Markers for breast cancer diagnosis

Abstract

The invention relates to the field of biological medicine. Specifically, the present application relates to the use of an agent for determining the level of a combination of markers in a biological sample in the preparation of a kit for predicting the risk of a subject having breast cancer, or for diagnosing whether a subject has breast cancer, or for monitoring the progression of breast cancer in a subject, or is used for diagnosing whether tumor cells exist in the body of a subject or not. The invention further provides a kit for diagnosing whether a subject suffers from the breast cancer or not.

Description

Technical Field

[0001] The present application relates to the field of biomedicine. Specifically, the present application relates to the use of a reagent for determining the level of a combination of markers in a biological sample in the preparation of a kit for predicting the risk of a subject suffering from breast cancer, or for diagnosing whether a subject suffers from breast cancer, or for monitoring the progression of breast cancer in a subject, or for diagnosing whether a subject has tumor cells in the body. The present application also provides a kit for diagnosing whether a subject suffers from breast cancer. Background Art

[0002] According to statistics from the World Health Organization, in 2020, approximately 2.3 million women worldwide were diagnosed with breast cancer, and 685,000 died. The incidence and mortality rate of breast cancer ranked first among female cancers, and breast cancer has become the second most common malignant tumor in the world. In my country, the incidence of breast cancer is gradually increasing, especially in the eastern coastal areas and economically developed cities. In terms of the age of onset, the incidence rate begins to gradually increase after the age of 20, reaching a peak at 45-50 years old.

[0003] There are many treatments for breast cancer, usually including surgical resection, radiotherapy and drug therapy (hormone therapy, chemotherapy, targeted biological therapy) combined, which are expensive, traumatic, and difficult for patients to accept. Although the mortality rate of breast cancer patients has gradually decreased worldwide with the continuous optimization of treatment strategies, in China, especially in rural areas, the downward trend of breast cancer mortality is still not obvious. If patients are diagnosed with breast cancer in the early stage, early detection and early treatment will greatly increase the prognosis and significantly reduce the mortality rate. Clinical statistics show that the cure rate of breast cancer detected early can reach more than 90%.

[0004] Therefore, early diagnosis is of great significance, and finding biomarkers with high sensitivity and accuracy is of great significance. Summary of the invention

[0005] The applicant of the present application obtained a marker combination that can effectively determine whether a subject has breast cancer by analyzing the lipidomes of two groups of subject samples (breast cancer subjects, healthy subjects) from two different cohorts (a screening cohort of 1,393 subjects and a validation cohort of 333 subjects).

[0006] Therefore, in a first aspect, the present application provides a use of a reagent for determining the level of a marker combination in a biological sample in the preparation of a kit, wherein the kit is used to predict the risk of a subject having breast cancer, or to diagnose whether a subject has breast cancer, or to monitor the progression of breast cancer in a subject, or to diagnose whether a subject has tumor cells (e.g., breast cancer cells) in the body;

[0007] Wherein, the marker combination is selected from any 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of monoglyceride (MG 22:3), acylcarnitine (CAR 18:1), ceramide (Cer 18:1; 2O / 22:0), sphingomyelin (SM 18:1; 2O / 24:0), phosphatidylcholine (PC 18:1_19:1), lysophosphatidylethanolamine (LPE 21:3), sphingomyelin (SM 18:2; 2O / 17:1), oxidized fatty acid (OxFA 16:0; (2OH)), ceramide (Cer 18:2; 2O / 23:0), and sphingomyelin (SM 18:1; 2O / 22:0).

[0008] In certain embodiments, the marker combination comprises: Cer 18:1; 2O / 22:0 and OxFA 16:0; (2OH).

[0009] In certain embodiments, the marker combination comprises or consists of Cer 18:1;2O / 22:0, SM 18:1;2O / 24:0, LPE 21:3, and OxFA 16:0;(2OH).

[0010] In certain embodiments, the marker combination comprises or consists of MG 22:3, CAR 18:1, Cer 18:1; 2O / 22:0, LPE 21:3, and OxFA 16:0; (2OH).

[0011] In certain embodiments, the marker combination comprises or consists of Cer 18:1; 2O / 22:0, PC 18:1_19:1, SM 18:2; 2O / 17:1, OxFA 16:0; (2OH), Cer 18:2; 2O / 23:0 and SM 18:1; 2O / 22:0.

[0012] In certain embodiments, the marker combination comprises or consists of MG 22:3, CAR 18:1, Cer 18:1; 2O / 22:0, SM 18:1; 2O / 24:0, PC 18:1_19:1, LPE 21:3, SM 18:2; 2O / 17:1, OxFA 16:0; (2OH), Cer 18:2; 2O / 23:0 and SM 18:1; 2O / 22:0.

[0013] In certain embodiments, the structural formula of the markers herein and the cytoplasmic-to-nuclear ratio of the corresponding fragmented forms are shown in the table below. In certain embodiments, the names of the markers herein are derived from the MS-DIAL database.

[0014]

[0015]

[0016] In certain embodiments, the marker combination of the present application is in tumor cells (e.g., breast cancer cells) compared with non-tumor cells (e.g., non-breast cancer cells) and has a difference in level. For example, in certain embodiments, one or more markers in the marker combination exist in tumor cells (e.g., breast cancer cells), and are missing in non-tumor cells (e.g., non-breast cancer cells). In other embodiments, one or more markers in the marker combination are missing in tumor cells (e.g., breast cancer cells), but exist in non-tumor cells (e.g., non-breast cancer cells). In further embodiments, one or more markers in the marker combination have a difference in tumor cells (e.g., breast cancer cells) compared with non-tumor cells (e.g., non-breast cancer cells) compared. For example, one or more markers in a marker panel may be present at any differential level, but generally will be elevated by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, In some embodiments, the present invention relates to a polypeptide that is present at a level that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% reduced (e.g., deleted).

[0017] In certain embodiments, the breast cancer cells include primary breast cancer cells, recurrent breast cancer cells, and tumor cells derived from breast cancer (eg, tumor cells resulting from breast cancer metastasis).

[0018] In certain embodiments, the breast cancer includes breast tube in situ carcinoma, infiltrative lobular in situ cancer, tubular / cribriform cancer, mucus (glial) cancer, myeloid cancer, papillary cancer, and metaplastic cancer.

