Application of reagent for detecting blood metabolites in preparation of product for diagnosing colorectal cancer

Abstract

The invention belongs to the technical field of disease diagnosis reagents, and particularly relates to application of a reagent for detecting blood metabolites in preparation of a product for diagnosing colorectal cancer, and the blood metabolites comprise one or more of BNO3, BP01, TLCA or LCA. The blood metabolite can be used for distinguishing healthy people from colorectal lesion patients and can be used as a colorectal cancer diagnosis marker.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease diagnostic reagents, and particularly relates to the application of a reagent for detecting blood metabolites in the preparation of a product for diagnosing colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is a common malignant tumor and one of the main causes of death among cancer patients. Currently, colonoscopy is the main method for diagnosing and screening CRC, but its invasiveness and patient compliance limit its widespread application. The fecal occult blood test is a simple and rapid CRC screening item, and CEA is also a commonly used CRC serological index, but their sensitivity and specificity still need to be improved.

[0003] With the development of metabolomics analysis technology, metabolite markers identified by liquid chromatography-mass spectrometry (LC-MS / MS), such as lipid metabolites like fatty acids and bile acids, are expected to become new indicators for CRC.

[0004] Due to the better advantages of blood metabolites compared to fecal metabolites in terms of sample collection and direct influence of diet, the clinical application of blood metabolites in CRC has received more attention. Summary of the Invention

[0005] The present invention discovers that there is a relationship between blood metabolites (one or more of BN03 (9,12,13-trihydroxy-9-octadecenoic acid lactone), BP01 (myristoyl-L-carnitine), TLCA (taurolithocholic acid), or LCA (lithocholic acid)) and the occurrence and development of colorectal cancer, and they can be used as markers for diagnosing colorectal cancer.

[0006] To achieve the above object, the present invention can adopt the following technical solutions:

[0007] On the one hand, the present invention provides the application of a reagent for detecting blood metabolites in the preparation of a product for diagnosing colorectal cancer, and the blood metabolites include one or more of BN03 (9,12,13-trihydroxy-9-octadecenoic acid lactone), BP01 (myristoyl-L-carnitine), TLCA (taurolithocholic acid), or LCA (lithocholic acid).

[0008] Preferably, in the above application, the reagent for detecting blood metabolites is a reagent based on the LC-MS / MS detection method.

[0009] Preferably, in the above application, the blood metabolites include BN03 (9,12,13-trihydroxy-9-octadecenoic acid lactone) and LCA (lithocholic acid).

[0010] Preferably, in the above application, the colorectal cancer is in the abnormal colorectal stage, stage I of colorectal cancer or stage II of colorectal cancer.

[0011] Preferably, in the above application, the product is a detection kit or a detection reagent.

[0012] The present invention also provides a system for diagnosing colorectal cancer, comprising:

[0013] An analysis unit for obtaining the results of biomarkers of a subject, the biomarkers including one or more of BN03 (9,12,13-trihydroxy-9-octadecenoic acid lactone), BP01 (myristoyl-L-carnitine), TLCA (taurolithocholic acid) or LCA (lithocholic acid);

[0014] An evaluation unit for assigning corresponding evaluation scores to obtain a total score according to the obtained results of the biomarkers; an output unit for outputting the colorectal cancer diagnosis of the subject according to the total score obtained by the evaluation unit

[0015] Preferably, in the above system, the biomarkers are BN03 (9,12,13-trihydroxy-9-octadecenoic acid lactone), BP01 (myristoyl-L-carnitine), TLCA (taurolithocholic acid) and LCA (lithocholic acid); the total score y = (-8.24) + 2.0456×BN03 + 0.0730×BP01 + (-0.0078)×LCA + (-0.2206)×TLCA.

[0016] Preferably, the biomarkers in the above system further include CEA (carcinoembryonic antigen).

[0017] Preferably, in the above system, the colorectal cancer is in the abnormal colorectal stage, stage I of colorectal cancer or stage II of colorectal cancer.

