Detection and treatment methods for prostate cancer

Abstract

Methods and associated kits are provided for detecting early stage prostate cancer and determining the risk of being at risk for progression of prostate cancer.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 049,521, filed 8 July 2020, and U.S. Provisional Patent Application No. 63 / 067,601, filed 19 August 2020, the entire contents of which are incorporated herein by reference.

[0002] This patent or application file includes at least one drawing made in color. A copy of this patent or patent application publication accompanied by the color drawing will be provided by the Office upon request and payment of the necessary fees.

[0003] Statement on federally funded research This invention was made with government support under grant number CA223527, issued by the National Institutes of Health. The government has certain rights to this invention. [Background technology]

[0004] Elevated serum Cav-1 levels are associated with high-risk prostate cancer, castration resistance, and biochemical recurrence after prostatectomy. Increased plasma Cav-1 has previously been demonstrated to be associated with early disease reclassification in prostate cancer patients who first present with clinically localized disease. Cav-1 is the eponymous protein component of caveolae (bulb-shaped 50-100 nm plasma membrane invaginations, rich in sphingoglycolipids and cholesterol). Cav-1 also functions in organizing membrane microdomain compositions and regulating transmembrane signaling. Growing evidence suggests that Cav-1 functions as an essential lipid chaperone, promoting cellular lipid transport and homeostasis, endocytosis and exocytosis, and mechanoprotection of the cell membrane. Cav-1 is known to transport molecules including insulin, chemokines, albumin, and low-density and high-density lipoproteins (LDL and HDL). Recently, it has been discovered that Cav-1, which contains extracellular vesicles in white adipose tissue, transports proteins and lipids between endothelial cells and adipocytes in response to the metabolic state of the system.

[0005] In cancer, the role of Cav-1 is dynamic and context-dependent. Cav-1 has been shown to modulate and promote the activity of receptor tyrosine kinases, G protein-coupled receptors, integrins, and cadherins. Cav-1 expression is closely associated with invasive phenotypes in various tumor types and is linked to epithelial-mesenchymal plasticity, tumor invasion and metastasis potential, and radioresistance and multidrug resistance.

[0006] While Cav-1 is associated with metabolic alterations in prostate cancer, the mechanisms by which Cav-1 influences metabolic rewiring have not been previously understood. Investigations into Cav-1 function in the context of prostate tumor metabolism revealed an integrated metabolic program of enhanced lipid scavenging and differential ceramide metabolism in prostate tumors exhibiting Grease-grade progression, following initial registration for active surveillance. Importantly, this metabolic phenotype provides a biomarker for disease progression and identifies points of treatment sensitivity. Key features of the metabolic program supporting this tumor include Cav-1-mediated lipid uptake, increased tumor catabolism of extracellular sphingomyelin (SM), altered ceramide metabolism linked to increased sphingoglycolipid synthesis, and the efflux of circulating Cav-1-sphingolipid particles, which cross over with mitochondrial repair. Based on these mechanistic findings, potential actionable metabolic vulnerabilities are being tested by targeting Cav-1-mediated lipid scavenging and metabolism in a mouse model of invasive prostate cancer.

[0007] Metabolmic profiling of baseline plasma from a longitudinal prospective cohort of participants in active surveillance (AS) for prostate cancer identified changes in plasma sphingolipids as a prominent feature of patients with advanced AS. Combining the properties of these metabolites may yield a predictive signature for disease progression in early prostate cancer. Previous studies have shown that baseline plasma caveolin-1 (Cav-1) is an independent predictor of disease classification in a similar AS cohort. This study presupposed a well-established role of tissue-localized and secreted Cav-1 in invasive and potentially drug-resistant prostate cancer. Plasma Cav-1 can further be integrated with plasma sphingolipid properties to form a combined predictive signature. Mechanistic studies have been conducted to elucidate the key biological processes involved in tumor-supporting tumor metabolism underlying the observed plasma signature properties. Using a syngeneic RM-9 mouse model of prostate cancer and established human prostate cancer cell lines, we have found that Cav-1 promotes the rewiring of cancer cell lipid metabolism toward programs of exogenous lipid cleavage and vesicle biosynthesis that cross with sphingolipid metabolism; activation of this program is demonstrated in plasma signatures; and we have evidence that this program exhibits metabolic vulnerability that makes it targetable as an antitumor therapy. [Overview of the project] [Means for solving the problem]

