A set of markers for breast cancer diagnosis

Abstract

The present application relates to the field of biological medicine. Specifically, the present application relates to the use of reagents for determining the levels of a combination of markers in a biological sample in the manufacture of a kit for predicting the risk of a subject of 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 present application also provides a kit for diagnosing whether a subject has breast cancer.

Description

Technical Field

[0001] This application relates to the field of biomedicine. Specifically, this application relates to the use of reagents for determining the levels of a combination of biomarkers in a biological sample in the preparation of a kit for predicting the risk of a subject having breast cancer, diagnosing whether a subject has breast cancer, monitoring the progression of breast cancer in a subject, or diagnosing the presence of tumor cells in a subject. This application also provides a kit for diagnosing whether a subject has breast cancer. Background Technology

[0002] According to the World Health Organization, approximately 2.3 million women worldwide were diagnosed with breast cancer in 2020, and 685,000 died from it. Breast cancer has the highest incidence and mortality rate among women's cancers, making it the second most common malignant tumor globally. In my country, the incidence of breast cancer is gradually increasing, particularly in the eastern coastal areas and economically developed large cities. In terms of age of onset, the incidence begins to rise gradually after age 20, peaking between 45 and 50 years of age.

[0003] Currently, there are many treatment options for breast cancer, typically including surgical resection, radiation therapy, and a combination of drug therapy (hormone therapy, chemotherapy, targeted biotherapy), which are expensive, invasive, and difficult for patients to accept. Although the global mortality rate of breast cancer patients has gradually decreased with the continuous optimization of treatment strategies, in China, especially in vast rural areas, the downward trend in breast cancer mortality is still not significant. If patients are diagnosed with breast cancer early and receive early treatment, the prognosis will be greatly improved, and the mortality rate will be significantly reduced. Clinical statistics show that the cure rate for breast cancer detected early can reach over 90%.

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

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

[0006] Therefore, in a first aspect, this application provides the use of a reagent for determining the level of a combination of biomarkers 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 for diagnosing whether a subject has tumor cells (e.g., breast cancer cells) in their body.

[0007] The biomarker combination is selected from any 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of the following: mono-fatty acid glycerides (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 acids (OxFA 16:0; (2OH)), ceramide (Cer 18:2; 2O / 23:0), and sphingomyelin (SM 18:1; 2O / 22:0).

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

[0009] In some 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 some 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 some 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 some 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 some embodiments, the structural formulas of the markers described herein and the corresponding fragmented nuclei are shown in the table below. In some embodiments, the names of the markers described herein are derived from the MS-DIAL database.

[0014]

[0015]

[0016] In some embodiments, the biomarker combination of this application exhibits different levels in tumor cells (e.g., breast cancer cells) compared to non-tumor cells (e.g., non-breast cancer cells). For example, in some embodiments, one or more biomarkers in the biomarker combination are present in tumor cells (e.g., breast cancer cells) but absent in non-tumor cells (e.g., non-breast cancer cells). In other embodiments, one or more biomarkers in the biomarker combination are absent in tumor cells (e.g., breast cancer cells) but present in non-tumor cells (e.g., non-breast cancer cells). In a further embodiment, one or more biomarkers in the biomarker combination exhibit different levels in tumor cells (e.g., breast cancer cells) compared to non-tumor cells (e.g., non-breast cancer cells). For example, one or more markers in a combination of markers may be present at any level of variation, but generally at an increase of 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%, or at least 12%. It is present at a level of 0%, at least 130%, at least 140%, at least 150% or more, or generally at a level reduced 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%, or 100% (e.g., missing).

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

[0018] In some 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 understandable 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, in situ or invasive cancer cells in the male breast.

[0020] Therefore, in some embodiments, the breast cancer cells include breast cancer cells of the male breast (e.g., in situ or invasive cancer cells of the male breast).

[0021] The biomarker combinations described herein may be used in conjunction with other biomarkers or combinations of biomarkers for identifying breast cancer, or in conjunction with other cancer biomarkers. For example, prostate cancer biomarkers include, but are not limited to: AMACR / P504S (US Patent No. 6,262,245); PCA3 (US Patent No. 7,008,765); PCGEM1 (US Patent No. 6,828,429); prostein / P501S, P503S, P504S, P509S, P510S, prostate / P703P, P710P (US Publication No. 20030185830).