[0019] It is understood that not only women can have breast cancer cells in their breasts, but men can also have breast cancer cells in their breasts, for example, cancer cells in situ or invasively in the male breast.

[0020] Thus, in certain embodiments, the breast cancer cells include breast cancer cells of the male breast (eg, in situ or invasive cancer cells of the male breast).

[0021] Marker combination as described herein can be used in conjunction with other markers or marker combinations for identifying breast cancer, or, used in conjunction with other cancer markers. For example, prostate cancer markers include but are not limited to: AMACR / P504S (U.S. Patent number 6,262,245); PCA3 (U.S. Patent number 7,008,765); PCGEM1 (U.S. Patent number 6,828,429); prostein / P501S, P503S, P504S, P509S, P510S, prostate / P703P, P710P (U.S. Publication No. 20030185830).

[0022] Any suitable method can be used to detect the markers of the present application, including but not limited to liquid and gas chromatography, mass spectrometry alone or coupled (see, e.g., the experimental section below), NMR (see, e.g., U.S. Patent Publication 20070055456, incorporated herein by reference), immunoassays, chemical assays, spectroscopy, etc. In certain embodiments, commercial systems for chromatography and NMR analysis are used.

[0023] In other embodiments, metabolites (e.g., biomarkers and their derivatives) are detected using optical imaging techniques, such as magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), CAT scans, ultrasound, MS-based tissue imaging, or X-ray detection methods (e.g., energy dispersive X-ray fluorescence detection).

[0024] Furthermore, the level of one or more additional substances may be measured indirectly to determine the level of a marker of the present application, for example by using a compound (or compounds) that measures the level of a marker that correlates with the level of the marker to be measured.

[0025] Thus, in certain embodiments, the reagent (e.g., the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and / or tenth or a combination of reagents) determines the level of a biomarker in the biological sample by chromatography and / or mass spectrometry, fluorescence, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near infrared spectroscopy (near IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS) and turbidimetry.

[0026] In certain embodiments, the reagent measures the level of the biomarker in the biological sample by spectroscopy, liquid or gas chromatography, mass spectrometry, or liquid or gas chromatography coupled with mass spectrometry.

[0027] In certain embodiments, the kit further comprises reagents and / or consumables for chromatography, reagents and / or consumables for mass spectrometry, or any combination thereof.

[0028] In certain embodiments, the reagents and / or consumables for chromatography are selected from chromatographic columns, aqueous acetonitrile solutions (e.g., 10% aqueous acetonitrile solutions, 50% aqueous acetonitrile solutions, 60% aqueous acetonitrile solutions), ammonium acetate, ammonium formate, formic acid, or any combination thereof.

[0029] In certain embodiments, the reagents and / or consumables for mass spectrometry are selected from mass spectrometry columns, formic acid, acetonitrile, or any combination thereof.

[0030] In certain embodiments, the subject is a mammal, such as a human.

[0031] In certain embodiments, the kit is used to distinguish between subjects who have breast cancer and subjects who do not have breast cancer.

[0032] In certain embodiments, the breast cancer includes stage 0, stage 1, stage 2, stage 3, stage 4, and stage 5 of breast cancer; more preferably, the kit is used to distinguish subjects suffering from stage 0, stage 1, stage 2, stage 3, stage 4, and stage 5 of breast cancer.

[0033] In certain embodiments, the biological sample is selected from urine, body fluid, serum, whole blood, plasma, human secretions, cerebrospinal fluid, or any combination thereof.

[0034] In certain embodiments, the biological sample is selected from whole blood (eg, peripheral blood), serum, plasma, or any combination thereof.

[0035] In certain embodiments, the kit further comprises reagents for pre-treating the biological sample (eg, reagents for centrifugation, immunocapture and / or cell lysis).

[0036] In a second aspect, the present application provides a kit for predicting the risk of a subject having breast cancer, or for diagnosing whether a subject has breast cancer, or for monitoring the progression of breast cancer in a subject, or for diagnosing whether a subject has tumor cells in the body, the kit comprising reagents for determining the level of a marker combination in a biological sample;

[0037] Wherein, the marker combination is selected from any 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of monoglyceride (MG 22:3), acylcarnitine (CAR 18:1), ceramide (Cer 18:1; 2O / 22:0), sphingomyelin (SM 18:1; 2O / 24:0), phosphatidylcholine (PC 18:1_19:1), lysophosphatidylethanolamine (LPE 21:3), sphingomyelin (SM 18:2; 2O / 17:1), oxidized fatty acid (OxFA 16:0; (2OH)), ceramide (Cer 18:2; 2O / 23:0), and sphingomyelin (SM 18:1; 2O / 22:0).

[0038] In certain embodiments, the marker combination comprises: Cer 18:1; 2O / 22:0 and OxFA 16:0; (2OH).

[0039] In certain embodiments, the marker combination comprises or consists of Cer 18:1;2O / 22:0, SM 18:1;2O / 24:0, LPE 21:3, and OxFA 16:0;(2OH).

[0040] In certain embodiments, the marker combination comprises or consists of MG 22:3, CAR 18:1, Cer 18:1; 2O / 22:0, LPE 21:3, and OxFA 16:0; (2OH).

[0041] In certain embodiments, the marker combination comprises or consists of Cer 18:1; 2O / 22:0, PC 18:1_19:1, SM 18:2; 2O / 17:1, OxFA 16:0; (2OH), Cer 18:2; 2O / 23:0 and SM 18:1; 2O / 22:0.

[0042] In certain embodiments, the marker combination comprises or consists of MG 22:3, CAR 18:1, Cer 18:1; 2O / 22:0, SM 18:1; 2O / 24:0, PC 18:1_19:1, LPE 21:3, SM 18:2; 2O / 17:1, OxFA 16:0; (2OH), Cer 18:2; 2O / 23:0 and SM 18:1; 2O / 22:0.

[0043] In certain embodiments, the breast cancer cells include primary breast cancer cells, recurrent breast cancer cells, and tumor cells derived from breast cancer (eg, tumor cells resulting from breast cancer metastasis).