[0008] A method and kit for evaluating the status of prostate cancer are provided. The method and kit utilize multiple assays of biomarkers contained in biological samples obtained from subjects. Analysis of one or more biomarkers, including CAV-1, SM(40:2), SM(44:2), lactosylceramide (32:0) ("LacCer32:0"), lactosylceramide (36:0) ("LacCer36:0"), trihexosylceramide (34:1) ("TriHexCer"), and hexosylceramide (40:0) ("HexCer40:0"), provides highly accurate risk assessment and prognosis regarding the progression of prostate cancer.

[0009] Regression models were identified that could predict the risk of prostate cancer progression in subjects based on one or more levels of the biomarkers CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 found in biological samples from the subjects.

[0010] Accordingly, provided herein is a method for determining and / or quantifying the risk of pancreatic cancer progression in a subject, comprising measuring the levels of one or more of the biomarkers CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a sample from the subject.

[0011] Methods for treating or preventing the progression of prostate cancer in subjects classified as being at risk of progression based on the levels of one or more biomarkers: CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0.

[0012] A corresponding kit is also provided for determining the presence of indicators of prostate cancer progression in a sample from a subject, for determining the risk of prostate cancer progression in a subject, and for determining and / or quantifying the risk of prostate cancer progression in a subject, comprising materials for measuring one or more of the biomarkers CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a sample.

[0013] In some embodiments, the biomarker is measured in a blood sample taken from the subject. In some embodiments, the presence or amount of the biomarker in the biological sample can be determined. In some embodiments, the level of the biomarker in the biological sample can be quantified.

[0014] In some embodiments, a surface is provided for analyzing a biological sample. In some embodiments, the biomarker of interest is adsorbed nonspecifically to this surface. In some embodiments, a receptor specific to the biomarker of interest is incorporated into this surface. In some embodiments, the surface is bound to particles, such as beads.

[0015] In some embodiments, a biomarker can bind to a specific receptor molecule, allowing for the determination of the presence or quantity of the biomarker receptor complex. In some embodiments, the quantity of the biomarker receptor complex can be quantified. In some embodiments, the receptor molecule can be linked to an enzyme to facilitate detection and quantification.

[0016] In some embodiments, a biomarker binds to a specific relay molecule, and the biomarker-relay molecule complex binds to a receptor molecule. In some embodiments, the presence or amount of the biomarker-relay receptor complex can be determined. In some embodiments, the amount of the biomarker-relay receptor complex can be quantified. In some embodiments, the receptor molecule is linked to an enzyme to facilitate detection and quantification.

[0017] In some embodiments, the biological sample is analyzed sequentially for individual biomarkers. In some embodiments, the biological sample is divided into separate parts to allow for simultaneous analysis of multiple biomarkers. In some embodiments, the biological sample is analyzed for multiple biomarkers in a single process.

[0018] In some embodiments, the presence or absence of a biomarker may be determined by visual inspection. In some embodiments, the amount of a biomarker may be determined by the use of spectroscopic techniques. In some embodiments, the spectroscopic technique is mass spectrometry. In some embodiments, the spectroscopic technique is UV / Vis spectroscopy. In some embodiments, the spectroscopic technique is excitation / emission techniques such as fluorescence spectroscopy. In some embodiments, the spectroscopic technique is mass spectrometry. In some embodiments, the spectroscopic technique is combined with chromatographic techniques. In some embodiments, the chromatographic technique is liquid chromatography. In some embodiments, the chromatographic technique is high-performance liquid chromatography ("HPLC"). In some embodiments, the chromatographic technique is gas chromatography ("GC").

[0019] In some embodiments, the analysis of biomarkers CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 may be combined with the analysis of additional biomarkers. In some embodiments, the additional biomarkers may be protein biomarkers. In some embodiments, the additional biomarkers may be non-protein biomarkers.

