Early breast cancer detection using blood-based metabolomics analysis

Abstract

Described herein are blood-based metabolites and metabolic markers for informatively distinguishing between healthy controls and early stage breast cancer (e.g., Stage I or II), Stage I breast cancer, Stage II breast cancer, Stage III breast cancer, ductal carcinoma (e.g., Stage I or II), lobular carcinoma (e.g., Stage I or II), and luminal breast cancer (e.g., luminal A type).

Description

[0001] This specification relates to blood-based metabolites for the detection of early-stage breast cancer. It also relates to blood-based metabolites capable of differentiating breast cancer stages and tumor subtypes.

[0002] This manual cites several references, the contents of which are incorporated into this document in their entirety through citation. Background Technology

[0003] Breast cancer is a leading cause of cancer death among women, and early diagnosis is crucial for the successful treatment, management, and care of breast cancer patients. Current breast cancer screening methods primarily rely on imaging techniques such as mammography; however, these techniques would greatly benefit from informative biomarkers that can help identify early-stage breast cancer and differentiate breast cancer stages and / or subtypes. Summary of the Invention

[0004] In a first aspect, this article describes a method for processing human clinical blood samples, the method comprising obtaining a blood sample from a subject clinically assessed as having or suspected of having breast cancer, and quantifying an informative group of metabolites in the blood sample that distinguish breast cancer from healthy or non-breast cancer controls. In some embodiments, the metabolite group described herein may comprise at least one blood-based metabolite or consist substantially of at least one blood-based metabolite, said at least one blood-based metabolite being: propionic acid (propionate), asparagine, butyric acid, CO (carnitine), C3 (propionylcarnitine), C3OH (hydroxypropionylcarnitine), C4 (butyrylcarnitine), C4:1 (butenoylcarnitine), C5OH (hydroxyvalerate carnitine; C3-DC-M), C6.1 (hexenoylcarnitine), C12 (dodecanoylcarnitine), C14 (tetradecanoylcarnitine), C14:2OH (hydroxytetradecanoylcarnitine), C16:1OH (hydroxyhexadecanoylcarnitine), C16:2OH (hydroxyhexadecanoylcarnitine), C18:1OH (hydroxyoctadecanoylcarnitine), fumaric acid (fumarate), glucose, kynurenine, lysoPC a C18:1 (lysophosphatidylcholine acyl C18:1), methylmalonic acid (methylmalonate), PC aa C36:0 (phosphatidylcholine diacyl C36:0), PC aa C36:6 (phosphatidylcholine diacyl C36:6), PC aa C38:0 (phosphatidylcholine diacyl C38:0), PC aa C40:1 (phosphatidylcholine diacyl C40:1), PC aa C40:2 (phosphatidylcholine diacyl C40:2), PCae C40:6 (phosphatidylcholine acyl-alkyl C40:6), phenylalanine, serotonin, SM C16:0 (sphingomyelin C16:0), SMC18:0 (sphingomyelin C18:0), SM C18:1 (sphingomyelin C18:1), SM C20:2 (sphingomyelin C20:2), SM(OH) C16:1 (hydroxysphingomyelin C16:1), SM(OH)C22:1 (hydroxysphingomyelin C22:1), SM(OH)C22:2 (hydroxysphingomyelin C22:2), SM(OH)C24:1 (hydroxysphingomyelin C24:1), spermidine, succinic acid (succinate), taurine, or any combination thereof.

[0005] On the other hand, this document describes a method for assessing breast cancer in subjects, the method comprising: processing human clinical blood samples according to the method described herein, and comparing the levels of a quantified group of metabolites with corresponding reference values ​​indicating healthy or non-breast cancer subjects. In some embodiments, a decrease in the level of one or more of the following metabolites indicates breast cancer: propionic acid (propionate), C6.1 (hexenocarnitine), C18:1OH (hydroxyoctadecenocarnitine), fumaric acid (fumarate), kynurenine, or succinic acid (succinate). In some implementations, elevated levels of one or more of the following metabolites indicate breast cancer: asparagine, butyric acid, C0 (carnitine), C3 (propionylcarnitine), C3OH (hydroxypropionylcarnitine), C4 (butyrylcarnitine), C4:1 (butenoylcarnitine), C5OH (hydroxyvalerate carnitine; C3-DC-M), C12 (dodecanoylcarnitine), C14 (tetradecanoylcarnitine), C14:2OH (hydroxytetradecanoylcarnitine), C16:1OH (hydroxyhexadecanoylcarnitine), C16:2OH (hydroxyhexadecanoylcarnitine), glucose, lysoPC a C18:1 (lysophosphatidylcholine acyl C18:1), methylmalonic acid (methylmalonate), PC aa C36:0 (phosphatidylcholine diacyl C36:0), PC aa C36:6 (phosphatidylcholine diacyl C36:6), PC aa C38:0 (phosphatidylcholine diacyl C38:0), PC aa C40:1 (phosphatidylcholine diacyl C40:1), PC aa C40:2 (phosphatidylcholine diacyl C40:2), PC ae C40:6 (phosphatidylcholine acyl-alkyl C40:6), phenylalanine, serotonin, SM C16:0 (sphingomyelin C16:0), SM C18:0 (sphingomyelin C18:0), SM C18:1 (sphingomyelin C18:1), SM C20:2 (sphingomyelin C20:2), SM(OH) C16:1 (hydroxysphingomyelin C16:1), SM(OH) C22:1 (hydroxysphingomyelin C22:1), SM(OH) C22:2 (hydroxysphingomyelin C22:2), SM(OH) C24:1 (hydroxysphingomyelin C24:1), spermidine, or taurine.

[0006] On the other hand, this document describes a method for implementing or improving a breast cancer screening program (e.g., an existing breast cancer screening program), the method comprising: incorporating a group of metabolites from a quantitative sample of a subject into the screening program, the group of metabolites comprising or consisting substantially of at least one blood-based metabolite as described herein.

[0007] On the other hand, this article describes a method for treating a subject with breast cancer, the method comprising: assessing the subject's breast cancer status according to the method described herein, and administering breast cancer treatment to the subject based on that assessment.

[0008] On the other hand, this article describes a biomarker used in a group of blood-based biomarkers for assessing breast cancer in subjects, said biomarker being at least one blood-based metabolite as described herein.

[0009] In another aspect, this paper describes a group of breast cancer biomarkers comprising at least one blood-based metabolite as described herein.

[0010] General definition Titles and other identifiers (e.g., (a), (b), (i), (ii), etc.) are provided solely for ease of reading the specification and claims. The use of titles or other identifiers in the specification or claims does not necessarily require that steps or elements be performed in alphabetical or numerical order or in the order in which they are presented.

[0011] When used in conjunction with the term "comprising" in the claims and / or description, the word "a (or an)" may mean "one," but may also mean "one or more," "at least one," and "one or more."

[0012] When used herein, the term “about” means that a value includes the standard deviation of the error of the apparatus or method used to determine that value. Typically, the term “about” is intended to indicate a possible variation of up to 10%. Therefore, the term “about” covers variations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10% of the value. Unless otherwise stated, the use of the term “about” before a range applies to both ends of that range.

[0013] As used in this specification and claims, the terms “comprising” (and any of its forms, such as “comprise” and “comprises”), “having” (and any of its forms, such as “have” and “has”), “including” (and any of its forms, such as “includes” and “include”), or “containing” (and any of its forms, such as “contains” and “contain”) are inclusive or open-ended and do not exclude other unmentioned elements or method steps.