[0044] In certain embodiments, the breast cancer includes breast tube in situ carcinoma, infiltrative lobular in situ cancer, tubular / cribriform cancer, mucus (glial) cancer, myeloid cancer, papillary cancer, and metaplastic cancer.

[0045] In certain embodiments, the tumor cells include male breast tumor cells (eg, male breast in situ or invasive tumor cells).

[0046] In certain embodiments, the reagent (e.g., the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and / or tenth or a combination of reagents) determines the level of a biomarker in the biological sample by chromatography and / or mass spectrometry, fluorescence, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near infrared spectroscopy (near IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS) and turbidimetry.

[0047] In certain embodiments, the reagent measures the level of the biomarker in the biological sample by spectroscopy, liquid or gas chromatography, mass spectrometry, or liquid or gas chromatography coupled with mass spectrometry.

[0048] In certain embodiments, the kit further comprises reagents and / or consumables for chromatography, reagents and / or consumables for mass spectrometry, or any combination thereof.

[0049] In certain embodiments, the reagents and / or consumables for chromatography are selected from chromatographic columns, aqueous acetonitrile solutions (e.g., 10% aqueous acetonitrile solutions, 50% aqueous acetonitrile solutions, 60% aqueous acetonitrile solutions), ammonium acetate, ammonium formate, formic acid, or any combination thereof.

[0050] In certain embodiments, the reagents and / or consumables for mass spectrometry are selected from mass spectrometry columns, formic acid, acetonitrile, or any combination thereof.

[0051] In certain embodiments, the subject is a mammal, such as a human.

[0052] In certain embodiments, the kit is used to distinguish between subjects who have breast cancer and subjects who do not have breast cancer.

[0053] In certain embodiments, the breast cancer includes stage 0, stage 1, stage 2, stage 3, stage 4, and stage 5 of breast cancer; more preferably, the kit is used to distinguish subjects suffering from stage 0, stage 1, stage 2, stage 3, stage 4, and stage 5 of breast cancer.

[0054] In certain embodiments, the biological sample is selected from urine, body fluid, serum, whole blood, plasma, human secretions, cerebrospinal fluid, or any combination thereof.

[0055] In certain embodiments, the biological sample is selected from whole blood (eg, peripheral blood), serum, plasma, or any combination thereof.

[0056] In certain embodiments, the kit further comprises reagents for pre-treating the biological sample (eg, reagents for centrifugation, immunocapture and / or cell lysis).

[0057] In a third aspect, the present application provides a method for predicting the risk of a subject having breast cancer, or diagnosing whether a subject has breast cancer, or monitoring the progression of breast cancer in a subject, or detecting the prognosis of a subject having breast cancer, the method comprising:

[0058] (1) obtaining a biological sample containing a marker combination from a subject;

[0059] (2) determining the level of the marker combination in the biological sample; and

[0060] (3) diagnosing whether the subject has breast cancer based on the level of the marker combination;

[0061] Wherein, the marker combination is selected from any 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of monoglyceride (MG 22:3), acylcarnitine (CAR 18:1), ceramide (Cer 18:1; 2O / 22:0), sphingomyelin (SM 18:1; 2O / 24:0), phosphatidylcholine (PC 18:1_19:1), lysophosphatidylethanolamine (LPE 21:3), sphingomyelin (SM 18:2; 2O / 17:1), oxidized fatty acid (OxFA 16:0; (2OH)), ceramide (Cer 18:2; 2O / 23:0), and sphingomyelin (SM 18:1; 2O / 22:0).

[0062] In certain embodiments, the marker combination comprises: Cer 18:1; 2O / 22:0 and OxFA 16:0; (2OH).

[0063] In certain embodiments, the marker combination comprises or consists of Cer 18:1;2O / 22:0, SM 18:1;2O / 24:0, LPE 21:3, and OxFA16:0;(2OH).

[0064] In certain embodiments, the marker combination comprises or consists of MG 22:3, CAR 18:1, Cer 18:1; 2O / 22:0, LPE 21:3, and OxFA 16:0; (2OH).

[0065] In certain embodiments, the marker combination comprises or consists of Cer 18:1; 2O / 22:0, PC 18:1_19:1, SM 18:2; 2O / 17:1, OxFA 16:0; (2OH), Cer 18:2; 2O / 23:0 and SM 18:1; 2O / 22:0.

[0066] In certain embodiments, the marker combination comprises or consists of MG 22:3, CAR 18:1, Cer 18:1; 2O / 22:0, SM 18:1; 2O / 24:0, PC 18:1_19:1, LPE 21:3, SM 18:2; 2O / 17:1, OxFA 16:0; (2OH), Cer 18:2; 2O / 23:0 and SM 18:1; 2O / 22:0.

[0067] In certain embodiments, in step (3), by comparing the level of the marker combination with a reference value or the level of the marker combination of a healthy subject, the risk of the subject suffering from breast cancer is predicted, or whether the subject suffers from breast cancer is diagnosed, or the progression of breast cancer in the subject is monitored, or the prognosis of a subject suffering from breast cancer is detected. In certain embodiments, the reference value is the level or range of the marker in a biological sample obtained from a normal population.

[0068] In certain embodiments, the breast cancer includes breast tube in situ carcinoma, infiltrative lobular in situ cancer, tubular / cribriform cancer, mucus (glial) cancer, myeloid cancer, papillary cancer, and metaplastic cancer.

[0069] In certain embodiments, the present invention also provides one or more treatment methods for use in conjunction with the above methods.

[0070] For example, in certain embodiments, the present invention provides one or more compounds targeting the above-mentioned marker combinations. These compounds can be, for example, by interfering with the synthesis of the marker combination or its precursors or metabolites (e.g., by hindering the transcription or translation of the enzyme involved in the synthesis of the metabolite, by passivating the enzyme involved in the synthesis of the metabolite (e.g., by post-translational modification or binding with an irreversible inhibitor) or by inhibiting the enzyme activity involved in the synthesis of the metabolite), by combining the function of inhibiting the marker combination to adjust (e.g., increase or decrease) the level of the marker combination.

[0071] In certain embodiments, the method further comprises the step of treating the subject based on the diagnosis of breast cancer; wherein the treatment is selected from surgery, radiation, chemotherapy, hormone therapy, targeted drug therapy, immunotherapy, or any combination thereof.