[0022] The biomarkers of this application can be detected using any suitable method, including but not limited to liquid and gas chromatography, mass spectrometry alone or in combination (see, for example, the Experimental Section below), NMR (see, for example, U.S. Patent Publication 20070055456, incorporated herein by reference), immunoassay, chemiluminescence, spectrophotometry, and the like. In some embodiments, commercial systems using chromatographic and NMR analysis are employed.

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

[0024] In addition, the level of one or more other substances can be measured indirectly to determine the level of the marker of this application, for example by measuring the level of a compound (or compound) related to the level of the marker to be measured.

[0025] Therefore, in some embodiments, the reagents (e.g., the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and / or tenth reagents or combinations thereof) determine the level of biomarkers in the biological sample by methods such as: chromatographic and / or mass spectrometry, fluorescence assay, 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 some embodiments, the reagent is used to determine the level of biomarkers in the biological sample by spectroscopy, liquid or gas chromatography, mass spectrometry, or liquid or gas chromatography coupled with mass spectrometry.

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

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

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

[0030] In some implementations, the subject is a mammal, such as a human.

[0031] In some implementations, the kit is used to distinguish between subjects with breast cancer and subjects without breast cancer.

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

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

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

[0035] In some embodiments, the kit also includes reagents for pretreating the biological sample (e.g., reagents for centrifugation, immune capture, and / or cell lysis).

[0036] In a second aspect, this 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 their body, the kit comprising reagents for determining the level of a combination of biomarkers in a biological sample;

[0037] The biomarker combination is selected from any 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of the following: mono-fatty acid glycerides (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 acids (OxFA 16:0; (2OH)), ceramide (Cer 18:2; 2O / 23:0), and sphingomyelin (SM 18:1; 2O / 22:0).

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

[0039] In some 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 some 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 some 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 some 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 some embodiments, the breast cancer cells include primary breast cancer cells, recurrent breast cancer cells, and tumor cells derived from breast cancer (e.g., tumor cells resulting from breast cancer metastasis).

[0044] In some 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 some embodiments, the tumor cells include tumor cells of the male breast (e.g., in situ or invasive tumor cells of the male breast).

[0046] In some embodiments, the reagents (e.g., the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and / or tenth reagents or combinations thereof) determine the level of biomarkers in the biological sample by methods such as: chromatographic and / or mass spectrometry, fluorescence assay, 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 some embodiments, the reagent is used to determine the level of biomarkers in the biological sample by spectroscopy, liquid or gas chromatography, mass spectrometry, or liquid or gas chromatography coupled with mass spectrometry.

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

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

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

[0051] In some implementations, the subject is a mammal, such as a human.

[0052] In some implementations, the kit is used to distinguish between subjects with breast cancer and subjects without breast cancer.

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

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

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

[0056] In some embodiments, the kit also includes reagents for pretreating the biological sample (e.g., reagents for centrifugation, immune capture, and / or cell lysis).

[0057] In a third aspect, this 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 with breast cancer, the method comprising:

[0058] (1) Obtain biological samples containing a combination of biomarkers from the subjects;

[0059] (2) Determining the levels of biomarker combinations in biological samples; and

[0060] (3) Based on the levels of the combination of biomarkers, diagnose whether the subject has breast cancer;

[0061] The biomarker combination is selected from any 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of the following: mono-fatty acid glycerides (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 acids (OxFA 16:0; (2OH)), ceramide (Cer 18:2; 2O / 23:0), and sphingomyelin (SM 18:1; 2O / 22:0).

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

[0063] In some 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 some 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 some 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 some 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 some embodiments, in step (3), the risk of a subject having breast cancer is predicted, or whether a subject has breast cancer is diagnosed, or the progression of breast cancer in a subject is monitored, or the prognosis of a subject with breast cancer is assessed, by comparing the level of the biomarker combination with a reference value or the level of the biomarker combination in a healthy subject. In some embodiments, the reference value is the level or range of the biomarker in a biological sample obtained from a normal population.

