Early breast cancer detection using blood-based metabolomic profiling

Abstract

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

Description

[0001] EARLY BREAST CANCER DETECTION USING BLOOD-BASED METABOLOMIC PROFILING

[0002] The present description relates to blood-based metabolites for detecting early stage breast cancer. The present description also relates to blood-based metabolites that are able to distinguish between breast cancer stages and tumor subtypes.

[0003] The present description refers to a number of documents, the contents of which are herein incorporated by reference in their entirety.

[0004] BACKGROUND

[0005] Breast cancer is a leading cause of cancer death among women and early diagnosis is key to the successful treatment, management, and care of breast cancer patients. Current breast cancer screening methods rely mainly on imagining techniques such as mammography, yet such techniques would greatly benefit from informative biomarkers that can help identify early stage breast cancers, and distinguish between breast cancer stages and / or subtypes.

[0006] SUMMARY

[0007] In a first aspect, described herein is a method for processing a human clinical blood sample, the method comprising obtaining a blood sample from a subject clinically assessed as having or suspected of having breast cancer, and quantifying a panel of metabolites in said blood sample that are informative distinguishing breast cancer from healthy or non-breast cancer controls. In some embodiments, the panel of metabolites described herein may comprise or consist essentially of at least one blood-based metabolite which is: propionic acid (propionate), asparagine, butyric acid, CO (carnitine), C3 (propionylcamitine), C30H (hydroxypropionylcamitine), C4 (butyrylcamitine), C4: l (butenylcamitine), C50H (hydroxy valerylcarnitine; C3-DC-M), C6.1 (hexenoylcamitine), C12 (dodecanoylcamitine), C14 (tetradecanoylcamitine), C14:2OH (hydroxytetradecadienylcamitine), C16: 10H (hydroxyhexadecenoylcamitine), C16:2OH (hydroxyhexadecadienylcamitine), C18: 10H (hydroxyoctadecenoylcamitine), fumaric acid (fumarate), glucose, kynurenine, lysoPC a Cl 8: 1 (lysophosphatidylcholine acyl Cl 8: 1), methylmalonic acid (methyhnalonate), 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 Cl 8: 1), SM C20:2 (sphingomyelin C20:2), SM(OH) Cl 6: 1 (hydroxysphingomyeline C16: 1), SM(OH) C22: l (hydroxysphingomyeline C22: l), SM(OH) C22:2 (hydroxysphingomyeline C22:2), SM(OH) C24: l (hydroxysphingomyeline C24: l), spermidine, succinic acid (succinate), taurine, or any combination thereof.

[0008] In a further aspect, described herein is a method of assessing breast cancer in a subject, the method comprising processing a human clinical blood sample according to a method described herein, and comparing the levels of the quantified panel of metabolites to corresponding reference values indicative of a healthy or non-breast cancer subject. In some embodiments, a lower level of one or more of the following metabolites is indicative of breast cancer: propionic acid (propionate), C6.1 (hexenoylcamitine), C18: 1OH (hydroxyoctadecenoylcamitine), fumaric acid (fumarate), kynurenine, or succinic acid (succinate). In some embodiments, an elevated level of one or more of the following metabolites is indicative of breast cancer: asparagine, butyric acid, CO (carnitine), C3 (propionylcamitine), C30H (hydroxypropionylcamitine), C4 (butyrylcamitine), C4: l (butenylcamitine), C50H (hydroxyvalerylcamitine; C3-DC-M), C12 (dodecanoylcamitine), C14 (tetradecanoylcamitine), C14:2OH (hydroxytetradecadienylcamitine), C16: 10H (hydroxyhexadecenoylcamitine), C16:2OH (hydroxyhexadecadienylcamitine), glucose, lysoPC a C18: l (lysophosphatidylcholine acyl C18: 1), methylmalonic acid (methyhnalonate), 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: l), 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: l (hydroxysphingomyeline C16: 1), SM(OH) C22: l (hydroxysphingomyeline C22: l), SM(OH) C22:2 (hydroxysphingomyeline C22:2), SM(OH) C24: l (hydroxysphingomyeline C24: l), spermidine, or taurine.

[0009] In a further aspect, described herein is a method for implementing or modifying a breast cancer screening program (e.g., an already existing breast cancer screening program), the method comprising incorporating into said screening program the quantification of a panel of metabolites in a blood sample of a subject, the panel comprising or consists essentially of at least one blood-based metabolite as described herein.

[0010] In a further aspect, described herein is a method for treating a breast cancer subject, the method comprising assessing the breast cancer status of a subject according to a method described herein, and administering a breast cancer treatment to the subject based thereon.

[0011] In a further aspect, described herein is a biomarker for use in a blood-based biomarker panel for assessing breast cancer in a subject, the biomarker being at least one blood-based metabolite as described herein. In a further aspect, described herein is a breast cancer biomarker panel comprising at least one of the blood-based metabolites as described herein.

[0012] General Definitions

[0013] Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0014] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one” but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”.

[0015] The term “about”, when used herein, indicates that a value includes the standard deviation of error for the device or method being employed in order to determine the value. In general, the terminology “about” is meant to designate a possible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term “about”. Unless indicated otherwise, use of the term “about” before a range applies to both ends of the range.

[0016] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0017] Other objects, advantages and features of the present description will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In the appended drawings:

[0020] Fig. 1 depicts the composition of the present study population, including the number of samples from breast cancer vs healthy controls, and the different breast cancer subgroups included (Fig. 1A), as well as the different breast cancer stages included (Fig. IB).

[0021] Fig. 2 shows relative values of particularly informative metabolites for distinguishing breast cancer from healthy controls.

[0022] Fig. 3 shows the results of a partial least squares discriminant analysis (PLS-DA) scores plot using a set of five metabolite plus age, illustrating the spatial separation of breast cancer samples from healthy controls.