[0072] Definition of terms

[0073] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the chemical, biochemical, metabolomics, lipidomics and other operating steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0074] As used herein, the term "marker" refers to a biochemical index that can mark changes or possible changes in the structure or function of a system, organ, tissue, cell, or subcellular structure, and has a very wide range of uses. Markers can be used to diagnose diseases, determine disease stages, or evaluate the safety and effectiveness of new drugs or new therapies in target populations.

[0075] As used herein, the term "breast cancer" refers to a disease in which abnormal breast cells grow out of control and form tumors. If not controlled, the tumors may spread throughout the body. Breast cancer cells begin in the milk ducts and / or milk-producing lobules in the breast. The earliest form (in situ) is not life-threatening and can be detected in the early stages. The cancer cells spread to nearby breast tissue (invasion). This forms a tumor, causing a lump or thickening. Invasive cancer can spread to nearby lymph nodes or other organs (metastasis). Metastasis can be life-threatening and fatal. Treatment is based on the patient's individual condition, the type of cancer, and how far it has spread. Treatment methods combine surgery, radiation therapy, and medication.

[0076] As used herein, the term "reference value" refers to a predetermined value of a compound, which is derived from the level of a compound in a control sample (e.g., obtained from a healthy subject). The reference value can be used as a threshold to distinguish between subjects who may have breast cancer and healthy subjects. The reference value can be a relative value, a numerical range with an upper and lower limit, an average value, an intermediate value, etc. Those skilled in the art can select a suitable control sample, measure, and obtain a reference value according to methods disclosed in the prior art.

[0077] In certain embodiments, a significant difference in the level of the marker compared to a reference value range of the marker level in a healthy subject (e.g., MG 22:3; CAR 18:1; Cer18:1; 2O / 22:0; SM 18:1; 2O / 24:0; PC 18:1_19:1; LPE 21:3; SM 18:2; 2O / 17:1; OxFA 16:0; (2OH); Cer 18:2; 2O / 23:0; SM 18:1; 2O / 22:0) outside the normal range indicates that the subject is at risk for breast cancer, has already had breast cancer, or has had a recurrence of breast cancer.

[0078] As used herein, the term "lipid" refers to a substance produced in a human chemical or physical process. Lipids are mainly divided into 16 categories, and the abbreviations herein are represented in brackets, including acylcarnitines (CAR), ceramides (Cer), cardiolipins (CL), ether-linked phosphatidylcholines (EtherPC), ether-linked phosphatidylethanolamines (EtherPE), free fatty acids (FA), lysophosphatidylcholines (LPC), lysophosphatidylethanolamines (LPE), phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylglycerols (PG), phosphatidylinositols (PI), phosphatidylserines (PS), sphingolipids (SM), triglycerides (TG) and other classes (Others). Relative to other metabolisms, lipids are important substances for cell signaling in the body, and they have uniqueness and functional specificity, and their dynamic changes also indicate changes in the metabolic mechanism of the disease.

[0079] As used in this article, the term "lipidomics" refers to the science of using chromatography-mass spectrometry to study the changes in lipids with different chemical properties in organisms. Its research content mainly includes determining lipid structure (for example: number of atoms, carbon chain length, number and position of double bonds, etc.), accurately quantifying various lipids to discover lipid markers, and revealing the interactions between various lipids and lipids and metabolites.

[0080] As used herein, the term "mass spectrometry (MS)" is a technique for measuring and analyzing molecules that involves fragmenting a target molecule and then analyzing the fragments based on their mass-to-charge ratio to produce a mass spectrum, as a "molecular fingerprint." The mass-to-charge ratio of an object is determined by determining the wavelength at which electromagnetic energy is absorbed by the object. There are several common methods for determining the mass-to-charge ratio of ions, some methods use electromagnetic waves to measure the interaction of ion orbits, other methods measure the time it takes for ions to travel a specific distance, or both. Data from these fragment mass measurements can be queried in a database to obtain the exact identification of the target molecule. Mass spectrometry is also widely used in other fields of chemistry, such as petrochemicals or other fields such as pharmaceutical quality control.

[0081] As used herein, the term "subject" refers to mammals, including but not limited to humans, rodents (mice, rats, guinea pigs), dogs, horses, cows, cats, pigs, monkeys, chimpanzees, etc. In certain embodiments, the subject is a human.

[0082] As used herein, the term "prognosis" refers to the prediction of the likelihood of death or progression from cancer, including the progression of metastatic breast cancer. The term "prediction" as used herein refers to the likelihood that a patient will respond favorably or unfavorably to a drug or group of drugs.

[0083] As used herein, the term "tumor" refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0084] As used herein, the term "area under the curve" or "AUC" refers to the area under the curve of the receiver operating characteristic (ROC) curve, both of which are well known in the art. The AUC measurement can be used to compare the accuracy of the classifier within the entire data range. A classifiers with a larger AUC have greater ability to correctly classify unknowns between two groups of interest (e.g., breast cancer samples and normal or control samples). The ROC curve can be used to plot the performance of a specific feature (e.g., any one of the biomarkers described herein and / or any additional biomedical information items) in distinguishing two populations (e.g., cases with breast cancer and controls without breast cancer). Typically, based on the value of a single feature, the feature data of the entire population (e.g., cases and controls) are sorted in ascending order. Then, for each value of the feature, the true positive rate and false positive rate of the data are calculated. The true positive rate is determined by counting the number of cases higher than the value of the feature, and then divided by the total number of cases. The false positive rate is determined by counting the number of controls higher than the value of the feature, and then divided by the total number of controls. The ROC curve of the present invention is the characteristic of the present invention.Although this definition refers to the situation that the characteristic in the case is increased compared with the control, this definition is also applicable to the situation that the characteristic in the case is reduced compared with the control (in such case, the sample of the value below the characteristic is counted).The ROC curve can be generated for a single feature and other single outputs, for example, the combination of two or more features can be mathematically combined (for example, adding, subtracting, multiplying, etc.) to provide a single sum value, and this single sum value can be plotted in the ROC curve.Additionally, any combination of multiple features can be drawn in the ROC curve, wherein this combination derives a single output value.The combination of these features can constitute a test.The ROC curve is a chart of the true positive rate (sensitivity) of the test to the false positive rate (1-specificity) of the test.

