BIOMARKER FOR PROSTATE CANCER

Abstract

Provided is a method of accurate and sensitive characterization and prognosis of prostate cancer in a subject. The method includes obtaining a biological sample from the subject and determining the level of identified biomarkers.

Claims

1. A method to characterize prostate cancer in a subject in need thereof, comprising detecting a level of a prostate cancer marker in a biological sample from the subject, wherein the prostate cancer marker comprises one or more of metabolite markers in Tables 1, 2, 5 and 6.

2. The method of claim 1, wherein the prostate cancer marker comprises at least one metabolite marker selected from the group consisting of Ethanimidic acid, N-(trimethylsilyl)-, trimethylsilyl ester; ethanolamine; Glycine, di-TMS; pyruvic acid; Beta-alanine 1; L-(+) lactic acid; 2-hydroxypyridine; Diethanolamine, 3TMS derivative; glyceric acid; Pentenoic acid, 4-[(trimethylsilyl)oxy]-, trimethylsilyl ester; guanidinoacetic acid 2; tartronic acid; Butanoic acid, 2,4-bis[(trimethylsilyl)oxy]-, trimethylsilyl ester; L-pyroglutamic acid; DL-isoleucine 2; 1H-Indole, 1-(trimethylsilyl)-5-[(trimethylsilyl)oxy]; 2,3,4-Trihydroxybutyric acid tetrakis(trimethylsilyl) deriv., (, (R*,R*)); 1-Deoxypentitol, 4TMS derivative; 4-hydroxybenzoic acid; 4-acetamidobutyric acid 1; L-glutamine 2; D-lyxose 2; Arabinofuranose, 1,2,3,5-tetrakis-O-(trimethylsilyl); xanthine; Ribitol TMS; xylitol; L-()-Arabitol, 5TMS derivative; Furan, tetrahydro-2,5-dipropyl-; 1,5-anhydro-D-sorbitol; L-Phenylalanine, 2TMS derivative; 3,4-Dihydroxyphenylacetic Acid, 3TMS derivative; DL-4-hydroxymandelic acid; 3-methyl-L-histidine; trans-aconitic acid; Ethyl (E)-1-penten-3-ynesulfonate; D-allose 2; D-allose 1; L-tyrosine 2; quinic acid; galacturonic acid 2; Ononitol TMS; D-Gluconic acid, 6TMS derivative; pantothenic acid 2; N-acetyl-D-mannosamine 1; D-Allose, pentakis(trimethylsilyl) ether, ethyloxime (isomer 2); Pseudo uridine penta-tms; palmitic acid; 2-phenyl-3,5,7-tris(trimethylsilyloxy)-1-benzopyran-4-one; stearic acid; Guanosine, N,N-dimethyl-1-(trimethylsilyl)-2,3,5-tris-O-(trimethylsilyl)-; 1-Monopalmitin, 2TMS derivative; lactose 1; 2-Monostearin, 2TMS derivative; 1-stearoyl-rac-glycerol; and 3-Phenyl-5,10-secocholesta-1(10),2-dien-5-one.

3. The method of claim 2, wherein the biological sample is peripheral blood, sera, plasma, urine, semen, prostatic fluid, Cowper's fluid, pre-ejaculatory fluid, or any combination thereof.

4. The method of claim 3, further comprising detecting a level of prostate specific antigen in the biological sample from the subject.

5. The method of claim 4, further comprising grouping the subject by NCCN risk classification into six groups of different severities of prostate cancer, wherein the six groups are benign group, very low-risk/low-risk prostate cancer, favorable-intermediate-risk prostate cancer, unfavorable-intermediate-risk prostate cancer, high-risk/very high-risk prostate cancer, and metastasis prostate cancer group.

6. The method of claim 5, further comprising distinguishing the severity of prostate cancer in the subject in one group from the other groups.

7. The method of claim 6, further comprising distinguishing the severity of prostate cancer in the subject in more than one group from the other groups.

8. A method for determining a need of biopsy for prostate cancer diagnosis in a subject in need thereof, comprising detecting a level of a prostate cancer marker in a biological sample from the subject, wherein the prostate cancer marker is selected from the group consisting of panels in Tables 3, 4, 7 and 8.

9. The method of claim 8, wherein the prostate cancer marker is selected from the group consisting of panel 1, panel 2, panel 3, panel 4, and any combination thereof, and wherein: panel 1 is selected from the group consisting of C.sub.10H.sub.21N.sub.4O.sub.2, C.sub.12H.sub.17NO, C.sub.12H.sub.2NOPS, C.sub.12H.sub.9O.sub.9P, C.sub.13H.sub.19N.sub.5O.sub.5, C.sub.17H.sub.32N.sub.3O.sub.7, C.sub.18H.sub.16N.sub.6O.sub.3, C.sub.18H.sub.33NO.sub.4, C.sub.18H.sub.43N.sub.4O.sub.3, C.sub.19H.sub.35NO.sub.5, C.sub.19H.sub.38N.sub.2O.sub.3, C.sub.24H.sub.42N.sub.7O.sub.3, C.sub.27H.sub.12N.sub.9, C.sub.34H.sub.23N.sub.7O.sub.5, C.sub.5H.sub.1 NO, C.sub.51H.sub.29N.sub.5O.sub.4, C.sub.6H.sub.15N, C.sub.8H.sub.9N, C.sub.9H.sub.4N.sub.5O.sub.9, C.sub.9H.sub.8O.sub.2, and any combination thereof; panel 2 is selected from the group consisting of C.sub.11H.sub.5NOPS, C.sub.12H.sub.16NO.sub.7, C.sub.12H.sub.2NOPS, C.sub.12H.sub.9O.sub.9P, C.sub.13H.sub.25NO.sub.2, C.sub.13H.sub.25NO.sub.3, C.sub.14H.sub.30N.sub.4O.sub.2, C.sub.16H.sub.13N.sub.3O.sub.3P, C.sub.17H.sub.41N.sub.4O.sub.3, C.sub.19H.sub.19N.sub.8, C.sub.22H.sub.45NO.sub.4, C.sub.26H.sub.51N.sub.4O.sub.5, C.sub.26H.sub.58N.sub.13P, C.sub.27H.sub.12N.sub.9, C.sub.27H.sub.55N.sub.8O.sub.3, C.sub.30H.sub.57NO.sub.7, C.sub.30H.sub.64N.sub.15O.sub.2P, C.sub.41H.sub.23N.sub.11O.sub.2, C.sub.5, C.sub.5H.sub.7NO.sub.3, C.sub.6HCl.sub.5, C.sub.6H.sub.16N.sub.3O.sub.5, C.sub.8H.sub.16NO.sub.5, and any combination thereof; panel 3 is selected from the group consisting of C.sub.10H.sub.18N.sub.2O.sub.5, C.sub.12H.sub.21NO.sub.4, C.sub.13H.sub.23NO.sub.6, C.sub.13H.sub.25NO.sub.3, C.sub.14H.sub.30N.sub.4O.sub.2, C.sub.15H.sub.30N.sub.10OP, C.sub.16H.sub.13N.sub.3O.sub.3P, C.sub.19H.sub.31N.sub.6O.sub.2, C.sub.22H.sub.45NO.sub.4, C.sub.27H.sub.12N.sub.9, C.sub.28H.sub.57N.sub.8O.sub.4, C.sub.30H.sub.61N.sub.8O.sub.5, C.sub.30H.sub.64N.sub.15O.sub.2P, C.sub.35H.sub.71N.sub.8O.sub.7, C.sub.40H.sub.38N.sub.22O.sub.4, C.sub.41H.sub.23N.sub.11O.sub.2, C.sub.43H.sub.40N.sub.20O.sub.3, C.sub.5H.sub.11NO, C.sub.5H.sub.11NO.sub.2S, C.sub.5H.sub.2O.sub.2P, C.sub.8H.sub.16NO.sub.5, and any combination thereof; and panel 4 is selected from the group consisting of C.sub.11H.sub.20O.sub.2, C.sub.11H.sub.5NOPS, C.sub.12H.sub.2NOPS, C.sub.12H.sub.25NO.sub.4P, C.sub.12H.sub.9O.sub.9P, C.sub.13H.sub.25NO.sub.2, C.sub.13H.sub.25NO.sub.3, C.sub.14H.sub.30N.sub.4O.sub.2, C.sub.16H.sub.30N.sub.3O.sub.2, C.sub.17H.sub.41N.sub.4O.sub.3, C.sub.18H.sub.34O.sub.5, C.sub.19H.sub.31N.sub.6O.sub.2, C.sub.21H.sub.36N.sub.4O.sub.3, C.sub.22H.sub.45NO.sub.4, C.sub.23H.sub.47N.sub.8O.sub.2, C.sub.24H.sub.41N.sub.14O.sub.8, C.sub.27H.sub.12N.sub.9, C.sub.30H.sub.57NO.sub.7, C.sub.30H.sub.64N.sub.15O.sub.2P, C.sub.35H.sub.71N.sub.8O.sub.7, C.sub.43H.sub.40N.sub.20O.sub.3, C.sub.5H.sub.11NO, C.sub.5H.sub.11NO.sub.2S, C.sub.6H.sub.14N.sub.2O.sub.5P, and any combination thereof.

