Colorectal cancer biomarker and use thereof

Abstract

The present invention provides a colorectal cancer biomarker capable of simply and non-invasively determining the presence or absence of colorectal cancer with excellent sensitivity and specificity.　The colorectal cancer biomarker according to the present invention is at least one urinary protein selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, MME and ACP2.

Description

Colorectal cancer biomarkers and their uses

[0001] The present invention relates to a colorectal cancer biomarker and its use. This application claims priority to Japanese Patent Application No. 2022-106363, filed with the Japan Patent Office on June 30, 2022, the entire contents of which are incorporated by reference.

[0002] The gold standard for diagnosing colorectal cancer is lower gastrointestinal endoscopy and the accompanying tissue diagnosis. However, lower gastrointestinal endoscopy is a time-consuming, highly invasive procedure that causes discomfort to the subject during endoscope insertion and observation, and is expensive. Additionally, there are risks associated with the procedure, such as bleeding and perforation during the procedure, and intestinal obstruction due to pre-treatment laxatives. Therefore, lower gastrointestinal endoscopy is not recommended for screening tests such as health checks for healthy individuals.

[0003] Therefore, fecal occult blood tests are widely used as a test for colorectal cancer (e.g., Non-Patent Documents 1 and 2). It is generally believed that fecal occult blood tests can determine whether or not a person has colorectal cancer and can reduce the mortality rate from colorectal cancer. However, fecal occult blood tests have low sensitivity for detecting early-stage colorectal cancer. While the sensitivity of fecal occult blood tests for advanced colorectal cancer is relatively high at approximately 80%, some reports state that the sensitivity for early-stage colorectal cancer is only about 28.1%.

[0004] In addition, fecal occult blood tests have an extremely high probability of false positives, meaning that healthy subjects are mistakenly identified as having colorectal cancer. This means that fecal occult blood tests lack specificity. Positive results in fecal occult blood tests can be due to injuries to the anus or mucous membranes. For this reason, an endoscopic examination, which is highly invasive and costly, may be required. Another issue with fecal occult blood tests is the complicated handling of samples.

[0005] For these reasons, there is a need for the development of biomarkers that enable minimally invasive testing. Examples of such biomarkers that have been proposed include CEA (carcinoembryonic antigen) and CA19-9 (carbohydrate antigen 19-9). However, CEA and CA19-9 also have low sensitivity for detecting colorectal cancer, and therefore their use in the diagnosis of colorectal cancer is not recommended.

[0006] National Cancer Center, "Cancer Information Service" (URL: https: / / ganjoho.jp / med_pro / pre_scr / screening / screening_colon.html, accessed June 13, 2022) Igaku Shoin, "Weekly Medical World Newspaper, No. 2940, August 8, 2011" (URL: http: / / www.igaku-shoin.co.jp / paperDetail.do?id=PA02940_05, accessed June 13, 2022)

[0007] The present invention provides a colorectal cancer biomarker that can easily and non-invasively determine the presence or absence of colorectal cancer with excellent sensitivity and specificity.

[0008] According to one aspect of the present invention, there is provided a colorectal cancer biomarker, which is at least one urinary protein selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, MME, and ACP2.

[0009] According to the present invention, a colorectal cancer biomarker is provided that can easily and non-invasively determine the presence or absence of colorectal cancer with excellent sensitivity and specificity.

[0010] 1 is an explanatory diagram showing an overview of an example. FIG. 1 is an explanatory diagram showing an overview of comprehensive relative quantitative mass spectrometry (iTRAQ (registered trademark)) using a discovery cohort. FIG. 2 shows the results of the comprehensive relative quantitative mass spectrometry outlined in FIG. 3 is a heat map showing proteins abnormally expressed in colorectal cancer patients. FIG. 2 is a diagram showing a comparison of the expression levels of DPEP1 in urine between healthy subjects and colorectal cancer patients. For colorectal cancer patients, the expression levels are shown in order of progression stage. The expression levels in the diagram are standardized values ​​obtained by dividing by the urinary creatinine concentration. FIG. 1 is a diagram showing a comparison of the expression levels of TFF1 in urine between healthy subjects and colorectal cancer patients. For colorectal cancer patients, the expression levels are shown in order of progression stage. The expression levels in the diagram are standardized values ​​obtained by dividing by the urinary creatinine concentration. FIG. 1 is a diagram showing a comparison of the expression levels of ANPEP in urine between healthy subjects and colorectal cancer patients. For colorectal cancer patients, the expression levels are shown in order of progression stage. The expression levels in the diagram are standardized values ​​obtained by dividing by the urinary creatinine concentration. 1 is a diagram comparing the expression levels of LGALS3 in urine between healthy subjects and colorectal cancer patients. For colorectal cancer patients, the expression levels are shown in order of progression stage. The expression levels in the diagram are standardized values ​​divided by the urinary creatinine concentration. This diagram compares the expression levels of DPEP1 in urine between healthy subjects and colorectal cancer patients. For colorectal cancer patients, the expression levels are shown in order of progression stage. The expression levels in the diagram are absolute values ​​before standardization. This diagram compares the expression levels of TFF1 in urine between healthy subjects and colorectal cancer patients. For colorectal cancer patients, the expression levels are shown in order of progression stage. The expression levels in the diagram are absolute values ​​before standardization. This diagram compares the expression levels of ANPEP in urine between healthy subjects and colorectal cancer patients. For colorectal cancer patients, the expression levels are shown in order of progression stage. The expression levels in the diagram are absolute values ​​before standardization. This diagram compares the expression levels of LGALS3 in urine between healthy subjects and colorectal cancer patients. For colorectal cancer patients, the expression levels are shown in order of progression stage. The expression levels in the diagram are absolute values ​​before standardization. 1 is a diagram comparing the expression levels of DPEP1 in urine between healthy subjects, adenoma patients, and colorectal cancer patients. The expression levels in the diagram are standardized values ​​divided by the creatinine concentration in urine. 1 is a diagram comparing the expression levels of TFF1 in urine between healthy subjects, adenoma patients, and colorectal cancer patients. The expression levels in the diagram are standardized values ​​divided by the creatinine concentration in urine.1 is a diagram comparing the expression levels of DPEP1 in urine between healthy subjects, adenoma patients, and colorectal cancer patients. The expression levels in the diagram are absolute values ​​before normalization. FIG. 1 is a diagram comparing the expression levels of TFF1 in urine between healthy subjects, adenoma patients, and colorectal cancer patients. The expression levels in the diagram are absolute values ​​before normalization. FIG. 1 is a diagram comparing the expression levels of DPEP1 in urine between healthy subjects, adenoma patients, and colorectal cancer patients. For colorectal cancer patients, the expression levels are shown in order of progression stage. The expression levels in the diagram are standardized values ​​obtained by dividing by the urinary creatinine concentration. FIG. 1 is a diagram comparing the expression levels of TFF1 in urine between healthy subjects, adenoma patients, and colorectal cancer patients. For colorectal cancer patients, the expression levels are shown in order of progression stage. The expression levels in the diagram are standardized values ​​obtained by dividing by the urinary creatinine concentration. FIG. 1 is a diagram comparing the expression levels of DPEP ... absolute values ​​before normalization. 1 is a diagram comparing the expression levels of TFF1 in urine between healthy individuals, adenoma patients, and colorectal cancer patients. For colorectal cancer patients, the expression levels are shown in order of progression stage. The expression levels in the diagram are absolute values ​​before standardization. An ROC curve created to determine the presence or absence of early colorectal cancer (adenoma) is shown. The expression levels are standardized values ​​divided by the urinary creatinine concentration. An ROC curve created to determine the presence or absence of early colorectal cancer (adenoma) is shown. The expression levels are absolute values ​​before standardization. An ROC curve created based on the measured values ​​of protein concentration in serum samples to determine the presence or absence of colorectal cancer is shown. The expression levels are absolute values ​​before standardization.