[0085] As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and include fluids, liquids, tissues, and gases. Biological samples include blood products, such as plasma, serum, and the like. However, this example should not be construed as limiting the sample types available for the present invention.

[0086] As used herein, the term "biological sample" can be an animal (including humans), a fluid, a liquid (e.g., feces) or a tissue, as well as liquid and solid food and feed products, and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples can be obtained from domestic animals of all families and wild and wild animals, including but not limited to ungulates, bears, fish, lagomorphs, rodents, etc. The biological sample can contain any biological material suitable for detecting the desired biomarker, and can include cells and / or non-cellular materials from the subject. The sample can be isolated from any suitable biological tissue or fluid, for example, prostate tissue, blood, plasma, urine, or cerebrospinal fluid (CSF).

[0087] Advantageous Effects of the Invention

[0088] The present application obtains a marker combination that can accurately determine whether a subject has breast cancer by analyzing the lipid groups of two groups of subject samples (breast cancer subjects and healthy subjects) of two different cohorts (screening cohort and validation cohort). That is, MG 22:3, CAR 18:1, Cer18:1; 2O / 22:0, SM 18:1; 2O / 24:0, PC 18:1_19:1, LPE 21:3, SM 18:2; 2O / 17:1, OxFA 16:0; (2OH), Cer 18:2; 2O / 23:0, SM 18:1; Any 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of 2O / 22:0. These marker combinations have very high diagnostic accuracy for breast cancer (higher than 90%, up to 96%, and any single marker is basically between 55%-65%).

[0089] Therefore, the marker combination of the present application has great potential in the diagnosis, investigation, screening and treatment of breast cancer.

[0090] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 The lipids in clinical serum and their relationship with diseases are shown. Figure 1 Figure A shows an overview of the number and types of lipids detected in the serum of two groups of breast cancer subjects (BC) and healthy subjects (HD). Figure 1B in the figure shows the partial least squares discriminant analysis PLS-DA analysis between the two groups of the test cohort; Figure 1 C in the figure shows the partial least squares discriminant analysis PLS-DA analysis between the two groups of the validation cohort; Figure 1 Panel D in Figure 2 shows the ROC analysis of lipids that overlap between the test and validation cohorts.

[0092] Figure 2 The screened lipid markers and their validation results were displayed. Figure 2 A in the figure shows the TOP 10 differential lipids screened by VIP value, and it can be found that MG 22:3; CAR 18:1; Cer 18:1; 2O / 22:0; SM 18:1; 2O / 24:0; PC 18:1_19:1; LPE 21:3; SM 18:2; 2O / 17:1; OxFA 16:0; (2OH); Cer 18:2; 2O / 23:0; SM18:1; 2O / 22:0 have the most significant changes; Figure 2 B is a bar graph showing the total differences of TOP 10 lipids in the breast cancer (BC) group and the healthy subject (HD) group in the test cohort; Figure 2 C in the figure is a separate box plot of the TOP 10 lipids in the breast cancer (BC) group and the healthy subject (HD) group in the test cohort; Figure 2 D in the figure is a box plot of the total difference of TOP 10 lipids in the breast cancer (BC) group and the healthy subject (HD) group in the validation cohort; Figure 2 Figure E is a separate box plot of the TOP 10 lipids in the breast cancer (BC) group and the healthy subject (HD) group in the validation cohort; Figure 2 F in FIG. 1 is the ROC curve of the TOP 10 lipids and the overall combined analysis.

[0093] Figure 3 The screened lipid marker combinations and their validation results are shown. Figure 3 A in the figure is the ROC curve of the combined analysis of four lipids including Cer 18:1; 2O / 22:0, SM 18:1; 2O / 24:0, LPE 21:3 and OxFA 16:0; (2OH) as markers; Figure 3 B is the ROC curve of the combined analysis of five lipid markers: MG 22:3, CAR 18:1, Cer 18:1; 2O / 22:0, LPE 21:3 and OxFA16:0; (2OH); Figure 3C in the figure is the ROC curve of the combined analysis of six lipid markers, namely Cer 18:1; 2O / 22:0, PC 18:1_19:1, SM 18:2; 2O / 17:1, OxFA 16:0; (2OH), Cer 18:2; 2O / 23:0 and SM 18:1; 2O / 22:0. DETAILED DESCRIPTION

[0094] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0095] Unless otherwise specified, the experiments and methods described in the embodiments are basically carried out according to conventional methods well known in the art and described in various references. For example, conventional techniques such as chemistry, biochemistry, metabolomics, lipidomics used in the present invention can be found in Birendra N. Pramanik, AK Ganguly, etc., "Electrospray Mass Spectrometry Application Technology" (2005); Xu Guowang, etc., "Metabolomics-Methods and Applications" (2008); Jia Wei, etc., "Medical Metabolomics" (2011).

[0096] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0097] Example 1. Acquisition of serum lipid samples from healthy subjects and breast cancer subjects This application uses liquid chromatography tandem mass spectrometry (LC / MS) to explore the lipid levels in the serum of two groups of subjects: breast cancer subjects (BC) and healthy subjects (HD).

[0098] The clinical serum samples used in this experiment were all from the Union Hospital Affiliated to Fujian Medical University, and a total of 1,726 serum samples were collected. Among them, there were 1,393 cases in the test cohort (698 breast cancer subjects and 695 healthy subjects) and 333 cases in the validation cohort (142 breast cancer subjects and 191 healthy subjects). Among them, breast cancer subjects were diagnosed with breast cancer by doctors based on clinical manifestations, physical examinations, imaging examinations, histopathology, etc. The whole blood collected from the subjects was immediately centrifuged to harvest the serum and stored in a -80°C refrigerator. After that, it was transported to the laboratory on dry ice for lipid extraction.