[0018] The beneficial effects of the present invention include:

[0019] (1) When BN03 (9,12,13-trihydroxy-9-octadecenoic acid lactone) is used as a biomarker for diagnosing colorectal cancer, the area under the curve for distinguishing colorectal lesions and apparently healthy individuals is 0.930 (95% CI: 0.899 - 0.962), and the area under the curve for distinguishing early colorectal cancer, high-risk adenomas from low-risk adenomas and apparently healthy individuals is 0.882 (95% CI: 0.836 - 0.927). When the cut-off value is 3.865 ng / ml, its sensitivity and specificity are 85.62% and 91.95% respectively, which are higher than the AUC of carcinoembryonic antigen (CEA) (0.778, 95% CI: 0.718–0.838).

[0020] (2) When BN03 (9,12,13 - trihydroxy - 9 - octadecenoic acid lactone) is combined as a CEA for diagnosing colorectal cancer, the sensitivity and specificity reach 89.31% and 85.87% respectively.

[0021] (3) When LCA (lithocholic acid) is used as a marker for diagnosing colorectal cancer, when LCA exceeds 88.09 ng / mL, the sensitivity is 50.33% and the specificity is 96.55%.

[0022] (4) When BN03 (9,12,13 - trihydroxy - 9 - octadecenoic acid lactone) is combined with LCA (lithocholic acid) as a marker for diagnosing colorectal cancer, the sensitivity and specificity are increased to 86.93% and 89.66%, which is better than using LCA alone as a marker. Description of the Drawings

[0023] Figure 1 Shows the concentrations of BN03, BP01, TLCA, and LCA in CRC patients;

[0024] Figure 2 Shows the detection of different marker combinations;

[0025] Figure 3 Shows the metabolic models of patients with colorectal abnormalities and healthy individuals. Detailed Embodiments

[0026] The examples given are for better illustration of the present invention, but the content of the present invention is not limited only to the examples given. Therefore, those skilled in the art who make non - essential improvements and adjustments to the implementation solutions based on the above - mentioned invention content still fall within the protection scope of the present invention.

[0027] The terms used herein are only for describing specific embodiments and are not intended to limit the present disclosure. Unless there is an obvious different meaning in the context, the singular form of the expression includes the plural form of the expression. As used herein, it should be understood that terms such as "including", "having", "containing" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations. The terms of the present invention are disclosed in the specification, and it is not intended to exclude the possibility of the existence or addition of one or more other features, numbers, operations, components, parts, elements, materials, or their combinations. As used herein, depending on the context, " / " can be interpreted as "and" or "or".

[0028] An embodiment of the present invention provides an application of a reagent for detecting blood metabolites in the preparation of a product for diagnosing colorectal cancer, and the blood metabolites include one or more of BN03 (9,12,13-trihydroxy-9-octadecenoic acid lactone), BP01 (myristoyl-L-carnitine), TLCA (taurolithocholic acid), or LCA (lithocholic acid).

[0029] It should be noted that the present invention has found that compared with healthy individuals, the concentrations of BN03, BP01, TLCA, and LCA in CRC (colorectal cancer) patients are significantly increased, indicating that BN03, BP01, TLCA, and LCA can be used as markers for diagnosing colorectal cancer; among them, BN03 shows the best performance, and its area under the curve for distinguishing colorectal lesions and apparently healthy individuals is 0.930 (95% CI: 0.899 - 0.962), and the area under the curve for distinguishing early colorectal cancer (including stage I and stage II colorectal cancer), high-risk adenomas (high-risk adenomas can be considered pre-cancerous lesions), low-risk adenomas (low-risk adenomas have a certain possibility of canceration), and apparently healthy individuals is 0.882 (95% CI: 0.836 - 0.927). When the cut-off value is 3.865 ng / ml, its sensitivity and specificity are 85.62% and 91.95% respectively. In addition, the present invention has established a method for detecting lipid metabolites in serum based on LC-MS / MS and confirmed that the detection performance meets the requirements of CLSI.

[0030] In some specific examples, in the above application, the reagent for detecting blood metabolites is a reagent based on the LC-MS / MS detection method.

[0031] It should be noted that the reagent for detecting blood metabolites in the present invention is all the reagents well-known in the art that can detect the above blood metabolites in a blood sample, such as reagents based on the LC-MS / MS detection method, such as mobile phases or buffers, etc.

[0032] It should also be noted that LC-MS / MS is a commonly used method for metabolite analysis and can simultaneously measure multiple metabolites; the sensitivity of the blood metabolites identified by LC-MS / MS for CRC diagnosis can reach 93.6%, and the specificity can reach 80.2%.