[0020] In some embodiments, kits are provided for the analysis of biological samples. In some embodiments, the kit may contain the chemicals and reagents necessary to perform the analysis. In some embodiments, the kit includes means for handling the biological sample to minimize the necessary operator intervention. In some embodiments, the kit may digitally record the results of the analysis. In some embodiments, the kit may perform any necessary mathematical processing of the data generated by the analysis.

[0021] In another aspect, the present disclosure provides a method for determining the risk of prostate cancer progression in a subject using a biomarker panel and a protein biomarker panel, wherein the biomarker panel comprises one or more of the biomarkers CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0; wherein the method comprises performing the step of measuring the levels of the biomarkers and protein biomarkers in a biological sample obtained from the subject; wherein the amounts of the biomarkers and protein biomarkers determine the risk of prostate cancer progression in the subject.

[0022] In another aspect, the present disclosure provides a kit for the methods described herein, comprising a first reagent solution comprising a first solute for the detection of CAV-1; a second reagent solution comprising a second solute for the detection of SM(40:2), a third reagent solution comprising a third solute for the detection of SM(44:2), a fourth reagent solution comprising a fourth solute for the detection of LacCer32:0, a fifth reagent solution comprising a fifth solute for the detection of LacCer36:0, a sixth reagent solution comprising a sixth solute for the detection of TriHexCer34:1, and a seventh reagent solution comprising a seventh solute for the detection of HexCer40:0.

[0023] In one embodiment, such a kit comprises a device for contacting the reagent solution with a biological sample. In another embodiment, such a kit comprises at least one surface having means for binding at least one biomarker. In another embodiment, the at least one biomarker is selected from the group consisting of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0.

[0024] In another embodiment, the disclosure provides a method for treating a subject suspected to be at risk of progression of prostate cancer, comprising the steps of: analyzing the subject for risk of progression of prostate cancer using the method described herein; and administering a therapeutic dose effective for treating prostate cancer. In one embodiment, the therapeutic dose is surgery, chemotherapy, radiotherapy, targeted therapy, or a combination thereof. In another embodiment, such a method comprises at least one receptor molecule that selectively binds to one or more biomarkers selected from the group consisting of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0. In another embodiment, the detection of the amounts of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 comprises the use of solid particles. In another embodiment, the solid particles are beads. In another embodiment, at least one reporter molecule is linked to an enzyme. In another embodiment, at least one protein or metabolite marker generates a detectable signal. In another embodiment, the detectable signal is detectable by spectroscopy. In another embodiment, the spectroscopy is mass spectrometry. In another embodiment, such a method includes including patient history information in assigning whether or not a patient is at risk of progression of prostate cancer. In another embodiment, such a method includes administering at least one alternative diagnostic test to patients assigned as being at risk of progression of prostate cancer.

[0025] In another embodiment, the disclosure provides a method for treating or preventing the progression of prostate cancer in a subject, comprising one or more steps of: administering a chemotherapeutic agent to a subject having prostate cancer; administering therapeutic radiation to a subject having prostate cancer; and performing surgery for partial or complete surgical removal of cancerous tissue in a patient having prostate cancer, wherein levels of one or more biomarkers CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 identify the risk of prostate cancer progression in the subject. In one embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are elevated. In another embodiment, levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are elevated compared to levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a reference group or group that is not at risk of prostate cancer progression. In another embodiment, the reference group or group is healthy. In another embodiment, the reference group or group has inactive prostate cancer. In another embodiment, levels of TriHexCer34:1 and SM40:2 are elevated in the subjects compared to healthy subjects. In another embodiment, levels of TriHexCer34:1 and SM40:2 are elevated compared to levels in a reference group or group that does not have invasive prostate cancer. In another embodiment, the levels of TriHexCer34:1 and SM40:2 are elevated compared to the levels in a reference subject or group with inactive prostate cancer.