[0068] In some 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 some embodiments, the present invention also provides one or more treatment methods used in conjunction with the methods described above.

[0070] For example, in some embodiments, the present invention provides one or more compounds that target the aforementioned combination of biomarkers. These compounds can modulate (e.g., increase or decrease) the level of the biomarker combination by, for example, interfering with the synthesis of the biomarker combination or its precursors or metabolites (e.g., by inhibiting the transcription or translation of enzymes involved in metabolite synthesis, by inactivating enzymes involved in metabolite synthesis (e.g., by post-translational modification or binding to an irreversible inhibitor), or otherwise by inhibiting the activity of enzymes involved in metabolite synthesis), or by binding to the function of inhibiting the biomarker combination.

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

[0072] Terminology Definition

[0073] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the chemical, biochemical, metabolomics, and lipidomics procedures used herein are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0074] As used herein, the term "marker" refers to a biochemical indicator that can mark changes or potential changes in the structure or function of systems, organs, tissues, cells, and subcellular structures, and has a very wide range of applications. Markers can be used for disease diagnosis, disease staging, or to evaluate the safety and efficacy of new drugs or therapies in target populations.

[0075] As used in this article, the term "breast cancer" refers to a disease in which abnormal breast cells grow uncontrollably and form tumors. If left untreated, these tumors can spread throughout the body. Breast cancer cells originate in the milk ducts and / or lobules of the breast. The earliest form (in situ) is not life-threatening and can be detected at an early stage. Cancer cells can spread to nearby breast tissue (invasiveness). 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 individual patient's condition, the type of cancer, and its extent. Treatment methods combine surgery, radiation therapy, and medication.

[0076] As used herein, the term "reference value" refers to a predetermined value for a compound, derived from the level of the compound in a control sample (e.g., obtained from healthy subjects). Reference values ​​can be used as thresholds to distinguish between subjects who may have breast cancer and healthy subjects. Reference values ​​can be relative values, numerical ranges with upper and lower limits, averages, medians, etc. Those skilled in the art can select suitable control samples, determine, and obtain reference values ​​according to methods disclosed in the prior art.

[0077] In some implementations, a significant difference in the level of the biomarker from the reference range for the biomarker in healthy subjects (e.g., 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; SM 18:1; 2O / 22:0) indicates that the subject has a risk of developing breast cancer, or already has breast cancer, or has experienced a recurrence of breast cancer.

[0078] As used herein, the term "lipid" refers to substances produced during chemical or physical processes in the human body. Lipids are primarily classified into 16 classes, with abbreviations in parentheses, including acylcarnitines (CAR), ceramides (Cer), cardiolipins (CL), ether-phosphatidylcholine derivatives (EtherPC), ether-phosphatidylethanolamine derivatives (EtherPE), free fatty acids (FA), lysophosphatidylcholine derivatives (LPC), lysophosphatidylethanolamine derivatives (LPE), phosphatidylcholine derivatives (PC), phosphatidylethanolamine derivatives (PE), phosphatidylglycerol derivatives (PG), phosphatidylinositol derivatives (PI), phosphatidylserine derivatives (PS), sphingolipids (SM), triglycerides (TG), and others. Compared to other metabolic processes, lipids, as crucial substances for cellular signal transduction, possess unique characteristics and functional specificity; their dynamic changes also foreshadow alterations in disease metabolic mechanisms.

[0079] As used in this article, the term "lipomics" refers to the science of studying the changes in lipids with different chemical properties in organisms using chromatography-mass spectrometry. Its research content mainly includes determining lipid structure (e.g., number of atoms, carbon chain length, number and position of double bonds, etc.), accurately quantifying various lipids, discovering lipid biomarkers, 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 target molecules and then analyzing the fragments based on their mass-to-charge ratios to produce a mass spectrum, serving as a "molecular fingerprint." The mass-to-charge ratio of an object is determined by specifying the wavelengths in which electromagnetic energy is absorbed by that object. Several common methods exist for determining the mass-to-charge ratio of ions; some methods use electromagnetic waves to measure the interactions of ion orbitals, others measure the time it takes for ions to travel a specific distance, or both. Data from these fragment mass measurements can be retrieved from databases to obtain the precise identification of the target molecule. Mass spectrometry is also widely used in other areas of chemistry, such as petrochemicals or 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, cattle, cats, pigs, monkeys, chimpanzees, etc. In some embodiments, the subject is a human.