10. The method of claim 8, wherein the prostate cancer marker is selected from the group consisting of panel 5, panel 6, panel 7, panel 8, and any combination thereof, and wherein: panel 5 is selected from the group consisting of C.sub.10H.sub.16O.sub.4, C.sub.10H.sub.18N.sub.2O.sub.4, C.sub.11H.sub.20NO.sub.3P.sub.3, C.sub.12H.sub.7N.sub.4O.sub.2, C.sub.15H.sub.28N.sub.6OP.sub.2, C.sub.16H.sub.39N.sub.8OP, C.sub.19H.sub.14O.sub.3, C.sub.21H.sub.33N.sub.3O.sub.3, C.sub.23H.sub.27O.sub.11S, C.sub.23H.sub.42N.sub.7O, C.sub.25H.sub.46N.sub.7O.sub.3, C.sub.27H.sub.48P.sub.2, C.sub.27H.sub.54O.sub.6, C.sub.33H.sub.22O.sub.7, C.sub.34H.sub.73N.sub.8O.sub.2P, C.sub.38H.sub.48O.sub.12, C.sub.40H.sub.85NOP.sub.3, C.sub.5H.sub.4O.sub.3, C.sub.5H.sub.3N.sub.2O.sub.2, C.sub.6H.sub.11N.sub.4O.sub.3P, C.sub.6H.sub.13N.sub.4OP.sub.2, C7H.sub.10O.sub.4, C7H.sub.17O.sub.7P.sub.2, C7H.sub.6O.sub.6S, C.sub.8H.sub.14O.sub.4, and any combination thereof; panel 6 is selected from the group consisting of C.sub.10H.sub.16O.sub.4, C.sub.11H.sub.16N.sub.4O.sub.4, C.sub.12H.sub.7N.sub.4O.sub.2, C.sub.14H.sub.20N.sub.2O.sub.5, C.sub.16H.sub.32O.sub.2, C.sub.17H.sub.34N.sub.9O.sub.2, C.sub.21H.sub.33N.sub.3O.sub.3, C.sub.23H.sub.27O.sub.11S, C.sub.26H.sub.43NO.sub.6, C.sub.27H.sub.48P.sub.2, C.sub.28H.sub.52N.sub.7O, C.sub.34H.sub.73N.sub.8O.sub.2P, C.sub.38H.sub.48O.sub.12, C.sub.39H.sub.26O.sub.7, C.sub.4H.sub.6O.sub.4, C.sub.40H.sub.85NOP.sub.3, C.sub.5H.sub.4N.sub.4O.sub.2, C.sub.5H.sub.3N.sub.2O.sub.2, C.sub.6H.sub.11N.sub.4O.sub.2P, C.sub.6H.sub.13N.sub.4OP.sub.2, C.sub.6H.sub.6N.sub.4O.sub.2, C.sub.6H.sub.8N.sub.2O.sub.4, C7H.sub.10O.sub.4, C7H.sub.17O.sub.7P.sub.2, C7H.sub.6O.sub.6S, C.sub.9H.sub.16O.sub.4, C.sub.9H.sub.9NO.sub.3, and any combination thereof; panel 7 is selected from the group consisting of C.sub.10H.sub.18N.sub.2O.sub.4, C.sub.10H.sub.19N.sub.5P.sub.3, C.sub.12H.sub.7N.sub.4O.sub.2, C.sub.15H.sub.28N.sub.6O P.sub.2, C.sub.16H.sub.32O.sub.2, C.sub.17H.sub.34N.sub.9O.sub.2, C.sub.19H.sub.14O.sub.3, C.sub.21H.sub.33N.sub.3O.sub.3, C.sub.21H.sub.39N.sub.4OP, C.sub.27H.sub.48P.sub.2, C.sub.38H.sub.48O.sub.12, C.sub.39H.sub.26O.sub.7, C.sub.40H.sub.85NOP.sub.3, C.sub.5H.sub.10N.sub.2O.sub.3, C.sub.5H.sub.4N.sub.4O.sub.2, C.sub.6H.sub.10O.sub.4S, C.sub.6H.sub.1N.sub.4O.sub.2P, C.sub.6H.sub.13N.sub.4OP.sub.2, C.sub.6H.sub.15O.sub.8P, C7H.sub.10O.sub.4, C7H.sub.17O.sub.7P.sub.2, C7H.sub.21N.sub.3OP.sub.3, C7H.sub.22N.sub.4O.sub.9PS, C7H.sub.8O.sub.6S, C.sub.8H.sub.9O.sub.6, C.sub.9H.sub.16O.sub.4, C.sub.9H.sub.17NO.sub.4S, C.sub.9H.sub.9NO.sub.3, and any combination thereof; and panel 8 is selected from the group consisting of C.sub.10H.sub.19N.sub.5P.sub.3, C.sub.12H.sub.7N.sub.4O.sub.2, C.sub.15H.sub.28N.sub.6OP.sub.2, C.sub.17H.sub.34N.sub.9O.sub.2, C.sub.17H.sub.42N.sub.5OP.sub.2, C.sub.21H.sub.33N.sub.3O.sub.3, C.sub.22H.sub.38N.sub.7O, C.sub.24H.sub.40N.sub.4O.sub.3, C.sub.25H.sub.46N.sub.7O.sub.3, C.sub.25H.sub.50O.sub.6, C.sub.27H.sub.54O.sub.6, C.sub.39H.sub.26O.sub.7, C.sub.39H.sub.78O.sub.6, C.sub.40H.sub.85NOP.sub.3, C.sub.6H.sub.11N.sub.4O.sub.2P, C.sub.6H.sub.15O.sub.8P, C.sub.6H.sub.5N.sub.2OP, C.sub.6H.sub.8O.sub.6S, C7H.sub.10O.sub.4, C7H.sub.17O.sub.7P.sub.2, C7H.sub.21N.sub.3OP.sub.3, C.sub.8H.sub.16N.sub.2O.sub.5P, C.sub.8H.sub.18NO.sub.6P, C.sub.8H.sub.4N.sub.4O.sub.3, C.sub.9H.sub.16O.sub.4, C.sub.9H.sub.9NO.sub.3, and any combination thereof.

11. The method of claim 8, wherein the prostate cancer marker is selected from the group consisting of panel 9, panel 10, panel 11, panel 12, and any combination thereof, and wherein: panel 9 is selected from the group consisting of Pyruvic acid, 4-Acetamidobutyric acid, 1,5-Anhydro-D-glucitol, Beta-Alanine, Glyceric acid, D-Lyxose, Galacturonic acid, D-Allose, L-Tyrosine, 3-Methyl-L-histidine, L-Glutamine, L-Pyroglutamic acid, Guanidinoacetic acid, Lactose, 2-Hydroxypyridine, N-acetyl-D-mannosamine, Palmitic acid, 1-Deoxy-d-ribitol, Monopalmitin, 2-Stearoylglycerol, Galangin, 6-ethoxyiminohexane-1,2,3,4,5-pentol, D-Gluconic acid, N,N-Dimethylguanosine, Pseudouridine, Ribitol, and any combination thereof; panel 10 is selected from the group consisting of Pyruvic acid, Xanthine, 4-Acetamidobutyric acid, 1,5-Anhydro-D-glucitol, Beta-Alanine, 1-Stearoyl-rac-glycerol, Glyceric acid, Galacturonic acid, Quinic acid, Xylitol, L-Pyroglutamic acid, Guanidinoacetic acid, Lactose, Ononitol, 5-Hydroxyindole, Monopalmitin, Galangin, 3,4-Dihydroxyphenylacetic acid, 3-Phenyl-5,10-secocholesta-1 (10),2-dien-5-one, Acetamide, Ethyl 1-penten-3-ynesulfonate, 2,5-Dipropyltetrahydrofuran, L-Phenylalanine, Pseudouridine, and any combination thereof; panel 11 is selected from the group consisting of L-Lactic acid, Xanthine, 4-Acetamidobutyric acid, Beta-Alanine, 1-Stearoyl-rac-glycerol, 4-hydroxymandelic acid, trans-Aconitic acid, D-Allose, Tartronic acid, Stearic acid, L-Tyrosine, Quinic acid, Ethanolamine, Guanidinoacetic acid, DL-isoleucine, Palmitic acid, Monopalmitin, Arabinofuranose, 2,4-Dihydroxybutanoic acid, Diethanolamine, Acetamide, 2,5-Dipropyltetrahydrofuran, Glycine, L-Arabinitol, Levulinic acid, Pseudouridine, and any combination thereof; and panel 12 is selected from the group consisting of Pyruvic acid, Xanthine, 4-Hydroxybenzoic acid, Beta-Alanine, 1-Stearoyl-rac-glycerol, Galacturonic acid, D-Allose, Tartronic acid, Quinic acid, Pantothenic acid, Xylitol, Guanidinoacetic acid, 1-Deoxy-d-ribitol, Monopalmitin, Threonic acid, Galangin, Arabinofuranose, 2,4-Dihydroxybutanoic acid, D-Gluconic acid, 2,5-Dipropyltetrahydrofuran, Levulinic acid, Pseudouridine, and any combination thereof.