[0011] The following terms have the following meanings in this specification. "Mutant" refers to a natural variant due to polymorphism, mutation, etc., or a variant containing deletion, substitution, addition, or insertion of one or more bases. "Sensitivity" means (number of true positives) / (number of true positives + number of false negatives). Higher sensitivity makes it easier to detect colorectal cancer early, contributing to complete resection of cancer-affected areas and a lower recurrence rate. As a result, it may also contribute to a lower mortality rate from colorectal cancer. "Specificity" means (number of true negatives) / (number of true negatives + number of false positives). Higher specificity makes it easier to prevent healthy subjects from being mistakenly identified as colorectal cancer patients, preventing the implementation of unnecessary additional tests, reducing the burden on patients and contributing to a reduction in medical costs. "Accuracy" means (number of true positives + number of true negatives) / (total number of samples). The acts specified by the terms "inspection," "discrimination," and "test" do not include medical acts by physicians (e.g., acts of diagnosing human diseases, acts of treating human diseases, etc.) in Japan and in countries and regions where medical acts are excluded from patentability. The symbol "~" indicating a range of values ​​means that the values ​​before and after it are included as the lower and upper limits.

[0012] <Colorectal Cancer Biomarker> The colorectal cancer biomarker of the present invention is at least one urinary protein (hereinafter sometimes simply referred to as "urinary protein") selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, MME, and ACP2.

[0013] The present inventors have conceived the idea of ​​establishing proteins that exhibit abnormal expression in the urine of colorectal cancer patients as biomarkers for a simple, non-invasive test for the presence or absence of colorectal cancer. Urine samples can be collected non-invasively. Furthermore, obtaining and handling test samples is simpler than fecal occult blood tests. Thus, urine as a specimen material offers numerous advantages, including the ease with which anyone can collect a sample, the lack of special equipment or reagents required for collection and storage, the ability to collect a sufficient amount of sample at one time, the lack of pain or risk associated with collection, and the ability to reflect the body's circulatory dynamics. For these reasons, urine samples are considered to be the most suitable specimen for health checkups and home screening.

[0014] In the present invention, a urine sample from a subject having the above advantages is used, and urinary proteins and / or combinations thereof present in trace amounts in urine are used as colorectal cancer biomarkers. This allows for non-invasive testing of the presence or absence of colorectal cancer. Meanwhile, various screening kits using urine samples, such as Testape for diabetes and pregnancy tests, are widely used in general clinical practice and daily life. However, because a colorectal cancer biomarker that can be detected in a urine sample has not yet been established, no practical colorectal cancer screening kit using a urine sample has yet been put to practical use.

[0015] While urine samples have such excellent advantages as screening samples, it is difficult to say that these advantages are being fully utilized in the field of colorectal cancer testing. In light of this background, the colorectal cancer biomarker of the present invention enables colorectal cancer to be tested non-invasively, simply, and even at home. Therefore, the colorectal cancer biomarker of the present invention is extremely useful, particularly in the field of medical examinations and primary screening.

[0016] As shown in the Examples below, the present inventors comprehensively analyzed the protein concentrations of urine samples collected from colorectal cancer patients. As a result of a cohort study using statistical methods, the present inventors discovered that 16 specific urinary proteins are abnormally expressed in colorectal cancer patients. By using these urinary proteins as colorectal cancer biomarkers, the presence or absence of colorectal cancer can be non-invasively tested.

[0017] The present inventors have identified 16 types of urinary colorectal cancer biomarkers: DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, MME, and ACP2. These colorectal cancer biomarkers can determine the presence or absence of colorectal cancer with excellent sensitivity and specificity. As shown in the Examples below, the colorectal cancer biomarkers of the present invention can detect not only early colorectal cancer but also colorectal adenomas with excellent sensitivity.

[0018] Each protein will be described in turn below. In the following description, the "concentration" of a protein in urine can be substantially synonymous with terms such as "content," "abundance," "expression amount," and "level" of the protein in urine.

[0019] DPEP1 is a polypeptide having the amino acid sequence of SEQ ID NO: 1. DPEP1 is an abbreviation for dipeptidase 1. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-P16444. DPEP1 may be a mutant thereof.

[0020] The present inventors have found that DPEP1 is significantly highly expressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the DPEP1 concentration in urine shows a positive correlation with the incidence of colorectal cancer. Therefore, if the DPEP1 concentration in a urine sample is higher than the cutoff value, the subject can be determined to have colorectal cancer. On the other hand, if the DPEP1 concentration in a urine sample is lower than the cutoff value, the subject can be determined to not have colorectal cancer.

[0021] TFF1 is a polypeptide having the amino acid sequence of SEQ ID NO: 2. TFF1 is an abbreviation for trefoil factor 1. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-P04155. TFF1 may be a mutant thereof.

[0022] The present inventors have found that TFF1 is significantly highly expressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the concentration of TFF1 in urine shows a positive correlation with the incidence of colorectal cancer. Therefore, if the TFF1 concentration in a urine sample is higher than the cutoff value, the subject can be determined to have colorectal cancer. On the other hand, if the TFF1 concentration in a urine sample is lower than the cutoff value, the subject can be determined to not have colorectal cancer.

[0023] ANPEP is a polypeptide having the amino acid sequence of SEQ ID NO: 3. ANPEP is an abbreviation for alanyl aminopeptidase. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-P15144. ANPEP may be a variant thereof.

[0024] The present inventors have found that ANPEP is significantly highly expressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the concentration of ANPEP in urine shows a positive correlation with the incidence of colorectal cancer. Therefore, if the ANPEP concentration in a urine sample is higher than the cutoff value, the subject can be determined to have colorectal cancer. On the other hand, if the ANPEP concentration in a urine sample is lower than the cutoff value, the subject can be determined to not have colorectal cancer.

[0025] LGALS3 is a polypeptide having the amino acid sequence of SEQ ID NO: 4. LGALS3 is an abbreviation for galectin-3. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-P17931. LGALS3 may be a mutant thereof.