[0082] As used herein, the term "prognosis" refers to the predicted likelihood of death or progression from cancer, including the development of metastatic breast cancer. The term "prediction," as used here, refers to the likelihood that a patient will have a favorable or unfavorable response to a drug or a group of drugs.

[0083] As used in this article, the term “tumor” refers to the growth and proliferation of all tumor cells, whether malignant or benign, as well as 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 receiver operating characteristic (ROC) curve, both of which are well known in the art. The AUC measure can be used to compare the accuracy of classifiers across the entire data range. A classifier with a larger AUC has a greater ability to correctly classify an unknown between two groups of interest (e.g., breast cancer samples and normal or control samples). ROC curves can be used to plot the performance of a specific feature (e.g., any 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, feature data for the entire population (e.g., cases and controls) are sorted in ascending order based on the values ​​of individual features. Then, for each value of that 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 with values ​​higher than that feature and then dividing by the total number of cases. The false positive rate is determined by counting the number of controls with values ​​higher than that feature and then dividing by the total number of controls. Although this definition refers to a case where a characteristic is elevated compared to a control, it also applies to cases where a characteristic is decreased compared to a control (in such cases, samples with values ​​below that characteristic will be counted). ROC curves can be generated for individual characteristics as well as other individual outputs. For example, combinations of two or more characteristics can be mathematically combined (e.g., added, subtracted, multiplied, etc.) to provide a single sum, which can be plotted on an ROC curve. Additionally, any combination of multiple characteristics can be plotted on an ROC curve, where this combination yields a single output value. These combinations of characteristics can constitute a test. An ROC curve is a graph of the true positive rate (sensitivity) of a test versus the false positive rate (1-specificity) of a test.

[0085] As used herein, the term "sample" is used in its broadest sense. In one sense, it refers to 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, etc. However, this example should not be construed as limiting the types of samples that can be used with respect to this invention.

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

[0087] Beneficial effects of the invention

[0088] This application obtained a combination of biomarkers capable of accurately determining whether a subject has breast cancer by analyzing the lipidome of two groups of subjects (breast cancer subjects and healthy subjects) in two different cohorts (screening cohort and validation cohort). Specifically, it includes any two, three, four, five, six, seven, eight, nine, or all ten biomarkers listed in the following combination: 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, SM 18:1; 2O / 22:0. These biomarker combinations have a very high diagnostic accuracy for breast cancer (above 90%, reaching up to 96%, while any single biomarker typically has an accuracy between 55% and 65%).

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

[0090] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0091] Figure 1 This study demonstrates lipids in clinical serum and their association with disease. Among them, 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 diagram illustrates the partial least squares discriminant analysis (PLS-DA) analysis between the two groups in the test queue; Figure 1 C in the diagram illustrates the partial least squares discriminant analysis (PLS-DA) analysis between the two groups in the verification queue; Figure 1 The D-plot in the diagram shows the ROC analysis of lipids with overlap between the test and validation cohorts.

[0092] Figure 2 The screened lipid biomarkers and their validation results are presented. Among them, Figure 2 Figure A shows the top 10 differentially expressed lipids selected based on VIP values. The most significant changes were observed in the following lipids: 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; SM 18:1; and 2O / 22:0. Figure 2 B in the figure is a bar chart showing the total differences in the top 10 lipids between the breast cancer (BC) group and the healthy subjects (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 subjects (HD) group in the test cohort; Figure 2 D in the figure is a box plot of the total differences in the top 10 lipids between the breast cancer (BC) group and the healthy subjects (HD) group in the validation cohort; Figure 2 E in the figure is a separate box plot of the top 10 lipids in the breast cancer (BC) group and the healthy subjects (HD) group in the validation cohort; Figure 2 F in the figure represents the ROC curve of the combined analysis of the top 10 lipids and the total lipids.

