Metabolic markers for early auxiliary diagnosis of colorectal cancer and their applications

Abstract

This invention discloses metabolic markers for the early auxiliary diagnosis of colorectal cancer in the field of biomedical technology and their applications. The development and utilization of such metabolic markers will provide technical support for the early auxiliary diagnosis of colorectal cancer. Using a non-targeted metabolomics detection method, this invention screened and verified the application value of 10 colorectal cancer metabolites in differentiating colorectal cancer patients from healthy controls, and the application value of 7 metabolites in differentiating precancerous lesions from healthy controls, which can significantly improve the level of early diagnosis of colorectal cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to metabolic markers for early auxiliary diagnosis of colorectal cancer and applications thereof. Background Art

[0002] Colorectal cancer is the third most common malignant tumor worldwide. According to the latest data from the International Agency for Research on Cancer, there are 1.93 million new cases of colorectal cancer and 940,000 deaths from colorectal cancer each year worldwide. In my country, the incidence and mortality rates of colorectal cancer have jumped to second and fourth place among malignant tumors, respectively. New cases increased from 388,000 in 2015 to 555,000 in 2020, climbing rapidly at an annual rate of 7.4%. Furthermore, the incidence and mortality rates of colorectal cancer are trending younger, placing a heavy burden on society and families. Effectively curbing the incidence and mortality of colorectal cancer is key to achieving the goal of "increasing the overall five-year cancer survival rate by 15%" as outlined in the "Healthy China 2030" plan.

[0003] Due to the insidious onset of colorectal cancer, 85% of patients are already in the middle or late stages at the time of clinical diagnosis, resulting in poor treatment efficacy. The five-year survival rate for patients in the late stages is less than 15%. In comparison, the five-year survival rate for patients with early-stage colorectal cancer can reach over 90%. Therefore, timely detection and elimination of early cancer and precancerous lesions through population screening is a key measure to reduce the morbidity and mortality of colorectal cancer. However, current screening methods for colorectal cancer mainly include fecal occult blood testing (FOBT) and colonoscopy. The FOBT has poor specificity and low sensitivity for detecting early cancer and precancerous lesions (reported in the literature to be less than 40%). The FOBT is invasive and painful for the patient, leading to low compliance. It also requires dietary and bowel preparation, is time-consuming and labor-intensive, and is more likely to be accompanied by complications (such as bleeding and perforation). Furthermore, serum tumor markers such as CEA and CA19-9 have poor sensitivity and specificity for the early diagnosis of colorectal cancer (reported in the literature to be less than 20%), making them only suitable for clinical diagnosis and treatment monitoring of colorectal cancer. Therefore, new non-invasive methods with high sensitivity and specificity are needed to improve the early detection of colorectal cancer.

[0004] Metabolomics is a new omics technology that has emerged after genomics, transcriptomics, and proteomics. It can quantitatively analyze thousands of intermediates and end products involved in biochemical reactions within organisms and has widespread applications in fields such as etiology, diagnostics, biological function research, and drug development. Compared with other omics research methods, metabolomics has the following advantages: ① The metabolome is downstream of the regulation of life networks, and the characteristics it reflects are closer to changes in disease phenotypes; ② Small changes in gene and protein expression at the functional level can be amplified by metabolites, and metabolite detection can more easily identify key molecular events that lead to disease; ⑤ The samples used for measurement can be biological fluids (such as blood and urine), which are easily accessible and easy to promote. Therefore, metabolomics technology can help discover early events in colorectal cancer and identify biomarkers with population-wide application value, thereby improving the early diagnosis of colorectal cancer.