[0099] Example 2. Lipidomics experiment of test cohort serum

[0100] Extraction of serum lipids:

[0101] After the samples were thawed and vortexed, 50 μL of serum was taken from each sample into the corresponding 1.5 mL centrifuge tube. Then, 400 μL of pre-cooled mass spectrometry grade methanol containing internal standard was added, vortexed for 2 min, 1 mL of tert-butyl methyl ether (MTBE) was added, the metal bath was pre-cooled with nitrogen, and the mixture was shaken at 1000 rpm for 20 min. 400 μL of ultrapure water was added, vortexed for 1 min, and allowed to stand for 2 min. Then, the samples were centrifuged at 7 °C at high speed (11000 rpm, 10 min). 150 μL of the lower layer supernatant was taken into a new centrifuge tube and dried with nitrogen. For the preparation of mixed samples, 50 μL of the lower layer supernatant was taken from each sample and combined into one sample, which was carefully vortexed to form a mixed sample as a reference sample.

[0102] (1) Mass spectrometry acquisition conditions:

[0103] Instrument: Thermo Scientific Orbitrap Exploris 120

[0104] The ion source parameters were set as follows: H-ESI ion source; Sheath Gas (Arb): 25; Aux Gas (Arb): 10; Sweep Gas (Arb): 1; Ion Transfer Tube Temp (℃): 320; Vaporizer Temp (℃): 350; Orbitrap Resolution: 120000; Mass spectrometry scanning range: m / z 150-2000Da.

[0105] (2) Chromatographic conditions:

[0106] Chromatographic column: Waters ACQUITY UPLC BEH C8 column (100 x 2.1 mm, 1.7 μm);

[0107] Mobile phase: A: (6:4 v / v) acetonitrile-water (10 mM ammonium acetate); B: (9:1 v / v) isopropanol-acetonitrile (10 mM ammonium acetate); column temperature: 55°C, flow rate: 0.26 mL / min; elution gradient: t=0 min, 32% B; t=1.5 min, 32% B; t=15.5 min, 85% B; t=15.6 min, 97% B; t=18 min, 97% B; t=18.1 min, 32% B; t=20 min, 32% B.

[0108] Example 3. Lipidomics experiment of serum from validation cohort

[0109] Extraction of serum lipids:

[0110] After the sample was thawed and vortexed, 50 μL of serum was taken from each sample into the corresponding 1.5 mL centrifuge tube. Then, 400 μL of pre-cooled tert-butyl methyl ether (MTBE) was added, vortexed for 30 seconds, and 80 μL of pre-cooled methanol containing internal standard was added, vortexed for 30 seconds, ultrasonicated on ice for 10 minutes, and then the sample was centrifuged at 4 ° C (3000 rpm, 15 minutes). 300 μL of supernatant was drawn into a new centrifuge tube and blown dry with nitrogen. For the preparation of mixed samples, 20 μL of supernatant was drawn from each sample during reconstitution and combined into one sample, which was carefully vortexed to form a mixed sample as a reference sample.

[0111] (1) Mass spectrometry acquisition conditions:

[0112] Instrument: Thermo Scientific Orbitrap Exploris 240

[0113] The ion source parameters were set as follows: H-ESI ion source; Sheath Gas (Arb): 40; Aux Gas (Arb): 10; Sweep Gas (Arb): 10; Ion Transfer Tube Temp (℃): 320; Vaporizer Temp (℃): 350; Orbitrap Resolution (MS1): 120000; Orbitrap Resolution (MS2): 150000; Mass spectrometry scanning range: m / z 100-1500Da.

[0114] (2) Chromatographic conditions:

[0115] Chromatographic column: BEH C8 column (2.1×100mm with 1.7μm particle size, Waters, Milford, MA, USA);

[0116] Mobile phase: A: (6:4 v / v) acetonitrile-water (2 mM ammonium formate); B: (9:1 v / v) isopropanol-acetonitrile (10 mM ammonium acetate); column temperature: 55°C, flow rate: 0.26 mL / min; elution gradient: t=0 min, 32% B; t=1.5 min, 32% B; t=15.5 min, 85% B; t=15.6 min, 97% B; t=18 min, 97% B; t=18.1 min, 32% B; t=23 min, 32% B.

[0117] Example 4. Screening and verification of markers

[0118] The relative quantitative results were processed by the XCMSR language package as the basis for relative quantification. The data must be corrected first, including total peak area correction, internal standard correction, and QC correction. The stattarget package was used for QC correction, CV was set to 30%, the 80% principle was used for metabolomics data screening, and the KNN algorithm was selected for missing value filling. Statistical analysis was performed after data correction.

[0119] Experimental Results

[0120] The results of lipidomics screening and validation experiments are as follows Figures 1 to 3 The detailed information of the 10 lipid markers screened is shown in Table 1.

[0121] Figure 1 Figure A shows an overview of the number and categories of lipids detected in the serum of two groups of breast cancer subjects (BC) and healthy subjects (HD). Figure 1 B in the figure shows the partial least squares discriminant analysis PLS-DA analysis between the two groups of the test cohort; Figure 1 C in the figure shows the partial least squares discriminant analysis PLS-DA analysis between the two groups of the validation cohort; Figure 1 Figure D shows the lipid ROC analysis of the intersection of the test cohort and the validation cohort.

[0122] Figure 2 A in the figure shows the TOP 10 differential lipids screened by VIP value, and it can be found that MG 22:3; CAR 18:1; Cer 18:1; 2O / 22:0; SM 18:1; 2O / 24:0; PC 18:1_19:1; LPE 21:3; SM 18:2; 2O / 17:1; OxFA16:0; (2OH); Cer18:2; 2O / 23:0; SM 18:1; 2O / 22:0 have the most significant changes; Figure 2 B is a bar graph showing the difference in total content of TOP 10 lipids in the subjects of the breast cancer (BC) group and the healthy subjects (HD) group in the test cohort; Figure 2 Panel C is a separate box plot of the TOP 10 lipids in the breast cancer (BC) group and the healthy subjects (HD) group in the test cohort.

[0123] After the above analysis, the markers with significant level differences in the breast cancer (BC) group and the healthy subjects (HD) group were obtained through the test cohort screening, namely, MG 22:3; CAR 18:1; Cer 18:1; 2O / 22:0; SM 18:1; 2O / 24:0; PC 18:1_19:1; LPE 21:3; SM 18:2; 2O / 17:1; OxFA 16:0; (2OH); Cer 18:2; 2O / 23:0 and SM18:1; 2O / 22:0. Further, the markers obtained by the above screening were verified in the validation cohort. The results of the validation cohort are as follows: Figure 2 As shown in D to F.