[0033] In some specific examples, in the above application, the blood metabolites include BN03 (9,12,13-trihydroxy-9-octadecenoic acid lactone) and LCA (lithocholic acid).

[0034] It should be noted that the present invention has found that when BN03 and LCA are combined as markers for diagnosing colorectal cancer, the sensitivity and specificity are increased to 86.93% and 89.66%, which is better than using LCA alone as a marker.

[0035] In some specific examples, in the above application, the colorectal cancer is in the abnormal colorectal period, stage I of colorectal cancer, or stage II of colorectal cancer.

[0036] In some specific examples, in the above application, the product is a detection kit or a detection reagent.

[0037] It should be noted that the product for diagnosing colorectal cancer in the present invention can be a detection kit or a detection reagent, generally a detection kit, which is more convenient for transportation and storage.

[0038] The embodiment of the present invention also provides a system for diagnosing colorectal cancer, including:

[0039] An analysis unit for obtaining the results of biomarkers of a subject, where the biomarkers include one or more of BN03 (9,12,13-trihydroxy-9-octadecenoic acid lactone), BP01 (myristoyl-L-carnitine), TLCA (taurolithocholic acid), or LCA (lithocholic acid);

[0040] An evaluation unit for assigning corresponding evaluation scores according to the obtained results of the biomarkers to obtain a total score; an output unit for outputting the colorectal cancer diagnosis situation of the subject according to the total score obtained by the evaluation unit.

[0041] In some specific examples, in the above system, the biomarkers are BN03 (9,12,13-trihydroxy-9-octadecenoic acid lactone), BP01 (myristoyl-L-carnitine), TLCA (taurolithocholic acid), and LCA (lithocholic acid); the total score y = (-8.24) + 2.0456×BN03 + 0.0730×BP01 + (-0.0078)×LCA + (-0.2206)×TLCA.

[0042] In some specific examples, the biomarker in the above system further includes CEA.

[0043] It should be noted that when the CEA cut-off value is 5 ng / ml, the sensitivity and specificity are 33.33% and 98.85% respectively; after combining the BN03 and CEA indicators, the sensitivity and specificity reach 89.31% and 85.87% respectively.

[0044] It should also be noted that the present invention established a method for detecting lipid metabolites in serum based on LC-MS / MS and confirmed that the detection performance meets the requirements of CLSI, and found that the combined detection of BN03 and CEA indicators can improve the detection sensitivity.

[0045] In some specific examples, the colorectal cancer in the above system is in the abnormal colorectal period, stage I of colorectal cancer, or stage II of colorectal cancer.

[0046] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with specific examples, but the content of the present invention is not limited to the following examples only.

[0047] In the following examples, high-performance liquid chromatography (HPLC)-grade acetonitrile (ACN), methanol (MeOH), isopropanol (IPA), ammonium formate, and formic acid (FA) were purchased from Thermo Fisher. Deionized water (>18.2 MΩ·cm) was from Watson; glycocholic acid (GLCA), glycochenodeoxycholic acid (GCA), deoxycholic acid (DCA), cholic acid (CA), tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), taurodeoxycholic acid (TCA), glycoursodeoxycholic acid (GCDCA), taurochenodeoxycholic acid (TCDCA), glycochenodeoxycholic acid monohydrate (GDCA), lithocholic acid (LCA), chenodeoxycholic acid (CDCA), glycoursodeoxycholic acid (GUDCA), (±)15-HETE (BN01), 12,13-DiHome (BN02), 9,12,13-TriHOME (BN03), myristoyl-L-carnitine (BP01), trans-2-octanoyl-L-carnitine (BP02), trans-2-hexadecenoyl-L-carnitine (BP03), and (±)-hexenoyl chloride (BP04) were from Shanghai Yuanye Biotechnology, Alfa Aesar, National Institutes for Food and Drug Control, zzstandard, BePuro, ISOREAG, MCE, sigma, and TRC respectively; purchased from ISOREAG and BePure.

[0048] In the following examples, the 22 blood metabolites were GLCA, GCA, DCA, CA, TUDCA, UDCA, TCA, TDCA, GCDCA, TCDCA, GDCA, LCA, CDCA, GUDCA, TLCA, BN01, BN02, BN03, BP01, BP02, BP03, and BP04; the specific information is shown in Table 1 below.