[0014] Other objectives, advantages, and features of this specification will become more apparent after reading the following non-limiting description of specific embodiments given by way of example only and with reference to the accompanying drawings. Attached Figure Description

[0015] In the attached diagram: Figure 1 depicts the composition of the study population, including the sample size from breast cancer and healthy controls, and the different breast cancer subgroups included. Figure 1A ), and the different breast cancer stages included ( Figure 1B ).

[0016] Figure 2 The relative values ​​of metabolites that are particularly informative for distinguishing breast cancer from healthy controls are shown.

[0017] Figure 3 The results of partial least squares discriminant analysis (PLS-DA) score plots using a small group of five metabolites plus age are shown, demonstrating the spatial separation between breast cancer samples and healthy controls.

[0018] Figure 4 The receiver operating characteristic (ROC) curves generated for all stages of breast cancer patients using the optimal logistic regression model when the included age is shown.

[0019] Figure 5 The image shows a heatmap constructed to distinguish stage I breast cancer patients from healthy controls.

[0020] Figure 6 The image shows a heatmap constructed to distinguish stage II breast cancer patients from healthy controls.

[0021] Figure 7 ROC curves based on six metabolites plus age were shown to distinguish between stage I and stage II breast cancer patients and healthy controls. Detailed Implementation

[0022] In a first aspect, this document describes a method for processing human clinical blood samples, the method comprising obtaining a blood sample from a subject clinically assessed as having or suspected of having breast cancer, and quantifying a metabolite group in the blood sample. In some embodiments, the metabolite group comprises or consists substantially of blood-based metabolites that informatively distinguish between healthy controls and early-stage breast cancer (e.g., stage I or II), stage I breast cancer, stage II breast cancer, stage III breast cancer, ductal carcinoma (e.g., stage I or II), lobular carcinoma (e.g., stage I or II), and luminal breast cancer (e.g., luminal type A).

[0023] In some embodiments, the metabolite group comprises at least one blood-based metabolite or consists substantially of at least one blood-based metabolite, said blood-based metabolite being: propionic acid (propionate), asparagine, butyric acid, CO (carnitine), C3 (propionylcarnitine), C3OH (hydroxypropionylcarnitine), C4 (butyrylcarnitine), C4:1 (butenoylcarnitine), C5OH (hydroxyvalerate carnitine; C3-DC-M), C6.1 (hexenoylcarnitine), C12 (dodecanoylcarnitine), C14 (tetradecanoylcarnitine), C14:2OH (hydroxytetradecanoylcarnitine), C16:1OH (hydroxyhexadecanoylcarnitine), C16:2OH (hydroxyhexadecanoylcarnitine), C18:1OH (hydroxyoctadecanoylcarnitine), fumaric acid (fumarate), glucose, kynurenine, lysoPC a C18:1 (lysophosphatidylcholine acyl C18:1), methylmalonic acid (methylmalonate), PC aa C36:0 (phosphatidylcholine diacyl C36:0), PC aa C36:6 (phosphatidylcholine diacyl C36:6), PC aa C38:0 (phosphatidylcholine diacyl C38:0), PC aa C40:1 (phosphatidylcholine diacyl C40:1), PC aa C40:2 (phosphatidylcholine diacyl C40:2), PC ae C40:6 (phosphatidylcholine acyl-alkyl C40:6), phenylalanine, serotonin, SM C16:0 (sphingomyelin C16:0), SM C18:0 (sphingomyelin C18:0), SM C18:1 (sphingomyelin C18:1), SM C20:2 (sphingomyelin C20:2), SM(OH) C16:1 (hydroxysphingomyelin C16:1), SM(OH)C22:1 (hydroxysphingomyelin C22:1), SM(OH)C22:2 (hydroxysphingomyelin C22:2), SM(OH)C24:1 (hydroxysphingomyelin C24:1), spermidine, succinic acid (succinate), taurine, or any combination thereof. In some embodiments, at least one blood-based metabolite may comprise propionic acid (propionate). In some embodiments, the metabolite group may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 of the blood-based metabolites.

[0024] As used herein, the term "subjects clinically assessed as having or suspected of having breast cancer" refers to a subset of subjects who, based on, for example, multiple risk factors (e.g., patient symptoms, age, family history of breast cancer, genetic testing, imaging results (e.g., mammography, MRI, ultrasound) or biopsy results), have a reasonable likelihood of having or developing breast cancer. In some implementations, this term may refer to subjects who have been evaluated by a healthcare professional and recommended for further breast cancer screening, or subjects already enrolled in an existing breast cancer screening program.

[0025] As used herein, in the context of the biomarker group described herein, the expression "consistently of" means a metabolite included in the group and sufficient to achieve the intended purpose (e.g., assessing breast cancer or differentiating different breast cancer stages and / or subtypes), but does not exclude the inclusion of other metabolites that do not materially affect the method or the performance of the biomarker group for breast cancer.

[0026] In some embodiments, the blood sample described herein may be from a subject clinically assessed as having or suspected of having early-stage breast cancer, wherein the early-stage breast cancer is stage I or II breast cancer, and wherein the metabolite group quantified in the blood sample comprises or consists substantially of at least one blood-based metabolite, wherein the at least one blood-based metabolite is: propionic acid, asparagine, CO, C3, C3OH, C4, C4:1, C5OH, C6.1, C12, C14, C14:2OH, C16:1OH, C16:2OH, fumaric acid, glucose, kynurenine, lysoPC aC18:1, methylmalonic acid, PC aa C36:0, PC aa C36:6, PC aa C38:0, PC aa C40:1, PC aa C40:2, PC ae C40:6, phenylalanine, serotonin, SM C16:0, SM C18:0, SM C18:1, SM C20:2, SM(OH)C16:1, SM(OH)C22:1, SM(OH)C22:2, SM(OH)C24:1, spermidine, succinic acid, taurine, or any combination thereof. In some embodiments, the metabolite group quantified in the blood sample may comprise at least one blood-based metabolite or consist substantially of at least one blood-based metabolite, said at least one blood-based metabolite being: propionic acid, asparagine, CO, C5OH, C16:2OH, fumaric acid, glucose, kynurenine, PCaa C40:1, PCaa C40:2, PCae C40:6, phenylalanine, serotonin, SM(OH)C24:1, spermidine, succinic acid, or any combination thereof.

[0027] In some embodiments, the blood sample described herein may be from a subject clinically assessed as having or suspected of having stage I breast cancer, wherein the metabolite group quantified in the blood sample comprises or is primarily composed of at least one blood-based metabolite, said at least one blood-based metabolite being: asparagine, CO, C3, C3OH, C4, C5OH, C6.1, C12, C14, C16:1OH, C16:2OH, fumaric acid, glucose, lysoPC aC18:1, methylmalonic acid, PC aaC36:0, PC aaC36:6, PC aaC40:1, PC aaC40:2, PC aeC40:6, phenylalanine, SM C18:0, SM C20:2, SM(OH) C16:1, SM(OH) C22:1, SM(OH) C22:2, spermidine, or any combination thereof.