[0124] Figure 2 D in the figure is a box plot of the total difference of TOP 10 lipids in the breast cancer (BC) group and the healthy subject (HD) group in the validation cohort; Figure 2 Figure E is a separate box plot of the TOP 10 lipids in the breast cancer (BC) group and the healthy subject (HD) group in the validation cohort; Figure 2 F in the figure is the ROC curve of the TOP 10 lipids and the overall joint analysis. It can be found that the AUC value of the ROC curve of the joint analysis of ten lipids is as high as 0.96, which has a high credibility. Compared with any single lipid in the ten lipid combinations (mostly between 0.55-0.65), the AUC value of the lipid combination of the present application has been significantly improved.

[0125] Figure 3 A in the figure is the ROC curve of the combined analysis of four lipid combination markers: Cer 18:1; 2O / 22:0, SM 18:1; 2O / 24:0, LPE 21:3, OxFA 16:0; (2OH); the AUC value of the ROC curve of the combined analysis of these four lipids is as high as 0.8986. Figure 3 B in the figure is the ROC curve of the combined analysis of five lipid combination biomarkers: MG 22:3, CAR 18:1, Cer 18:1; 2O / 22:0, LPE 21:3, OxFA 16:0; (2OH); the AUC value of the R OC curve of the combined analysis of these five lipids is as high as 0.9332. Figure 3 C in the figure is the ROC curve of the joint analysis of six lipid combination markers: Cer 18:1; 2O / 22:0, PC 18:1_19:1, SM 18:2; 2O / 17:1, OxFA 16:0; (2OH), Cer 18:2; 2O / 23:0, SM 18:1; 2O / 22:0. The AUC value of the ROC curve for the joint analysis of these six lipid combinations is as high as 0.9116.

[0126] In summary, according to Figure 3 It can be seen that the ROC curves of the combined analysis of these markers are close to or greater than 0.9, which has a high degree of credibility.

[0127] Table 1 Lipid marker information

[0128]

[0129]

[0130] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. Use of a reagent for determining the level of a marker combination in a biological sample in the preparation of a kit for predicting the risk of a subject suffering from breast cancer, or for diagnosing whether a subject suffers from breast cancer, or for monitoring the progression of breast cancer in a subject, or for diagnosing whether a subject has tumor cells (e.g., breast cancer cells) in the body; in, The marker combination is selected from any 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of monoglyceride (MG 22:3), acylcarnitine (CAR 18:1), ceramide (Cer 18:1; 2O / 22:0), sphingomyelin (SM 18:1; 2O / 24:0), phosphatidylcholine (PC 18:1-19:1), lysophosphatidylethanolamine (LPE 21:3), sphingomyelin (SM 18:2; 2O / 17:1), oxidized fatty acid (OxFA 16:0; (2OH)), ceramide (Cer 18:2; 2O / 23:0), and sphingomyelin (SM 18:1; 2O / 22:0); Preferably, the marker combination comprises or consists of Cer 18:1;2O / 22:0, SM 18:1;2O / 24:0, LPE 21:3 and OxFA 16:0;(2OH); Preferably, the marker combination comprises or consists of MG 22:3, CAR 18:1, Cer18:1; 2O / 22:0, LPE 21:3 and OxFA 16:0; (2OH); Preferably, the marker combination comprises or consists of Cer 18:1; 2O / 22:0, PC 18:1_19:1, SM 18:2; 2O / 17:1, OxFA 16:0; (2OH), Cer 18:2; 2O / 23:0 and SM 18:1; 2O / 22:

0.

2. The use according to claim 1, wherein The breast cancer cells include primary breast cancer cells, recurrent breast cancer cells, and tumor cells derived from breast cancer (e.g., tumor cells generated by breast cancer metastasis); Preferably, the breast cancer includes breast tube in situ cancer, infiltrative lobular in situ cancer, tubular / cribriform cancer, mucus (glial) cancer, myeloid cancer, papillary cancer and metaplastic cancer; Preferably, the breast cancer cells include male breast breast cancer cells (eg, male breast in situ or invasive cancer cells).

3. The use of claim 1 or 2, wherein the reagent (e.g., the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and / or tenth or a combination of reagents) determines the level of a biomarker in the biological sample by chromatography and / or mass spectrometry, fluorescence, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near infrared spectroscopy (near IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS) and turbidimetry; Preferably, the reagent measures the level of the biomarker in the biological sample by spectroscopy, liquid or gas chromatography, mass spectrometry, or liquid or gas chromatography coupled with mass spectrometry; Preferably, the kit further comprises reagents and / or consumables for chromatography, reagents and / or consumables for mass spectrometry, or any combination thereof; Preferably, the reagents and / or consumables for chromatography are selected from chromatographic columns, acetonitrile aqueous solution (e.g., 10% acetonitrile aqueous solution, 50% acetonitrile aqueous solution, 60% acetonitrile aqueous solution), ammonium acetate, ammonium formate, formic acid, or any combination thereof; Preferably, the reagents and / or consumables for mass spectrometry are selected from mass spectrometry columns, formic acid, acetonitrile, or any combination thereof.

4. The use according to any one of claims 1 to 3, wherein The subject is a mammal, such as a human; Preferably, the kit is used to distinguish between subjects suffering from breast cancer and subjects not suffering from breast cancer; Preferably, the breast cancer includes stage 0, stage 1, stage 2, stage 3, stage 4 and stage 5 of breast cancer; more preferably, the kit is used to distinguish subjects suffering from stage 0, stage 1, stage 2, stage 3, stage 4 and stage 5 of breast cancer; Preferably, the biological sample is selected from urine, body fluid, serum, whole blood, plasma, human secretions, cerebrospinal fluid, or any combination thereof; Preferably, the biological sample is selected from whole blood (e.g., peripheral blood), serum, plasma, or any combination thereof; Preferably, the kit further comprises reagents for pre-treating the biological sample (eg, reagents for centrifugation, immunocapture and / or cell lysis).