[0026] The present inventors have found that LGALS3 is significantly highly expressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the concentration of LGALS3 in urine shows a positive correlation with the incidence of colorectal cancer. Therefore, if the LGALS3 concentration in a urine sample is higher than the cutoff value, the subject can be determined to have colorectal cancer. On the other hand, if the LGALS3 concentration in a urine sample is lower than the cutoff value, the subject can be determined to not have colorectal cancer.

[0027] PRG4 is a polypeptide having the amino acid sequence of SEQ ID NO: 5. PRG4 is an abbreviation for proteoglycan 4. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-Q92954. PRG4 may be a mutant thereof.

[0028] The present inventors have found that PRG4 is significantly highly expressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the PRG4 concentration in urine shows a positive correlation with the incidence of colorectal cancer. Therefore, if the PRG4 concentration in a urine sample is higher than the cutoff value, the subject can be determined to have colorectal cancer. On the other hand, if the PRG4 concentration in a urine sample is lower than the cutoff value, the subject can be determined to not have colorectal cancer.

[0029] CDH17 is a polypeptide having the amino acid sequence of SEQ ID NO: 6. CDH17 is an abbreviation for cadherin-17. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-Q12864. CDH17 may be a mutant thereof.

[0030] The present inventors have found that CDH17 is significantly highly expressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the concentration of CDH17 in urine shows a positive correlation with the incidence of colorectal cancer. Therefore, if the CDH17 concentration in a urine sample is higher than the cutoff value, the subject can be determined to have colorectal cancer. On the other hand, if the CDH17 concentration in a urine sample is lower than the cutoff value, the subject can be determined not to have colorectal cancer.

[0031] PIGR is a polypeptide having the amino acid sequence of SEQ ID NO: 7. PIGR is an abbreviation for polymeric immunoglobulin receptor. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-P01833. PIGR may be a mutant thereof.

[0032] The present inventors have found that PIGR is significantly highly expressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the urinary PIGR concentration shows a positive correlation with the incidence of colorectal cancer. Therefore, if the PIGR concentration in a urine sample is higher than the cutoff value, the subject can be determined to have colorectal cancer. On the other hand, if the PIGR concentration in a urine sample is lower than the cutoff value, the subject can be determined to not have colorectal cancer.

[0033] TNFRSF10C is a polypeptide having the amino acid sequence of SEQ ID NO: 8. TNFRSF10C is an abbreviation for Tumor necrosis factor receptor superfamily member 10c. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-014798. TNFRSF10C may be a mutant thereof.

[0034] The present inventors have found that TNFRSF10C is significantly highly expressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the concentration of TNFRSF10C in urine shows a positive correlation with the incidence of colorectal cancer. Therefore, if the concentration of TNFRSF10C in a urine sample is higher than the cutoff value, the subject can be determined to be suffering from colorectal cancer. On the other hand, if the concentration of TNFRSF10C in a urine sample is lower than the cutoff value, the subject can be determined not to be suffering from colorectal cancer.

[0035] MEP1A is a polypeptide having the amino acid sequence of SEQ ID NO: 9. MEP1A is an abbreviation for Meprin A subunit alpha. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-Q16819. MEP1A may be a mutant thereof.

[0036] The present inventors have found that MEP1A is significantly highly expressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the concentration of MEP1A in urine shows a positive correlation with the incidence of colorectal cancer. Therefore, if the MEP1A concentration in a urine sample is higher than the cutoff value, the subject can be determined to have colorectal cancer. On the other hand, if the MEP1A concentration in a urine sample is lower than the cutoff value, the subject can be determined to not have colorectal cancer.

[0037] LGALS3BP is a polypeptide having the amino acid sequence of SEQ ID NO: 10. LGALS3BP is an abbreviation for galectin-3 binding protein. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-Q08380. LGALS3BP may be a mutant thereof.

[0038] The present inventors have found that LGALS3BP is significantly highly expressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the concentration of LGALS3BP in urine shows a positive correlation with the incidence of colorectal cancer. Therefore, if the LGALS3BP concentration in a urine sample is higher than the cutoff value, the subject can be determined to have colorectal cancer. On the other hand, if the LGALS3BP concentration in a urine sample is lower than the cutoff value, the subject can be determined not to have colorectal cancer.

[0039] APOA1 is a polypeptide having the amino acid sequence of SEQ ID NO: 11. APOA1 is an abbreviation for apolipoprotein A-1. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-P02647. APOA1 may be a mutant thereof.

[0040] The present inventors have found that APOA1 is significantly highly expressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the concentration of APOA1 in urine shows a positive correlation with the incidence of colorectal cancer. Therefore, if the APOA1 concentration in a urine sample is higher than the cutoff value, the subject can be determined to have colorectal cancer. On the other hand, if the APOA1 concentration in a urine sample is lower than the cutoff value, the subject can be determined to not have colorectal cancer.

[0041] LRG1 is a polypeptide having the amino acid sequence of SEQ ID NO: 12. LRG1 is an abbreviation for leucine rich alpha-2-glycoprotein 1. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-P02750. LRG1 may be a mutant thereof.

[0042] The present inventors have found that LRG1 is significantly highly expressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the concentration of LRG1 in urine shows a positive correlation with the incidence of colorectal cancer. Therefore, if the LRG1 concentration in a urine sample is higher than the cutoff value, the subject can be determined to have colorectal cancer. On the other hand, if the LRG1 concentration in a urine sample is lower than the cutoff value, the subject can be determined to not have colorectal cancer.

[0043] ENPEP is a polypeptide having the amino acid sequence of SEQ ID NO: 13. ENPEP is an abbreviation for Glutamyl aminopeptidase. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-Q07075. ENPEP may be a variant thereof.

[0044] The present inventors have found that ENPEP is significantly highly expressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the concentration of ENPEP in urine shows a positive correlation with the incidence of colorectal cancer. Therefore, if the concentration of ENPEP in a urine sample is higher than the cutoff value, the subject can be determined to have colorectal cancer. On the other hand, if the concentration of ENPEP in a urine sample is lower than the cutoff value, the subject can be determined not to have colorectal cancer.

[0045] S100A11 is a polypeptide having the amino acid sequence of SEQ ID NO: 14. S100A11 is an abbreviation for Protein S100-A11. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-P31949. S100A11 may be a mutant thereof.

[0046] The present inventors have found that S100A11 is significantly highly expressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the concentration of S100A11 in urine shows a positive correlation with the incidence of colorectal cancer. Therefore, if the S100A11 concentration in a urine sample is higher than the cutoff value, the subject can be determined to have colorectal cancer. On the other hand, if the S100A11 concentration in a urine sample is lower than the cutoff value, the subject can be determined not to have colorectal cancer.

[0047] MME is a polypeptide having the amino acid sequence of SEQ ID NO: 15. MME is an abbreviation for membrane metalloendopeptidase. MME is also called neprilysin. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-P08473. MME may be a mutant thereof.

[0048] The present inventors have found that MME is significantly highly expressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the concentration of MME in urine shows a positive correlation with the incidence of colorectal cancer. Therefore, if the MME concentration in a urine sample is higher than the cutoff value, the subject can be determined to have colorectal cancer. On the other hand, if the MME concentration in a urine sample is lower than the cutoff value, the subject can be determined not to have colorectal cancer.