12. The method of claim 8, wherein the prostate cancer marker is selected from the group consisting of panel 13, panel 14, panel 15, panel 16, and any combination thereof, and wherein: panel 13 is selected from the group consisting of 1-Methoxymethyl-2-phenylthioindole-3-carbaldehyde, 2,3-Dihydroxybutanoic acid, 2-Hydroxypyridine, 2-Stearoylglycerol, 3-Hydroxyphenylacetic acid, 3-Indoleacetic acid, 3-Methyl-L-histidine, 4-Acetamidobutyric acid, 4-hydroxymandelic acid, 6-ethoxyiminohexane-1,2,3,4,5-pentol, alpha-Hydroxyisobutyric acid, D-Altrose, D-Gluconic acid, D-Lyxose, Galacturonic acid, Galangin, Glyceric acid, Lactose, L-Fucose, L-Pyroglutamic acid, Monopalmitin, Ononitol, Oxamide, Palmitic acid, Pseudouridine, Pyruvic acid, Ribitol, trans-Aconitic acid and any combination thereof; panel 14 is selected from the group consisting of 1-Stearoyl-rac-glycerol, 2,5-Dipropyltetrahydrofuran, 3,4-Dihydroxyphenylacetic acid, Acetamide, Beta-Alanine, Cyclohexylamine, Ethyl 1-penten-3-ynesulfonate, Galacturonic acid Galangin, Glyceric acid, Guanidinoacetic acid, Levulinic acid, Monopalmitin, Ononitol, Palmitic acid, p-Tolyl-beta-D-glucopyranosid-uronsaeure, Quinic acid, Stearic acid, Sucrose, Uric acid, Xanthine, Xylitol, and any combination thereof; panel 15 is selected from the group consisting of (22S,23S,25R)-3-methoxy-16,23:22,26-diepoxy-5-cholestane, 1-Methoxymethyl-2-phenylthioindole-3-carbaldehyde, 1-Stearoyl-rac-glycerol, 2,4-Dihydroxybutanoic acid, 2,5-Dipropyltetrahydrofuran, 4-hydroxymandelic acid, Acetamide, Arabinofuranose, Beta-Alanine, Daidzein, D-Allose, DL-isoleucine, D-tagatofuranose, Ethanolamine, Galangin, Guanidinoacetic acid, L-Arabinitol, Levulinic acid, L-Lactic acid, Monopalmitin, Palmitic acid, Pseudouridine, Quinic acid, Stearic acid, Sucrose, Tartronic acid, Xanthine, and any combination thereof; and panel 16 is selected from the group consisting of (4RS,5SR)-5-hydroperoxy-4-decanol, 2,5-Dipropyltetrahydrofuran, 3,4,5-Trihydroxypentanoic acid, 4-Hydroxybenzoic acid, 6-ethoxyiminohexane-1,2,3,4,5-pentol, Acetamide, Arabinofuranose, Beta-Alanine, D-Allose, DL-4-Hydroxy-3-methoxymandelic acid, Ethyl 1-penten-3-ynesulfonate, Galacturonic acid, Galangin, Glyceric acid, Guanidinoacetic acid, Hippuric Acid, Levulinic acid, L-Pyroglutamic acid, Pantothenic acid, Pseudouridine, Pyruvic acid, Quinic acid, Tartronic acid, Uric acid, Xanthine, and any combination thereof.

13. A method for monitoring a prostate cancer subject on active surveillance (AS), comprising detecting a level of a prostate cancer marker in a biological sample from the subject, wherein the prostate cancer marker is selected from the group consisting of markers in Tables 1, 2, 5 and 6.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present disclosure provides a method and biomarkers to diagnose, stratify, prognosticate and monitor prostate cancer in a subject in need thereof by analyzing the levels of one or more biomarker in a sample obtained from the subject. All terms including descriptive or technical terms which are used herein should be construed as having meanings that are obvious to one of ordinary skill in the art. However, the terms may have different meanings according to an intention of one of ordinary skill in the art, case precedents, or appearance of new technologies. Also, some terms may be arbitrarily selected by the applicant, and in this case, the meaning of the selected terms will be described in detail in the comprehensive descriptions of the present disclosure. Thus, the terms used herein have to be defined based on meaning of the terms together with descriptions throughout the specification.

[0036] Also, when a part includes or comprises a component or a step, unless there is a particular description contrary thereto, the part can further include other components or other steps, not excluding the others.

[0037] It is further noted that, as used in this disclosure, the singular forms a, an, and the include plural referents unless expressly and unequivocally limited to one referent. The term or is used interchangeably with the term and/or unless the context clearly indicates otherwise.

[0038] The term to characterize in a subject or individual may include, but is not limited to, to provide the diagnosis of a disease or a condition, to determine the stratification of a disease risk, to assess the risk of a disease, to provide the prognosis of a disease or a condition, to determine a disease stage or a condition stage, to determine the severity of a disease, to evaluate the malignancy potential of a disease, to monitor a recurrence of cancer, to evaluate a drug efficacy, to describe a physiological condition, to evaluate an organ distress or organ rejection, to monitor disease or condition progression, to determine therapy-related association to a disease or a condition, or to describe a specific physiological or biological state.

[0039] As used herein, prognosis of cancer may include predicting the clinical outcome of the patient, assessing the risk of cancer recurrence, determining treatment modality, or determining treatment efficacy.

[0040] As used herein, the term metastasis describes the spread of a cancer from one part of the body to another. A tumor formed by cells that have spread can be called a metastatic tumor or a metastasis. The metastatic tumor often contains cells that are similar to those in the original (primary) tumor, and have, but not limited to, genomic, epigenetic, transcriptomic, and metabolic alterations.

[0041] As used herein, the term progression describes the course of a disease, such as a cancer, as it becomes worse or spreads in the body.

[0042] The terms subject, patient and individual are used interchangeably herein and refer to a warm-blooded animal, such as a mammal that is afflicted with, or suspected of having, at risk for or being pre-disposed to, or being screened for cancer, e.g., actual or suspected cancer. These terms include, but are not limited to, domestic animals, sports animals, primates and humans. For example, the terms refer to a human.

[0043] The term detect, detecting or detection includes assaying, or otherwise establishing the presence or absence of the target biomarker(s), subunits, or combinations of reagent-bound targets, and the like, or assaying for ascertaining, establishing, characterizing, predicting or otherwise determining one or more factual characteristics of a cancer such as stage, aggressiveness, metastatic potential or patient survival, or assisting with the same. A cut-off value or a standard may correspond to levels quantitated for samples from control healthy subjects with no disease or low-grade cancer or from other samples of the subject.

[0044] As used herein, the term marker or biomarker is a biological molecule, or a panel of biological molecules, whose altered level in a tissue, cell or sample as compared to its level in normal or healthy tissue, cell or sample is associated with a disease state, such as an abnormal prostate state, including disease in an early stage, e.g., prior to the detection of one or more symptoms associated with the disease. In an aspect of the disclosure, prostate cancer may be characterized by identifying and measuring the level of one or more biomarkers listed in Tables 1 to 8 in a biological sample.

[0045] The biological sample obtained from the subject may be any bodily fluid. For example, the biological sample can be peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, cerumen, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, pus, sebum, vomit, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates or other lavage fluids.

[0046] In one embodiment, the marker is detected in a urine sample. In another embodiment, the marker is detected in a blood sample, e.g., serum or plasma. In one embodiment, the marker is detected in serum. In one embodiment, the marker is detected in plasma. In some embodiments, the serum or plasma can be further processed to remove abundant blood proteins (e.g., albumin) or irrelevant proteins that are not marker proteins prior to analysis.

[0047] Examples of biomarkers include, but are not limited to, polypeptides, peptides, polypeptide fragments, antibodies, hormones, polynucleotides, RNA or RNA fragments, microRNAs (miRNAs), lipids, metabolites, or polysaccharides. In some embodiments, biomarker may be a metabolite marker. In one embodiment, the severity of prostate cancer in a subject can be determined or predicted by a panel of biomarkers or by a combination of two panels that are established by metabolites markers, respectively, through the processes including, but not limited to, K-fold cross validation, forward selection, reverse selection, logistic regression, and/or decision tree analysis. As such, the disclosure can effectively improve the predication index such as, but not limited to, area under the curve (AUC), sensitivity, specificity, positive predictive value (PPV), or negative predictive value (NPV). In some embodiment, the efficacy of combination of two panels of metabolite markers may be better than that of individual panel thereof. As used herein, panel refers to a particular combination of biomarkers that is used to determine or predict severity of prostate cancer in a subject, and assign subjects into different groups according to the severity of prostate cancer. As used herein, group refers to a selection of subjects being determined to have similar condition or severity of prostate cancer. As used herein, a model refers to use of different panels of biomarkers in determining and assigning potential prostate cancer patients in different groups of severity.

[0048] In some embodiments, the biomarker involves in the pathway such as, but not limited to, fatty acid biosynthesis, purine metabolism, tryptophan metabolism, pyrimidine metabolism, arginine proline metabolism, pentose and glucuronate interconversion, valine degradation/pyrimidine metabolism, glyoxylate metabolism, ubiquinone biosynthesis, or any combination thereof. In one embodiment, the biomarker is a metabolite marker.

EXAMPLE

[0049] Exemplary embodiments of the present disclosure are further described in the following examples, which do not limit the scope of the present disclosure.

Example 1: Grouping of Prostate Cancer Patients

[0050] For efficient identification of prostate cancer patients with different severities, Gleason's pattern scale (from grade 1 to 5) is assigned to each prostate tissue biopsy core by experienced pathologist. Grade 1 is given to cells that look like normal prostate tissue while the grade 5 is assigned to cancer cells with very abnormal growth patterns. Most prostate cancers score a grade of 3 or higher. Grade 1 and 2 are not used in the biopsy reports. Prostate tumors are often made up of multiple foci with different grades. Two grades are usually assigned for each patient to give rise to a Gleason sum or Gleason scores. A primary grade is given to describe the cells that make up the largest area of the tumor, and a second grade is given to describe cells of the next largest area. Based on patient's disease risk and severities, six different groups were used to stratify patients in a more precise manner.

[0051] The first group is a benign group where no cancer was found; the second group is the metastatic prostate cancer group (mPC) with cancer cells breaking the prostate capsule barrier and invading into other organs (e.g., lymph nodes or bones); the third group is very-low-risk/low-risk prostate cancer group (VLR/LR PC) with all below criteria: Gleason score less than or equal to 6 (e.g., 3+3, the first 3 is the primary grade and the second 3 is the secondary grade), clinical T1 to T2a stage, and PSA of 10 ng/mL or less; the fourth group is high-risk/very-high-risk prostate cancer group (HR/VHR PC) with one of the below criteria: clinical T3a or more, Gleason sum of 8 or more, and PSA of more than 20 ng/mL; the fifth and sixth groups are intermediate-risk prostate cancer group with at least one intermediate-risk criteria below: clinical T2b-2c, Gleason score of 4+3 or 3+4, and PSA of 10 to 20 ng/mL. Among them, the fifth group is favorable-intermediate-risk prostate cancer group (FIR PC) with the below three criteria: only one intermediate-risk factor, Gleason score of 3+4 or less, and less than 50% biopsy cores positive for prostate cancer. The sixth group is the unfavorable-intermediate-risk prostate cancer group (UIR PC) with one of the below three criteria: 2 or 3 intermediate-risk factors, Gleason score of 4+3, and 50% or more of biopsy cores positive for prostate cancer.