5. A kit for predicting the risk of a subject having breast cancer, or for diagnosing whether a subject has breast cancer, or for monitoring the progression of breast cancer in a subject, or for diagnosing whether a subject has tumor cells (e.g., breast cancer cells) in the body, the kit comprising reagents for determining the level of a marker combination in a biological sample; in, The marker combination is selected from any 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of monoglyceride (MG 22:3), acylcarnitine (CAR 18:1), ceramide (Cer 18:1; 2O / 22:0), sphingomyelin (SM 18:1; 2O / 24:0), phosphatidylcholine (PC 18:1-19:1), lysophosphatidylethanolamine (LPE 21:3), sphingomyelin (SM 18:2; 2O / 17:1), oxidized fatty acid (OxFA 16:0; (2OH)), ceramide (Cer 18:2; 2O / 23:0), and sphingomyelin (SM 18:1; 2O / 22:0); Preferably, the marker combination comprises or consists of Cer 18:1;2O / 22:0, SM 18:1;2O / 24:0, LPE 21:3 and OxFA 16:0;(2OH); Preferably, the marker combination comprises or consists of MG 22:3, CAR 18:1, Cer18:1; 2O / 22:0, LPE 21:3 and OxFA 16:0; (2OH); Preferably, the marker combination comprises or consists of Cer 18:1; 2O / 22:0, PC 18:1_19:1, SM 18:2; 2O / 17:1, OxFA 16:0; (2OH), Cer 18:2; 2O / 23:0 and SM 18:1; 2O / 22:

0.

6. The kit according to claim 5, wherein The breast cancer cells include primary breast cancer cells, recurrent breast cancer cells, and tumor cells derived from breast cancer (e.g., tumor cells generated by breast cancer metastasis); Preferably, the breast cancer includes breast tube in situ cancer, infiltrative lobular in situ cancer, tubular / cribriform cancer, mucus (glial) cancer, myeloid cancer, papillary cancer and metaplastic cancer; Preferably, the breast cancer cells include male breast breast cancer cells (eg, male breast in situ or invasive cancer cells).

7. The kit of claim 5 or 6, wherein the reagent (e.g., the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and / or tenth or a combination of reagents) is for determining the level of a biomarker in the biological sample by chromatography and / or mass spectrometry, fluorescence, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near infrared spectroscopy (near IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS) and turbidimetry; Preferably, the reagent measures the level of the biomarker in the biological sample by spectroscopy, liquid or gas chromatography, mass spectrometry, or liquid or gas chromatography coupled with mass spectrometry; Preferably, the kit further comprises reagents and / or consumables for chromatography, reagents and / or consumables for mass spectrometry, or any combination thereof; Preferably, the reagents and / or consumables for chromatography are selected from chromatographic columns, acetonitrile aqueous solution (e.g., 10% acetonitrile aqueous solution, 50% acetonitrile aqueous solution, 60% acetonitrile aqueous solution), ammonium acetate, ammonium formate, formic acid, or any combination thereof; Preferably, the reagents and / or consumables for mass spectrometry are selected from mass spectrometry columns, formic acid, acetonitrile, or any combination thereof.

8. The kit according to any one of claims 5 to 7, wherein The subject is a mammal, such as a human; Preferably, the kit is used to distinguish between subjects suffering from breast cancer and subjects not suffering from breast cancer; Preferably, the breast cancer includes stage 0, stage 1, stage 2, stage 3, stage 4 and stage 5 of breast cancer; more preferably, the kit is used to distinguish subjects suffering from stage 0, stage 1, stage 2, stage 3, stage 4 and stage 5 of breast cancer; Preferably, the biological sample is selected from urine, body fluid, serum, whole blood, plasma, human secretions, cerebrospinal fluid, or any combination thereof; Preferably, the biological sample is selected from whole blood (e.g., peripheral blood), serum, plasma, or any combination thereof; Preferably, the kit further comprises reagents for pre-treating the biological sample (eg, reagents for centrifugation, immunocapture and / or cell lysis).

9. A method for predicting the risk of a subject suffering from breast cancer, or diagnosing whether a subject suffers from breast cancer, or monitoring the progression of breast cancer in a subject, or detecting the prognosis of a subject suffering from breast cancer, the method comprising: (1) obtaining a biological sample containing a marker combination from a subject; (2) determining the level of a marker combination in a biological sample; and (3) diagnosing whether the subject has breast cancer based on the level of the marker combination; Wherein, the marker combination is selected from any 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of monoglyceride (MG 22:3), acylcarnitine (CAR 18:1), ceramide (Cer 18:1; 2O / 22:0), sphingomyelin (SM 18:1; 2O / 24:0), phosphatidylcholine (PC 18:1_19:1), lysophosphatidylethanolamine (LPE 21:3), sphingomyelin (SM 18:2; 2O / 17:1), oxidized fatty acid (OxFA 16:0; (2OH)), ceramide (Cer 18:2; 2O / 23:0), and sphingomyelin (SM 18:1; 2O / 22:0); Preferably, the marker combination comprises or consists of Cer 18:1;2O / 22:0, SM 18:1;2O / 24:0, LPE 21:3 and OxFA 16:0;(2OH); Preferably, the marker combination comprises or consists of MG 22:3, CAR 18:1, Cer18:1; 2O / 22:0, LPE 21:3 and OxFA 16:0; (2OH); Preferably, the marker combination comprises or consists of Cer 18:1;2O / 22:0, PC 18:1_19:1, SM 18:2;2O / 17:1, OxFA 16:0;(2OH), Cer 18:2;2O / 23:0 and SM 18:1;2O / 22:0; Preferably, in step (3), the risk of the subject suffering from breast cancer is predicted, or whether the subject has breast cancer is diagnosed, or the progression of breast cancer in the subject is monitored, or the prognosis of a subject with breast cancer is detected by comparing the level of the marker combination with a reference value or the level of the marker combination of a healthy subject.

10. The method of claim 9, wherein: The breast cancer includes breast tube in situ cancer, infiltrative lobular in situ cancer, tubular / cribriform cancer, mucus (glial) cancer, myeloid cancer, papillary cancer and metaplastic cancer. Preferably, the method further comprises the step of treating the subject based on the diagnosis of breast cancer; wherein the treatment is selected from surgery, radiation, chemotherapy, hormone therapy, targeted drug therapy, immunotherapy, or any combination thereof.

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