[0028] In some embodiments, the blood sample described herein may be from a subject clinically assessed as having or suspected of having stage II breast cancer, wherein the metabolite group quantified in the blood sample comprises or is substantially composed of at least one blood-based metabolite, said at least one blood-based metabolite being: CO, C3OH, C4, C4:1, C5OH, C12, C14, C14:2OH, fumaric acid, glucose, lysoPC a C18:1, PC aa C36:0, PC aa C36:6, PC aa C38:0, PC aa C40:1, PC aa C40:2, PC ae C40:6, serotonin, SM C16:0, SM C18:0, SM C18:1, SM C20:2, SM(OH) C16:1, SM(OH) C22:1, SM(OH) C22:2, spermidine, succinic acid, taurine, or any combination thereof.

[0029] In some embodiments, the blood sample described herein may be from a subject clinically assessed as having or suspected of having stage III breast cancer, wherein the metabolite group quantified in the blood sample comprises or consists substantially of at least one blood-based metabolite, said at least one blood-based metabolite being: butyric acid, PC aa C40:2, or a combination thereof.

[0030] In some embodiments, the blood sample described herein may be from a subject clinically assessed as having or suspected of having ductal carcinoma (e.g., stage I or II), wherein the metabolite group quantified in the blood sample comprises or consists substantially of at least one blood-based metabolite, said at least one blood-based metabolite being: asparagine, fumaric acid, glucose, kynurenine, serotonin, spermidine, or any combination thereof.

[0031] In some embodiments, the blood sample described herein may be from a subject who has been clinically assessed as having or suspected of having lobular carcinoma (e.g., stage I or II), wherein the metabolite group quantified in the blood sample comprises or consists substantially of at least one blood-based metabolite, said at least one blood-based metabolite being: asparagine, glucose, PC aa C40:1, or any combination thereof.

[0032] In some embodiments, the blood sample described herein may be from a subject clinically assessed as having or suspected of having luminal breast cancer (e.g., luminal type A), wherein the metabolite group quantified in the blood sample comprises or consists substantially of at least one blood-based metabolite, said at least one blood-based metabolite being: CO, C18:1OH, fumaric acid, kynurenine, PCaaC40:2, spermidine, succinic acid, or any combination thereof.

[0033] On the other hand, this document describes a method for assessing breast cancer in subjects, the method comprising: processing human clinical blood samples according to the method described herein, and comparing the levels of a quantified group of metabolites with corresponding reference values ​​indicating healthy or non-breast cancer subjects. In some embodiments, a decrease in the level of one or more of the following metabolites indicates breast cancer: propionic acid (propionate), C6.1 (hexenocarnitine), C18:1OH (hydroxyoctadecenocarnitine), fumaric acid (fumarate), kynurenine, or succinic acid (succinate). In some implementations, elevated levels of one or more of the following metabolites indicate breast cancer: asparagine, butyric acid, C0 (carnitine), C3 (propionylcarnitine), C3OH (hydroxypropionylcarnitine), C4 (butyrylcarnitine), C4:1 (butenoylcarnitine), C5OH (hydroxyvalerate carnitine; C3-DC-M), C12 (dodecanoylcarnitine), C14 (tetradecanoylcarnitine), C14:2OH (hydroxytetradecanoylcarnitine), C16:1OH (hydroxyhexadecanoylcarnitine), C16:2OH (hydroxyhexadecanoylcarnitine), glucose, lysoPC a C18:1 (lysophosphatidylcholine acyl C18:1), methylmalonic acid (methylmalonate), PC aa C36:0 (phosphatidylcholine diacyl C36:0), PC aa C36:6 (phosphatidylcholine diacyl C36:6), PC aa C38:0 (phosphatidylcholine diacyl C38:0), PC aa C40:1 (phosphatidylcholine diacyl C40:1), PC aa C40:2 (phosphatidylcholine diacyl C40:2), PC ae C40:6 (phosphatidylcholine acyl-alkyl C40:6), phenylalanine, serotonin, SM C16:0 (sphingomyelin C16:0), SM C18:0 (sphingomyelin C18:0), SM C18:1 (sphingomyelin C18:1), SM C20:2 (sphingomyelin C20:2), SM(OH) C16:1 (hydroxysphingomyelin C16:1), SM(OH) C22:1 (hydroxysphingomyelin C22:1), SM(OH) C22:2 (hydroxysphingomyelin C22:2), SM(OH) C24:1 (hydroxysphingomyelin C24:1), spermidine, or taurine.

[0034] In some implementations, a method for assessing breast cancer in subjects may include: distinguishing between stage I or II breast cancer subjects and healthy controls or non-breast cancer subjects based on the metabolite groups described herein (e.g., summarized in Example 6); distinguishing between stage I breast cancer subjects and healthy controls or non-breast cancer subjects; distinguishing between stage II breast cancer subjects and healthy controls or non-breast cancer subjects; distinguishing between stage III breast cancer subjects and healthy controls or non-breast cancer subjects; distinguishing between ductal carcinoma (e.g., stage I or II) subjects and healthy controls or non-breast cancer subjects; distinguishing between lobular carcinoma (e.g., stage I or II) subjects and healthy controls or non-breast cancer subjects; distinguishing between luminal breast cancer (e.g., luminal type A) subjects and healthy controls or non-breast cancer subjects; or any combination thereof.

[0035] In some embodiments, a method for assessing breast cancer in a subject may further include: determining a breast cancer probability score based on a quantitative metabolite panel combined with the subject's age. In some embodiments, a method for assessing breast cancer in a subject may further include: providing breast cancer treatment recommendations based on the breast cancer assessment.

[0036] On the other hand, this document describes a method for implementing or improving a breast cancer screening program, the method comprising: incorporating quantification of a metabolite group in a subject's blood sample or a breast cancer assessment method as described herein into the screening program, the metabolite group comprising or substantially consisting of at least one blood-based metabolite as described herein.

[0037] In some embodiments, the blood sample as described herein may be plasma. In some embodiments, the blood sample as described herein may be serum.

[0038] In some implementations, the subjects described herein are clinically assessed as having or suspected of having breast cancer via mammography, breast magnetic resonance imaging (MRI), breast ultrasound, breast biopsy, or any combination thereof. In some implementations, the methods described herein may be used as part of an early breast cancer screening program that includes mammography, breast magnetic resonance imaging (MRI), breast ultrasound, breast biopsy, or any combination thereof.

[0039] On the other hand, this article describes a method for treating a subject with breast cancer, the method comprising: assessing the subject's breast cancer status according to the method described herein, and administering breast cancer treatment (e.g., early breast cancer treatment) to the subject based on this assessment.

[0040] On the other hand, this document describes a biomarker for use in a blood-based biomarker panel for assessing breast cancer in subjects, said biomarker being at least one blood-based metabolite as described herein. In some embodiments, assessing breast cancer in subjects may include: distinguishing between stage I or II breast cancer and healthy controls or non-breast cancer subjects based on the metabolite panel described herein (e.g., summarized in Example 6); distinguishing between stage I breast cancer and healthy controls or non-breast cancer subjects; distinguishing between stage II breast cancer and healthy controls or non-breast cancer subjects; distinguishing between stage III breast cancer and healthy controls or non-breast cancer subjects; distinguishing between ductal carcinoma (e.g., stage I or II) and healthy controls or non-breast cancer subjects; distinguishing between lobular carcinoma (e.g., stage I or II) and healthy controls or non-breast cancer subjects; distinguishing between luminal breast cancer (e.g., luminal type A) and healthy controls or non-breast cancer subjects; or any combination thereof.