[0049] ACP2 is a polypeptide having the amino acid sequence of SEQ ID NO: 16. ACP2 is an abbreviation for acid phosphatase 2. ACP2 is also called lysosomal acid phosphatase. The sequence of this protein is well known in the art and can be obtained, for example, from UniProtKB-P11117. ACP2 may be a mutant thereof.

[0050] The present inventors have found that ACP2 is significantly underexpressed in the urine of colorectal cancer patients. According to data obtained by the inventors, the concentration of ACP2 in urine shows a negative correlation with the incidence of colorectal cancer. Therefore, if the ACP2 concentration in a urine sample is lower than the cutoff value, the subject can be determined to have colorectal cancer. On the other hand, if the ACP2 concentration in a urine sample is higher than the cutoff value, the subject can be determined to not have colorectal cancer.

[0051] As a colorectal cancer biomarker, the above-mentioned 16 kinds of urinary proteins may be used alone, or two or more kinds may be used in combination.Among them, when urinary DPEP1 is used alone, the sensitivity and specificity are extremely high, and the reproducibility is also high.However, the colorectal cancer biomarker is not limited to DPEP1.For example, at least one urinary protein selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, PIGR and TNFRSF10C is also relatively useful as a colorectal cancer biomarker in terms of reproducibility.

[0052] Preferred combinations of colorectal cancer biomarkers include, for example, the following (1) and (2): (1): A combination of DPEP1 and at least one selected from the group consisting of TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C; (2): A combination of TFF1 and at least one selected from the group consisting of DPEP1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C.

[0053] However, the combination of colorectal cancer biomarkers is not limited to the above (1) and (2). In addition to these, various other combinations of urinary proteins may also be useful.

[0054] Some examples of the use of the colon cancer biomarker will be described below. However, the following descriptions are provided as representative examples of embodiments of the colon cancer biomarker of the present invention. The scope of application of the colon cancer biomarker of the present invention is not limited to the following descriptions.

[0055] <Method for testing for colon cancer> The method for testing for colon cancer of the present invention (hereinafter referred to as "the method") comprises detecting at least one urinary protein selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, MME, and ACP2.

[0056] (Detection of urinary proteins) Detection of urinary proteins can be performed by a method that can be commonly selected by a person skilled in the art, such as ELISA, immunochromatography, quantitative mass spectrometry, Western blotting, immunoassay, radioimmunoassay, protein quantification methods using a spectrophotometer such as the BCA method or the Bradford method, or aptamer detection methods. In addition, a method that visually notifies the detection of urinary proteins, such as pregnancy test drugs that are already in practical use, can also be used.

[0057] When detecting urinary proteins, standardization may be performed by dividing the urinary protein concentration by the expression level of a protein that is thought to be constitutively expressed in urine. Alternatively, the absolute value (raw data) of the urinary protein concentration may be used. In either method, colorectal cancer can be detected based on the magnitude of the measured protein concentration. An example of a protein that is constitutively expressed in urine for use in standardization is urinary creatinine. However, from the viewpoint of simplicity, it is preferable to use the amount of a colorectal cancer biomarker as an absolute value (raw data) without standardizing it by the expression level of the protein.

[0058] (Cutoff Value) In one example of the present method, when the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, and MME, if the concentration of the detected urinary protein is equal to or greater than the cutoff value, the subject can be determined to have colorectal cancer. Furthermore, when the urinary protein is ACP2, if the concentration of the detected urinary protein is equal to or less than the cutoff value, the subject can be determined to have colorectal cancer. The cutoff value can be appropriately set by those skilled in the art taking into consideration sensitivity, specificity, diagnostic accuracy, positive predictive value, negative predictive value, etc. For example, the cutoff value may be a value determined on a case-by-case basis based on the concentration of urinary protein in a urine sample collected from a subject not suffering from colorectal cancer, or may be a predetermined value.

[0059] The cutoff value is a value that can vary depending on the measurement method and determination method. The cutoff value can also vary depending on whether emphasis is placed on improving sensitivity or specificity. The cutoff value may be set by taking into account the balance between sensitivity and specificity. Also, depending on the purpose of the test, for example, if the main purpose is to detect early colorectal cancer, the cutoff value may be set by prioritizing improving sensitivity over specificity.

[0060] For example, when DPEP1 is used alone, the cutoff value is considered to be preferably 1 to 1000 ng / g Cr in terms of the relative urinary creatinine (Cr) value, more preferably 20 to 190 ng / g Cr, and even more preferably 35 to 128 ng / g Cr. The cutoff value is considered to be preferably 1 to 1000 pg / ml in terms of the absolute urinary value, more preferably 35 to 130 pg / ml, and even more preferably 50 to 105 pg / ml. When the cutoff value for DPEP1 is within the above-mentioned ranges, the presence or absence of colorectal cancer can be determined with even greater sensitivity and specificity.

[0061] When the purpose is to detect early colorectal cancer of stage 0 or stage I, the cutoff value when using DPEP1 alone is considered to be a relative urinary Cr value of preferably 1 to 1000 ng / g Cr, more preferably 20 to 190 ng / g Cr, and even more preferably 35 to 130 ng / g Cr. The absolute urinary value is considered to be a preferred range of 1 to 1000 pg / ml, more preferably 35 to 130 pg / ml, and even more preferably 50 to 105 pg / ml.

[0062] A method using a discriminant can also be used to determine the presence or absence of colorectal cancer. A discriminant can distinguish between colorectal cancer and healthy individuals. A discriminant can be obtained by any discriminant analysis method. Examples of discriminant analysis methods include Fisher's discriminant analysis, nonlinear discriminant analysis using Mahalanobis distance, neural networks, and Support Vector Machines (SVMs), but are not limited to these examples. Other methods that can be used for discrimination include, for example, k-nearest neighbor analysis, decision trees, and logistic regression analysis.

[0063] In one example of this method, the expression level of a colorectal cancer biomarker may be compared with a reference value for testing and / or diagnosis. By comparing the expression level of a colorectal cancer biomarker with the reference value, it is possible to determine whether a subject is suffering from colorectal cancer. The reference value may be a value above or below which the subject is suspected of having colorectal cancer. When determining the reference value, the expression level of a colorectal cancer biomarker in a healthy subject may be used as a reference. Typically, the reference value is the expression level of a colorectal cancer biomarker in a healthy subject. The reference value may be set on a case-by-case basis, or a predetermined value may be used as the reference value. The reference value used for determination can be set appropriately depending on conditions such as the age and sex of the subject, the type of colorectal cancer biomarker used, the collection method, and the type of sample.

[0064] Although the biological species of the subject is mainly assumed to be humans (Homo sapiens), the scope of application of the colon cancer biomarker of the present invention is not necessarily limited to humans. For example, if each protein of the colon cancer biomarker described above has an ortholog in various mammals such as monkeys, mice, rats, dogs, cats, and rabbits, the urinary protein of the ortholog may be useful as a colon cancer biomarker in these animals as well as in humans.