[0023] Fig. 4 shows a receiver operating characteristic curve (ROC) generated using an optimal logistic regression model for the breast cancer patients at all stages when age is added.

[0024] Fig. 5 shows a heat map constructed to distinguish breast cancer stage I patients from healthy controls.

[0025] Fig. 6 shows a heat map constructed to distinguish breast cancer stage II patients from healthy controls.

[0026] Fig. 7 shows a ROC curve based on six metabolites plus age to distinguish breast cancer stage I and stage II patients from healthy controls.

[0027] DETAILED DESCRIPTION

[0028] In a first aspect, described herein is a method for processing a human clinical blood sample, the method comprising obtaining a blood sample from a subject clinically assessed as having or suspected of having breast cancer, and quantifying a panel of metabolites in said blood sample. In some embodiments, the panel of metabolites comprise or consist essentially of blood-based metabolites that are informative for distinguishing between healthy controls and early stage breast cancer (e.g., stage I or stage II), stage I breast cancer, stage II breast cancer, stage III breast cancer, ductal carcinoma (e.g., stage I or stage II), lobular carcinoma (e.g., stage I or stage II), and luminal breast cancer (e.g., luminal A).

[0029] In some embodiments, the panel of metabolites comprise or consist of blood-based metabolites at least one blood-based metabolite which is: propionic acid (propionate), asparagine, butyric acid, CO (carnitine), C3 (propionylcamitine), C30H (hydroxypropionylcamitine), C4 (butyrylcamitine), C4: l (butenylcamitine), C50H (hydroxyvalerylcamitine; C3-DC-M), C6.1 (hexenoylcamitine), C12 (dodecanoylcamitine), C14 (tetradecanoylcamitine), C14:2OH (hydroxytetradecadienylcamitine), C16: 10H (hydroxyhexadecenoylcarnitine), C16:2OH (hydroxyhexadecadienylcamitine), C18: 10H (hydroxyoctadecenoylcamitine), fumaric acid (fumarate), glucose, kynurenine, lysoPC a Cl 8: 1 (lysophosphatidylcholine acyl Cl 8: 1), methylmalonic acid (methyhnalonate), 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 Cl 8: 1), SM C20:2 (sphingomyelin C20:2), SM(OH) Cl 6: 1 (hydroxysphingomyeline C16: 1), SM(OH) C22: l (hydroxysphingomyeline C22: l), SM(OH) C22:2 (hydroxysphingomyeline C22:2), SM(OH) C24: l (hydroxysphingomyeline C24: l), spermidine, succinic acid (succinate), taurine, or any combination thereof. In some embodiments, the at least one blood-based metabolite may comprise propionic acid (propionate). In some embodiments, the panel may comprise 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 said blood-based metabolites.

[0030] As used herein, the expression “subject clinically assessed as having or suspected of having breast cancer” refers to a subset of subjects with a reasonable likelihood of having or of developing breast cancer, based for example on a number of risk factors such as patient symptoms, age, family history of breast cancer, genetic testing, imaging results (e.g., mammography, MRI, ultrasound), or biopsy results. In some embodiments, the expression may refer to subjects that have been evaluated by a medical professional and who have been recommended for further breast cancer screening, or who are already part of an existing breast cancer screening program.

[0031] As used herein, the expression “consisting essentially of’ in the context of biomarker panels described herein, refers to metabolites comprised in the panels that are sufficient for the intended purpose (e.g., assess breast cancer or to distinguish between different breast cancer stages and / or subtypes), but does not exclude the inclusion of other metabolites that do not materially affect the performance of the method or biomarker panel with respect to breast cancer.

[0032] 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 said early stage breast cancer is stage I or stage II breast cancer, and wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: propionic acid, asparagine, CO, C3, C3OH, C4, C4: l, C5OH, C6.1, C12, C14, C14:2OH, C16: 1OH, C16:2OH, fumaric acid, glucose, kynurenine, lysoPC a C18: 1, methylmalonic acid, PC aa C36:0, PC aa C36:6, PC aa C38:0, PC aa C40: l, PC aa C40:2, PC ae C40:6, phenylalanine, serotonin, SM Cl 6:0, SM Cl 8:0, SM Cl 8: 1, SM C20:2, SM(OH) C16: l, SM(OH) C22: l, SM(OH) C22:2, SM(OH) C24: l, spermidine, succinic acid, taurine, or any combination thereof. In some embodiments, the panel of metabolites quantified in said blood sample may comprise or consist essentially of at least one blood-based metabolite which is: propionic acid, asparagine, CO, C5OH, C16:2OH, fumaric acid, glucose, kynurenine, 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.

[0033] 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 panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: asparagine, CO, C3, C3OH, C4, C5OH, C6.1, C12, C14, C16: 1OH, C16:2OH, fumaric acid, glucose, lysoPC a C18: 1, methylmalonic acid, PC aa C36:0, PC aa C36:6, PC aa C40: 1, PC aa C40:2, PC ae C40:6, phenylalanine, SM C18:0, SM C20:2, SM(OH) C16: l, SM(OH) C22: l, SM(OH) C22:2, spermidine, or any combination thereof.

[0034] 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 panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: CO, C30H, C4, C4: l, C50H, C12, C14, C14:2OH, fumaric acid, glucose, lysoPC a C18: l, 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: l, SM C20:2, SM(OH) C16: l, SM(OH) C22: l, SM(OH) C22:2, spermidine, succinic acid, taurine, or any combination thereof.

[0035] In some embodiments, the blood samples described herein may be from a subject clinically assessed as having or suspected of having stage III breast cancer, wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: butyric acid, PC aa C40:2, or a combination thereof.