[0052] Currently, assessing and dividing potential prostate cancer patients into these six groups as mentioned above rely on invasive needle biopsy. With the present disclosure herewith, efficient assessment of patients can be made with the use of corresponding panel of biomarkers with a proper method of analysis. Different models, through the use of different panels of biomarkers, to determine and distinguish potential prostate cancer patients in different groups of severity is adopted and useful under various clinical scenarios. These models identify and distinguish a potential prostate cancer patient in one or more severity groups from the rest of the groups. For example, patients can be distinguished between the benign group versus the rest groups, VLR/LR PC, FIR PC, UIR PC, HR/VHR PC and mPC, for population screening or general health checkup. In another population screening or health check-up, VLR/LR PC can be regarded as benign and divided the subjects under test into a group of benign and VLR/LR PC versus another group consisting of FIR PC, UIR PC, HR/VHR PC and mPC. In another clinical scenario with elder prostate cancer patients, such as those older than 75 years old, an analysis to distinguish between the group of benign, VLR/LR PC or FIR PC versus the group of UIR PC, HR/VHR PC and mPC would be meaningful, considering the risk derived from VLR/LR PC or FIR PC may unlikely blunt a life-span expectation of a man older than 75 years old.

[0053] While in a clinical scenario involving a new positive biopsy that the patient is in need of risk stratification and prognosis, an analysis dividing the subject between the group of VLR/LR PC or FIR PC versus the group of UIR PC, HR/VHR PC and mPC is required. Another clinical scenario that could find this analysis useful is for monitoring prostate cancer among patients with VLR/LR PC or FIR PC under active surveillance (AS).

[0054] Furthermore, when there is a young prostate cancer patient with a new positive biopsy, then an analysis on whether he belongs to VLR/LR PC versus FIR/UIR/HR/VHR PC or mPC group is useful, considering that the risk of FIR/UIR/HR/VHR PC or mPC may significantly blunt his life-span expectation and impair his social-economical contribution, if not diagnosed in time and properly treated. This analysis that distinguishes an VLR/LR PC group from FIR/UIR/HR/VHR PC or mPC groups is also meaningful for a young prostate cancer patient seeking for AS options.

[0055] Therefore, for monitoring patients under AS, different models of comparison and/or panels of markers can be used based on the age of the patient, other physiological condition or clinical manifestations, e.g., PSA level. Doctors can decide which model of comparison and/or panels of markers to be used to allow the best AS option for each patient. For example, for elder patients such as those aged greater than 75-year-old, the AS will adopt the model of comparison that distinguishes benign, VLR/LR PC or FIR PC from those of UIR/HR/VHR PC or mPC, and for younger patients such as those aged less than 60-year-old, the AS will adopt the model of comparison that distinguishes benign, VLR/LR PC from those of FIR/UIR/HR/VHR PC or mPC.

Example 2: Identification of Metabolite Markers for assessing prostate cancer Risk and Assigning Patients in Different Group of Severity Using Liquid Chromatography-Mass Spectrometry (LC/MS) Analysis

[0056] Two modes of metabolite analysis were carried out with different columns using liquid chromatography-mass spectrometry (LC/MS) analysis, which are the positive mode with BEH C18 column and negative mode with HILIC column. For positive mode, the urine samples were diluted with water (1:10 vol/vol), and then centrifuged at 4 C. and 13200 rpm for 10 minutes. The supernatants were then transferred to the new sample vial for LC/MS analysis with respective columns.

[0057] The LC/MS system used is Agilent 1290 Infinity II ultra-performance liquid chromatography (UPLC) system (Agilent Technologies, Palo Alto, CA, USA) coupled online to the Dual AJS electrospray ionization (ESI) source of an Agilent 6545 quadrupole time-of-flight (Q-TOF) mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). The sample was separated by using ACQUITY UPLC BEH C18 column (1.7 m, 2.1100 mm, Waters Corp., Milford, MA, USA) and ACQUITY UPLC BEH amide column (1.7 m, 2.1100 mm, Waters Corp., Milford, MA, USA). The column temperature was 40 C. The mobile phase for BEH C18 column was H.sub.2O (eluent A) and acetonitrile (eluent B), both eluents with 0.1% formic acid. The gradient condition was: 0 to 1 min, 2% B; 1 to 4 min, 2 to 40% B; 4 to 8 min, 40 to 70% B; 8 to 10 min, 70 to 95% B; 10 to 12 min, 95% B; 12 to 13 min, 95 to 2% B; 13 to 16 min, 2% B. The flow rate was 400 L/min, and the injection volume of sample was 1 L. The mobile phase for BEH amide column was H.sub.2O (eluent A) and 90% acetonitrile (eluent B), both eluents with 15 mM ammonium acetate and 0.3% NH.sub.4OH. The gradient condition was: 0 to 7 min, 90% B; 7 to 8 min, 70 to 50% B; 8 to 10 min, 50% B; 10 to 11 min, 50 to 90% B; 11 to 16 min, 90% B. The post time of elution was 4 min. The flow rate was 300 L/min, and the injection volume of sample was 2 L. The instrument was operated in positive full-scan mode with BEH C18 column and negative full-scan mode with BEH amide column, both methods collected from an m/z of 60 to 1700. The MS operating conditions were optimized as follows: Vcap voltage, 3.5 kV; nozzle voltage, 0.5 kV; nebulizer, 45 psi; gas temperature, 300 C.; sheath gas temperature, 325 C.; sheath gas flow (nitrogen), 8 L/min; drying gas (nitrogen), 8 L/min.

[0058] For negative mode with HILIC column, the urine samples were diluted with acetonitrile (1:10 vol/vol), then centrifuged at 4 C. and 13200 rpm for 10 min. The supernatants were transferred to the new sample vial for LC/MS analysis. The LC/MS method is same with BEH C18 column as described above.

[0059] The chromatogram acquisition, detection of mass spectral peaks, and their waveform processing were performed using Agilent Qualitative Analysis 10.0 and Agilent Profinder 10.0 software (Agilent, USA).

[0060] To identify and select the specific metabolites as markers for distinguishing different groups, a univariate logistic regression to select differentially accumulated metabolites with P values less than 0.1. The identified differential compounds were further analyzed by Receiver operating characteristic (ROC), using Medcalc software version 11.2 (Medcalc Software, Belgium). Furthermore, a K-fold cross validation and a followed reverse selection-based logistic regression were applied to select discriminator sets with improved AUC performance.

[0061] To find urine biomarkers that distinguish prostate tumors with different malignancy potential, four different models of comparison were performed by analyzing metabolites from LC/MS with BEH C18 column. The union of all these four sets of markers from LC/MS with BEH C18 column is listed in Table 1 below.