[0093] Figure 3 The selected lipid biomarker combinations and their validation results are presented. Among them, Figure 3 A in the figure is the ROC curve of a combination of four lipids as biomarkers: Cer 18:1; 2O / 22:0, SM 18:1; 2O / 24:0, LPE 21:3, and OxFA 16:0; (2OH). Figure 3 B in the figure is the ROC curve of the combined analysis of five lipids as biomarkers: MG 22:3, CAR 18:1, Cer 18:1; 2O / 22:0, LPE 21:3, and OxFA 16:0; (2OH). Figure 3The C in the figure is the ROC curve of a combination analysis of six lipids as biomarkers: 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 Implementation

[0094] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0095] Unless otherwise specified, the experiments and methods described in the examples are generally performed in accordance with conventional methods well known in the art and described in various references. For example, conventional techniques such as chemistry, biochemistry, metabolomics, and lipidomics used in this invention can be found in Birendra N. Pramanik, AK Ganguly, et al., *Applications of Electrospray Mass Spectrometry* (2005); Xu Guowang, et al., *Metabolomics - Methods and Applications* (2008); and Jia Wei, et al., *Medical Metabolomics* (2011).

[0096] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

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

[0098] All clinical serum samples used in this experiment were collected from Fujian Medical University Union Hospital, totaling 1726 samples. The testing cohort consisted of 1393 participants (698 breast cancer patients and 695 healthy individuals), while the validation cohort consisted of 333 participants (142 breast cancer patients and 191 healthy individuals). The breast cancer patients were diagnosed by physicians based on a combination of clinical manifestations, physical examinations, imaging studies, and histopathological examinations. The collected whole blood was immediately centrifuged to harvest serum, which was then stored at -80°C. The serum was then 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 thawing and vortexing the samples, 50 μL of serum from each sample was transferred to a corresponding 1.5 mL centrifuge tube. Then, 400 μL of pre-chilled mass spectrometry-grade methanol containing internal standard was added, and the mixture was vortexed for 2 min. Next, 1 mL of tert-butyl methyl ether (MTBE) was added, the metal bath was pre-chilled under nitrogen, and the mixture was shaken at 1000 rpm for 20 min. Then, 400 μL of ultrapure water was added, the mixture was vortexed for 1 min, allowed to stand for 2 min, and then centrifuged at 7 °C (11000 rpm, 10 min). 150 μL of the supernatant was transferred to a new centrifuge tube and dried under nitrogen. For the preparation of mixed samples, 50 μL of the supernatant from each sample was combined into one sample, carefully vortexed, and used 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 (°C): 320; Vaporizer Temp (°C): 350; Orbitrap Resolution: 120000; Mass spectrometry scan range: m / z 150-2000 Da.

[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℃; 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 Experiments Using Validation Cohort Serum

[0109] Extraction of serum lipids:

[0110] After thawing and vortexing the samples, 50 μL of serum from each sample was transferred to a corresponding 1.5 mL centrifuge tube. Then, 400 μL of pre-chilled tert-butyl methyl ether (MTBE) was added, and the mixture was vortexed for 30 seconds. Next, 80 μL of pre-chilled methanol containing an internal standard was added, and the mixture was vortexed for 30 seconds. The samples were then sonicated on ice for 10 min, and finally centrifuged at 4°C (3000 rpm, 15 min). 300 μL of the supernatant was transferred to a new centrifuge tube and dried under nitrogen. For the preparation of mixed samples, 20 μL of supernatant from each sample was combined into one sample during reconstitution, and the mixture was carefully vortexed to form a mixed sample, which served 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 (°C): 320; Vaporizer Temp (°C): 350; Orbitrap Resolution (MS1): 120000; Orbitrap Resolution (MS2): 150000; Mass spectrometry scan range: m / z 100-1500 Da.

[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℃; 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 Validation of Biomarkers

[0118] The relative quantitative results, based on the XCMSR language package, require data correction, including total peak area correction, internal standard correction, and QC correction. QC correction is performed using the stattarget package, with a CV setting of 30%. Metabolomics data screening follows an 80% principle, and KNN algorithm is used for missing value imputation. Statistical analysis is then performed after data correction.