[0041] In another aspect, this document describes a breast cancer biomarker panel comprising at least one blood-based metabolite as described herein. In some embodiments, the breast cancer biomarker panel can be used, based on the metabolite panel described herein (e.g., summarized in Example 6), to distinguish between stage I or II breast cancer and healthy controls or non-breast cancer subjects; to distinguish between stage I breast cancer and healthy controls or non-breast cancer subjects; to distinguish between stage II breast cancer and healthy controls or non-breast cancer subjects; to distinguish between stage III breast cancer and healthy controls or non-breast cancer subjects; to distinguish between ductal carcinoma (e.g., stage I or II) and healthy controls or non-breast cancer subjects; to distinguish between lobular carcinoma (e.g., stage I or II) and healthy controls or non-breast cancer subjects; to distinguish between luminal breast cancer (e.g., luminal type A) and healthy controls or non-breast cancer subjects; or any combination thereof.

[0042] On the other hand, this document describes a group of breast cancer biomarkers comprising at least one, two, three, four, five, six, seven, eight, nine, ten, or all of the following blood-based metabolites: SM(OH) C22:2, SMC18:0, CO, C3OH, C14:2OH, C16:2OH, lysoPCa C18:1, PCaa C36:0, asparagine, kynurenine, and tryptophan (e.g., summarized in Example 7). In some embodiments, the breast cancer biomarker group may also comprise the kynurenine:tryptophan ratio and / or age. In some embodiments, the breast cancer biomarker panel described herein may include a simplified metabolomics biomarker that detects no more than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 13, 12, 11, or 10 metabolites. In some embodiments, the simplified metabolomics biomarker may include a breast cancer probability or predictive score calculated based on the levels of any combination of the above metabolites, or may consist of a breast cancer probability or predictive score calculated based on the levels of any combination of the above metabolites.

[0043] project In some implementations, this document describes one or more of the following items: 1. A method for processing human clinical blood samples, the method comprising obtaining a blood sample from a subject clinically assessed as having or suspected of having breast cancer, and quantifying a metabolite group in the blood sample, the metabolite group comprising or consisting substantially of at least one blood-based metabolite, said at least one blood-based metabolite being: propionic acid (propionate), asparagine, butyric acid, CO (carnitine), C3 (propionylcarnitine), C3OH (hydroxypropionylcarnitine), C4 (butyryl... Carnitine), C4:1 (butenoyl carnitine), C5OH (hydroxyvalerate carnitine; C3-DC-M), C6.1 (hexenoyl carnitine), C12 (dodecanoyl carnitine), C14 (tetradecanoyl carnitine), C14:2OH (hydroxytetradecadienoyl carnitine), C16:1OH (hydroxyhexadecadienoyl carnitine), C16:2OH (hydroxyhexadecadienoyl carnitine), C18:1OH (hydroxyoctadecadienoyl carnitine), fumaric acid (fumarate), glucose, kynurenine, lysoPC a C18:1 (lysophosphatidylcholine acyl C18:1), methylmalonic acid (methylmalonate), PC aa C36:0 (phosphatidylcholine diacyl C36:0), PC aa C36:6 (phosphatidylcholine diacyl C36:6), PC aa C38:0 (phosphatidylcholine diacyl C38:0), PC aa C40:1 (phosphatidylcholine diacyl C40:1), PC aa C40:2 (phosphatidylcholine diacyl C40:2), PC ae C40:6 (phosphatidylcholine acyl-alkyl C40:6), phenylalanine, serotonin, SM C16:0 (sphingomyelin C16:0), SM C18:0 (sphingomyelin C18:0), SM C18:1 (sphingomyelin C18:1), SM C20:2 (sphingomyelin C20:2), SM(OH) C16:1 (hydroxysphingomyelin C16:1), SM(OH)C22:1 (hydroxysphingomyelin C22:1), SM(OH)C22:2 (hydroxysphingomyelin C22:2), SM(OH)C24:1 (hydroxysphingomyelin C24:1), spermidine, succinic acid (succinate), taurine, or any combination thereof.

[0044] 2. The method of Project 1, wherein the at least one blood-based metabolite comprises propionic acid (propionate); and / or wherein the at least one blood-based metabolite comprises at least one, two, three, four, five, six, seven, eight, nine, ten, or all of the following: SM(OH) C22:2, SM C18:0, CO, C3OH, C14:2OH, C16:2OH, lysoPC a C18:1, PC aa C36:0, asparagine, kynurenine, and tryptophan.

[0045] 3. The method as described in item 1 or 2, wherein the group of metabolites comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 of the blood-based metabolites.

[0046] 4. The method of any one of items 1 to 3, wherein the blood sample is from a subject clinically assessed as having or suspected of having early breast cancer, wherein the early breast cancer is stage I or II breast cancer, and wherein the metabolite group quantified in the blood sample comprises or consists substantially of at least one blood-based metabolite, wherein the at least one blood-based metabolite is: propionic acid, asparagine, CO, C3, C3OH, C4, C4:1, C5OH, C6.1, C12, C14, C14:2OH, C16:1OH, C16:2OH, fumaric acid, glucose, kynurenine, lysoPCa C18:1, methylmalonic acid, PCaa C36:0, PCaa C36:6, PCaa C38:0, PCaa C40:1, PCaa C40:2, PCae C40:6, phenylalanine, serotonin, SM C16:0, SM C18:0, SM C18:1, SM C20:2, SM(OH) C16:1, SM(OH) C22:1, SM(OH) C22:2, SM(OH) C24:1, spermidine, succinic acid, taurine, or any combination thereof.

[0047] 5. The method of Project 4, wherein the metabolite group quantified in the blood sample comprises or consists substantially of at least one blood-based metabolite, said at least one blood-based metabolite being: propionic acid, asparagine, CO, C3OH, C5OH, C16:2OH, fumaric acid, glucose, kynurenine, lysoPCa C18:1, PCaa C40:1, PCaa C40:2, PCae C40:6, phenylalanine, serotonin, SM(OH) C24:1, spermidine, succinic acid, or any combination thereof.

[0048] 6. The method of any one of items 1 to 5, wherein the blood sample is obtained from a subject clinically assessed as having or suspected of having stage I breast cancer, wherein the metabolite group quantified in the blood sample comprises or is composed primarily of at least one blood-based metabolite, said at least one blood-based metabolite being: asparagine, CO, C3, C3OH, C4, C5OH, C6.1, C12, C14, C16:1OH, C16:2OH, fumaric acid, glucose, lysoPCa C18:1, methylmalonic acid, PCaa C36:0, PCaa C36:6, PCaa C40:1, PCaa C40:2, PCae C40:6, phenylalanine, SM C18:0, SM C20:2, SM(OH) C16:1, SM(OH) C22:1, SM(OH) C22:2, spermidine, or any combination thereof.

[0049] 7. The method of any one of items 1 to 6, wherein the blood sample is obtained from a subject clinically assessed as having or suspected of having stage II breast cancer, wherein the metabolite group quantified in the blood sample comprises or consists substantially of at least one blood-based metabolite, said at least one blood-based metabolite being: CO, C4, C4:1, C3OH, C5OH, C12, C14, C14:2OH, fumaric acid, glucose, lysoPC a C18:1, PC aa C36:0, PC aa C36:6, PC aa C38:0, PC aa C40:1, PC aa C40:2, PC ae C40:6, serotonin, SM C16:0, SM C18:0, SM C18:1, SM C20:2, SM(OH) C16:1, SM(OH) C22:1, SM(OH) C22:2, spermidine, succinic acid, taurine, or any combination thereof.