[0049] Table 1 Information of 22 blood metabolites

[0050] Metabolite English name Chinese name GLCA Lithocholylglycine Lithocholylglycine GCA glycocholicacid Glycocholic acid DCA deoxycholicacid Deoxycholic acid CA cholicacid Cholic acid TUDCA tauroursodeoxycholicacid Tauroursodeoxycholic acid UDCA ursodeoxycholicacid Ursodeoxycholic acid TCA taurocholicacid Taurocholic acid TDCA taurodeoxycholicacid Taurodeoxycholic acid GCDCA glycochenodeoxycholicacid Glycochenodeoxycholic acid TCDCA taurochenodeoxycholicacid Taurochenodeoxycholic acid GDCA glycodeoxycholicacidmonohydrate Glycodeoxycholic acid monohydrate LCA lithocholicacid Lithocholic acid CDCA chenodeoxycholicacid Chenodeoxycholic acid GUDCA glycoursodeoxycholicacid Glycoursodeoxycholic acid TLCA Taurolithocholicacid Taurolithocholic acid BN01 (±)15-HETE 15-Hydroxyeicosatetraenoic acid BN02 12,13-DiHOME 12,13-Dihydroxy-9-octadecenoic acid lactone BN03 9,12,13-TriHOME 9,12,13-Trihydroxy-9-octadecenoic acid lactone BP01 myristoyl-L-carnitine Myristoyl-L-carnitine BP02 trans-2-octenoyl-L-carnitine trans-2-Octenoyl-L-carnitine BP03 trans-2-hexadecenoy-1-L-carnitine trans-2-Hexadecenoyl-L-carnitine BP04 (±)-hexanoylcamitinechloride (±)-Hexanoyl carnitine chloride

[0051] In the following examples, the serum metabolites were detected according to the following steps:

[0052] (1) Sample preparation: Serum metabolite extraction: Add 10 μL of internal standard mixture (the internal standard mixture of the above metabolites, which is the isotope-labeled substance corresponding to these compounds) to 80 μL of serum, then add 150 μL of a mixed solution of acetonitrile and isopropanol (the volume ratio of acetonitrile to isopropanol is 4:1, ThermoFisher) and 50 μL of ammonium formate (0.5 g / mL), vortex centrifuge at 17949×g for 10 min, and use 150 μL of HPLC-grade water to dilute 60 μL of the supernatant before use.

[0053] (2) Liquid chromatography-tandem mass spectrometry analysis procedure: LC-MS / MS uses an ABSCIEXTripleQuad TM 4500 system; the injection volume for each mode is 10 μL; by infusing the corresponding standards, electrospray ionization (ESI) is carried out in positive and negative ion modes to optimize the mass spectrometer parameters of each metabolite (see Table 2 below). Serum metabolites are eluted from a Shim-packvelox column (C18, 2.7 μm, 50 x 2.1 mm) at a flow rate of 0.075 mL / min; mobile phase A (water containing 0.1% formic acid) and mobile phase B (acetonitrile containing 0.1% formic acid) are gradient eluted: 0 - 0.2 min: 25% B phase, 0.2 - 0.8 min: 25% - 40% B phase, 0.8 - 2 min: 40% - 45% B phase, 2 - 2.5 min: 45% - 60% B phase, 2.5 min - 3.6 min: 60% - 70% B phase,

[0054] 3.6 min - 5 min: 70% - 98% B phase, 5 min - 5.1 min: 98% - 25% B phase; The metabolite peaks are integrated using SciexAnalyst 1.6.3 software and multiQuant 3.0.2 software.

[0055] Table 2 Mass spectrometer parameters for each metabolite

[0056]

[0057]

[0058] In the following examples, the statistical analysis is as follows: Data with a normal distribution are shown as mean ± standard deviation, and results with a non-normal distribution are shown as median ± interquartile range; The Shapiro-Wilk test is used for normal distribution. For pairwise comparisons among three groups with a non-normal distribution, the Kruskal-Wallis test is used, and a post hoc Bonferroni correction is performed; GraphPad Prism (9.5) or R language is used for statistical analysis and curve plotting; *, p < 0.05; **, p < 0.01; ***, p < 0.001; n, no significance.