[0026] In another embodiment, the Disclosure provides a method for treating or preventing the progression of prostate cancer in a subject, comprising the steps of: administering a chemotherapeutic agent to a subject having prostate cancer; administering therapeutic radiation to a subject having prostate cancer; and performing one or more surgeries for partial or complete surgical removal of cancerous tissue in a patient having prostate cancer, wherein levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 identify the subject as having or being at risk of progression of prostate cancer. In one embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are elevated. In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are elevated compared to the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a reference subject or group that is not at risk of prostate cancer progression. In another embodiment, the reference subject or group is healthy. In another embodiment, the reference subject or group has inactive prostate cancer. In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are elevated compared to the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a reference subject or group with adenocarcinoma.In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are elevated compared to the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a reference subject or group having squamous cell carcinoma. In another embodiment, the subject is at high risk of progression of prostate cancer.

[0027] In another aspect, the disclosure provides a method of treating a subject suspected of having a risk of progression of prostate cancer, comprising analyzing the subject for a risk of progression of prostate cancer using the methods disclosed herein; and administering a therapeutically effective amount of a therapy for treating prostate cancer. In one embodiment, the therapy is surgery, chemotherapy, radiation therapy, targeted therapy, or a combination thereof.

[0028] (a) Using an in vitro assay, one or more of the biomarkers caveolin-1 (CAV-1), sphingomyelin 40:2 (SM40:2), sphingomyelin 44:2 (SM44:2), lactosylceramide 32:0 (LacCer32:0), lactosylceramide 36:0 (LacCer36:0), trihexosylceramide 34:1 (TriHexCer34:1), and hexosylceramide 40:0 (HexCer40:0) are detected in the biological sample from the subject. A method for classifying a subject with prostate cancer as either at risk of developing invasive prostate cancer or not, predicting the subject's predisposition to invasive prostate cancer, comprising the steps of (b) measuring the level of a number and (b) comparing one or more levels of the biomarkers CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in the sample to a reference, and predicting the subject's predisposition to invasive prostate cancer. Methods are also provided for diagnosing invasive prostate cancer in subjects with prostate cancer, determining the risk of a subject developing invasive prostate cancer, predicting the likelihood of prostate cancer progression in subjects with prostate cancer, providing prognosis for subjects with prostate cancer, or selecting subjects with prostate cancer for treatment with anticancer therapy, wherein the changed amounts of one or more of the biomarkers CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 compared to the aforementioned reference provide indices selected from a group consisting of indices indicating whether a subject is at risk of developing invasive prostate cancer or not, indices indicating the subject's predisposition to invasive prostate cancer, indices indicating the likelihood of prostate cancer progression in a subject, indices indicating the subject's progression-free survival, indices indicating the probable outcome of treatment for prostate cancer, and indices indicating that a subject is a candidate for treatment with anticancer therapy.

[0029] A method comprising (a) measuring the levels of sphingomyelin 40:2 (SM40:2), sphingomyelin 44:2 (SM44:2), lactosylceramide 32:0 (LacCer32:0), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 (TriHexCer34:1) in a biological sample from the subject using an in vitro assay, and (b) comparing the levels of SM40:2, SM44:2, LacCer32:0, LacCer36:0, and TriHexCer34:1 in the sample with a reference, for classifying a subject with prostate cancer as either at risk of developing invasive prostate cancer or not, predicting the subject's predisposition to invasive prostate cancer, and diagnosing invasive prostate cancer in a subject with prostate cancer. The invention also provides a method for determining the risk of a subject having invasive prostate cancer, predicting the likelihood of prostate cancer progression in subjects with prostate cancer, providing a prognosis for subjects with prostate cancer, or selecting subjects with prostate cancer for treatment with anticancer therapy, wherein changes in the amounts of SM40:2, SM44:2, LacCer32:0, LacCer36:0, and TriHexCer34:1 relative to the aforementioned references provide an index selected from a group consisting of an index indicating whether a subject is at risk of developing invasive prostate cancer or not, an index indicating the subject's predisposition to invasive prostate cancer, an index indicating the likelihood of prostate cancer progression in the subject, an index indicating the subject's progression-free survival, an index indicating the probable outcome of treatment for prostate cancer, and an index indicating whether the subject is a candidate for treatment with anticancer therapy.