[0065] The condition of the subject is not particularly limited, and examples thereof include a specimen for which it is unknown whether it has colon cancer, a specimen that has already been determined by another method to have colon cancer, a specimen that has already been determined by another method to not have colon cancer, and a specimen undergoing treatment for colon cancer.

[0066] In order to more clearly distinguish the above-described methods for testing for colorectal cancer from methods for diagnosing colorectal cancer, which include a doctor's judgment, the methods for testing for colorectal cancer can also be considered synonymous with the following methods: a method for testing for colorectal cancer, a method for collecting data for diagnosing colorectal cancer, a method for diagnosing colorectal cancer, an in vitro method for assisting in the diagnosis of colorectal cancer, and a test method for diagnosing colorectal cancer.

[0067] According to the present invention, by utilizing objective indicators without relying on the judgment of a doctor, it is possible to provide information useful for diagnosing colorectal cancer. In addition, the presence or absence of colorectal cancer can be determined automatically or mechanically without relying on the judgment of a person with specialized knowledge such as a doctor or a medical technician.

[0068] <Colon Cancer Test Kit> The colon cancer test kit is a kit used for testing for colon cancer. The colon cancer test kit includes a container for collecting urine from a subject and a reagent for detecting at least one urinary protein selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, MME, and ACP2.

[0069] The container is not particularly limited. Various urine collection containers, droppers, cups, centrifuge tubes, etc. can be used. The details and preferred embodiments of the colorectal cancer biomarkers, i.e., specific urinary proteins, are as described above.

[0070] Reagents for detecting urinary proteins are preferably used experimentally or practically in methods commonly selected by those skilled in the art, such as ELISA, immunochromatography, quantitative mass spectrometry, Western blotting, immunoassay, radioimmunoassay, protein quantification methods using spectrophotometers such as the BCA method and the Bradford method, and aptamer detection methods. Reagents that utilize a method for visually notifying the detection of urinary proteins, such as pregnancy tests already in practical use, can also be used. It would be extremely useful to develop a simple kit, such as a urine pregnancy test, to enable home screening.

[0071] In one example, the reagent for detecting urinary proteins may be either or both of Reagent α and Reagent β below. Reagent α: A reagent that notifies a subject of colorectal cancer by a qualitative method if the concentration of the detected urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, and MME and is equal to or greater than a threshold. Reagent β: A reagent that notifies a subject of colorectal cancer by a qualitative method if the concentration of the detected urinary protein is ACP2 and is equal to or less than a threshold.

[0072] In the case of a detection reagent using a qualitative method such as immunochromatography, a threshold for a positive test can be set. This threshold can be appropriately set by a person skilled in the art taking into consideration sensitivity, specificity, diagnostic accuracy, positive predictive value, negative predictive value, etc. For example, the threshold can be a value predetermined based on the concentration of urinary protein in a urine sample collected from a subject not suffering from colorectal cancer.

[0073] However, the threshold value may vary depending on whether emphasis is placed on improving sensitivity or specificity. The threshold value may be set by taking into account the balance between sensitivity and specificity. Furthermore, depending on the purpose of the test, for example, if the main purpose is to detect early colorectal cancer, the threshold value of a detection reagent using a qualitative method such as immunochromatography may be set by prioritizing sensitivity over specificity.

[0074] The colorectal cancer biomarkers in the present invention are urinary proteins. Considering the fact that existing test tapes using immunochromatography and the like are commercially available, the barrier to creating a simple kit is considered low for those skilled in the art. Therefore, the creation of a simple kit is easily achievable by those skilled in the art.

[0075] The colon cancer test kit may be used in a facility or at home. When the kit is used in a facility, the subject undergoes the test at the facility and collects urine in a container such as a urine sample collection cup. The concentration of the protein in the urine is measured at the facility. For concentration measurement, a method commonly selected by those skilled in the art, such as an ELISA method, can be applied. Even when used in a facility, the test can be performed quickly and easily using a simple kit such as TesTape.

[0076] In the case of a kit for home use, the colon cancer test kit may include a urine sample container for mailing, dry ice, packaging material, an instruction manual, etc. If it is a simple kit, it may be possible to purchase it at a drugstore or the like and use the kit at home.

[0077] <Method for treating colorectal cancer> The method for treating colorectal cancer of the present invention includes diagnosing a subject using the above-mentioned colorectal cancer biomarker. In one embodiment, if the subject is diagnosed with colorectal cancer, a therapeutic drug for colorectal cancer is administered to the subject. In a further embodiment, if the subject is diagnosed with colorectal cancer, surgery is performed on the subject.

[0078] The therapeutic agent for colon cancer is not particularly limited. As the therapeutic agent for colon cancer, a drug whose efficacy and medicinal effect have been medically confirmed is preferable. The therapeutic agent may be an anticancer agent, a molecular targeted therapeutic agent, or an immune checkpoint inhibitor.

[0079] Examples of anticancer drugs include fluorouracil, tegafur / uracil combination drug, tegafur / gimeracil / oteracium potassium combination drug, capecitabine, irinotecan, oxaliplatin, and trifluridine / tipiracil hydrochloride.

[0080] Examples of molecularly targeted therapeutic agents include cetuximab, panitumumab, bevacizumab, ramucirumab, aflibercept, regorafenib, encorafenib, binimetinib, trastuzumab, and pertuzumab.

[0081] Immune checkpoint inhibitors include, for example, nivolumab, ipilimumab, and pembrolizumab.

[0082] However, therapeutic agents are not limited to these examples. In countries or regions other than Japan, various therapeutic agents for colorectal cancer approved in those countries or regions may be used.

[0083] The method of administering the drug is not particularly limited. Oral administration, infusion, etc. are usually selected. However, the method of administering the drug is not limited to these examples. The surgery performed on the subject is not particularly limited. Endoscopic surgery, surgical resection, radiation therapy, etc. are usually selected. However, the surgery performed on the subject is not limited to these examples.

[0084] The colon cancer treatment method of the present invention uses the above-mentioned colon cancer biomarkers, allowing subjects to be treated based on diagnostic results with excellent sensitivity and specificity. Furthermore, the method also has good sensitivity for detecting early-stage colon cancer. Therefore, colon cancer can be treated at an appropriate time, which can contribute to reducing the mortality rate from colon cancer.

[0085] <Mechanism of Action> The colon cancer biomarkers of the present invention, as explained above using several application examples, use the concentrations of the above-mentioned urinary proteins and / or combinations thereof as indicators of the presence or absence of colon cancer or early-stage colon cancer. Therefore, the presence or absence of colon cancer can be determined with excellent sensitivity and specificity. As shown in the examples described below, the colon cancer biomarkers of the present invention can detect not only early-stage colon cancer but also colon adenomas with excellent sensitivity. Furthermore, since the detection of urinary proteins is performed using the subject's urine, a non-invasive test method can be provided. This makes it easy to eliminate risks such as complications associated with highly invasive tests. Furthermore, the burden on patients associated with the test can be reduced. Furthermore, handling of urine samples is advantageously less complicated than that of fecal occult blood test samples. Therefore, the colon cancer biomarkers of the present invention can easily and non-invasively determine the presence or absence of colon cancer with excellent sensitivity and specificity.