[0005] Previous plasma metabolomics studies of colorectal cancer have been limited by small sample sizes or lack of external validation, and few studies have examined metabolic markers in patients with precancerous lesions, resulting in a lack of high-quality theoretical and technical support for translational applications. Therefore, systematic screening and external validation of auxiliary diagnostic markers for colorectal cancer and precancerous lesions using high-quality population-based sample resources, including patients with colorectal cancer and precancerous lesions, and employing non-targeted research strategies are crucial for achieving a dual reduction in colorectal cancer morbidity and mortality in my country. Summary of the Invention

[0006] To address the above shortcomings, the present invention provides a class of plasma metabolites that assist in the early diagnosis of colorectal cancer and their applications. This method can be applied to the detection of new biomarkers related to the early diagnosis of colorectal cancer.

[0007] The technical solutions of the present invention are as follows:

[0008] The first object of the present invention is to provide a marker combination associated with colorectal cancer and / or colorectal precancerous lesions, wherein the marker combination is a combination of one or more of the following 16 compounds:

[0009] (9S,10E,12Z,15Z)-9-Hydroxy-10,12,15-octadecatrienoic acid

[0010] sauroidal acid

[0011] Diacylglycerol trimethylhomoserine (2:0 / 20:1)

[0012] Lysophosphatidylcholine (19:0)

[0013] Lysophosphatidylinositol (18:0 / 0:0)

[0014] Methyl jasmonate

[0015] 3,4-Dihydroxybenzoic acid

[0016] 2-Hydroxyxanthone

[0017] Ceramide (d14:2 / 13:1)

[0018] 4-Methyl-2-oxopentanoic acid

[0019] Acylcarnitines (18:3)

[0020] Palmitoylethanolamide

[0021] Phytosphingosine

[0022] Phosphatidylinositol (16:0 / 18:3)

[0023] 14-Hydroxysteroids

[0024] Sulfatide (d29:1).

[0025] Furthermore, the marker combination associated with colorectal cancer is a combination of one or more of the following 10 compounds:

[0026] (9S,10E,12Z,15Z)-9-Hydroxy-10,12,15-octadecatrienoic acid

[0027] sauroidal acid

[0028] Diacylglycerol trimethylhomoserine (2:0 / 20:1)

[0029] Lysophosphatidylcholine (19:0)

[0030] Lysophosphatidylinositol (18:0 / 0:0)

[0031] Methyl jasmonate

[0032] 3,4-Dihydroxybenzoic acid

[0033] 2-Hydroxyxanthone

[0034] Ceramide (d14:2 / 13:1)

[0035] 4-Methyl-2-oxopentanoic acid.

[0036] Furthermore, the marker combination associated with colorectal precancerous lesions is a combination of one or more of the following seven compounds:

[0037] (9S,10E,12Z,15Z)-9-Hydroxy-10,12,15-octadecatrienoic acid

[0038] Acylcarnitines (18:3)

[0039] Palmitoylethanolamide

[0040] Phytosphingosine

[0041] Phosphatidylinositol (16:0 / 18:3)

[0042] 14-Hydroxysteroids

[0043] Sulfatide (d29:1).

[0044] Furthermore, the colorectal precancerous lesion is an advanced adenoma.

[0045] The second object of the present invention is to provide a product for detecting the aforementioned marker combination for use in preparing a product for diagnosing and / or predicting the risk of colorectal cancer and / or colorectal precancerous lesions.

[0046] Furthermore, the marker combination is derived from plasma.

[0047] The third object of the present invention is to provide the use of the aforementioned marker combination in screening drugs for treating and / or alleviating colorectal cancer and / or colorectal precancerous lesions.

[0048] Furthermore, the marker combination is derived from plasma.

[0049] The fourth object of the present invention is to provide the use of the aforementioned marker combination in the preparation of a detection kit for the diagnosis and / or risk prediction of colorectal cancer and / or colorectal precancerous lesions.

[0050] Furthermore, the marker combination is derived from plasma.