[0059] In the following example, serum metabolite extraction includes: adding 10 μL of internal standard mixture into 80 μL of serum (or blank matrix plasma), adding 150 μL of acetonitrile: isopropanol (volume ratio 4:1, Thermo Fisher) and 50 μL of ammonium formate (0.5 g / mL), vortexing and centrifuging at 17949×g for 10 min, and diluting 60 μL of the supernatant with 150 μL of HPLC-grade water before use.

[0060] I. Biomarker Screening

[0061] In the present invention, serum specimens of 50 colorectal cancer patients and healthy controls who visited the Cancer Institute and Hospital, Chinese Academy of Medical Sciences (CICAMS) during the period from March 2024 to July 2024 were detected for blood metabolites according to the following method. It was found that the concentrations of BN03, BP01, TLCA, and LCA in CRC patients were significantly higher than those in healthy individuals, indicating that BN03, BP01, TLCA, and LCA have the potential to be used as biomarkers for diagnosing CRC patients.

[0062] II. Biomarker Validation

[0063] (I) Clinical Specimens

[0064] Serum specimens of 247 colorectal cancer patients and healthy controls who visited the Cancer Institute and Hospital, Chinese Academy of Medical Sciences (CICAMS) during the period from March 2024 to July 2024 (different from the 50 samples in the above biomarker screening) were collected, stored at -80 °C for later detection, and the CEA test results of each individual were recorded. All colorectal cancer patients were untreated newly diagnosed patients and pathologically confirmed, excluding patients with secondary colorectal cancer and those with other tumors or medical histories. The baseline characteristics of all patients and apparent healthy controls are shown in Table 3.

[0065] Table 3 Baseline Characteristics of All Patients and Apparent Healthy Controls

[0066] CRC(N=117) CRA(N=36) NC(N=87) Gender Male 75 18 48 Female 42 18 39 Age(Average±SD) 61.55±11.33 64.08±10.11 52.79±12.59 Stage I-II 28 III-IV 32 unknow 57

[0067] The concentrations of 18 blood metabolites (BN02, BN03, BP01, BP04, CA, DCA, CDCA, GCA, GUDCA, GLCA, TCA, TDCA, TCDCA, TUDCA, and TLCA) in 247 samples were detected according to the above serum metabolite detection method. The results showed that these serum metabolites all met the standards of linear range, lower limit of quantitation, precision, and accuracy; and, compared with healthy individuals, the concentrations of BN03, BP01, TLCA, and LCA in CRC patients were significantly higher (P < 0.05, see Table 4 and Figure 1) It is illustrated that BN03, BP01, TLCA and LCA can be used as markers for diagnosing CRC patients.

[0068] Table 4 Concentrations of BN03, BP01, TLCA and LCA in CRC patients

[0069] Blood metabolite NC AA CRC BN03 2.05-5.26 5.77(4.10-10.68) 7.65(4.29-10.72) BP01 4.42-15.27 12.23(7.79-16.07) 11.16(7.87-14.35) TLCA 0.00-4.61 0.98(0.00-1.51) 1.24(0.40-1.87) LCA 0.00-121.40 40.76(0.00-325.50) 103.80(22.06-346.40)

[0070] (II) AUC value calculation

[0071] The AUC values of the above four markers (BN03, BP01, TLCA and LCA) were analyzed respectively. The results showed that among these four differential metabolites, the area under the curve (AUC) of BN03 in distinguishing colorectal abnormalities from normal controls (NC) was 0.930 (95% CI: 0.899–0.962), which was higher than the AUC of carcinoembryonic antigen (CEA) (0.778, 95% CI: 0.718–0.838)( Figure 2 A); In addition, the AUC of BN03 in distinguishing early CRC (stage I / II) from NC was 0.825 (95% CI: 0.759–0.869)( Figure 2 B). In addition, adenomas can be divided into high-grade adenomas and low-grade adenomas. Among them, patients with high-grade adenomas are more likely to progress to CRC and require more clinical intervention. When distinguishing colorectal abnormalities from NC, the AUC of BN03 was 0.882 (95% CI: 0.759–0.869), which was better than that of CEA (AUC: 0.783, 95% CI: 0.723–0.842)( Figure 2 C).