TABLE-US-00001 TABLE 1 Mass to charge ratio (m/z) of the union of all sets of potential metabolite markers from LC/MS with BEH C18 column for distinguishing patients of prostate tumor-with different malignancy potential Marker Retention CAS metabolites m/z time (min) Annotation by SIRIUS Number C.sub.5 59.9999 0.6 C.sub.5H.sub.11NO 101.0842 13.2 N-Butylformamide 871-71-6 C.sub.6H.sub.15N 101.1201 13.2 C.sub.8H.sub.9N 119.0732 1.9 Isoindoline 496-12-8 C.sub.5H.sub.2O.sub.2P 124.9791 0.6 C.sub.5H.sub.7NO.sub.3 129.0427 3.2 5-Oxo-D-proline 4042-36-8 C9H.sub.8O.sub.2 148.052 7.4 Pyruvophenone 579-07-07 C.sub.5H.sub.11NO.sub.2S 149.0505 0.9 Methionine 59-51-8 C.sub.11H.sub.20O.sub.2 184.1458 5.4 Undecylenic acid 112-38-9 C.sub.12H.sub.17NO 191.1302 5.6 Hexanilide 621-15-8 C.sub.8H.sub.16NO.sub.5 206.1025 2.5 C.sub.6H.sub.16N.sub.3O.sub.5 210.1089 0.7 C.sub.6H.sub.14N.sub.2O.sub.5P 225.0635 2.7 Vanilloylglycine 1212-04-0 C.sub.13H.sub.25NO.sub.2 227.1883 5.7 Undecylenamide MEA 20545-92-0 C.sub.10H.sub.21N.sub.4O.sub.2 229.1673 5 N-decanoylglycine 14305-32-9 C.sub.11H.sub.5NOPS 229.9828 0.9 C.sub.12H.sub.2NOPS 238.9582 0.6 C.sub.12H.sub.21NO.sub.4 243.1467 3 3-(Cyclobutanecarbonyloxy)-4- (trimethylazaniumyl)butanoate C.sub.13H.sub.25NO.sub.3 243.1826 5.1 N-Undecanoylglycine 83871-09-4 C.sub.10H.sub.18N.sub.2O.sub.5 246.1209 2.7 C.sub.6HCl.sub.5 247.8566 13.4 C.sub.12H.sub.25NO.sub.4P 278.1509 9 Dibutyl phthalate 84-74-2 C.sub.12 H.sub.16NO.sub.7 286.0931 3.2 C.sub.14H.sub.30N.sub.4O.sub.2 286.2362 0.7 C.sub.13H.sub.23NO.sub.6 289.1519 2.4 O-adipoyl-L-carnitine 102636-83-9 C.sub.16H.sub.30N.sub.3O.sub.2 296.2339 8.1 10-Hydroxyoctadeca-12,15- 34932-14-4 dienoic acid C.sub.13H.sub.19N.sub.5O.sub.5 325.1379 2 N(2),N(2),7-trimethylguanosine C.sub.9H.sub.4N.sub.5O.sub.9 325.9998 6.6 C.sub.16H.sub.13N.sub.3O.sub.3P 326.0699 8.1 C.sub.18H.sub.33NO.sub.4 327.2396 7 C.sub.12H.sub.9O.sub.9P 327.9969 6.6 C.sub.18H.sub.34O.sub.5 330.2395 5.7 C.sub.19H.sub.38N.sub.2O.sub.3 342.2868 6.3 Cocamidopropyl betaine 4292/10/8 C.sub.17H.sub.41N.sub.4O.sub.3 349.3176 10.3 C.sub.19H.sub.35NO.sub.5 357.2518 6.3 [3-carboxy-2-[(Z)-3- hydroxydodec-9- enoyl]oxypropyl]- trimethylazanium C.sub.19H.sub.19N.sub.8 359.1742 8.5 N-(1,4-Dihydroxy-4- 2058332-33-3 methylpentan-2-YL)-3-hydroxy- 5-oxo-6-phenylhexanamide C.sub.18H.sub.43N.sub.4O.sub.3 363.3334 10.8 1,2-Propanediol, 3-((2- 34719-62-5 hydroxyheptadecyl)oxy)- C.sub.18H.sub.16N.sub.6O.sub.3 364.1293 8.6 C.sub.19H.sub.31N.sub.6O.sub.2 375.2519 6.6 1,3,5-Tris(2,2- 745070-61-5 dimethylpropionylamino)benzene C.sub.22H.sub.45NO.sub.4 387.3335 11.6 C.sub.17H.sub.32N.sub.3O.sub.7 390.2231 2.5 C.sub.21H.sub.36N.sub.4O.sub.3 392.2789 6.6 (2R)-N-[(2S)-1-amino-3- 1212507-31-7 cyclohexyl-1-oxopropan-2-yl]-1- (cyclohexanecarbonyl)piperazine- 2-carboxamide C.sub.15H.sub.30N.sub.10OP 397.2335 6.6 Lysylthreonyllysine 106326-71-0 C.sub.27H.sub.12N.sub.9 462.1229 10 C.sub.23H.sub.47N.sub.8O.sub.2 467.3828 9.8 C.sub.24H.sub.42N.sub.7O.sub.3 476.3347 6.6 C.sub.26H.sub.51N.sub.4O.sub.5 499.388 10.2 C.sub.27H.sub.55N.sub.8O.sub.3 539.4401 10.7 C.sub.30H.sub.57NO.sub.7 543.4126 10.2 C.sub.28H.sub.57N.sub.8O.sub.4 569.4509 10.2 C.sub.26H.sub.58N.sub.13P 583.4642 10.7 C.sub.34H.sub.23N.sub.7O.sub.5 609.1765 12.1 C.sub.30H.sub.61N.sub.8O.sub.5 613.477 10.2 C.sub.24H.sub.41N.sub.14O.sub.8 653.3222 10.4 C.sub.30H.sub.64N.sub.15O.sub.2P 697.5124 10.7 C.sub.41H.sub.23N.sub.11O.sub.2 701.2021 12 C.sub.35H.sub.71N.sub.8O.sub.7 715.5414 10.6 C.sub.51H.sub.29N.sub.5O.sub.4 775.2244 12.9 C.sub.43H.sub.40N.sub.20O.sub.3 884.3598 11.2 C.sub.40H.sub.38N.sub.22O.sub.4 890.3456 12.4

[0062] To find urine biomarkers that distinguish prostate tumors with different malignancy potential, metabolites from LC/MS with HILIC column in four different models of comparison were analyzed. The union of all these four sets of markers from LC/MS with HILIC column for distinguishing different malignancy potential of prostate tumors is listed in Table 2 below.

TABLE-US-00002 TABLE 2 Mass to charge ratio (m/z) of the union of all sets of potential metabolite markers from LC/MS with HILIC column for distinguishing prostate tumors with different malignancy potential Retention Marker time Annotation by CAS metabolites m/z (min) SIRIUS Number C.sub.5H.sub.4O.sub.3 112.016 5.1 2-Furoate 88-14-2 C.sub.4H.sub.6O.sub.4 118.0263 4 Succinate 110-15-6 C.sub.5H.sub.8N.sub.2O.sub.2 128.0578 1.6 1,3-Diazepane- 75548-99-1 2,4-dione C.sub.5H.sub.10N.sub.2O.sub.3 146.0691 1.6 Alanylglycine 1188-01-8 C.sub.6H.sub.5N.sub.2OP 152.0134 1.8 C.sub.5H.sub.4N.sub.4O.sub.2 152.0328 2.4 Xanthine 69-89-6 C.sub.7H.sub.10O.sub.4 158.0575 3.7 Hept-2- 1085697- enedioic acid 38-6 C.sub.6H.sub.6N.sub.4O.sub.2 166.0491 1.6 1-Methylxanthine 6136-37-4 C.sub.6H.sub.8N.sub.2O.sub.4 172.0481 2.6 Hydantoin- 5624-26-0 propionate C.sub.8H.sub.14O.sub.4 174.089 3.4 Suberic acid 505-48-6 C.sub.6H.sub.10O.sub.4S 178.03 1.5 3,3- Thiodipropanoate C.sub.9H.sub.9NO.sub.3 179.0584 1.4 Hippurate 495-69-2 C.sub.9H.sub.16O.sub.4 188.1045 3.2 Azelaic acid 123-99-9 C.sub.10H.sub.16O.sub.4 200.1049 1 Radioplex 10018-78-7 C.sub.8H.sub.9O.sub.6 201.0396 1.4 C.sub.6H.sub.11N.sub.4O.sub.2P 202.0602 2.3 C.sub.8H.sub.4N.sub.4O.sub.3 204.0299 1.6 C.sub.6H.sub.8O.sub.6S 208.0033 1 C7H.sub.6O.sub.6S 217.9884 1.6 Salicylsulfuric 89-45-2 acid C.sub.6H.sub.11N.sub.4O.sub.3P 218.055 3.4 C.sub.6H.sub.13N.sub.4OP.sub.2 219.0564 1.9 S-(3-Oxopropyl)- 140226-30-8 N-acetylcysteine C.sub.7H.sub.8O.sub.6S 220.0041 0.9 1-Methyl- pyrogallol-3-O- sulphate C.sub.10H.sub.18N.sub.2O.sub.4 230.1258 2 DI-Acetyl-lysine 499-86-5 C.sub.9H.sub.17NO.sub.4S 235.0868 1.6 S-(D- 1632078- Carboxybutyl)-L- 43-3 homocysteine C.sub.12H.sub.7N.sub.4O.sub.2 239.0556 1.9 C.sub.6H.sub.15O.sub.8P 246.0504 3.4 C.sub.8H.sub.16N.sub.2O.sub.5P 251.0787 1.5 N- 1220-05-9 Feruloylglycine C.sub.8H.sub.18NO.sub.6P 255.0875 2.1 Pantothenate 79-83-4 C7H.sub.21N.sub.3OP.sub.3 256.0895 4.5 C.sub.16H.sub.32O.sub.2 256.2403 0.9 Hexadecanoic 1957/10/3 acid C.sub.11H.sub.16N.sub.4O.sub.4 268.1169 3.1 Acetylcarnosine C7H.sub.17O.sub.7P.sub.2 275.0449 1 L-Tyrosine 81660-41-5 methyl ester 4- sulfate C.sub.19H.sub.14O.sub.3 290.0942 0.8 C.sub.14H.sub.20N.sub.2O.sub.5 296.1362 1.5 C.sub.10H.sub.19N.sub.5P.sub.3 302.0856 1.6 C.sub.11H.sub.20NO.sub.3P.sub.3 307.0652 2.7 C.sub.7H.sub.22N.sub.4O.sub.9PS 369.084 2.8 C.sub.15H.sub.28N.sub.6OP.sub.2 370.18 0.9 DHT-sulfate 2641-48-7 C.sub.21H.sub.33N.sub.3O.sub.3 375.2509 0.9 C.sub.16H.sub.39N.sub.8OP 390.2988 0.9 C.sub.21H.sub.39N.sub.4OP 394.2848 1.7 C.sub.17H.sub.42N.sub.5OP.sub.2 394.2849 0.9 C.sub.17H.sub.34N.sub.9O.sub.2 396.2825 1.7 C.sub.22H.sub.38N.sub.7O 416.3138 1 C.sub.24H.sub.40N.sub.4O.sub.3 432.3084 0.9 C.sub.23H.sub.42N.sub.7O 432.3413 0.9 C.sub.27H.sub.48P.sub.2 434.3237 0.9 C.sub.25H.sub.50O.sub.6 446.3605 0.9 C.sub.26H.sub.43NO.sub.6 465.3085 1.7 C.sub.27H.sub.54O.sub.6 474.3916 0.9 C.sub.25H.sub.46N.sub.7O.sub.3 492.3639 0.9 C.sub.28H.sub.52N.sub.7O 502.4229 0.9 C.sub.23H.sub.27O.sub.11S 511.1262 1.4 C.sub.33H.sub.22O.sub.7 530.1361 0.8 C.sub.39H.sub.26O.sub.7 606.1673 0.8 C.sub.39H.sub.78O.sub.6 642.5768 0.9 C.sub.34H.sub.73N.sub.8O.sub.2P 656.5583 0.9 C.sub.40H.sub.85NOP.sub.3 688.5843 1 C.sub.38H.sub.48O.sub.12 696.3159 0.9

[0063] In each of different models of comparison designed for diverse clinical scenarios, a representative panel of metabolite markers was identified from LC/MS with BEH C18 column, as shown in Table 3 below, with their prediction ability evaluated by AUC analysis. Sensitivity and specificity of the prediction are also shown in bracket following AUC.