[0030] A method comprising the steps of (a) measuring the levels of sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 (TriHexCer34:1) in a biological sample from the subject using an in vitro assay, and (b) comparing the levels of SM40:2, LacCer36:0, and TriHexCer34:1 in the sample with a reference, classifies a subject with prostate cancer as either at risk of developing invasive prostate cancer or not, predicts the subject's predisposition to invasive prostate cancer, diagnoses invasive prostate cancer in a subject with prostate cancer, and determines the risk of a subject having invasive prostate cancer. Methods are also provided for determining, predicting the likelihood of prostate cancer progression in subjects with prostate cancer, providing prognosis for subjects with prostate cancer, or selecting subjects with prostate cancer for treatment with anticancer therapy, wherein changes in the amounts of SM40:2, LacCer36:0, and TriHexCer34:1 relative to the aforementioned reference provide an index selected from a group consisting of an index indicating whether a subject is at risk of developing invasive prostate cancer or not, an index indicating the subject's predisposition to invasive prostate cancer, an index indicating the likelihood of prostate cancer progression in the subject, an index indicating the subject's progression-free survival, an index indicating the probable outcome of treatment for prostate cancer, and an index indicating that the subject is a candidate for treatment with anticancer therapy.

[0031] A method comprising the steps of (a) measuring the level of trihexosylceramide 34:1 (TriHexCer34:1) in a biological sample from the subject using an in vitro assay, and (b) comparing the level of TriHexCer34:1 in the sample to a reference, classifies a subject with prostate cancer as either at risk of developing invasive prostate cancer or not, predicts the subject's predisposition to invasive prostate cancer, diagnoses invasive prostate cancer in a subject with prostate cancer, determines the subject's risk of developing invasive prostate cancer, and Methods are also provided for predicting the likelihood of progression, providing prognosis for subjects with prostate cancer, or selecting subjects with prostate cancer for treatment with anticancer therapy, wherein a change in the amount of TriHexCer34:1 relative to the aforementioned reference provides an index selected from a group consisting of an index indicating whether a subject is at risk of developing invasive prostate cancer or not, an index indicating the subject's predisposition to invasive prostate cancer, an index indicating the likelihood of prostate cancer progression in the subject, an index indicating the subject's progression-free survival, an index indicating the probable outcome of treatment for prostate cancer, and an index indicating that the subject is a candidate for treatment with anticancer therapy.

[0032] A method comprising the steps of (a) measuring the level of sphingomyelin 40:2 (SM40:2) in a biological sample from the subject using an in vitro assay, and (b) comparing the level of SM40:2 in the sample to a reference, classifies a subject with prostate cancer as either at risk of developing invasive prostate cancer or not, predicts the subject's predisposition to invasive prostate cancer, diagnoses invasive prostate cancer in a subject with prostate cancer, determines the subject's risk of developing invasive prostate cancer, and assesses the possibility of prostate cancer progression in a subject with prostate cancer. Methods are also provided for predicting sex, providing prognosis for subjects with prostate cancer, or selecting subjects with prostate cancer for treatment with anticancer therapy, wherein a change in the amount of SM40:2 relative to the aforementioned reference provides an index selected from a group consisting of an index indicating whether a subject is at risk of developing invasive prostate cancer or not, an index indicating the subject's predisposition to invasive prostate cancer, an index indicating the likelihood of prostate cancer progression in the subject, an index indicating the subject's progression-free survival, an index indicating the probable outcome of treatment for prostate cancer, and an index indicating that the subject is a candidate for treatment with anticancer therapy.

[0033] A method comprising the steps of (a) measuring the levels of sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1; and / or trihexosylceramide 34:1; and / or sphingomyelin 40:2 (SM40:2) in a biological sample from the subject using an in vitro assay, and (b) comparing the level of SM40:2 in the sample with a reference, to classify a subject with prostate cancer as being at risk of developing invasive prostate cancer or not, predict the predisposition to invasive prostate cancer in the subject, diagnose invasive prostate cancer in a subject with prostate cancer, and if the subject is invasive Methods are also provided for determining the risk of having invasive prostate cancer, predicting the likelihood of prostate cancer progression in subjects with prostate cancer, providing prognosis for subjects with prostate cancer, or selecting subjects with prostate cancer for treatment with anticancer therapy, wherein a change in the amount of SM40:2 relative to the aforementioned reference provides an index selected from a group consisting of an index indicating whether a subject is at risk of developing invasive prostate cancer or not, an index indicating the subject's predisposition to invasive prostate cancer, an index indicating the likelihood of prostate cancer progression in the subject, an index indicating the subject's progression-free survival, an index indicating the probable outcome of treatment for prostate cancer, and an index indicating that the subject is a candidate for treatment with anticancer therapy.