[0072] (III) Combined analysis of metabolites and CEA based on LC-MS / MS technology

[0073] To further explore the clinical effect of combining BN03 with other indicators, the ability of BN03, LCA and CEA to distinguish colorectal abnormalities from healthy individuals at their respective cut-off values was analyzed. The results showed that:

[0074] As Figure 2 shown in D, when BN03 was greater than the cut-off value of 3.865 ng / mL, the sensitivity was 85.62% and the specificity was 91.95%; when LCA exceeded 88.09 ng / mL, the sensitivity was 50.33% and the specificity was 96.55%; when CEA was at the cut-off value of 5 ng / mL, the sensitivity was 33.33% and the specificity was 98.85%; The combined analysis of BN03 and LCA increased the sensitivity and specificity to 86.93% and 89.66%, which was better than using LCA alone; when BN03 was combined with CEA, the sensitivity was 89.31% and the specificity was 85.87%.

[0075] In addition, as Figure 2 shown in E, among 86 CRC patients with negative CEA, 72 showed elevated BN03 levels; among 22 CRC patients with negative BN03, 8 showed elevated CEA levels. Therefore, the combined analysis of BN03 and CEA improves the sensitivity and specificity of CRC detection.

[0076] (IV) Construction of metabolic characteristics

[0077] A metabolic model was constructed using LASSO regression analysis to distinguish colorectal abnormal patients from healthy individuals ( Figure 3 ); the formula of the metabolic model is as follows: y = (-8.24) + 2.0456×BN03 + 0.0730×BP01 + (-0.0078)×LCA + (-0.2206)×TLCA; where, in the formula, BN03, BP01, LCA, and TLCA are the corresponding detected concentrations; the coefficients of each metabolite were calculated using the R software package "glmnet".

[0078] The AUC of this model for distinguishing colorectal abnormalities from NC was 0.939 (95% CI: 0.907–0.970), the sensitivity was 92.00%, and the specificity was 87.00%. In addition, the AUC of the combined analysis of the model constructed with 4 metabolites and CEA was 0.969 (95% CI: 0.949–0.988), the sensitivity was 92.00%, and the specificity was 90.70%. These results indicate that the combined analysis of CRC-related blood metabolites quantified by LC-MS / MS technology can improve the accuracy of colorectal abnormality diagnosis.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. Use of a reagent for detecting blood metabolites in the preparation of a product for diagnosing colorectal cancer, wherein the blood metabolites include one or more of BN03 (9,12,13-trihydroxy-9-octadecenoic acid lactone), BP01 (myristoyl L-carnitine), TLCA (taurolithocholic acid) or LCA (lithocholic acid).

2. The use according to claim 1, characterized in that: The reagent for detecting blood metabolites is a reagent based on the LC-MS / MS detection method.

3. The use according to claim 1 or 2, characterized in that: Blood metabolites include BN03 (9,12,13-trihydroxy-9-octadecenoic acid lactone) and LCA (lithocholic acid).

4. The use according to claim 1 or 2, characterized in that: Colorectal cancer is stage abnormal colorectal cancer, stage I colorectal cancer, or stage II colorectal cancer.

5. The use according to claim 1 or 2, characterized in that: The product is a test kit or test reagent.

6. A system for diagnosing colorectal cancer, characterized in that: include: An analysis unit, for obtaining a result of a biomarker of a subject, wherein the biomarker comprises one or more of BN03 (9,12,13-trihydroxy-9-octadecenoic acid lactone), BP01 (myristoyl L-carnitine), TLCA (taurolithocholic acid) or LCA (lithocholic acid); An evaluation unit, configured to assign corresponding evaluation scores to obtain a total score according to the results of obtaining the markers; The output unit is used to output the colorectal cancer diagnosis of the subject according to the total score obtained by the evaluation unit.

7. The system according to claim 6, characterized in that The biomarkers are BN03 (9,12,13-trihydroxy-9-octadecenoic acid lactone), BP01 (myristoyl L-carnitine), TLCA (taurolithocholic acid) and LCA (lithocholic acid); the total score y = (-8.24) + 2.0456 × BN03 + 0.0730 × BP01 + (-0.0078) × LCA + (-0.2206) × TLCA.

8. The system according to claim 6 or 7, characterized in that: Biomarkers also include CEA.

9. The system according to claim 6 or 7, characterized in that: Colorectal cancer is stage abnormal colorectal cancer, stage I colorectal cancer, or stage II colorectal cancer.

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