[0086] Summary of Embodiments Embodiments of the present invention include the following [1] to

[14] . However, the embodiments of the present invention are not limited to the following: [1] A method for testing for colorectal cancer, the method comprising detecting at least one urinary protein selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, MME, and ACP2. [2] The method of [1], further comprising: determining that the subject has colorectal cancer if the concentration of the detected urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, and MME, and determining that the subject has colorectal cancer if the concentration of the detected urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C, and if the urinary protein is ACP2, determining that the subject has colorectal cancer if the concentration of the detected urinary protein is at most a cutoff value. [3] The method of [1] or [2], wherein the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C. [4] The method of any one of [1] to [3], wherein the urinary protein is a combination of DPEP1 and at least one selected from the group consisting of TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C. [5] The method of any one of [1] to [4], wherein the urinary protein is a combination of TFF1 and at least one selected from the group consisting of DPEP1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C. [6] The method of any one of [1] to [5], wherein the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, and LGALS3. [7] The method of any one of [1] to [6], wherein the colorectal cancer is early colorectal cancer.[8] A kit for use in colon cancer testing, comprising: a container for collecting urine from a subject; and a reagent for detecting at least one urinary protein selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, MME, and ACP2. [9] The colon cancer testing kit according to [8], wherein the reagent is either or both of Reagent α and Reagent β below. Reagent α: a reagent that notifies a subject of the presence of colorectal cancer by a qualitative method if the concentration of the detected urinary protein is equal to or higher than a threshold value when the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, and MME. Reagent β: a reagent that notifies a subject of the presence of colorectal cancer by a qualitative method if the concentration of the detected urinary protein is equal to or lower than a threshold value when the urinary protein is ACP2.

[10] The colon cancer test kit according to [8] or [9], wherein the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C.

[11] The colon cancer test kit according to any of [8] to

[10] , wherein the urinary protein is a combination of DPEP1 and at least one selected from the group consisting of TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C.

[12] The colon cancer test kit according to any of [8] to

[11] , wherein the urinary protein is a combination of TFF1 and at least one selected from the group consisting of DPEP1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C.

[13] The colon cancer test kit according to any one of [8] to

[12] , wherein the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, and LGALS3.

[14] The colon cancer test kit according to any one of [8] to

[13] , wherein the colon cancer is early colon cancer.

[0087] Further embodiments of the present invention include the following

[15] to

[23] . However, the embodiments of the present invention are not limited to the following:

[15] A method for diagnosing colorectal cancer, comprising detecting at least one urinary protein selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, MME, and ACP2.

[16] The method of

[15] , further comprising: when the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, and MME, determining that the subject has colorectal cancer if the concentration of the detected urinary protein is equal to or higher than a cutoff value; and when the urinary protein is ACP2, determining that the subject has colorectal cancer if the concentration of the detected urinary protein is equal to or lower than a cutoff value.

[17] The method according to

[15] or

[16] , wherein the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C.

[18] The method according to any one of

[15] to

[17] , wherein the urinary protein is a combination of DPEP1 and at least one selected from the group consisting of TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C.

[19] The method according to any one of

[15] to

[18] , wherein the urinary protein is a combination of TFF1 and at least one selected from the group consisting of DPEP1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C.

[20] The method according to any one of

[15] to

[19] , wherein the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, and LGALS3.

[21] The method according to any one of

[15] to

[20] , wherein the colorectal cancer is early-stage colorectal cancer.

[22] A method for treating colorectal cancer, comprising: diagnosing a subject using the method of any one of

[15] to

[21] ; and, if the subject is diagnosed as having colorectal cancer, administering a therapeutic drug for colorectal cancer to the subject.

[23] A method for treating colorectal cancer, comprising: diagnosing a subject using the method of any one of

[15] to

[22] ; and, if the subject is diagnosed as having colorectal cancer, performing surgery on the subject.

[0088] Further embodiments of the present invention include the following

[24] to

[29] . However, the embodiments of the present invention are not limited to the following.

[24] A colorectal cancer biomarker, which is at least one urinary protein selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, MME, and ACP2.

[25] The colorectal cancer biomarker according to

[24] , wherein the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C.

[26] The colon cancer biomarker according to

[24] or

[25] , wherein the urinary protein is a combination of DPEP1 and at least one selected from the group consisting of TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C.

[27] The colon cancer biomarker according to any of

[24] to

[26] , wherein the urinary protein is a combination of TFF1 and at least one selected from the group consisting of DPEP1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C.

[28] The colon cancer biomarker according to any of

[24] to

[27] , wherein the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, and LGALS3.

[29] The colon cancer biomarker according to any one of

[24] to

[28] , wherein the colon cancer is early colon cancer.

[0089] Further embodiments of the present invention include the following

[30] to

[35] . However, the embodiments of the present invention are not limited to the following.

[30] Use of a urinary protein for the examination and / or diagnosis of colorectal cancer, wherein the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, MME, and ACP2.

[31] The use according to

[30] , wherein the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C.

[32] The use according to

[31] or

[32] , wherein the urinary protein is a combination of DPEP1 and at least one selected from the group consisting of TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C.

[33] The use according to any of

[30] to

[32] , wherein the urinary protein is a combination of TFF1 and at least one selected from the group consisting of DPEP1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C.

[34] The use according to any of

[30] to

[33] , wherein the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, and LGALS3.

[35] The use according to any of

[30] to

[34] , wherein the colorectal cancer is early colorectal cancer.

[0090] Although several embodiments have been described above, the present invention is not limited to the embodiments disclosed in this specification and can be appropriately modified and implemented without departing from the spirit of the invention. The embodiments disclosed in this specification can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention.

[0091] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following description.

[0092] <Explanation of terms> n: number of cases median: median IQR: interquartile range P value: p-value AUC: Area under the ROC curve 95% CI: 95% confidence interval HC: Health control CRC: colorectal cancer patient Univariate analysis: univariate analysis Multivariate analysis: multivariate analysis In the tables and figures shown below, the prefix "u" for each protein means that it is a urinary protein.

[0093] <Cohort Construction> Three types of cohorts were constructed randomly, matched by age and gender, from urine samples collected from 175 age- and gender-matched colorectal cancer patients with clinical stage 0, stage I, stage II, and stage III colorectal cancer and 299 healthy individuals. Discovery cohort: 32 cases, consisting of 16 colorectal cancer patients and 16 healthy individuals. Training cohort: 220 cases, consisting of 110 colorectal cancer patients and 110 healthy individuals. Validation cohort: 111 cases, consisting of 62 colorectal cancer patients and 49 healthy individuals.