TABLE-US-00003 TABLE 3 Representative panels of metabolite markers from LC/MS with BEH C18 column for distinguishing patients of prostate tumors with different malignancy potential AUC AUC (sensitivity/ (sensitivity/ Models of Metabolite markers specificity) specificity) No comparison (N = Number of markers) without PSA with PSA 1 Benign N = 20 0.89 (90%/55%) 0.91 (90%/64%) vs. C.sub.10H.sub.21N.sub.4O.sub.2 VLR PC/LR C.sub.12H.sub.17NO PC/FIR PC/UIR C.sub.12H.sub.2NOPS PC/HR PC/VHR C.sub.12H.sub.9O.sub.9P PC/mPC C.sub.13H.sub.19N.sub.5O.sub.5 C.sub.17H.sub.32N.sub.3O.sub.7 C.sub.18H.sub.16N.sub.6O.sub.3 C.sub.18H.sub.33NO.sub.4 C.sub.18H.sub.43N.sub.4O.sub.3 C.sub.19H.sub.35NO.sub.5 C.sub.19H.sub.38N.sub.2O.sub.3 C.sub.24H.sub.42N.sub.7O.sub.3 C.sub.27H.sub.12N.sub.9 C.sub.34H.sub.23N.sub.7O.sub.5 C.sub.5H.sub.11NO C.sub.51H.sub.29N.sub.5O.sub.4 C.sub.6H.sub.15N C.sub.8H.sub.9N C.sub.9H.sub.4N.sub.5O.sub.9 C.sub.9H.sub.8O.sub.2 2 Benign/VLR N = 23 0.84 (90%/49%) 0.89 (90%/62%) PC/LR PC C.sub.11H.sub.5NOPS vs. C.sub.12H.sub.16NO.sub.7 FIR PC/UIR C.sub.12H.sub.2NOPS PC/HR PC/VHR C.sub.12H.sub.9O.sub.9P PC/mPC C.sub.13H.sub.25NO.sub.2 C.sub.13H.sub.25NO.sub.3 C.sub.14H.sub.30N.sub.4O.sub.2 C.sub.16H.sub.13N.sub.3O.sub.3P C.sub.17H.sub.41N.sub.4O.sub.3 C.sub.19H.sub.19N.sub.8 C.sub.22H.sub.45NO.sub.4 C.sub.26H.sub.51N.sub.4O.sub.5 C.sub.26H.sub.58N.sub.13P C.sub.27H.sub.12N.sub.9 C.sub.27H.sub.55N.sub.8O.sub.3 C.sub.30H.sub.57NO.sub.7 C.sub.30H.sub.64N.sub.15O.sub.2P C.sub.41H.sub.23N.sub.11O.sub.2 C.sub.5 C.sub.5H.sub.7NO.sub.3 C.sub.6HCl.sub.5 C.sub.6H.sub.16N.sub.3O.sub.5 C.sub.8H.sub.16NO.sub.5 3 Benign/VLR N = 21 0.78 (90%/47%) 0.86 (90%/61%) PC/LR PC/FIR PC C.sub.10H.sub.18N.sub.2O.sub.5 vs. C.sub.12H.sub.21NO.sub.4 UIR PC/HR C.sub.13H.sub.23NO.sub.6 PC/VHR PC/mPC C.sub.13H.sub.25NO.sub.3 C.sub.14H.sub.30N.sub.4O.sub.2 C.sub.15H.sub.30N.sub.10OP C.sub.16H.sub.13N.sub.3O.sub.3P C.sub.19H.sub.31N.sub.6O.sub.2 C.sub.22H.sub.45NO.sub.4 C.sub.27H.sub.12N.sub.9 C.sub.28H.sub.57N.sub.8O.sub.4 C.sub.30H.sub.61N.sub.8O.sub.5 C.sub.30H.sub.64N.sub.15O.sub.2P C.sub.35H.sub.71N.sub.8O.sub.7 C.sub.40H.sub.38N.sub.22O.sub.4 C.sub.41H.sub.23N.sub.11O.sub.2 C.sub.43H.sub.40N.sub.20O.sub.3 C.sub.5H.sub.11NO C.sub.5H.sub.11NO.sub.2S C.sub.5H.sub.2O.sub.2P C.sub.8H.sub.16NO.sub.5 4 Benign/PC with N = 24 0.82 (90%/48%) 0.86 (90%/57%) GS < 7 C.sub.11H.sub.20O.sub.2 vs. C.sub.11H.sub.5NOPS PG with GS >= 7 C.sub.12H.sub.2NOPS C.sub.12H.sub.25NO.sub.4P C.sub.12H.sub.9O.sub.9P C.sub.13H.sub.25NO.sub.2 C.sub.13H.sub.25NO.sub.3 C.sub.14H.sub.30N.sub.4O.sub.2 C.sub.16H.sub.30N.sub.3O.sub.2 C.sub.17H.sub.41N.sub.4O.sub.3 C.sub.18H.sub.34O.sub.5 C.sub.19H.sub.31N.sub.6O.sub.2 C.sub.21H.sub.36N.sub.4O.sub.3 C.sub.22H.sub.45NO.sub.4 C.sub.23H.sub.47N.sub.8O.sub.2 C.sub.24H.sub.41N.sub.14O.sub.8 C.sub.27H.sub.12N.sub.9 C.sub.30H.sub.57NO.sub.7 C.sub.30H.sub.64N.sub.15O.sub.2P C.sub.35H.sub.71N.sub.8O.sub.7 C.sub.43H.sub.40N.sub.20O.sub.3 C.sub.5H.sub.11NO C.sub.5H.sub.11NO.sub.2S C.sub.6H.sub.14N.sub.2O.sub.5P

[0064] In each of different models of comparison designed for diverse clinical scenarios, a representative panel of metabolite markers from LC/MS with HILIC column was also identified, as shown in Table 4 below. The prediction ability was evaluated by AUC analysis, with or without inclusion of PSA level in calculation. Sensitivity and specificity of the prediction is shown in bracket following AUC.

TABLE-US-00004 TABLE 4 Representative panels of metabolite markers from LC/MS with HILIC column for distinguishing prostate tumors with different malignancy potential AUC AUC (sensitivity/ (sensitivity/ Models of Metabolite markers specificity) specificity) No comparison (N = Number of markers) without PSA with PSA 1 Benign N = 25 0.86 (90%/51%) 0.88 (90%/65%) vs. C.sub.10H.sub.16O.sub.4 VLR PC/LR C.sub.10H.sub.18N.sub.2O.sub.4 PC/FIR PC/UIR C.sub.11H.sub.20NO.sub.3P.sub.3 PC/HR PC/VHR C.sub.12H.sub.7N.sub.4O.sub.2 PC/mPC C.sub.15H.sub.28N.sub.6OP.sub.2 C.sub.16H.sub.39N.sub.8OP C.sub.19H.sub.14O.sub.3 C.sub.21H.sub.33N.sub.3O.sub.3 C.sub.23H.sub.27O.sub.11S C.sub.23H.sub.42N.sub.7O C.sub.25H.sub.46N.sub.7O.sub.3 C.sub.27H.sub.48P.sub.2 C.sub.27H.sub.54O.sub.6 C.sub.33H.sub.22O.sub.7 C.sub.34H.sub.73N.sub.8O.sub.2P C.sub.38H.sub.48O.sub.12 C.sub.40H.sub.85NOP.sub.3 C.sub.5H.sub.4O.sub.3 C.sub.5H.sub.8N.sub.2O.sub.2 C.sub.6H.sub.11N.sub.4O.sub.3P C.sub.6H.sub.13N.sub.4OP.sub.2 C7H.sub.10O.sub.4 C7H.sub.17O.sub.7P.sub.2 C7H.sub.6O.sub.6S C.sub.8H.sub.14O.sub.4 2 Benign/VLR N = 27 0.80 (90%/46%) 0.86 (90%/53%) PC/LR PC C.sub.10H.sub.16O.sub.4 vs. C.sub.11H.sub.16N.sub.4O.sub.4 FIR PC/UIR C.sub.12H.sub.7N.sub.4O.sub.2 PC/HR PC/VHR C.sub.14H.sub.20N.sub.2O.sub.5 PC/mPC C.sub.16H.sub.32O.sub.2 C.sub.17H.sub.34N.sub.9O.sub.2 C.sub.21H.sub.33N.sub.3O.sub.3 C.sub.23H.sub.27O.sub.11S C.sub.26H.sub.43NO.sub.6 C.sub.27H.sub.48P.sub.2 C.sub.28H.sub.52N.sub.7O C.sub.34H.sub.73N.sub.8O.sub.2P C.sub.38H.sub.48O.sub.12 C.sub.39H.sub.26O.sub.7 C.sub.4H.sub.6O.sub.4 C.sub.40H.sub.85NOP.sub.3 C.sub.5H.sub.4N.sub.4O.sub.2 C.sub.5H.sub.8N.sub.2O.sub.2 C.sub.6H.sub.11N.sub.4O.sub.2P C.sub.6H.sub.13N.sub.4OP.sub.2 C.sub.6H.sub.6N.sub.4O.sub.2 C.sub.6H.sub.8N.sub.2O.sub.4 C7H.sub.10O.sub.4 C7H.sub.17O.sub.7P.sub.2 C7H.sub.6O.sub.6S C.sub.9H.sub.16O.sub.4 C.sub.9H.sub.9NO.sub.3 3 Benign/VLR N = 28 0.77 (90%/42%) 0.86 (90%/56%) PC/LR PC/FIR PC C.sub.10H.sub.18N.sub.2O.sub.4 vs. C.sub.10H.sub.19N.sub.5P.sub.3 UIR PC/HR C.sub.12H.sub.7N.sub.4O.sub.2 PC/VHR PC/mPC C.sub.15H.sub.28N.sub.6OP.sub.2 C.sub.16H.sub.32O.sub.2 C.sub.17H.sub.34N.sub.9O.sub.2 C.sub.19H.sub.14O.sub.3 C.sub.21H.sub.33N.sub.3O.sub.3 C.sub.21H.sub.39N.sub.4OP C.sub.27H.sub.48P.sub.2 C.sub.38H.sub.48O.sub.12 C.sub.39H.sub.26O.sub.7 C.sub.40H.sub.85NOP.sub.3 C.sub.5H.sub.10N.sub.2O.sub.3 C.sub.5H.sub.4N.sub.4O.sub.2 C.sub.6H.sub.10O.sub.4S C.sub.6H.sub.11N.sub.4O.sub.2P C.sub.6H.sub.13N.sub.4OP.sub.2 C.sub.6H.sub.15O.sub.8P C7H.sub.10O.sub.4 C7H.sub.17O.sub.7P.sub.2 C7H.sub.21N.sub.3OP.sub.3 C7H.sub.22N.sub.4O.sub.9PS C7H.sub.8O.sub.6S C.sub.8H.sub.9O.sub.6 C.sub.9H.sub.16O.sub.4 C.sub.9H.sub.17NO.sub.4S C.sub.9H.sub.9NO.sub.3 4 Benign/PC with N = 26 0.81 (90%/53%) 0.87 (90%/57%) GS < 7 C.sub.10H.sub.19N.sub.5P.sub.3 vs. C.sub.12H.sub.7N.sub.4O.sub.2 PG with GS >= 7 C.sub.15H.sub.28N.sub.6OP.sub.2 C.sub.17H.sub.34N.sub.9O.sub.2 C.sub.17H.sub.42N.sub.5OP.sub.2 C.sub.21H.sub.33N.sub.3O.sub.3 C.sub.22H.sub.38N.sub.7O C.sub.24H.sub.40N.sub.4O.sub.3 C.sub.25H.sub.46N.sub.7O.sub.3 C.sub.25H.sub.50O.sub.6 C.sub.27H.sub.54O.sub.6 C.sub.39H.sub.26O.sub.7 C.sub.39H.sub.78O.sub.6 C.sub.40H.sub.85NOP.sub.3 C.sub.6H.sub.11N.sub.4O.sub.2P C.sub.6H.sub.15O.sub.8P C.sub.6H.sub.5N.sub.2OP C.sub.6H.sub.8O.sub.6S C7H.sub.10O.sub.4 C7H.sub.17O.sub.7P.sub.2 C7H.sub.21N.sub.3OP.sub.3 C.sub.8H.sub.16N.sub.2O.sub.5P C.sub.8H.sub.18NO.sub.6P C.sub.8H.sub.4N.sub.4O.sub.3 C.sub.9H.sub.16O.sub.4 C.sub.9H.sub.9NO.sub.3