[0036] In some embodiments, the blood samples described herein may be from a subject clinically assessed as having or suspected of having ductal carcinoma (e.g., stage I or stage II), wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: asparagine, fumaric acid, glucose, kynurenine, serotonin, spermidine, or any combination thereof.

[0037] In some embodiments, the blood samples described herein may be from a subject clinically assessed as having or suspected of having lobular carcinoma (e.g., stage I or stage II), wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one bloodbased metabolite which is: asparagine, glucose, PC aa C40: 1, or any combination thereof.

[0038] In some embodiments, the blood samples described herein may be from a subject clinically assessed as having or suspected of having luminal breast cancer (e.g., luminal A), wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: CO, C18: 1OH, fumaric acid, kynurenine, PC aa C40:2, spermidine, succinic acid, or any combination thereof.

[0039] In a further aspect, described herein is a method of assessing breast cancer in a subject, the method comprising processing a human clinical blood sample according to a method described herein, and comparing the levels of the quantified panel of metabolites to corresponding reference values indicative of a healthy or non-breast cancer subject. In some embodiments, a lower level of one or more of the following metabolites is indicative of breast cancer: propionic acid (propionate), C6.1 (hexenoylcamitine), C18: 1OH (hydroxyoctadecenoylcamitine), fumaric acid (fumarate), kynurenine, or succinic acid (succinate). In some embodiments, an elevated level of one or more of the following metabolites is indicative of breast cancer: asparagine, butyric acid, CO (carnitine), C3 (propionylcamitine), C3OH (hydroxypropionylcamitine), C4 (butyrylcamitine), C4: l (butenylcamitine), C50H (hydroxyvalerylcamitine; C3-DC-M), C12 (dodecanoylcamitine), C14 (tetradecanoylcamitine), C14:2OH (hydroxytetradecadienylcamitine), C16: 10H (hydroxyhexadecenoylcamitine), C16:2OH (hydroxyhexadecadienylcamitine), glucose, lysoPC a C18: l (lysophosphatidylcholine acyl C18: l), 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: l), 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: l), SM C20:2 (sphingomyelin C20:2), SM(OH) C16: l (hydroxysphingomyeline C16: l), SM(OH) C22: l (hydroxysphingomyeline C22: l), SM(OH) C22:2 (hydroxysphingomyeline C22:2), SM(OH) C24: l (hydroxysphingomyeline C24: l), spermidine, or taurine.

[0040] In some embodiments, a method of assessing breast cancer in a subject may comprise distinguishing stage I or stage II breast cancer from a healthy control or a non-breast cancer subject, distinguishing stage I breast cancer from a healthy control or a non-breast cancer subject, distinguishing stage II breast cancer from a healthy control or a non-breast cancer subject, stage III breast cancer from a healthy control or a non-breast cancer subject, distinguishing ductal carcinoma (e.g., stage I or stage II) from a healthy control or a non-breast cancer subject, distinguishing lobular carcinoma (e.g., stage I or stage II) from a healthy control or a non-breast cancer subject, distinguishing luminal breast cancer (e.g., luminal A) from a healthy control or a non-breast cancer subject, or any combination thereof, based on metabolite panels described herein (e.g., summarized in Example 6).

[0041] In some embodiments, a method of assessing breast cancer in a subject may further comprise determining a breast cancer probability score based on the quantified panel of metabolites combined with the subject’s age. In some embodiments, a method of assessing breast cancer in a subject may further comprise providing a breast cancer treatment recommendation based on said breast cancer assessment.

[0042] In a further aspect, described herein is a method for implementing or modifying a breast cancer screening program, the method comprising incorporating into said screening program the quantification of a panel of metabolites in a blood sample of a subject, the panel comprising or consists essentially of at least one blood-based metabolite as described herein, or a breast cancer assessment method as described herein.

[0043] In some embodiments, the blood sample as described herein may be plasma. In some embodiments, the blood sample as described herein may be serum.

[0044] In some embodiments, the subject described herein was 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 embodiments, the methods described herein may be used as part of an early breast cancer screening program that comprises mammography, breast magnetic resonance imaging (MRI), breast ultrasound, breast biopsy, or any combination thereof.

[0045] In a further aspect, described herein is a method for treating a breast cancer subject, the method comprising assessing the breast cancer status of a subject according to a method described herein and administering a breast cancer treatment (e.g., an early-stage breast cancer treatment) to the subject based thereon.

[0046] In further aspect, described herein is a biomarker for use in a blood-based biomarker panel for assessing breast cancer in a subject, the biomarker being at least one blood-based metabolite as described herein. In some embodiments, assessing breast cancer in the subject may comprise distinguishing stage I or stage II breast cancer from a healthy control or a non-breast cancer subject, distinguishing stage I breast cancer from a healthy control or a non-breast cancer subject, distinguishing stage II breast cancer from a healthy control or a non-breast cancer subject, stage III breast cancer from a healthy control or a non-breast cancer subject, distinguishing ductal carcinoma (e.g., stage I or stage II) from a healthy control or a non-breast cancer subject, distinguishing lobular carcinoma (e.g., stage I or stage II) from a healthy control or a non-breast cancer subject, distinguishing luminal breast cancer (e.g., luminal A) from a healthy control or a non-breast cancer subject, or any combination thereof, based on metabolite panels described herein (e.g., summarized in Example 6).

[0047] In further aspect, described herein is a breast cancer biomarker panel comprising at least one of the blood-based metabolites as described herein. In some embodiments, the breast cancer biomarker panel may be for use in distinguishing stage I or stage II breast cancer from a healthy control or a non-breast cancer subject, distinguishing stage I breast cancer from a healthy control or a non-breast cancer subject, distinguishing stage II breast cancer from a healthy control or a non-breast cancer subject, stage III breast cancer from a healthy control or a non-breast cancer subject, distinguishing ductal carcinoma (e.g., stage I or stage II) from a healthy control or a non-breast cancer subject, distinguishing lobular carcinoma (e.g., stage I or stage II) from a healthy control or a non-breast cancer subject, distinguishing luminal breast cancer (e.g., luminal A) from a healthy control or a non-breast cancer subject, or any combination thereof, based on metabolite panels described herein (e.g., summarized in Example 6).