[0050] 8. The method of any one of items 1 to 7, wherein the blood sample is from a subject clinically assessed as having or suspected of having stage III breast cancer, wherein the metabolite group quantified in the blood sample comprises or consists substantially of at least one blood-based metabolite, said at least one blood-based metabolite being: butyric acid, PC aa C40:2, or a combination thereof.

[0051] 9. The method of any one of items 1 to 8, wherein the blood sample is obtained from a subject clinically assessed as having or suspected of having ductal carcinoma (e.g., stage I or II), wherein the metabolite group quantified in the blood sample comprises or consists substantially of at least one blood-based metabolite, said at least one blood-based metabolite being: asparagine, fumaric acid, glucose, kynurenine, serotonin, spermidine, or any combination thereof.

[0052] 10. The method of any one of items 1 to 9, wherein the blood sample is obtained from a subject clinically assessed as having or suspected of having lobular carcinoma (e.g., stage I or II), wherein the metabolite group quantified in the blood sample comprises or consists substantially of at least one blood-based metabolite, said at least one blood-based metabolite being: asparagine, glucose, PC aa C40:1, or any combination thereof.

[0053] 11. The method of any one of items 1 to 10, wherein the blood sample is from a subject clinically assessed as having or suspected of having luminal breast cancer (e.g., luminal type A), wherein the metabolite group quantified in the blood sample comprises or consists substantially of at least one blood-based metabolite, said at least one blood-based metabolite being: CO, C18:1OH, fumaric acid, kynurenine, PCaaC40:2, spermidine, succinic acid, or any combination thereof.

[0054] 12. A method for assessing breast cancer in a subject, the method comprising: processing a human clinical blood sample according to any one of items 1 to 11, and comparing the levels of a quantified group of metabolites with corresponding reference values ​​indicating healthy or non-breast cancer subjects, wherein: (a) a decrease in the level of one or more of the following metabolites indicates breast cancer: propionic acid (propionate), C6.1 (hexenoylcarnitine), C18:1OH (hydroxyoctadecenoylcarnitine), fumaric acid (fumarate), kynurenine, or succinic acid (succinate); and (b) the following Elevated levels of one or more metabolites indicate breast cancer: asparagine, butyric acid, C0 (carnitine), C3 (propionylcarnitine), C3OH (hydroxypropionylcarnitine), C4 (butyrylcarnitine), C4:1 (butenoylcarnitine), C5OH (hydroxyvalerate carnitine; C3-DC-M), C12 (dodecanoylcarnitine), C14 (tetradecanoylcarnitine), C14:2OH (hydroxytetradecadienoylcarnitine), C16:1OH (hydroxyhexadecadienoylcarnitine), C16:2OH (hydroxyhexadecadienoylcarnitine), glucose, lysoPC a C18:1 (lysophosphatidylcholine acyl C18:1), methylmalonic acid (methylmalonate), PC aa C36:0 (phosphatidylcholine diacyl C36:0), PC aa C36:6 (phosphatidylcholine diacyl C36:6), PC aa C38:0 (phosphatidylcholine diacyl C38:0), PC aa C40:1 (phosphatidylcholine diacyl C40:1), PC aa C40:2 (phosphatidylcholine diacyl C40:2), PC ae C40:6 (phosphatidylcholine acyl-alkyl C40:6), phenylalanine, serotonin, SM C16:0 (sphingomyelin C16:0), SM C18:0 (sphingomyelin C18:0), SM C18:1 (sphingomyelin C18:1), SM C20:2 (sphingomyelin C20:2), SM(OH) C16:1 (hydroxysphingomyelin C16:1), SM(OH) C22:1 (hydroxysphingomyelin C22:1), SM(OH) C22:2 (hydroxysphingomyelin C22:2), SM(OH) C24:1 (hydroxysphingomyelin C24:1), spermidine or taurine.

[0055] 13. The method of item 12, wherein: (a) assessing breast cancer in the subject includes distinguishing between stage I or II breast cancer and healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in item 4 or 5; (b) assessing breast cancer in the subject includes distinguishing between stage I breast cancer and healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in item 6; (c) assessing breast cancer in the subject includes distinguishing between stage II breast cancer and healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in item 7; (d) assessing breast cancer in the subject includes distinguishing between stage III breast cancer and healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in item 7. The metabolite groups quantified in the blood sample are as defined in item 8; (e) assessing breast cancer in the subject includes distinguishing ductal carcinoma (e.g., stage I or II) from healthy controls or non-breast cancer subjects, wherein the metabolite groups quantified in the blood sample are as defined in item 9; (f) assessing breast cancer in the subject includes distinguishing lobular carcinoma (e.g., stage I or II) from healthy controls or non-breast cancer subjects, wherein the metabolite groups quantified in the blood sample are as defined in item 10; (g) assessing breast cancer in the subject includes distinguishing luminal breast cancer (e.g., luminal type A) from healthy controls or non-breast cancer subjects, wherein the metabolite groups quantified in the blood sample are as defined in item 11; or (h) any combination of (a) to (g).

[0056] 14. The method described in Item 12 or 13 further includes determining a breast cancer probability score based on a quantitative metabolite panel combined with the subject's age.

[0057] 15. The method of any one of items 12 to 14 further includes providing breast cancer treatment recommendations based on the breast cancer assessment.

[0058] 16. A method for implementing or improving a breast cancer screening program, the method comprising: incorporating a metabolite group or the method of any one of projects 12 to 15 into the screening program, the metabolite group comprising or consisting substantially of at least one blood-based metabolite as defined in any one of projects 1 to 11.

[0059] 17. The method of any one of items 1 to 16, wherein the blood sample is plasma or serum.

[0060] 18. The method of any one of items 1 to 17, wherein the subject is clinically assessed as having or suspected of having breast cancer by mammography, breast magnetic resonance imaging (MRI), breast ultrasound, breast biopsy, or any combination thereof.

[0061] 19. The method of any one of items 1 to 18, wherein the method is used as part of an early breast cancer screening program, the early breast cancer screening program including mammography, breast magnetic resonance imaging (MRI), breast ultrasound, breast biopsy, or any combination thereof.

[0062] 20. A method for treating a subject with breast cancer, the method comprising: assessing the subject's breast cancer status according to any one of items 12 to 19, and administering breast cancer treatment to the subject accordingly.

[0063] 21. A biomarker for use in a group of blood-based biomarkers for assessing breast cancer in subjects, said biomarker being at least one blood-based metabolite as defined in any one of items 1 to 11.

[0064] 22. A biomarker for the use described in Item 21, wherein: (a) assessing breast cancer in the subject includes distinguishing stage I or II breast cancer from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in Item 4 or 5; (b) assessing breast cancer in the subject includes distinguishing stage I breast cancer from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in Item 6; (c) assessing breast cancer in the subject includes distinguishing stage II breast cancer from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in Item 7; (d) assessing breast cancer in the subject includes distinguishing stage III breast cancer from healthy controls or non-breast cancer subjects, wherein... The metabolite groups quantified in the blood sample are as defined in item 8; (e) assessing breast cancer in the subject includes distinguishing ductal carcinoma (e.g., stage I or II) from healthy controls or non-breast cancer subjects, wherein the metabolite groups quantified in the blood sample are as defined in item 9; (f) assessing breast cancer in the subject includes distinguishing lobular carcinoma (e.g., stage I or II) from healthy controls or non-breast cancer subjects, wherein the metabolite groups quantified in the blood sample are as defined in item 10; (g) assessing breast cancer in the subject includes distinguishing luminal breast cancer (e.g., luminal type A) from healthy controls or non-breast cancer subjects, wherein the metabolite groups quantified in the blood sample are as defined in item 11; or (h) any combination of (a) to (g).