[0119] Experimental results

[0120] The results of lipidomics screening and validation experiments are as follows: Figures 1 to 3 As shown in Table 1, the specific information of the 10 lipid biomarkers selected is as follows.

[0121] 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 1 B in the diagram illustrates the partial least squares discriminant analysis (PLS-DA) analysis between the two groups in the test queue; Figure 1 C in the diagram illustrates the partial least squares discriminant analysis (PLS-DA) analysis between the two groups in the verification queue; Figure 1 The D-plot in the diagram shows the overlapping lipid ROC analysis between the test and validation cohorts.

[0122] Figure 2 Figure A shows the top 10 differentially expressed lipids selected based on VIP values. The most significant changes were observed in the following lipids: 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; SM 18:1; and 2O / 22:0. Figure 2 B in the figure is a bar chart showing the difference in the total content of the top 10 lipids in the breast cancer (BC) group and the healthy subjects (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 subjects (HD) group in the test cohort.

[0123] Based on the above analysis, biomarkers showing significant differences in levels between the breast cancer (BC) group and the healthy subjects (HD) group were obtained through screening in the test cohort. These biomarkers are: 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. Furthermore, the biomarkers obtained from the above screening were validated in a validation cohort. The results of the validation cohort are as follows: Figure 2 As shown in D to F in the diagram.

[0124] Figure 2 D in the figure is a box plot of the total differences in the top 10 lipids between the breast cancer (BC) group and the healthy subjects (HD) group in the validation cohort; Figure 2 E in the figure is a separate box plot of the top 10 lipids in the breast cancer (BC) group and the healthy subjects (HD) group in the validation cohort; Figure 2 F in the figure represents the ROC curve of the combined analysis of the top 10 lipids and the total lipids. It can be seen that the AUC value of the combined ROC curve of the ten lipids is as high as 0.96, which has a high confidence level. Compared with any single lipid in the combination of ten lipids (most of which are between 0.55 and 0.65), the AUC value of the lipid combination in this application has been significantly improved.

[0125] Figure 3 A in the figure is the ROC curve of four lipidome biomarkers: 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 these four lipid biomarkers is as high as 0.8986. Figure 3 B in the figure represents the ROC curve of the combined analysis of five lipid biomarkers: MG 22:3, CAR 18:1, Cer 18:1; 2O / 22:0, LPE 21:3, OxFA 16:0; and (2OH). The AUC value of the ROC curve of the combined analysis of these five lipids is as high as 0.9332. Figure 3 In the figure, C represents the ROC curve of the combined analysis of six lipidome biomarkers: 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. The AUC value of the ROC curve for this combined analysis of six lipids is as high as 0.9116.

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

[0127] Table 1 Information on lipid biomarkers

[0128]

[0129]

[0130] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. The use of reagents for determining the level of a combination of biomarkers 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 for diagnosing whether a subject has tumor cells in his body; in, The biomarker combination is selected from any 2, 3, 4, 5, 6, 7, 8, 9, or all 10 of the following: mono-fatty acid glycerides (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 acids (OxFA 16:0;(2OH)), ceramide (Cer 18:2;2O / 23:0), and sphingomyelin (SM 18:1;2O / 22:0).

2. The use as described in claim 1, wherein, 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).

3. The use as described in claim 1, wherein, 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).

4. The use as described in claim 1, wherein, The marker combination includes 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.

5. The use as described in claim 1, wherein, The tumor cells are breast cancer cells.

6. The use as described in claim 5, wherein, The breast cancer cells include primary breast cancer cells, recurrent breast cancer cells, and tumor cells derived from breast cancer.

7. The use as described in claim 1, wherein, The breast cancers mentioned include ductal carcinoma in situ, invasive lobular carcinoma in situ, tubular / criteria carcinoma, mucinous carcinoma, myeloid carcinoma, papillary carcinoma, and metaplastic carcinoma.

8. The use as described in claim 5, wherein, The breast cancer cells mentioned include breast cancer cells from the male breast.

9. The use according to claim 1, wherein the reagent is used to determine the level of biomarkers in the biological sample by methods such as: chromatography and / or mass spectrometry, fluorescence assay, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy, fluorescence analysis, radiochemical analysis, near-infrared spectroscopy, nuclear magnetic resonance spectroscopy, light scattering analysis, and turbidimetry.