[0048] In further aspect, described herein is a breast cancer biomarker panel comprising at least one, two, three, four, five, six, seven, eight, nine, ten, or all of the blood-based metabolites: SM(OH) C22:2, SM CI8:0, CO, C30H, C14:2OH, C16:2OH, lysoPC a C18: l, PC aa C36:0, asparagine, kynurenine, and tryptophan (e.g., summarized in Example 7). In some embodiments, the breast cancer biomarker panel may further comprise a kynurenine : tryptophan ratio and / or age. In some embodiments, the breast cancer biomarker panel described herein may comprise the detection of a parsimonious metabolomic signature having 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 parsimonious metabolomic signature may comprise or consist of calculating a breast cancer probability or prediction score based on the levels of any combination of the aforementioned metabolites.

[0049] ITEMS

[0050] In some embodiments, described herein are one or more of the following items:

[0051] 1. A method for processing a human clinical blood sample, the method comprising obtaining a blood sample from a subject clinically assessed as having or suspected of having breast cancer, and quantifying a panel of metabolites in said blood sample, the panel comprising or consists essentially of at least one blood-based metabolite which is: propionic acid (propionate), asparagine, butyric acid, CO (carnitine), C3 (propionylcamitine), C3OH (hydroxypropionylcamitine), C4 (butyrylcamitine), C4: l (butenylcamitine), C5OH (hydroxyvalerylcamitine; C3-DC-M), C6.1 (hexenoylcamitine), C12 (dodecanoylcamitine), C14 (tetradecanoylcamitine), C14:2OH (hydroxytetradecadienylcamitine), C16: 1OH (hydroxyhexadecenoylcarnitine), C16:2OH (hydroxyhexadecadienylcamitine), C18: 1OH (hydroxyoctadecenoylcamitine), fumaric acid (fumarate), glucose, kynurenine, lysoPC a Cl 8: 1 (lysophosphatidylcholine acyl Cl 8: 1), methylmalonic acid (methyhnalonate), 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 Cl 8: 1), SM C20:2 (sphingomyelin C20:2), SM(OH) Cl 6: 1 (hydroxysphingomyeline C16: 1), SM(OH) C22: l (hydroxysphingomyeline C22: l), SM(OH) C22:2 (hydroxysphingomyeline C22:2), SM(OH) C24: l (hydroxysphingomyeline C24: l), spermidine, succinic acid (succinate), taurine, or any combination thereof.

[0052] 2. The method of item 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 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.

[0053] 3. The method of item 1 or 2, wherein the panel 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 said blood-based metabolites.

[0054] 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 stage breast cancer, wherein said early stage breast cancer is stage I or stage II breast cancer, and wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: propionic acid, asparagine, CO, C3, C30H, C4, C4: l, C50H, C6.1, C12, C14, C14:2OH, C16: 10H, C16:2OH, fumaric acid, glucose, kynurenine, lysoPC a C18: 1, methylmalonic acid, PC aa C36:0, PC aa C36:6, PC aa C38:0, PC aa C40: l, PC aa C40:2, PC ae C40:6, phenylalanine, serotonin, SM Cl 6:0, SM Cl 8:0, SM Cl 8: 1, SM C20:2, SM(OH) C16: l, SM(OH) C22: l, SM(OH) C22:2, SM(OH) C24: l, spermidine, succinic acid, taurine, or any combination thereof.

[0055] 5. The method of item 4, wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: 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.

[0056] 6. The method of any one of items 1 to 5, wherein the blood sample is from a subject clinically assessed as having or suspected of having stage I breast cancer, wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: asparagine, CO, C3, C3OH, C4, C5OH, C6.1, C12, C14, C16: 1OH, C16:2OH, fumaric acid, glucose, lysoPC a C18: 1, methylmalonic acid, PC aa C36:0, PC aa C36:6, PC aa C40: 1, PC aa C40:2, PC ae C40:6, phenylalanine, SM C18:0, SM C20:2, SM(OH) C16: l, SM(OH) C22: l, SM(OH) C22:2, spermidine, or any combination thereof.

[0057] 7. The method of any one of items 1 to 6, wherein the blood sample is from a subject clinically assessed as having or suspected of having stage II breast cancer, wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: CO, C4, C4: l, C3OH, C5OH, C12, C14, C14:2OH, fumaric acid, glucose, lysoPC a C18: l, 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: l, SM C20:2, SM(OH) C16: l, SM(OH) C22: l, SM(OH) C22:2, spermidine, succinic acid, taurine, or any combination thereof.

[0058] 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 panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: butyric acid, PC aa C40:2, or a combination thereof.

[0059] 9. The method of any one of items 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 stage II), wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: asparagine, fumaric acid, glucose, kynurenine, serotonin, spermidine, or any combination thereof.

[0060] 10. The method of any one of items 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 stage II), wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one bloodbased metabolite which is: asparagine, glucose, PC aa C40: 1, or any combination thereof.

[0061] 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 A), wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: CO, C18: 1OH, fumaric acid, kynurenine, PC aa C40:2, spermidine, succinic acid, or any combination thereof.