[0065] 23. A group of breast cancer biomarkers comprising at least one blood-based metabolite as defined in any one of items 1 to 11.

[0066] 24. The breast cancer biomarker group as described in Item 23, used for: (a) distinguishing stage I or II breast cancer from healthy controls or non-breast cancer subjects, wherein the metabolite group is as defined in Item 4 or 5; (b) distinguishing stage I breast cancer from healthy controls or non-breast cancer subjects, wherein the metabolite group is as defined in Item 6; (c) distinguishing stage II breast cancer from healthy controls or non-breast cancer subjects, wherein the metabolite group is as defined in Item 7; (d) distinguishing stage III breast cancer from healthy controls or non-breast cancer subjects, wherein the metabolite group is as defined in Item 8; (e) distinguishing ductal carcinoma (e.g., stage I or II) from healthy controls or non-breast cancer subjects, wherein the metabolite group is as defined in Item 9; (f) distinguishing lobular carcinoma (e.g., stage I or II) from healthy controls or non-breast cancer subjects, wherein the metabolite group is as defined in Item 10; (g) distinguishing luminal breast cancer (e.g., luminal type A) from healthy controls or non-breast cancer subjects, wherein the metabolite group is as defined in Item 11; or (h) any combination of (a) to (g).

[0067] Example Example 1: Method All biological samples were obtained from the Collaborative Human Tissue Network (CHTN) biobank. A quantitative mass spectrometry (MS)-based metabolomics approach was employed, using direct injection (DI) MS and reversed-phase high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS / MS) to analyze metabolites in plasma samples. Features with more than 80% missing values ​​were removed. The sample set was divided into a discovery set and a validation set. The discovery set was used for statistical testing, followed by cross-validation to obtain a logistic regression model. This model was used as the predictive model for the validation set. Optimal biomarker groups were determined using metabolite concentration data, clinical data, and hormone receptor status. The same biomarkers and regression model were used to evaluate the validation model. For each subgroup, the area under the receiver operating characteristic (AUROC), sensitivity, and specificity at selected cutoff points were calculated.

[0068] Example 2: Study population This study included 185 plasma samples from women diagnosed with breast cancer via biopsy and 56 plasma samples from healthy controls. The breast cancer study population subgroups included the following tumor types: estrogen receptor (ER)-positive and progesterone receptor (PR)-positive but HER2-negative tumors (luminal type A), ER-positive, PR-negative, and HER2-positive tumors (luminal type B), hormone receptor-negative but HER2-positive tumors, and triple-negative breast cancer (TNBC), such as... Figure 1A As shown. The study population included 98 samples from stage I breast cancer patients, 70 samples from stage II breast cancer patients, and 17 samples from stage III cancer patients. Figure 1B ).

[0069] Example 3: Metabolites used to informatively distinguish breast cancer from healthy controls As described in Example 1, a total of 138 metabolites were analyzed using a combination of direct injection (DI) mass spectrometry and reversed-phase high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS / MS). Figure 2 The results shown display the relative values ​​of particularly informative metabolites (coefficients greater than approximately 70) used to distinguish breast cancer from healthy controls. These informative metabolites include those found elevated in breast cancer samples compared to healthy control samples: C0 (carnitine), glucose, PCaa C40:2 (phosphatidylcholine diacyl C40:2), C16:2OH (hydroxyhexadecadienoyl carnitine), serotonin, spermidine, asparagine, C5OH (hydroxyvalerate carnitine / C3-DC-M), phenylalanine, SM(OH) C24:1 (hydroxysphingomyelin C24:1), and PCaa C40:1 (phosphatidylcholine diacyl C40:1). These informative metabolites also include those found decreased in breast cancer samples compared to healthy control samples: kynurenine, C18:1OH (hydroxyoctadecadienoyl carnitine), propionic acid (propionate), and succinic acid (succinate). Figure 2 ).

[0070] Example 4: Five metabolites + age differentiate breast cancer patients from healthy controls A logistic regression model was constructed using multiple metabolite groups to distinguish between breast cancer tumors and healthy controls. Figure 3Partial least squares discriminant analysis (PLS-DA) score plots show the spatial separation between breast cancer samples and healthy controls when using the following six biomarker subsets (combinations of five metabolites and age; values ​​in parentheses are relative): spermine (1.05), PC aaC40:2 (0.58), C0 (0.87), succinate (-0.75), kynurenine (-0.88), serotonin (0.81), and age (1.70). Receiver operating characteristic (ROC) curves were generated for all stages of breast cancer patients using the optimal logistic regression model at the included age. Figure 4 The area under the ROC curve (AUC) and 95% confidence interval were calculated using the same combination of biomarkers and age. The calculated AUC was 94.5%, the sensitivity was 90.3%, and the specificity was 86.8%. Figure 4 ).

[0071] Example 5: Metabolites for informative differentiation of breast cancer stages and subtypes This study explored the potential of using plasma metabolomics analysis and clinical data to detect breast cancer at different stages and subtypes. For example, to differentiate stage I breast cancer (… Figure 5 ) or stage II breast cancer ( Figure 6 Heatmaps were constructed using healthy controls. Different models were constructed using at least two of the most informative metabolites in combination with age (as a continuous variable) to distinguish between healthy controls and breast cancer cases of different stages and subtypes. Figure 7 ROC curves based on the following biomarkers are shown: fumarate (-1.3746), spermidine (0.8676), glucose (0.7505), asparagine (1.5345), serotonin (1.1838), kynurenine (-0.8679), and age (2.0220); the resulting AUC was 96.2%, sensitivity was 91.1%, and specificity was 89.3%. Accuracy reflects the performance of the binary classification prediction model for each stage and subtype compared to healthy controls. The table below summarizes examples of other metabolite groups and ROC curve analyses used for informatively differentiating different breast cancer stages and subtypes compared to healthy controls.

[0072] Table 1: Established Metabolite Groups Example 6: Summary of informative metabolites from Examples 2-5 "+" = elevated in breast cancer; "-" = decreased in breast cancer. Example 7: Biomarkers for breast cancer screening Small Group identification The primary objective of Example 7 is to construct a robust and concise set of metabolomics biomarkers for early breast cancer detection, based on the blood samples, study population, and metabolite library described in Examples 1-3. As described by Vaida et al. (2024), a variety of machine learning feature selection strategies are systematically combined to derive a small, robust, and reliable set of biomarkers. In short, unlike traditional methods relying on partial least squares (PLS) or LASSO regression, this method employs univariate Naive Bayes, L2-regularized support vector classifiers (SVC), principal component analysis (PCA), and feature engineering techniques to optimize and select the most informative features. Furthermore, the dataset imbalance problem is addressed through propensity score matching (PSM), ensuring reliable comparisons between the cancer group and the control group. The inclusion of a random sampling strategy further enhances the reliability of the selected set of biomarkers. The best-performing feature set included 11 biomarkers, including 9 metabolites [SM(OH)C22:2, SM C18:0, CO, C3OH, C14:2OH, C16:2OH, LysoPCa C18:1, PCaa C36:0, and asparagine], one metabolite ratio (kynurenine vs. tryptophan), and one demographic variable (age). It achieved a 98% (CI: 97.5%–98%) area under the ROC curve (AUC), reflecting a high discriminatory power between breast cancer cases and controls.