[0034] Also provided is a method for treating or preventing the progression of prostate cancer in a subject having prostate cancer, comprising one or more steps: administering an anticancer drug to the subject having prostate cancer; administering therapeutic radiation to the subject having prostate cancer; and performing surgery for partial or complete surgical removal of cancerous tissue in the patient having prostate cancer, wherein the levels of sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 are elevated compared to a reference without prostate cancer.

[0035] Also provided is a method for treating or preventing the progression of prostate cancer in a subject having prostate cancer, comprising one or more steps: administering an anticancer drug to the subject having prostate cancer; administering therapeutic radiation to the subject having prostate cancer; and performing surgery for partial or complete surgical removal of cancerous tissue in the patient having prostate cancer, wherein the level of trihexosylceramide 34:1 is elevated compared to a reference without prostate cancer.

[0036] Also provided is a method for treating or preventing the progression of prostate cancer in a subject having prostate cancer, comprising one or more steps: administering an anticancer drug to the subject having prostate cancer; administering therapeutic radiation to the subject having prostate cancer; and performing surgery for partial or complete surgical removal of cancerous tissue in the patient having prostate cancer, wherein the level of sphingomyelin 40:2 (SM40:2) is elevated compared to a reference without prostate cancer.

[0037] Also provided is a method for treating or preventing the progression of prostate cancer in a subject having prostate cancer, comprising the steps of: administering an anticancer drug to a subject having prostate cancer; administering therapeutic radiation to a subject having prostate cancer; and performing one or more surgeries for partial or complete surgical removal of cancerous tissue in a patient having prostate cancer, wherein the levels of (a) sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1; and / or (b) trihexosylceramide 34:1; and / or (c) sphingomyelin 40:2 (SM40:2) are elevated compared to a reference without prostate cancer.

[0038] A diagnostic panel for invasive prostate cancer is also provided, comprising caveolin-1 (CAV-1), sphingomyelin 40:2 (SM40:2), sphingomyelin 44:2 (SM44:2), lactosylceramide 32:0 (LacCer32:0), lactosylceramide 36:0 (LacCer36:0), trihexosylceramide 34:1 (TriHexCer34:1), and hexosylceramide 40:0 (HexCer40:0).

[0039] A diagnostic panel for invasive prostate cancer is also provided, comprising sphingomyelin 40:2 (SM40:2), sphingomyelin 44:2 (SM44:2), lactosylceramide 32:0 (LacCer32:0), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 (TriHexCer34:1).

[0040] A diagnostic panel for invasive prostate cancer is also provided, comprising sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 (TriHexCer34:1).

[0041] A diagnostic panel for invasive prostate cancer comprising trihexosylceramide 34:1 (TriHexCer34:1) is also provided.

[0042] A diagnostic panel for invasive prostate cancer comprising sphingomyelin 40:2 (SM40:2) is also provided.

[0043] A diagnostic panel for invasive prostate cancer is also provided, comprising sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1; and / or trihexosylceramide 34:1; and / or sphingomyelin 40:2 (SM40:2).

[0044] Also provided is a method for treating or preventing the progression of prostate cancer in a subject having prostate cancer, comprising one or more steps: administering an anticancer drug to the subject having prostate cancer; administering therapeutic radiation to the subject having prostate cancer; and performing surgery for partial or complete surgical removal of cancerous tissue in the patient having prostate cancer, wherein the levels of caveolin-1 (CAV-1), sphingomyelin 40:2 (SM40:2), sphingomyelin 44:2 (SM44:2), lactosylceramide 32:0 (LacCer32:0), lactosylceramide 36:0 (LacCer36:0), trihexosylceramide 34:1 and hexosylceramide 40:0 are elevated compared to a reference without prostate cancer.