[0062] 12. A method of assessing breast cancer in a subject, the method comprising processing a human clinical blood sample according to the method of any one of items 1 to 11, and comparing the levels of the quantified panel of metabolites to corresponding reference values indicative of a healthy or non-breast cancer subject, wherein: (a) a lower level of one or more of the following metabolites is indicative of breast cancer: propionic acid (propionate), C6.1 (hexenoylcamitine), C18: 1OH (hydroxyoctadecenoylcamitine), fumaric acid (fumarate), kynurenine, or succinic acid (succinate); and (b) an elevated level of one or more of the following metabolites is indicative of breast cancer: asparagine, butyric acid, CO (carnitine), C3 (propionylcamitine), C3OH (hydroxypropionylcamitine), C4 (butyrylcamitine), C4: l (butenylcamitine), C5OH (hydroxyvalerylcamitine; C3-DC-M), C12 (dodecanoylcamitine), C14 (tetradecanoylcamitine), C14:2OH (hydroxytetradecadienylcamitine), C16: 1OH (hydroxyhexadecenoylcarnitine), C16:2OH (hydroxyhexadecadienylcamitine), glucose, lysoPC a C18: 1 (lysophosphatidylcholine acyl C18: 1), methylmalonic acid (methyhnalonate), 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 Cl 8: 1), SM C20:2 (sphingomyelin C20:2), SM(OH) Cl 6: 1 (hydroxysphingomyeline C16: 1), SM(OH) C22: l (hydroxysphingomyeline C22: l), SM(OH) C22:2 (hydroxysphingomyeline C22:2), SM(OH) C24: l (hydroxysphingomyeline C24: l), spermidine, or taurine.

[0063] 13. The method of item 12, wherein: (a) assessing breast cancer in the subject comprises distinguishing stage I or stage II breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in item 4 or 5; (b) assessing breast cancer in the subject comprises distinguishing stage I breast cancer from a healthy control or a non- breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in item 6; (c) assessing breast cancer in the subject comprises distinguishing stage II breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in item 7; (d) assessing breast cancer in the subject comprises distinguishing stage III breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in item 8; (e) assessing breast cancer in the subject comprises distinguishing ductal carcinoma (e.g., stage I or stage II) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in item 9;

[0064] (f) assessing breast cancer in the subject comprises distinguishing lobular carcinoma (e.g., stage I or stage II) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in item 10; (g) assessing breast cancer in the subject comprises distinguishing luminal breast cancer (e.g., luminal A) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in item 11; or (h) any combination of (a) to (g).

[0065] 14. The method of item 12 or 13, further comprising determining a breast cancer probability score based on the quantified panel of metabolites combined with the subject’s age.

[0066] 15. The method of any one of items 12 to 14, further comprising providing a breast cancer treatment recommendation based on said breast cancer assessment.

[0067] 16. A method for implementing or modifying a breast cancer screening program, the method comprising incorporating into said screening program the quantification of a panel of metabolites in a blood sample of a subject, the panel comprising or consists essentially of at least one blood-based metabolite as defined in any one of items 1 to 11, or the method of any one of items 12 to 15.

[0068] 17. The method of any one of items 1 to 16, wherein the blood sample is plasma or serum.

[0069] 18. The method of any one of items 1 to 17, wherein the subject was 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.

[0070] 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 that comprises mammography, breast magnetic resonance imaging (MRI), breast ultrasound, breast biopsy, or any combination thereof.

[0071] 20. A method for treating a breast cancer subject, the method comprising assessing the breast cancer status of a subject according to the method of any one of items 12 to 19 and administering a breast cancer treatment to the subject based thereon.

[0072] 21. A biomarker for use in a blood-based biomarker panel for assessing breast cancer in a subject, the biomarker being at least one blood-based metabolite as defined in any one of items 1 to 11. 22. The biomarker for use of item 21, wherein: (a) assessing breast cancer in the subject comprises distinguishing stage I or stage II breast cancer from a healthy control or a non-breast cancer subject, and wherein the panel of metabolites quantified in said blood sample is as defined in item 4 or 5; (b) assessing breast cancer in the subject comprises distinguishing stage I breast cancer from a healthy control or a nonbreast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in item 6; (c) assessing breast cancer in the subject comprises distinguishing stage II breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in item 7; (d) assessing breast cancer in the subject comprises distinguishing stage III breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in item 8; (e) assessing breast cancer in the subject comprises distinguishing ductal carcinoma (e.g., stage I or stage II) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in item 9;

[0073] (f) assessing breast cancer in the subject comprises distinguishing lobular carcinoma (e.g., stage I or stage II) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in item 10; (g) assessing breast cancer in the subject comprises distinguishing luminal breast cancer (e.g., luminal A) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in item 11; or (h) any combination of (a) to (g).

[0074] 23. A breast cancer biomarker panel comprising at least one of the blood-based metabolites as defined in any one of items 1 to 11.

[0075] 24. The breast cancer biomarker panel of item 23, which is for: (a) distinguishing stage I or stage II breast cancer from a healthy control or a non-breast cancer subject, and wherein the panel of metabolites is as defined in item 4 or 5; (b) distinguishing stage I breast cancer from a healthy control or a non- breast cancer subject, wherein the panel of metabolites is as defined in item 6; (c) distinguishing stage II breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites is as defined in item 7; (d) distinguishing stage III breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites is as defined in item 8; (e) distinguishing ductal carcinoma (e.g., stage I or stage II) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites is as defined in item 9; (f) distinguishing lobular carcinoma (e.g., stage I or stage II) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites is as defined in item 10; (g) distinguishing luminal breast cancer (e.g., luminal A) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites is as defined in item 11; or (h) any combination of (a) to (g). EXAMPLES

[0076] Example 1: Methods

[0077] All biospecimens were obtained from the Cooperative Human Tissue Network (CHTN) biobank. A quantitative mass spectrometry (MS)-based metabolomics approach was used to analyze metabolites in plasma samples using a combination of direct injection (DI) MS and reverse-phase high performance liquid chromatography (HPLC) tandem mass spectrometry (MS / MS). Features with >80% missing values were removed. The sample set was split into a discovery set and validation set. The discovery set was used for statistical tests, followed by cross validation to obtain a logistic regression model. The model was used as a prediction model on the validation set. Metabolite concentration data, clinical data, and hormone receptor status were used to determine optimal biomarker sets. The same biomarkers and regression models were used and assessed on the validation models. The area under the receiver operator characteristic curves (AUROC), sensitivities, and specificities at selected cut off points were calculated for each subgroup.