[0073] SM(OH) C22:2 and SM C18:0 are both sphingomyelins involved in maintaining membrane structure, while SM(OH) C16:1 plays a crucial role in lipid metabolism and membrane integrity. C0 (carnitine) promotes fatty acid transport for energy production, while C3OH, C14:2OH, and C16:2OH are hydroxycarnitines involved in fatty acid oxidation. LysoPCa C18:1 contributes to membrane remodeling, while PCaa C36:0 is a key phospholipid within the cell membrane. Asparagine is an essential amino acid that supports protein synthesis. Finally, the ratio of kynurenine to tryptophan has been used as a marker of immune regulation and inflammation.

[0074] These results demonstrate the potential of this robust, cost-effective, and non-invasive breast cancer screening and diagnostic tool, providing important clinical value for early detection and personalized patient management.

[0075] References Vaida et al., “Identification of a Novel Biomarker Panel for BreastCancer Screening”. International Journal of Molecular Sciences, 2024, 25(21):11835. doi:10.3390 / ijms252111835。

Claims

1. A method for processing human clinical blood samples, the method comprising obtaining a blood sample from a subject clinically assessed as having or suspected of having breast cancer, and quantifying a metabolite group in the blood sample, the metabolite group comprising or consisting substantially of at least one blood-based metabolite, said at least one blood-based metabolite being: propionic acid (propionate), asparagine, butyric acid, CO (carnitine), C3 (propionylcarnitine), C3OH (hydroxypropionylcarnitine), C4 (butyric acid) Acylcarnitine), C4:1 (butenoylcarnitine), C5OH (hydroxypentanoylcarnitine; C3-DC-M), C6.1 (hexenoylcarnitine), C12 (dodecanoylcarnitine), C14 (tetradecanoylcarnitine), C14:2OH (hydroxytetradecadienoylcarnitine), C16:1OH (hydroxyhexadecadienoylcarnitine), C16:2OH (hydroxyhexadecadienoylcarnitine), C18:1OH (hydroxyoctadecadienoylcarnitine), fumaric acid (fumarate), glucose, kynurenine, lysoPC a C18:1 (lysophosphatidylcholine acyl C18:1), methylmalonic acid (methylmalonate), PC aa C36:0 (phosphatidylcholine diacyl C36:0), PC aa C36:6 (phosphatidylcholine diacyl C36:6), PC aa C38:0 (phosphatidylcholine diacyl C38:0), PC aa C40:1 (phosphatidylcholine diacyl C40:1), PC aa C40:2 (phosphatidylcholine diacyl C40:2), PC ae C40:6 (phosphatidylcholine acyl-alkyl C40:6), phenylalanine, serotonin, SM C16:0 (sphingomyelin C16:0), SM C18:0 (sphingomyelin C18:0), SM C18:1 (sphingomyelin C18:1), SM C20:2 (sphingomyelin C20:2), SM(OH) C16:1 (hydroxysphingomyelin C16:1), SM(OH)C22:1 (hydroxysphingomyelin C22:1), SM(OH)C22:2 (hydroxysphingomyelin C22:2), SM(OH)C24:1 (hydroxysphingomyelin C24:1), spermidine, succinic acid (succinate), taurine, or any combination thereof.

2. The method of claim 1, wherein the at least one blood-based metabolite comprises propionic acid (propionate); and / or wherein the at least one blood-based metabolite comprises at least one, two, three, four, five, six, seven, eight, nine, ten, or all of the following: SM(OH) C22:2, SM C18:0, CO, C3OH, C14:2OH, C16:2OH, lysoPC a C18:1, PC aa C36:0, asparagine, kynurenine, and tryptophan.

3. The method of claim 1 or 2, wherein the group of metabolites comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 of the blood-based metabolites.

4. The method of any one of claims 1 to 3, wherein the blood sample is from a subject clinically assessed as having or suspected of having early breast cancer, wherein the early breast cancer is stage I or II breast cancer, and wherein the metabolite group quantified in the blood sample comprises or is substantially composed of at least one of the following blood-based metabolites: propionic acid, asparagine, CO, C3, C3OH, C4, C4:1, C5OH, C6.1, C12, C14, C14:2OH, C16:1OH, C16:2OH, fumaric acid, glucose, kynurenine, lysoPCa C18:1, methylmalonic acid, PCaa C36:0, PCaa C36:6, PCaa C38:0, PCaa C40:1, PCaa C40:2, PCae C40:6, phenylalanine, serotonin, SM C16:0, SM C18:0, SM C18:1, SM C20:2, SM(OH) C16:1, SM(OH) C22:1, SM(OH) C22:2, SM(OH) C24:1, spermidine, succinic acid, taurine, or any combination thereof.

5. The method of claim 4, wherein the metabolite group quantified in the blood sample comprises or is substantially composed of at least one of the following blood-based metabolites: propionic acid, asparagine, CO, C3OH, C5OH, C16:2OH, fumaric acid, glucose, kynurenine, lysoPC a C18:1, PC aa C40:1, PC aa C40:2, PC ae C40:6, phenylalanine, serotonin, SM(OH) C24:1, spermidine, succinic acid, or any combination thereof.

6. The method of any one of claims 1 to 5, wherein the blood sample is derived from a subject clinically assessed as having or suspected of having stage I breast cancer, wherein the metabolite group quantified in the blood sample comprises or is composed primarily of at least one of the following blood-based metabolites: asparagine, CO, C3, C3OH, C4, C5OH, C6.1, C12, C14, C16:1OH, C16:2OH, fumaric acid, glucose, lysoPCa C18:1, methylmalonic acid, PCaa C36:0, PCaa C36:6, PCaa C40:1, PCaa C40:2, PCae C40:6, phenylalanine, SM C18:0, SM C20:2, SM(OH) C16:1, SM(OH) C22:1, SM(OH) C22:2, spermidine, or any combination thereof.

7. The method of any one of claims 1 to 6, wherein the blood sample is derived from a subject clinically assessed as having or suspected of having stage II breast cancer, wherein the metabolite group quantified in the blood sample comprises or is substantially composed of at least one of the following blood-based metabolites: CO, C4, C4:1, C3OH, C5OH, C12, C14, C14:2OH, fumaric acid, glucose, lysoPC a C18:1, PC aa C36:0, PC aa C36:6, PC aa C38:0, PC aa C40:1, PC aa C40:2, PC ae C40:6, serotonin, SM C16:0, SM C18:0, SMC18:1, SM C20:2, SM(OH) C16:1, SM(OH) C22:1, SM(OH) C22:2, spermidine, succinic acid, taurine, or any combination thereof.

8. The method of any one of claims 1 to 7, wherein the blood sample is from a subject clinically assessed as having or suspected of having stage III breast cancer, wherein the metabolite group quantified in the blood sample comprises or is substantially composed of at least one of the following blood-based metabolites: Butyric acid, PC aaC40:2, or a combination thereof.

9. The method of any one of claims 1 to 8, wherein the blood sample is from a subject clinically assessed as having or suspected of having ductal carcinoma (e.g., stage I or II), wherein the metabolite group quantified in the blood sample comprises or consists substantially of at least one of the following blood-based metabolites: asparagine, fumaric acid, glucose, kynurenine, serotonin, spermidine, or any combination thereof.

10. The method of any one of claims 1 to 9, wherein the blood sample is from a subject clinically assessed as having or suspected of having lobular carcinoma (e.g., stage I or II), wherein the metabolite group quantified in the blood sample comprises or is substantially composed of at least one of the following blood-based metabolites: Asparagine, glucose, PC aa C40:1, or any combination thereof.

