes.
  6. 　前記測定を質量分析法により行う、請求項１～５いずれか記載の方法。 The method according to any one of claims 1 to 5, wherein the measurement is performed by mass spectrometry.
  7. 　前記質量分析法がESI型LC-MS法である請求項６記載の方法。 The method according to claim 6, wherein the mass spectrometry is an ESI type LC-MS method.
  8. 　前記測定が抗体を用いて行う、請求項１～５いずれか記載の方法。 The method according to any one of claims 1 to 5, wherein the measurement is performed using an antibody.
  9. 　前記抗体を使用する方法がELISA法である請求項８記載の方法。 The method according to claim 8, wherein the method using the antibody is an ELISA method.
  10. 　ＣＥＡＣＡＭ８、ＣＥＡＣＡＭ１、ＣＤ１７７、及びＣ５ＡＲ１からなる群から選択される１つ又は２つ以上のタンパク質に対する抗体又はその断片を含有してなる、大腸がんの転移を検出するためのキット。 A kit for detecting metastasis of colorectal cancer, comprising an antibody against one or more proteins selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1, or a fragment thereof.
  11. 　ＣＥＡＣＡＭ８、ＣＥＡＣＡＭ１、ＣＤ１７７、及びＣ５ＡＲ１からなる群から選択される１つ又は２つ以上のタンパク質の転移のある大腸がんのマーカーとしての使用。 Use as a marker for colorectal cancer with metastasis of one or more proteins selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1.
  12. 　ＣＥＡＣＡＭ８、ＣＥＡＣＡＭ１、ＣＤ１７７、及びＣ５ＡＲ１からなる群から選択される１つ又は２つ以上のタンパク質を、被験者に由来する体液試料中から検出することを特徴とする、大腸がんの転移又は大腸がんの転移の疑いの情報を提供する方法。 Metastasis of colorectal cancer or colorectal cancer, wherein one or more proteins selected from the group consisting of CEACAM8, CEACAM1, CD177, and C5AR1 are detected from a body fluid sample derived from a subject. To provide information on suspicion of metastasis.
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WO2021132489A1 (en) 2019-12-27 2021-07-01 東ソー株式会社 Method for detecting protein in extracellular vesicles and extracellular vesicle membrane permeabilization treatment agent

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