[0078] Example 2: Study population

[0079] The present study included a total of 185 plasma samples from women with biopsy-confirmed breast cancer, and 56 plasma samples from healthy controls. The breast cancer study population subgroups included tumors that were estrogen receptor (ER)-positive and progesterone receptor (PR)- positive but negative for HER2 (Luminal A), ER-positive, PR-negative and HER2 -positive (Luminal B), hormone receptor-negative but HER2 positive, and triple -negative breast cancer (TNBC), as shown in Fig. 1A. The study population included 98 samples from breast cancer stage I patients, 70 samples from breast cancer stage II patients, and 17 samples from cancer stage III patients (Fig. IB).

[0080] Example 3: Metabolites informative for distinguishing breast cancer vs. healthy controls

[0081] A total of 138 metabolites were analyzed using a combination of direct injection (DI) mass spectrometry and reverse-phase high performance liquid chromatography (HPLC) tandem mass spectrometry (MS / MS) as described in Example 1. The results shown in Fig. 2 show the relative values of particularly informative metabolites (coefficients greater than about 70) for distinguishing breast cancer from healthy controls. The informative metabolites included ones that were found to be elevated in breast cancer samples as compared to in samples from healthy controls: CO (carnitine), glucose, PC aa C40:2 (phosphatidylcholine diacyl C40:2), C16:2OH (hydroxyhexadecadienylcamitine), serotonin, spermidine, asparagine, C5OH (hydroxyvalerylcamitine / C3-DC-M), phenylalanine, SM(OH) C24: l (hydroxysphingomyeline C24: 1), and PC aa C40: 1 (phosphatidylcholine diacyl C40: 1). The informative metabolites also included ones that were found to be lower in breast cancer samples as compared to in samples from healthy controls: kynurenine, C18: 1OH (hydroxyoctadecenoylcamitine), propionic acid (propionate), and succinic acid (succinate) (Fig. 2).

[0082] Example 4: Five metabolites + age distinguishes breast cancer vs. healthy controls

[0083] A logistic regression model was constructed with different sets of metabolites to discriminate breast cancer tumors from healthy controls. The partial least squares discriminant analysis (PLS-DA) scores plot in Fig. 3 shows the spatial separation of breast cancer samples from healthy controls using a set of the following six biomarkers (five metabolites combined with age; relative values in parentheses): spermidine (1.05), PC aa C40.2 (0.58), CO (0.87), succinic acid (-0.75), kynurenine (-0.88), serotonin (0.81), and age (1.70). A receiver operating characteristic curve (ROC) was generated using an optimal logistic regression model for the breast cancer patients at all stages when age is added (Fig. 4). The area under the ROC curves (AUC) and the 95% confidence intervals were calculated using the same biomarker combination and age. The calculated AUC was 94.5%, with a sensitivity of 90.3% and a specificity of 86.8% (Fig. 4).

[0084] Example 5: Metabolites informative for distinguishing breast cancer stages and subtypes

[0085] The potential of using plasma metabolomic profiling and clinical data for detecting breast cancer at different stages and subtypes was explored. Heat maps were constructed, for example to distinguish breast cancer stage I (Fig. 5) or stage II (Fig. 6) vs. healthy controls. Different models were constructed using at least two of the most informative metabolites combined with age, as a continuous variable, to discriminate between healthy controls and breast cancer cases at various breast cancer stages and subtypes. Fig. 7 shows a ROC curve based on the biomarkers: fumaric acid (-1.3746), spermidine (0.8676), glucose (0.7505), asparagine (1.5345), serotonin (1.1838), kynurenine (-0.8679), and age (2.0220); giving an AUC of 96.2%, with a sensitivity of 91. 1% and a specificity of 89.3%. The accuracy reflects the performance of the binary class prediction model of each of the stages and subtypes compared to healthy controls. Examples of sets of other metabolite panels and ROC curve analyses informative for distinguishing between different breast cancer stages and subtypes are summarized in the table below. Table 1: Established metabolite panels

[0086] Example 6: Summary of informative metabolites from Examples 2-5

[0087] “+” = elevated in breast cancer; = lower in breast cancer Example 7: Identification of Biomarker Panel for Breast Cancer Screening

[0088] The principal aim of Example 7 was to construct robust and parsimonious metabolomic signature(s) for early-stage breast cancer detection, based on the blood samples, study population, and pool of metabolites described in Examples 1-3. Multiple machine learning feature selection strategies were systematically combined as described in Vaida et al., 2024, to derive a small, robust, and reliable panel of biomarkers. Briefly, unlike conventional approaches that rely on Partial Least Squares (PLS) or LASSO regression, the present approach employed Univariate Naive Bayes, L2 -regularized Support Vector Classifier (SVC), Principal Component Analysis (PCA), and feature engineering techniques to refine and select the most informative features. Additionally, dataset imbalance was addressed by using propensity score matching (PSM), which ensured reliable comparisons between cancer and control groups. The inclusion of a random sampling strategy further enhanced the reliability of the selected biomarker sets. The best-performing feature set comprised 11 biomarkers, including 9 metabolites [SM(OH) C22:2, SM C18:0, CO, C3OH, C14:2OH, C16:2OH, LysoPC a C18:l, PC aa C36:0 and Asparagine], a metabolite ratio (Kynurenine-to-Tryptophan), and 1 demographic variable (Age), achieving an area under the ROC curve (AUC) of 98% (CI: 97.5%-98%), reflecting a high level of discriminatory power between breast cancer cases and controls.