[0094] <Narrowing down of biomarker candidates> In the present invention, relative quantitative mass spectrometry was used to narrow down the biomarkers. In addition, when establishing and verifying biomarkers, the concentration of individual urinary proteins was measured using the ELISA method, which utilizes an antigen-antibody reaction.

[0095] The discovery cohort was used to discover biomarker candidates. Comprehensive analysis was performed on 32 cases from the discovery cohort (comprehensive analysis cohort) using relative quantitative mass spectrometry (iTRAQ®). Urine sample concentration, protein reduction, alkylation, and digestion were performed according to standard methods. Next, as shown in Figure 2, high pH RP-HPLC separation and low pH nano LC-MS / MS were performed in this order, and data analysis was performed. As a result, 78 types of urinary proteins that were significantly abnormally expressed in the urine of colorectal cancer cases were identified (Figure 3). From these, 23 proteins that met gastrointestinal specificity and tumor specificity were narrowed down as biomarker candidates by database search.

[0096] <Establishment of Colorectal Cancer Biomarkers> The training cohort was used to establish colorectal cancer biomarkers. The urinary protein concentrations of 23 biomarker candidates narrowed down by analysis of the discovery cohort were measured by ELISA for 220 urine samples from the training cohort. For analysis convenience, the urinary protein concentrations were measured as absolute values, and also as relative urinary Cr values, standardized by dividing by the urinary creatinine concentration. Urinary creatinine is typically a protein widely expressed in the urine of the sample. The candidate protein concentrations were standardized by standardizing the protein concentrations of the biomarker candidates with the urinary creatinine concentration. The results are shown in Tables 1 to 4. In the data analysis results shown below, AUC is a statistical index ranging from 0 to 1. The closer the AUC value is to 1, the higher the discriminative ability, i.e., diagnostic ability, of the presence or absence of colorectal cancer.

[0097]

[0098]

[0099]

[0100]

[0101] From the analysis results of the training cohort, 15 colon cancer biomarkers were identified from 23 biomarker candidates, namely, DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, and MME were established based on the analysis results of urinary Cr relative values ​​(Tables 1 and 2). In addition, the analysis results of absolute values ​​identified 11 colon cancer biomarkers, namely, DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, LRG1, ACP2, S100A11, and MME (Tables 3 and 4).

[0102] As shown in Tables 1 to 4, these identified colorectal cancer biomarkers have lower p-values ​​than other candidate biomarker proteins. This indicates that colorectal cancer biomarkers are significantly abnormally expressed in the urine of colorectal cancer patients. Among them, 10 types - DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, LRG1, S100A11, and MME - have low p-values ​​regardless of whether or not they are normalized by urinary Cr, and therefore have high reproducibility.

[0103] <Verification of colorectal cancer biomarkers> Next, 111 cases of a validation cohort, independent of both the discovery cohort and the training cohort, were used. The validation cohort was used to verify the colorectal cancer biomarkers established through the discovery cohort and the training cohort. In this verification, the colorectal cancer diagnostic accuracy was verified for eight urinary proteins that showed high AUC in the analysis results of urinary Cr relative values, namely, DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, and TNFRSF10C. The results are shown in Tables 5 and 6.

[0104]

[0105]

[0106] As shown in Table 5, in terms of relative urinary Cr values, five urinary proteins, i.e., DPEP1, TFF1, ANPEP, LGALS3, and TNFRSF10C, were highly expressed in the urine of colorectal cancer patients compared to healthy individuals in the validation cohort. As shown in Table 6, in terms of absolute values, seven urinary proteins other than CDH17, i.e., DPEP1, TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C, were highly expressed in the urine of colorectal cancer patients compared to healthy individuals in the validation cohort.

[0107] These results are consistent with the discovery and training cohorts. Furthermore, in the validation cohort, as in the training cohort analysis, the AUC of urinary DPEP1 was the highest, with a urinary Cr relative value of 0.809 (Table 5) and an absolute value of 0.930 (Table 6). Thus, the reproducibility of urinary DPEP1 as a biomarker was the highest, enabling the diagnosis of colorectal cancer with extremely high accuracy.

[0108] The diagnostic accuracy of urinary DPEP1 was extremely high in both cohorts, with AUC = 0.802 (Table 1) and AUC = 0.809 (Table 5). The next best candidate, urinary TFF1, also showed high diagnostic accuracy in both cohorts, with AUC = 0.751 (Table 1) and AUC = 0.731 (Table 5).

[0109] <Diagnostic ability of colorectal cancer biomarkers> Table 7 shows examples of cutoff values ​​and diagnostic accuracy of various colorectal cancer biomarkers for stage 0-III colorectal cancer obtained from the analysis results of urinary Cr relative values. Table 8 shows examples of cutoff values ​​and diagnostic accuracy of various colorectal cancer biomarkers for stage 0-III colorectal cancer obtained from the analysis results of absolute values.

[0110] The cut-off values ​​shown in the table are merely examples applied in this analysis. The cut-off values ​​used in actual testing sites may not necessarily be the values ​​shown in the table below. The same applies to Tables 9 and 10 described below.

[0111] In this analysis, the cutoff value was calculated as follows. First, the values ​​of (sensitivity, specificity, 1 - specificity) were calculated for each factor value, and an ROC curve was drawn. The Youden Index of the ROC curve was used as the cutoff value. The Youden Index is the point at which the value of (sensitivity + specificity - 1) is maximized. Logistic regression analysis was used to calculate the cutoff value for a combination of two or more factors. A formula for predicting colorectal cancer was created using the combined factors as explanatory variables, and the predicted value was calculated. Similarly, the sensitivity, specificity, and 1 - specificity were calculated for each predicted value, an ROC curve was drawn, and the Youden Index was used as the cutoff value. However, the cutoff value used in actual testing situations is not necessarily limited to the Youden Index. The cutoff value can be set taking into account the purpose of colorectal cancer screening. For example, it may be preferable to set the cutoff value by prioritizing improved sensitivity.

[0112]

[0113]

[0114] As shown in Tables 7 and 8, when urinary DPEP1 was used alone, the sensitivity and specificity of the urinary Cr relative value were 80.4% and 64.5%, respectively, and the sensitivity and specificity of the absolute value were 85.4% and 64.5%, respectively. It is believed that urinary DPEP1 can determine colorectal cancer with excellent sensitivity and specificity.

[0115] Furthermore, the 16 types of urinary proteins can be used for diagnosis even when two or more types are combined. For example, various combination patterns centered on DPEP1 or TFF1 can be considered. Examples of such combination patterns and their sensitivity and specificity are shown in Tables 7 and 8. A combination centered on two proteins, urinary DPEP1 and TFF1, which are reproducible under all conditions, showed high AUCs of 0.846 in urinary Cr relative value and 0.870 in absolute value. In addition, the urinary Cr relative value showed high diagnostic accuracy with a sensitivity of 91.1% and a specificity of 62.8%, and absolute values ​​of 94.3% and 63.4%.