[0051] The present invention also protects the use of a product for detecting a combination of one or more of the following 10 compounds in the preparation of a preparation for colorectal cancer diagnosis and / or risk prediction:

[0052] (9S,10E,12Z,15Z)-9-Hydroxy-10,12,15-octadecatrienoic acid

[0053] sauroidal acid

[0054] Diacylglycerol trimethylhomoserine (2:0 / 20:1)

[0055] Lysophosphatidylcholine (19:0)

[0056] Lysophosphatidylinositol (18:0 / 0:0)

[0057] Methyl jasmonate

[0058] 3,4-Dihydroxybenzoic acid

[0059] 2-Hydroxyxanthone

[0060] Ceramide (d14:2 / 13:1)

[0061] 4-Methyl-2-oxopentanoic acid.

[0062] The present invention also protects the use of one or more combinations of the following 10 compounds in screening drugs for treating and / or alleviating colorectal cancer:

[0063] (9S,10E,12Z,15Z)-9-Hydroxy-10,12,15-octadecatrienoic acid

[0064] sauroidal acid

[0065] Diacylglycerol trimethylhomoserine (2:0 / 20:1)

[0066] Lysophosphatidylcholine (19:0)

[0067] Lysophosphatidylinositol (18:0 / 0:0)

[0068] Methyl jasmonate

[0069] 3,4-Dihydroxybenzoic acid

[0070] 2-Hydroxyxanthone

[0071] Ceramide (d14:2 / 13:1)

[0072] 4-Methyl-2-oxopentanoic acid.

[0073] The present invention also protects the use of a combination of one or more of the following 10 compounds in the preparation of a detection kit for colorectal cancer diagnosis and / or risk prediction:

[0074] (9S,10E,12Z,15Z)-9-Hydroxy-10,12,15-octadecatrienoic acid

[0075] sauroidal acid

[0076] Diacylglycerol trimethylhomoserine (2:0 / 20:1)

[0077] Lysophosphatidylcholine (19:0)

[0078] Lysophosphatidylinositol (18:0 / 0:0)

[0079] Methyl jasmonate

[0080] 3,4-Dihydroxybenzoic acid

[0081] 2-Hydroxyxanthone

[0082] Ceramide (d14:2 / 13:1)

[0083] 4-Methyl-2-oxopentanoic acid.

[0084] The present invention also protects the use of a product for detecting a combination of one or more of the following seven compounds in the preparation of a product for diagnosing and / or predicting the risk of colorectal precancerous lesions:

[0085] (9S,10E,12Z,15Z)-9-Hydroxy-10,12,15-octadecatrienoic acid

[0086] Acylcarnitines (18:3)

[0087] Palmitoylethanolamide

[0088] Phytosphingosine

[0089] Phosphatidylinositol (16:0 / 18:3)

[0090] 14-Hydroxysteroids

[0091] Sulfatide (d29:1).

[0092] The present invention also protects the use of one or more combinations of the following seven compounds in screening drugs for treating and / or alleviating colorectal precancerous lesions:

[0093] (9S,10E,12Z,15Z)-9-Hydroxy-10,12,15-octadecatrienoic acid

[0094] Acylcarnitines (18:3)

[0095] Palmitoylethanolamide

[0096] Phytosphingosine

[0097] Phosphatidylinositol (16:0 / 18:3)

[0098] 14-Hydroxysteroids

[0099] Sulfatide (d29:1).

[0100] The present invention also protects the use of a combination of one or more of the following seven compounds in the preparation of a detection kit for the diagnosis and / or risk prediction of colorectal precancerous lesions:

[0101] (9S,10E,12Z,15Z)-9-Hydroxy-10,12,15-octadecatrienoic acid

[0102] Acylcarnitines (18:3)

[0103] Palmitoylethanolamide

[0104] Phytosphingosine

[0105] Phosphatidylinositol (16:0 / 18:3)

[0106] 14-Hydroxysteroids

[0107] Sulfatide (d29:1).

[0108] Compared with the prior art, the present invention has the following beneficial effects:

[0109] 1. This invention adopts a rigorous screening, verification and evaluation system, based on a two-stage case-control study design, and includes a large sample of colorectal cancer patients, advanced adenoma patients and healthy controls. It independently screens and verifies non-invasive biomarkers for early auxiliary diagnosis of colorectal cancer, avoids false positive results, and the results are true and reliable.