[0089] SM(OH) C22:2 and SM C18:0, both sphingomyelins, are involved in maintaining membrane structure, while SM(OH) C16: 1 plays a key role in lipid metabolism and membrane integrity. CO (Carnitine) facilitates the transport of fatty acids for energy production, and C3OH, C14:2OH, and C16:2OH are hydroxy carnitines involved in fatty acid oxidation. LysoPC a C18: 1 contributes to membrane remodeling, and PC aa C36:0 is a key phospholipid within cellular membranes. Asparagine, an essential amino acid, supports protein synthesis. Lastly, the Kynurenine-to-Tryptophan ratio has been utilized as a marker for immune regulation and inflammation.

[0090] These results demonstrate the potential for a robust, cost-effective, and non-invasive breast cancer screening and diagnostic tool, offering significant clinical value for early detection and personalized patient management.

[0091] REFERENCES

[0092] Vaida et al., “Identification of a Novel Biomarker Panel for Breast Cancer Screening”. International Journal of Molecular Sciences, 2024, 25(21): 11835. doi: 10.3390 / ijms252111835

Claims

CLAIMS1. A method for processing a human clinical blood sample, the method comprising obtaining a blood sample from a subject clinically assessed as having or suspected of having breast cancer, and quantifying a panel of metabolites in said blood sample, the panel comprising or consists essentially of at least one blood-based metabolite which is: propionic acid (propionate), asparagine, butyric acid, CO (carnitine), C3 (propionylcamitine), C30H (hydroxypropionylcamitine), C4 (butyrylcamitine), C4: l (butenylcamitine), C5OH (hydroxyvalerylcamitine; C3-DC-M), C6.1 (hexenoylcamitine), C12 (dodecanoylcamitine), C14 (tetradecanoylcamitine), C14:2OH (hydroxytetradecadienylcamitine), C16: 1OH (hydroxyhexadecenoylcamitine), C16:2OH (hydroxyhexadecadienylcamitine), C18: 1OH (hydroxyoctadecenoylcamitine), fumaric acid (fumarate), glucose, kynurenine, lysoPC a Cl 8: 1 (lysophosphatidylcholine acyl Cl 8: 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: l (phosphatidylcholine diacyl C40:l), 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 C 18: 1 (sphingomyelin C 18: 1), SM C20:2 (sphingomyelin C20:2), SM(OH) C16:l (hydroxysphingomyeline C16: 1), SM(OH) C22: l (hydroxysphingomyeline C22: l), SM(OH) C22:2 (hydroxysphingomyeline C22:2), SM(OH) C24: l (hydroxysphingomyeline C24: l), 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 SM(OH) C22:2, SM C18:0, CO, C3OH, C14:2OH, C16:2OH, lysoPC a Cl 8: 1 , PC aa C36:0, asparagine, kynurenine, and tryptophan.

3. The method of claim 1 or 2, wherein the panel 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 said 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 stage breast cancer, wherein said early stage breast cancer is stage 1 or stage 11 breast cancer, and wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: propionic acid, asparagine, CO, C3, C3OH, C4, C4: l, C5OH, C6.1, C12, C14, C14:2OH, C16:1OH, C16:2OH, fumaricacid, glucose, kynurenine, lysoPC a Cl 8: 1 , methylmalonic acid, PC aa C36:0, PC aa C36:6, PC aa C38:0, PC aa C40: l, PC aa C40:2, PC ae C40:6, phenylalanine, serotonin, SM C16:0, SM C18:0, SM Cl 8: 1, SM C20:2, SM(OH) C16: l, SM(OH) C22: l, SM(OH) C22:2, SM(OH) C24: l, spermidine, succinic acid, taurine, or any combination thereof.

5. The method of claim 4, wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: propionic acid, asparagine, CO, C3OH, C5OH, C16:2OH, fumaric acid, glucose, kynurenine, lysoPC a C 18 : 1 , PC aa C40: l, PC aa C40:2, PC ae C40:6, phenylalanine, serotonin, SM(OH) C24:l, spermidine, succinic acid, or any combination thereof.

6. The method of any one of claims 1 to 5, wherein the blood sample is from a subject clinically assessed as having or suspected of having stage 1 breast cancer, wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: asparagine, CO, C3, C3OH, C4, C5OH, C6.1, C12, C14, C16:1OH, C16:2OH, fumaric acid, glucose, lysoPC a Cl 8: 1 , methylmalonic acid, PC aa C36:0, PC aa C36:6, PC aa C40: l, PC aa C40:2, PC ae C40:6, phenylalanine, SM C18:0, SM C20:2, SM(OH) C16: l, SM(OH) C22:l, 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 from a subject clinically assessed as having or suspected of having stage 11 breast cancer, wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: CO, C4, C4: l, 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: l, PC aa C40:2, PC ae C40:6, serotonin, SM C16:0, SM C18:0, SM Cl 8: 1, SM C20:2, SM(OH) C16: l, SM(OH) C22: l, 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 111 breast cancer, wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: butyric acid, PC aa C40:2, 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 1 or stage 11), wherein the panel ofmetabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: 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 1 or stage 11), wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one bloodbased metabolite which is: 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 A), wherein the panel of metabolites quantified in said blood sample comprises or consists essentially of at least one blood-based metabolite which is: CO, Cl 8: 1 OH, fumaric acid, kynurenine, PC aa C40:2, spermidine, succinic acid, or any combination thereof.