10. The use according to claim 9, wherein the reagent is used to determine 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.

11. The use as described in claim 1, wherein the kit further comprises reagents and / or consumables for chromatography, reagents and / or consumables for mass spectrometry, or any combination thereof.

12. The use as claimed in claim 11, wherein the reagents and / or consumables for chromatography are selected from chromatographic columns, aqueous acetonitrile solutions, ammonium acetate, ammonium formate, formic acid, or any combination thereof.

13. The use as described in claim 12, wherein the reagents and / or consumables for mass spectrometry are selected from mass spectrometry columns, formic acid, acetonitrile, or any combination thereof.

14. The use as described in claim 1, wherein, The subjects were mammals.

15. The use as described in claim 1, wherein, The subjects were humans.

16. The use as described in claim 1, wherein, The kit is used to distinguish between subjects with breast cancer and those without breast cancer.

17. The use as claimed in claim 1, wherein, The biological samples are selected from urine, body fluids, serum, whole blood, plasma, human secretions, cerebrospinal fluid, or any combination thereof.

18. The use as described in claim 1, wherein, The biological sample was selected from peripheral blood.

19. The use as described in claim 1, wherein, The kit also includes reagents for pretreating the biological sample.

20. 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 his body, said kit comprising reagents for determining the level of a combination of biomarkers in a biological sample; in, The biomarker combination is selected from any 2, 3, 4, 5, 6, 7, 8, 9, or all 10 of the following: mono-fatty acid glycerides (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 acids (OxFA 16:0;(2OH)), ceramide (Cer 18:2;2O / 23:0), and sphingomyelin (SM 18:1;2O / 22:0).

21. The kit of claim 20, wherein, The marker combination comprises or consists of Cer 18:1;2O / 22:0, SM18:1;2O / 24:0, LPE 21:3 and OxFA 16:0;(2OH).

22. The kit of claim 20, wherein, 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).

23. The kit of claim 20, wherein, The marker combination includes or consists of Cer 18:1;2O / 22:0, PC18:1_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.

24. The kit of claim 20, wherein, The tumor cells are breast cancer cells.

25. The kit of claim 24, wherein, The breast cancer cells include primary breast cancer cells, recurrent breast cancer cells, and tumor cells derived from breast cancer.

26. The kit of claim 20, wherein the breast cancer includes ductal carcinoma in situ, invasive lobular carcinoma in situ, tubular / cribriform carcinoma, mucinous carcinoma, myeloid carcinoma, papillary carcinoma, and metaplastic carcinoma.

27. The kit of claim 24, wherein the breast cancer cells comprise breast cancer cells from the male breast.

28. The kit of claim 20, wherein the reagent determines the level of biomarkers in the biological sample by means of: chromatography and / or mass spectrometry, fluorescence assay, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy, fluorescence analysis, radiochemical analysis, near-infrared spectroscopy, nuclear magnetic resonance spectroscopy, light scattering analysis, and turbidimetry.

29. The kit of claim 20, wherein the reagent determines 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.

30. The kit of claim 20, further comprising reagents and / or consumables for chromatography, reagents and / or consumables for mass spectrometry, or any combination thereof.

31. The kit of claim 20, wherein the reagents and / or consumables for chromatography are selected from chromatographic columns, aqueous acetonitrile solutions, ammonium acetate, ammonium formate, formic acid, or any combination thereof.

32. The kit of claim 20, wherein the reagents and / or consumables for mass spectrometry are selected from mass spectrometry columns, formic acid, acetonitrile, or any combination thereof.

33. The kit according to claim 20, wherein, The subjects were mammals.

34. The kit of claim 20, wherein, The subjects were humans.

35. The kit of claim 20, wherein the kit is used to distinguish between subjects with breast cancer and subjects without breast cancer.

36. The kit of claim 20, wherein the biological sample is selected from urine, body fluids, serum, whole blood, plasma, human secretions, cerebrospinal fluid, or any combination thereof.

37. The kit of claim 20, wherein the biological sample is selected from peripheral blood.

38. The kit of claim 20, further comprising reagents for pretreating the biological sample.

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