Example 3: Identification of Metabolite Markers for Assessing Prostate Cancer Risk and Grouping of Patients Using Gas Chromatography-Mass Spectrometry (GC/MS) Analysis

[0065] First, urine sample preparation started with incubating an individual urine sample with urease enzyme to deplete excess urea, as a high abundance of urea is a major chromatographic interference. 100 U of urease was added to 100 L of each human urine sample, followed by incubation at 37 C. with mild shaking at 650 rpm for 1 hour to decompose and remove excess urea. Subsequently, termination of urease activity and extraction of metabolites were carried out by admixing 1 mL of methanol with vortex for 30 seconds, and precipitated proteins were removed by centrifugation at 13,200 rpm for 15 min at 4 C. The supernatants were transferred to a 2-mL microcentrifugation tube and then dried in SpeedVac vacuum concentrators. The dried metabolic extract was derivatized by bis(trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS) and analyzed using GC/MS as explained below.

[0066] The derivatized samples were analyzed using Agilent 7890B gas chromatography coupled with 7250 quadrupole time-of-flight mass spectrometer (GC-Q-TOF/MS) equipped with electron ionization (EI). The separation was performed on Zorbax DB5-MS+10 m Duragard Capillary Column (30 m0.25 mm0.25 mm, Agilent). The GC temperature profile was held at 60 C. for 1 minute and then raised at 10 C./min to 325 C. and held at 325 C. for 10 minutes. The transfer line and the ion source temperature were set at 300 C. and 280 C., respectively. The mass range monitored was from 50 to 600 Daltons. Mass spectra were compared against the NIST 2017, Fiehn, and Wiley Registry 11th Edition mass spectral library.

[0067] A univariate logistic regression to select differentially accumulated metabolites with P values less than 0.1. The identified differential compounds were further analyzed by Receiver operating characteristic (ROC), using Medcalc software version 11.2 (Medcalc Software, Belgium). Furthermore, a K-fold cross validation and a followed reverse selection-based logistic regression were applied to select discriminator sets with improved AUC performance.

[0068] To identify urine biomarkers that distinguish prostate tumors with different malignancy potential, five different models of comparison analyzing metabolites from GC/MS were performed. The union of all these five sets of markers is listed in Table 5. For the patients with PSA less than 20 ng/ml, the union of these five sets of markers is shown in Table 6.

TABLE-US-00005 TABLE 5 The union of all sets of potential metabolite markers from GC/MS for distinguishing prostate tumors with different malignancy potential Metabolite markers CAS number MW g/mol Ethanimidic acid, N-(trimethylsilyl)-, trimethylsilyl ester 60-35-5 59.07 [700] ethanolamine [9.879] 141-43-5 61.08 Glycine, di-TMS 56-40-6 75.07 [1060] pyruvic acid [6.714] 127-17-3 88.06 [239] Beta-alanine 1 [12.044] 107-95-9 89.09 [107689] L-(+) lactic acid [6.851] 79-33-4 90.08 [8871] 2-hydroxypyridine [6.519] 142-08-05 95.1 Diethanolamine, 3TMS derivative 111-42-2 105.14 [439194] glyceric acid [10.735] 473-81-4 106.08 Pentenoic acid, 4-[(trimethylsilyl)oxy]-, trimethylsilyl ester 123-76-2 116.12 [763] guanidinoacetic acid 2 [14.751] 352-97-6 117.108 [45] tartronic acid [11.523] 80-69-3 120.06 Butanoic acid, 2,4-bis[(trimethylsilyl)oxy]-, trimethylsilyl ester 1518-62-3 120.1 [7405] L-pyroglutamic acid [13.218] 98-79-3 129.11 [791] DL-isoleucine 2 [10.225] 443-79-8 131.17 1H-Indole, 1-(trimethylsilyl)-5-[(trimethylsilyl)oxy]- 1953-54-4 133.15 2,3,4-Trihydroxybutyric acid tetrakis(trimethylsilyl) deriv., (, (R*,R*)-) 3909/12/4 136.1 1-Deoxypentitol, 4TMS derivative 13046-76-9 136.15 [135] 4-hydroxybenzoic acid [14.505] 99-96-7 138.12 [18189] 4-acetamidobutyric acid 1 [12.863] 3025-96-5 145.16 [738] L-glutamine 2 [14.083] 56-85-9 146.14 [439240] D-lyxose 2 [14.889] 1114-34-7 150.13 Arabinofuranose, 1,2,3,5-tetrakis-O-(trimethylsilyl)- 13221-22-2 150.13 [1188] xanthine [18.574] 69-89-6 152.11 Ribitol TMS 488-81-3 152.15 [6912] xylitol [15.376] 87-99-0 152.15 L-()-Arabitol, 5TMS derivative 7643-75-6 152.15 Furan, tetrahydro-2,5-dipropyl- 4457-62-9 156.26 [219984] 1,5-anhydro-D-sorbitol [16.967] 154-58-5 164.16 L-Phenylalanine, 2TMS derivative 63-91-2 165.19 3,4-Dihydroxyphenylacetic Acid, 3TMS derivative 102-32-9 168.15 [328] DL-4-hydroxymandelic acid [16.126] 1198-84-1 168.15 [64969] 3-methyl-L-histidine [16.423] 368-16-1 169.18 [444212] trans-aconitic acid [15.842] 4023-65-8 174.11 Ethyl (E)-1-penten-3-ynesulfonate 171816-65-2 174.22 [448388] D-allose 2 [17.521] 2595-97-3 180.156 [448388] D-allose 1 [17.278] 2595-97-3 180.16 [6057] L-tyrosine 2 [17.856] 60-18-4 181.19 [6508] quinic acid [17.076] 77-95-2 192.17 [445929] galacturonic acid 2 [18.105] 685-73-4 194.14 Ononitol TMS 6090-97-7 194.18 D-Gluconic acid, 6TMS derivative 526-95-4 196.16 [6613] pantothenic acid 2 [18.371] 79-83-4 219.23 [899] N-acetyl-D-mannosamine 1 [19.177] 7772-94-3 221.21 D-Allose, pentakis(trimethylsilyl) ether, ethyloxime (isomer 2) 2058302-87-5 223.22 Pseudo uridine penta-tms 1445-07-04 244.2 [985] palmitic acid [18.846] 1957/10/3 256.4 2-phenyl-3,5,7-tris(trimethylsilyloxy)-1-benzopyran-4-one 548-83-4 270.24 [5281] stearic acid [20.675] 1957/11/4 284.48 Guanosine, N,N-dimethyl-1-(trimethylsilyl)-2,3,5-tris-O-(trimethylsilyl)- 2140-67-2 311.29 1-Monopalmitin, 2TMS derivative 542-44-9 330.5 [84571] lactose 1 [24.386] 63-42-3 342.3 2-Monostearin, 2TMS derivative 621-61-4 358.56 [24699] 1-stearoyl-rac-glycerol [24.913] 123-94-4 358.6 3-Phenyl-5,10-secocholesta-1(10),2-dien-5-one * 460.74