12. A method of assessing breast cancer in a subject, the method comprising processing a human clinical blood sample according to the method of any one of claims 1 to 11, and comparing the levels of the quantified panel of metabolites to corresponding reference values indicative of a healthy or non-breast cancer subject, wherein:(a) a lower level of one or more of the following metabolites is indicative of breast cancer: propionic acid (propionate), C6.1 (hexenoylcamitine), Cl 8: 1 OH (hydroxyoctadecenoylcamitine), fumaric acid (fumarate), kynurenine, or succinic acid (succinate); and(b) an elevated level of one or more of the following metabolites is indicative of breast cancer: asparagine, butyric acid, CO (carnitine), C3 (propionylcamitine), C3OH (hydroxypropionylcamitine), C4 (butyrylcamitine), C4: l (butenylcamitine), C5OH (hydroxyvalerylcamitine; C3-DC-M), C12 (dodecanoylcamitine), C14 (tetradecanoylcamitine), C14:2OH (hydroxytetradecadienylcamitine), C16: 1OH (hydroxyhexadecenoylcamitine), C16:2OH (hydroxyhexadecadienylcamitine), glucose, lysoPC a Cl 8: 1 (lysophosphatidylcholine acyl Cl 8: 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:l (phosphatidylcholine diacyl C40: l), PC aa C40:2 (phosphatidylcholine diacyl C40:2), PC ae C40:6 (phosphatidylcholine acyl-alkyl C40:6), phenylalanine, serotonin, SM Cl 6:0(sphingomyelin Cl 6:0), SM Cl 8:0 (sphingomyelin Cl 8:0), SM Cl 8: 1 (sphingomyelin C18: 1), SM C20:2 (sphingomyelin C20:2), SM(OH) C16: l(hydroxysphingomyeline Cl 6: 1), SM(OH) C22:l (hydroxysphingomyeline C22: l), SM(OH) C22:2 (hydroxysphingomyeline C22:2), SM(OH) C24: l (hydroxysphingomyeline C24: l), spermidine, or taurine.

13. The method of claim 12, wherein:(a) assessing breast cancer in the subject comprises distinguishing stage 1 or stage 11 breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in claim 4 or 5;(b) assessing breast cancer in the subject comprises distinguishing stage 1 breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in claim 6;(c) assessing breast cancer in the subject comprises distinguishing stage 11 breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in claim 7;(d) assessing breast cancer in the subject comprises distinguishing stage 111 breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in claim 8;(e) assessing breast cancer in the subject comprises distinguishing ductal carcinoma (e.g., stage1 or stage 11) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in claim 9;(f) assessing breast cancer in the subject comprises distinguishing lobular carcinoma (e.g., stage 1 or stage 11) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in claim 10;(g) assessing breast cancer in the subject comprises distinguishing luminal breast cancer (e.g., luminal A) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in claim 11 ; or(h) any combination of (a) to (g).

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

15. The method of any one of claims 12 to 14, further comprising providing a breast cancer treatmentrecommendation based on said breast cancer assessment.

16. A method for implementing or modifying a breast cancer screening program, the method comprising incorporating into said screening program the quantification of a panel of metabolites in a blood sample of a subject, the panel comprising or consists essentially of at least one blood-based metabolite as defined in any one of claims 1 to 11, or the method of any one of claims 12 to 15.

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 was clinically assessed as having or suspected of having breast cancer via mammography, breast magnetic resonance imaging (MR1), 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 that comprises mammography, breast magnetic resonance imaging (MR1), breast ultrasound, breast biopsy, or any combination thereof.

20. A method for treating a breast cancer subject, the method comprising assessing the breast cancer status of a subject according to the method of any one of claims 12 to 19 and administering a breast cancer treatment to the subject based thereon.

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

22. The biomarker for use of claim 21 , wherein:(a) assessing breast cancer in the subject comprises distinguishing stage 1 or stage 11 breast cancer from a healthy control or a non-breast cancer subject, and wherein the panel of metabolites quantified in said blood sample is as defined in claim 4 or 5;(b) assessing breast cancer in the subject comprises distinguishing stage 1 breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in claim 6;(c) assessing breast cancer in the subject comprises distinguishing stage 11 breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in claim 7;(d) assessing breast cancer in the subject comprises distinguishing stage 111 breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in claim 8;(e) assessing breast cancer in the subject comprises distinguishing ductal carcinoma (e.g., stage1 or stage 11) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in claim 9;(f) assessing breast cancer in the subject comprises distinguishing lobular carcinoma (e.g., stage 1 or stage 11) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in claim 10;(g) assessing breast cancer in the subject comprises distinguishing luminal breast cancer (e.g., luminal A) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites quantified in said blood sample is as defined in claim 11 ; or(h) any combination of (a) to (g).

23. A breast cancer biomarker panel comprising at least one of the blood-based metabolites as defined in any one of claims 1 to 11.

24. The breast cancer biomarker panel of claim 23, which is for:(a) distinguishing stage 1 or stage 11 breast cancer from a healthy control or a non-breast cancer subject, and wherein the panel of metabolites is as defined in claim 4 or 5;(b) distinguishing stage 1 breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites is as defined in claim 6;(c) distinguishing stage 11 breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites is as defined in claim 7;(d) distinguishing stage 111 breast cancer from a healthy control or a non-breast cancer subject, wherein the panel of metabolites is as defined in claim 8;(e) distinguishing ductal carcinoma (e.g., stage 1 or stage 11) from a healthy control or a non- breast cancer subject, wherein the panel of metabolites is as defined in claim 9;(f) distinguishing lobular carcinoma (e.g., stage 1 or stage 11) from a healthy control or a non- breast cancer subject, wherein the panel of metabolites is as defined in claim 10;(g) distinguishing luminal breast cancer (e.g., luminal A) from a healthy control or a non-breast cancer subject, wherein the panel of metabolites is as defined in claim 11; or(h) any combination of (a) to (g).

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