[0110] 2. The present invention demonstrates that specific plasma metabolites can serve as a novel biomarker with a diagnostic sensitivity for precancerous lesions (>80%) significantly higher than that of currently commonly used primary screening techniques in clinical practice, including multi-target fecal DNA detection kits (sensitivity reported in the literature is 42%) and fecal occult blood tests (sensitivity reported in the literature is 40%), and thus has important value for the early diagnosis of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 The ability of 10 metabolites to distinguish colorectal cancer from healthy controls in samples from Guangzhou and Nanjing;

[0112] Figure 1 A is the discrimination ability in samples from Guangzhou area;

[0113] Figure 1 B is the discrimination ability in the samples from Nanjing area;

[0114] Figure 1 C is the ability to discriminate between stage 0-II colorectal cancer in Nanjing;

[0115] Figure 1 D is the ability to distinguish stage III-IV colorectal cancer in Nanjing area.

[0116] Figure 2 The ability of seven metabolites in samples from Guangzhou and Nanjing to distinguish advanced adenomas (precancerous lesions) from healthy controls;

[0117] Figure 2 A is the discrimination ability in samples from Guangzhou area;

[0118] Figure 2 B is the discrimination ability in samples from Nanjing area. DETAILED DESCRIPTION

[0119] The present invention is further explained below with reference to the following examples, but the examples do not limit the present invention in any form.

[0120] Example 1 Collection of samples and arrangement of sample data

[0121] 1. Sample selection: 107 patients with colorectal cancer, 107 patients with adenoma, and 107 healthy controls from the Sixth Affiliated Hospital of Sun Yat-sen University in Guangzhou were used as the discovery set, and 112 patients with colorectal cancer, 57 patients with adenoma, and 112 healthy controls from the Second Affiliated Hospital of Nanjing Medical University were used as the validation set.

[0122] 2. Extraction of plasma samples: Add 300ul of extraction solution (methanol) to 100ul of plasma to precipitate plasma proteins. After high-speed centrifugation, aspirate the supernatant.

[0123] Example 2 Plasma metabolome detection

[0124] 1. Prepare analytical samples: All patients fasted for at least 8 hours before blood collection. Samples were processed within 24 hours of collection and stored at -80°C until needed. Pipette 100 μL of sample into an EP tube, add 300 μL of extraction solution (methanol, containing an isotope-labeled internal standard mixture), and vortex mix for 30 seconds; sonicate for 10 minutes (in an ice-water bath); let stand at -40°C for 1 hour; centrifuge the sample at 4°C, 12,000 rpm for 15 minutes; precipitate plasma proteins, and after high-speed centrifugation, aspirate the supernatant into a sample injection bottle for analysis. Take an equal amount of supernatant from all samples and mix them into quality control (QC) samples for analysis.

[0125] 2. Sample detection: Ultra-high performance liquid chromatography (Vanquish, Thermo Fisher Scientific, Waltham, MA) coupled with high-resolution mass spectrometry (Orbitrap MS, Thermo Fisher Scientific) was used for detection.

[0126] (1) The target compounds were separated by chromatographic separation using a Waters ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column (Vanquish, Thermo Fisher Scientific, Waltham, MA). Phase A consisted of an aqueous phase containing 5 mmol / L ammonium acetate and 5 mmol / L acetic acid, and phase B consisted of acetonitrile. Injection volume was 2 μL.

[0127] (2) A high-resolution mass spectrometer (Orbitrap MS, Thermo Fisher Scientific) was used to collect primary and secondary mass spectrometric data under the control of Xcalibur software (version 4.4, Thermo). Mass spectrometric analysis was performed in the negative ion mode using electrospray ionization with the following parameters: sheath gas flow rate: 50 Arb, sweep gas: 15 Arb, capillary temperature: 320°C, full mass spectrum resolution: 60,000, MS / MS resolution: 15,000, collision energy: 10 / 30 / 60, and spray voltage: 3.8 kV and -3.4 kV.