11. The method of any one of claims 1 to 10, wherein the blood sample is from a subject clinically assessed as having or suspected of having luminal breast cancer (e.g., luminal type A), wherein the metabolite group quantified in the blood sample comprises or is substantially composed of at least one of the following blood-based metabolites: C0, C18:1OH, fumaric acid, kynurenine, PC, aa, C40:2, spermidine, succinic acid, or any combination thereof.

12. A method for assessing breast cancer in a subject, the method comprising: The method according to any one of claims 1 to 11 processes human clinical blood samples and compares the levels of quantified metabolite groups with corresponding reference values ​​indicating healthy or non-breast cancer subjects, wherein: (a) Decreased levels of one or more of the following metabolites indicate breast cancer: propionic acid (propionate), C6.1 (hexenoylcarnitine), C18:1OH (hydroxyoctadecenoylcarnitine), fumaric acid (fumarate), kynurenine, or succinic acid (succinate); and (b) Elevated levels of one or more of the following metabolites indicate breast cancer: asparagine, butyric acid, C0 (carnitine), C3 (propionylcarnitine), C3OH (hydroxypropionylcarnitine), C4 (butyrylcarnitine), C4:1 (butenoylcarnitine), C5OH (hydroxyvalerate carnitine; C3-DC-M), C12 (dodecanoylcarnitine), C14 (tetradecanoylcarnitine), C14:2OH (hydroxytetradecadienoylcarnitine), C16:1OH (hydroxyhexadecadienoylcarnitine), C16:2OH (hydroxyhexadecadienoylcarnitine), glucose, lysoPC aC18:1 (lysophosphatidylcholine acyl C18:1), methylmalonic acid (methylmalonate), PC aa C36:0 (phosphatidylcholine diacyl C36:0), PC aa C36:6 (phosphatidylcholine diacyl C36:6), PC aa C38:0 (phosphatidylcholine diacyl C38:0), PC aa C40:1 (phosphatidylcholine diacyl C40:1), PC aa C40:2 (phosphatidylcholine diacyl C40:2), PC ae C40:6 (phosphatidylcholine acyl-alkyl C40:6), phenylalanine, serotonin, SM C16:0 (sphingomyelin C16:0), SM C18:0 (sphingomyelin C18:0), SM C18:1 (sphingomyelin C18:1), SM C20:2 (sphingomyelin C20:2), SM(OH)C16:1 (hydroxysphingomyelin C16:1), SM(OH)C22:1 (hydroxysphingomyelin C22:1), SM(OH)C22:2 (hydroxysphingomyelin C22:2), SM(OH) C24:1 (hydroxysphingomyelin C24:1), spermidine, or taurine.

13. The method of claim 12, wherein: (a) Assessing breast cancer in the subject includes distinguishing stage I or II breast cancer from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in claim 4 or 5; (b) Assessing breast cancer in the subjects includes distinguishing stage I breast cancer from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in claim 6; (c) Assessing breast cancer in the subjects includes distinguishing stage II breast cancer from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in claim 7; (d) Assessing breast cancer in the subjects includes distinguishing stage III breast cancer from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in claim 8; (e) Assessing breast cancer in the subjects includes distinguishing ductal carcinoma (e.g., stage I or II) from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in claim 9; (f) Assessing breast cancer in the subjects includes distinguishing lobular carcinoma (e.g., stage I or II) from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in claim 10; (g) Assessing breast cancer in the subjects includes distinguishing luminal breast cancer (e.g., luminal type A) from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in claim 11; or Any combination of (h)(a) to (g).

14. The method of claim 12 or 13, further comprising determining a breast cancer probability score based on a quantitative metabolite panel combined with the subject's age.

15. The method of any one of claims 12 to 14, further comprising providing breast cancer treatment recommendations based on the breast cancer assessment.

16. A method for implementing or improving a breast cancer screening program, the method comprising: The screening program incorporates a group of metabolites in a quantitative blood sample of a subject or the method of any one of claims 12 to 15, wherein the group of metabolites comprises at least one blood-based metabolite as defined in any one of claims 1 to 11 or consists substantially of at least one blood-based metabolite as defined in any one of claims 1 to 11.

17. The method of any one of claims 1 to 16, wherein the blood sample is plasma or serum.

18. The method of any one of claims 1 to 17, wherein the subject is clinically assessed as having or suspected of having breast cancer by mammography, breast magnetic resonance imaging (MRI), breast ultrasound, breast biopsy, or any combination thereof.

19. The method of any one of claims 1 to 18, wherein the method is used as part of an early breast cancer screening program, the early breast cancer screening program including mammography, breast magnetic resonance imaging (MRI), breast ultrasound, breast biopsy, or any combination thereof.

20. A method for treating a subject with breast cancer, the method comprising: The method according to any one of claims 12 to 19 is used to assess the breast cancer status of a subject and, based on this assessment, to administer breast cancer treatment to the subject.

21. A biomarker for use in a group of blood-based biomarkers for assessing breast cancer in subjects, said biomarker being at least one blood-based metabolite as defined in any one of claims 1 to 11.

22. The biomarker used as described in claim 21, wherein: (a) Assessing breast cancer in the subject includes distinguishing stage I or II breast cancer from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in claim 4 or 5; (b) Assessing breast cancer in the subjects includes distinguishing stage I breast cancer from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in claim 6; (c) Assessing breast cancer in the subjects includes distinguishing stage II breast cancer from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in claim 7; (d) Assessing breast cancer in the subjects includes distinguishing stage III breast cancer from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in claim 8; (e) Assessing breast cancer in the subjects includes distinguishing ductal carcinoma (e.g., stage I or II) from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in claim 9; (f) Assessing breast cancer in the subjects includes distinguishing lobular carcinoma (e.g., stage I or II) from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in claim 10; (g) Assessing breast cancer in the subjects includes distinguishing luminal breast cancer (e.g., luminal type A) from healthy controls or non-breast cancer subjects, wherein the metabolite group quantified in the blood sample is as defined in claim 11; or Any combination of (h)(a) to (g).

23. A group of breast cancer biomarkers comprising at least one blood-based metabolite as defined in any one of claims 1 to 11.

24. The breast cancer biomarker group of claim 23, wherein it is used for: (a) Distinguishing between stage I or II breast cancer and healthy controls or non-breast cancer subjects, wherein the metabolite group is as defined in claim 4 or 5; (b) Distinguishing between stage I breast cancer and healthy controls or non-breast cancer subjects, wherein the metabolite group is as defined in claim 6; (c) Distinguishing between stage II breast cancer and healthy controls or non-breast cancer subjects, wherein the metabolite group is as defined in claim 7; (d) Distinguishing between stage III breast cancer and healthy controls or non-breast cancer subjects, wherein the metabolite group is as defined in claim 8; (e) Distinguishing ductal carcinoma (e.g., stage I or II) from healthy controls or non-breast cancer subjects, wherein the metabolite group is as defined in claim 9; (f) Distinguishing lobular carcinoma (e.g., stage I or II) from healthy controls or non-breast cancer subjects, wherein the metabolite group is as defined in claim 10; (d) Differentiating luminal breast cancer (e.g., luminal type A) from healthy controls or non-breast cancer subjects, wherein the metabolite group is as defined in claim 11; or Any combination of (h)(a) to (g).

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