TABLE-US-00006 TABLE 6 The union of all sets of potential metabolite markers from GC/MS for distinguishing prostate tumors (with PSA level less than 20 ng/mL) with different malignancy potential No. Name of metabolites 1 (4RS,5SR)-5-hydroperoxy-4-decanol 2 (22S,23S,25R)-3-methoxy-16,23:22,26-diepoxy-5-cholestane 3 1-Methoxymethyl-2-phenylthioindole-3-carbaldehyde 4 1-Stearoyl-rac-glycerol 5 2,3-Dihydroxybutanoic acid 6 2,4-Dihydroxybutanoic acid 7 2,5-Dipropyltetrahydrofuran 8 2-Hydroxypyridine 9 2-Stearoylglycerol 10 3,4,5-Trihydroxypentanoic acid 11 3,4-Dihydroxyphenylacetic acid 12 3-Hydroxyphenylacetic acid 13 3-Indoleacetic acid 14 3-Methyl-L-histidine 15 4-Acetamidobutyric acid 16 4-Hydroxybenzoic acid 17 4-hydroxymandelic acid 18 6-ethoxyiminohexane-1,2,3,4,5-pentol 19 Acetamide 20 alpha-Hydroxyisobutyric acid 21 Arabinofuranose 22 Beta-Alanine 23 Cyclohexylamine 24 Daidzein 25 D-Allose 26 D-Altrose 27 D-Gluconic acid 28 DL-4-Hydroxy-3-methoxymandelic acid 29 DL-isoleucine 30 D-Lyxose 31 D-tagatofuranose 32 Ethanolamine 33 Ethyl 1-penten-3-ynesulfonate 34 Galacturonic acid 35 Galangin 36 Glyceric acid 37 Guanidinoacetic acid 38 Hippuric Acid 39 Lactose 40 L-Arabinitol 41 Levulinic acid 42 L-Fucose 43 L-Lactic acid 44 L-Pyroglutamic acid 45 Monopalmitin 46 Ononitol 47 Oxamide 48 Palmitic acid 49 Pantothenic acid 50 Pseudouridine 51 p-Tolyl-beta-D-glucopyranosid-uronsaeure 52 Pyruvic acid 53 Quinic acid 54 Ribitol 55 Stearic acid 56 Sucrose 57 Tartronic acid 58 trans-Aconitic acid 59 Uric acid 60 Xanthine 61 Xylitol

Example 4: Metabolite Markers From GC/MS Analysis for Assessing Prostate Cancer Risk and Grouping of Patients

[0069] In each of different models of comparison designed for different patients, a representative panel of metabolite markers from GC/MS was identified, as shown in Table 7 below. The prediction ability was evaluated by AUC analysis, with or without inclusion of PSA level in calculation. Sensitivity and specificity of the prediction are also shown in bracket following AUC. For the patients with PSA less than 20 ng/ml, the representative panel of metabolite markers is listed in Table 8.

TABLE-US-00007 TABLE 7 Representative panels of GC/MS-derived metabolite markers for distinguishing the malignancy potential of patients with prostate tumors AUC AUC (sensitivity/ (sensitivity/ Models of Metabolite markers specificity) specificity) No comparison (N = Number of markers) without PSA with PSA 1 Benign N = 26 0.94 0.94 vs. Pyruvic acid (90%/79%) (90%/82%) VLR/LR/FIR/ 4-Acetamidobutyric acid UIR/HR/VHR 1,5-Anhydro-D-glucitol PC and mPC Beta-Alanine Glyceric acid D-Lyxose Galacturonic acid D-Allose L-Tyrosine 3-Methyl-L-histidine L-Glutamine L-Pyroglutamic acid Guanidinoacetic acid Lactose 2-Hydroxypyridine N-acetyl-D-mannosamine Palmitic acid 1-Deoxy-d-ribitol Monopalmitin 2-Stearoylglycerol Galangin 6-ethoxyiminohexane- 1,2,3,4,5-pentol D-Gluconic acid N,N-Dimethylguanosine Pseudouridine Ribitol 2 Benign and N = 24 0.85 0.90 VLR/LR PC Pyruvic acid (90%/63%) (90%/73%) vs. Xanthine FIR/UIR/HR/ 4-Acetamidobutyric acid VHR PC and 1,5-Anhydro-D-glucitol mPC Beta-Alanine 1-Stearoyl-rac-glycerol Glyceric acid Galacturonic acid Quinic acid Xylitol L-Pyroglutamic acid Guanidinoacetic acid Lactose Ononitol 5-Hydroxyindole Monopalmitin Galangin 3,4-Dihydroxyphenylacetic acid 3-Phenyl-5,10-secocholesta- 1(10),2-dien-5-one Acetamide Ethyl 1-penten-3-ynesulfonate 2,5-Dipropyltetrahydrofuran L-Phenylalanine Pseudouridine 3 Benign and N = 26 0.82 0.90 VLR/LR/FIR L-Lactic acid (90%/50%) (90%/67%) PC Xanthine vs. 4-Acetamidobutyric acid UIR/HR/VHR Beta-Alanine PC and mPC 1-Stearoyl-rac-glycerol 4-hydroxymandelic acid trans-Aconitic acid D-Allose Tartronic acid Stearic acid L-Tyrosine Quinic acid Ethanolamine Guanidinoacetic acid DL-isoleucine Palmitic acid Monopalmitin Arabinofuranose 2,4-Dihydroxybutanoic acid Diethanolamine Acetamide 2,5-Dipropyltetrahydrofuran Glycine L-Arabinitol Levulinic acid Pseudouridine 4 Benign + N = 22 0.80 0.85 GS < 7 PC Pyruvic acid (90%/46%) (90%/55%) vs. Xanthine GS 7 PC 4-Hydroxybenzoic acid Beta-Alanine 1-Stearoyl-rac-glycerol Galacturonic acid D-Allose Tartronic acid Quinic acid Pantothenic acid Xylitol Guanidinoacetic acid 1-Deoxy-d-ribitol Monopalmitin Threonic acid Galangin Arabinofuranose 2,4-Dihydroxybutanoic acid D-Gluconic acid 2,5-Dipropyltetrahydrofuran Levulinic acid Pseudouridine

TABLE-US-00008 TABLE 8 Representative panels of GC/MS metabolite markers for distinguishing the malignancy potential of patients with prostate tumors (PSA level less than 20 ng/mL) AUC AUC (sensitivity/ (sensitivity/ Models of Metabolite markers specificity) specificity) No comparison (N = Number of markers) without PSA with PSA 1 Benign N = 28 0.93 0.95 vs. 1-Methoxymethyl-2- (90%/73%) (90%/82%) VLR/LR/FIR/ phenylthioindole-3- UIR/HR/VHR carbaldehyde PC and mPC 2,3-Dihydroxybutanoic acid 2-Hydroxypyridine 2-Stearoylglycerol 3-Hydroxyphenylacetic acid 3-Indoleacetic acid 3-Methyl-L-histidine 4-Acetamidobutyric acid 4-hydroxymandelic acid 6-ethoxyiminohexane- 1,2,3,4,5-pentol alpha-Hydroxyisobutyric acid D-Altrose D-Gluconic acid D-Lyxose Galacturonic acid Galangin Glyceric acid Lactose L-Fucose L-Pyroglutamic acid Monopalmitin Ononitol Oxamide Palmitic acid Pseudouridine Pyruvic acid Ribitol trans-Aconitic acid 2 Benign and N = 22 0.87 0.90 VLR/LR PC 1-Stearoyl-rac-glycerol (90%/62%) (90%/72%) vs. 2,5-Dipropyltetrahydrofuran FIR/UIR/HR/ 3,4-Dihydroxyphenylacetic VHR PC and acid mPC Acetamide Beta-Alanine Cyclohexylamine Ethyl 1-penten-3-ynesulfonate Galacturonic acid Galangin Glyceric acid Guanidinoacetic acid Levulinic acid Monopalmitin Ononitol Palmitic acid p-Tolyl-beta-D- glucopyranosid-uronsaeure Quinic acid Stearic acid Sucrose Uric acid Xanthine Xylitol 3 Benign and N = 27 0.83 0.90 VLR/LR/FIR (22S,23S,25R)-3-methoxy- (90%/55%) (90%/70%) PC 16,23:22,26-diepoxy-5- vs. cholestane UIR/HR/VHR 1-Methoxymethyl-2- PC and mPC phenylthioindole-3- carbaldehyde 1-Stearoyl-rac-glycerol 2,4-Dihydroxybutanoic acid 2,5-Dipropyltetrahydrofuran 4-hydroxymandelic acid Acetamide Arabinofuranose Beta-Alanine Daidzein D-Allose DL-isoleucine D-tagatofuranose Ethanolamine Galangin Guanidinoacetic acid L-Arabinitol Levulinic acid L-Lactic acid Monopalmitin Palmitic acid Pseudouridine Quinic acid Stearic acid Sucrose Tartronic acid Xanthine 4 Benign + N = 25 0.78 0.82 GS < 7 PC (4RS,5SR)-5-hydroperoxy-4- (90%/38%) (90%/45%) vs. decanol GS 7 PC 2,5-Dipropyltetrahydrofuran 3,4,5-Trihydroxypentanoic acid 4-Hydroxybenzoic acid 6-ethoxyiminohexane- 1,2,3,4,5-pentol Acetamide Arabinofuranose Beta-Alanine D-Allose DL-4-Hydroxy-3- methoxymandelic acid Ethyl 1-penten-3-ynesulfonate Galacturonic acid Galangin Glyceric acid Guanidinoacetic acid Hippuric Acid Levulinic acid L-Pyroglutamic acid Pantothenic acid Pseudouridine Pyruvic acid Quinic acid Tartronic acid Uric acid Xanthine