[0128] Example 3 Data Analysis

[0129] 1. Data Processing: Raw data were converted to mzXML format using ProteoWizard software. Peak identification, extraction, alignment, and integration were performed using a proprietary R package (CAMERA). The data were then matched against a custom-built secondary mass spectrometry database (BiotreeDB (V2.1)) for substance annotation. The algorithm scoring cutoff value was set to 0.3. Off-line data were filtered for outliers, missing values, imputed, and normalized.

[0130] The plasma differential metabolites screened and verified between colorectal cancer, adenoma and control groups in Guangzhou and Nanjing are shown in Table 1.

[0131] Table 1.

[0132]

[0133]

[0134]

[0135] 2. Statistical analysis

[0136] In this two-stage case-control study, 891 metabolites were detected in baseline plasma using untargeted metabolomics technology.

[0137] Variance analysis was used to identify differential metabolites, lasso regression was used to screen metabolic markers with diagnostic value, and receiver operating characteristic analysis was used to evaluate the model's predictive accuracy and diagnostic efficacy. Ultimately, 10 metabolites were screened and verified to have application value in distinguishing colorectal cancer from healthy controls. The area under the curve in samples from Guangzhou was 0.955 (95% CI: 0.928-0.981), and the sensitivity and specificity reached 93.5% and 86.9%, respectively. Figure 1A), the area under the curve in Nanjing samples was 0.979 (95% CI: 0.965-0.993), and the sensitivity and specificity were 93.8% and 91.1%, respectively ( Figure 1 B), stratified analysis based on colorectal cancer stage found that these 10 metabolites have auxiliary diagnostic value for patients with early colorectal cancer (0-II), with a sensitivity and specificity of 98.1% and 85.7%, respectively ( Figure 1 C), the sensitivity and specificity of patients with intermediate and advanced stages (III-IV) were 98.3% and 92.9%, respectively ( Figure 1 D).

[0138] In addition, 7 metabolites were found to have application value in distinguishing precancerous lesions from healthy controls (one of which was the same as the previous 10 metabolites). The area under the curve in samples from Guangzhou was 0.877 (95% CI: 0.834-0.921), with a sensitivity and specificity of 83.2% and 75.7%, respectively. Figure 2 A), the area under the curve in Nanjing samples was 0.979 (95% CI: 0.965-0.993), and the sensitivity and specificity were 93.8% and 91.1%, respectively ( Figure 2 B).

[0139] The above examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the above examples are conventional conditions and methods unless otherwise specified. The raw materials in the above examples are all commercially available and, unless otherwise specified, can be obtained from commercial sources.

Claims

1. A marker combination associated with colorectal cancer, characterized in that: The marker combination associated with colorectal cancer is a combination of the following 10 compounds:

2. A combination of markers associated with colorectal precancerous lesions, characterized in that: The marker combination associated with colorectal precancerous lesions is a combination of the following 7 compounds:

3. The marker combination associated with colorectal precancerous lesions according to claim 2, characterized in that: The colorectal precancerous lesion is an advanced adenoma.

4. Use of a product for detecting the marker combination according to any one of claims 1 to 3 in the preparation of a product for diagnosing and / or predicting the risk of colorectal cancer and / or colorectal precancerous lesions.

5. The use according to claim 4, characterized in that The marker combination is derived from plasma.

6. Use of the marker combination according to any one of claims 1 to 3 in screening drugs for treating and / or alleviating colorectal cancer and / or colorectal precancerous lesions.

7. The use according to claim 6, characterized in that The marker combination is derived from plasma.

8. Use of the marker combination according to any one of claims 1 to 3 in the preparation of a detection kit for the diagnosis and / or risk prediction of colorectal cancer and / or colorectal precancerous lesions.

9. The use according to claim 8, characterized in that The marker combination is derived from plasma.

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