[0116] <Diagnostic ability in early colorectal cancer> Table 9 shows examples of cutoff values ​​and diagnostic accuracy of various colorectal cancer biomarkers for stage 0 / I colorectal cancer obtained from the analysis results of urinary Cr relative values. Table 10 shows examples of cutoff values ​​and diagnostic accuracy of various colorectal cancer biomarkers for stage 0 / I colorectal cancer obtained from the analysis results of absolute values.

[0117] The calculation methods for the cutoff values ​​shown in Tables 9 and 10 are the same as those shown in Tables 7 and 8, respectively. That is, the Youden Index of the ROC curve was used as the cutoff value. Furthermore, logistic regression analysis was used to calculate the cutoff value based on a combination of two or more factors. However, the cutoff values ​​used in actual testing are not necessarily limited to the values ​​shown in Tables 9 and 10. The cutoff value can be set taking into account the purpose of colorectal cancer screening. For example, it may be preferable to set the cutoff value by prioritizing improved sensitivity.

[0118]

[0119]

[0120] The established colon cancer biomarkers enable highly accurate diagnosis of early stage colon cancer (stage 0 / I), which can be resected by endoscopic treatment. This is particularly noteworthy. As shown in Figures 4-11, the expression levels of four urinary proteins (DPEP1, TFF1, ANPEP, and LGALS3), which show excellent sensitivity and specificity when used alone, were higher for each protein from stage 0 compared to healthy subjects. In addition, the expression levels of each protein remained higher in stages I-III compared to healthy subjects. Moreover, the graphs for each protein showed trends suitable for diagnosing early stage colon cancer.

[0121] The AUC for diagnosing early-stage colorectal cancer was 0.780 for relative urinary Cr values ​​and 0.842 for absolute values ​​for DPEP1 alone, and 0.792 for relative urinary Cr values ​​and 0.852 for absolute values ​​for DPEP1 + TFF1 (Tables 9 and 10).

[0122] <Diagnostic Ability for Adenoma> The concentrations of DPEP1 and TFF1 were measured in urine samples collected from 172 healthy subjects, 159 colorectal cancer patients, and 31 adenoma patients. The results are shown in Figures 12 to 19 and Tables 11 and 12. Table 11 shows the relative urinary Cr values ​​standardized by dividing each urinary protein concentration by the urinary creatinine concentration. Table 12 shows the absolute values ​​of each urinary protein concentration.

[0123]

[0124]

[0125] The expression of DPEP1 and TFF1 in urine samples was found to be significantly higher in adenoma patients than in healthy individuals. These results suggest the usefulness of this method for detecting adenomas as well as early-stage colorectal cancer.

[0126] Next, ROC curves were created to determine the presence or absence of adenoma from the measurement results of each urinary protein concentration ( Figures 20 and 21 ). Figure 20 shows an ROC curve based on the urinary Cr relative value, which was standardized by dividing each urinary protein concentration by the urinary creatinine concentration, and Figure 21 shows an ROC curve based on the absolute value of each urinary protein concentration. For the ROC curve of the urinary Cr relative value in Figure 20 , the AUC of DPEP1 was 0.618, and the AUC of TFF1 was 0.684. For the ROC curve of the absolute value in Figure 21 , the AUC of DPEP1 was 0.641, and the AUC of TFF1 was 0.668.

[0127] <Study of serum samples> The concentrations of DPEP1 and TFF1 were measured in serum samples collected from 68 healthy subjects and 68 colon cancer patients. The results are shown in Table 13. Table 13 shows the absolute values ​​of each urinary protein concentration. Figure 22 shows an ROC curve drawn based on the measured values ​​of each protein concentration. The AUC value was obtained from this ROC curve.

[0128]

[0129] Based on the above results, it is believed that a new and revolutionary colon cancer biomarker can be provided that can diagnose even early-stage colon cancer with high accuracy and non-invasiveness. High-accuracy, non-invasive diagnosis of early-stage colon cancer is difficult to achieve using existing fecal occult blood or serum tumor markers. It is expected that the use of the colon cancer biomarker of the present invention will make it possible to provide not only test kits for use in facilities, but also simple kits that can be used at home.

[0130] According to the present invention, a colorectal cancer biomarker is provided that can easily and non-invasively determine the presence or absence of colorectal cancer with excellent sensitivity and specificity.

Claims

1. A method for supporting colorectal cancer screening, A method comprising detecting at least one urinary protein selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, MME, and ACP2.

2. A method for supporting the diagnosis of colorectal cancer, A method comprising detecting at least one urinary protein selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, MME, and ACP2.

3. If the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, and MME, and the concentration of the detected urinary protein is above the cutoff value, it is determined that the subject has colorectal cancer. The method according to claim 1 or 2, further comprising determining that the subject has colorectal cancer if the urinary protein is ACP2 and the concentration of the detected urinary protein is below a cutoff value.

4. The method according to claim 1 or 2, wherein the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C.

5. The aforementioned urinary protein is DPEP1, At least one selected from the group consisting of TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C, The method according to claim 1 or 2, which is a combination of the two.

6. The aforementioned urinary protein is TFF1, At least one selected from the group consisting of DPEP1, ANPEP, LGALS3, PRG4, PIGR and TNFRSF10C, The method according to claim 1 or 2, which is a combination of the two.

7. The method according to claim 1 or 2, wherein the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, and LGALS3.

8. The method according to claim 1 or 2, wherein the colorectal cancer is early-stage colorectal cancer.

9. A kit used for screening for colorectal cancer, A container for collecting the subject's urine, A reagent for detecting at least one urinary protein selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, MME, and ACP2, A colorectal cancer screening kit equipped with [features / equipment].

10. The colorectal cancer screening kit according to claim 9, wherein the reagent is either one or both of the following reagents α and β. Reagent α: A reagent that, if the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, CDH17, PIGR, TNFRSF10C, MEP1A, LGALS3BP, APOA1, LRG1, ENPEP, S100A11, and MME, and the concentration of the detected urinary protein is above a threshold, notifies the subject of colorectal cancer by a qualitative method. Reagent β: If the urinary protein is ACP2, and the concentration of the detected urinary protein is below a threshold, this reagent qualitatively indicates that the subject has colorectal cancer.

11. The colorectal cancer screening kit according to claim 9 or 10, wherein the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C.

12. The aforementioned urinary protein is DPEP1, At least one selected from the group consisting of TFF1, ANPEP, LGALS3, PRG4, PIGR, and TNFRSF10C, A colorectal cancer screening kit according to claim 9 or 10, which is a combination of the above.

13. The aforementioned urinary protein is TFF1, At least one selected from the group consisting of DPEP1, ANPEP, LGALS3, PRG4, PIGR and TNFRSF10C, A colorectal cancer screening kit according to claim 9 or 10, which is a combination of the above.

14. The colorectal cancer screening kit according to claim 9 or 10, wherein the urinary protein is at least one selected from the group consisting of DPEP1, TFF1, ANPEP, and LGALS3.

15. The colorectal cancer screening kit according to claim 9 or 10, wherein the colorectal cancer is early-stage colorectal cancer.