[0045] A method is also provided for treating or preventing the progression of prostate cancer in a subject having prostate cancer, comprising one or more steps: administering an anticancer drug to the subject having prostate cancer; administering therapeutic radiation to the subject having prostate cancer; and performing surgery for partial or complete surgical removal of cancerous tissue in the patient having prostate cancer, wherein the levels of sphingomyelin 40:2 (SM40:2), sphingomyelin 44:2 (SM44:2), lactosylceramide 32:0 (LacCer32:0), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 are elevated compared to a reference without prostate cancer.

[0046] Also provided is a method for treating or preventing the progression of prostate cancer in a subject having prostate cancer, comprising one or more steps: administering an anticancer drug to the subject having prostate cancer; administering therapeutic radiation to the subject having prostate cancer; and performing surgery for partial or complete surgical removal of cancerous tissue in the patient having prostate cancer, wherein the levels of sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 are elevated compared to a reference without prostate cancer.

[0047] Also provided is a method for treating or preventing the progression of prostate cancer in a subject having prostate cancer, comprising one or more steps: administering an anticancer drug to the subject having prostate cancer; administering therapeutic radiation to the subject having prostate cancer; and performing surgery for partial or complete surgical removal of cancerous tissue in the patient having prostate cancer, wherein the level of trihexosylceramide 34:1 is elevated compared to a reference without prostate cancer.

[0048] Also provided is a method for treating or preventing the progression of prostate cancer in a subject having prostate cancer, comprising one or more steps: administering an anticancer drug to the subject having prostate cancer; administering therapeutic radiation to the subject having prostate cancer; and performing surgery for partial or complete surgical removal of cancerous tissue in the patient having prostate cancer, wherein the level of sphingomyelin 40:2 (SM40:2) is elevated compared to a reference without prostate cancer.

[0049] Also provided is a method for treating or preventing the progression of prostate cancer in a subject having prostate cancer, comprising the steps of: administering an anticancer drug to a subject having prostate cancer; administering therapeutic radiation to a subject having prostate cancer; and performing one or more surgeries for partial or complete surgical removal of cancerous tissue in a patient having prostate cancer, wherein the levels of sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1; and / or trihexosylceramide 34:1; and / or sphingomyelin 40:2 (SM40:2) are elevated compared to a reference without prostate cancer. [Brief explanation of the drawing]

[0050] [Figure 1](a) Individual ROC AUCs (light gray = baseline; dark gray = 12 months) of plasmosphingomyelin and sphingoglycolipids in early-stage prostate cancer patients experiencing disease progression are depicted. (i)SM(32:1)(ii)SM(32:2)(ii)SM(34:1)(iv)SM(34:2)(v)SM(36:1)(vi)SM(36:2)(vii )SM(40:2)(viii)SM(42:1)(ix)SM(42:3)(x)GlucosylCer(39:2)(xi)LactosylCer(32: 0)(xii)LactosylCer(32:1)(xiii)LactosylCer(34:1)(xiv)TrihexosylCer(d18:1 / 16:0)(xv)DihexosylCer(34:1)(xvi)DihexosylCer(36:1)(xvii)TrihexosylCer(34:1). (b) Volcano plot showing hazard ratios (horizontal axis) of individual plasma lipid species stratified by lipid domain when predicting disease progression using baseline plasma samples from a larger prospective cohort (n=459); vertical axis = -log(p value). (c) Kaplan-Meier survival curves showing progression-free survival (vertical axis) over time (months, horizontal axis) for participants with plasma sphingolipid signatures (plasma Cav-1 and six sphingolipids); plasma sphingolipid signature levels ≤4.33 or ≥4.33. [Figure 2] This describes intra-patient comparisons of sphingolipids identified in the discovery cohort. (i) Invasive baseline; (ii) Invasive 12M; (a) Glucosylceramide (38:2) (b) Lactosylceramide (32:0) (c) Lactosylceramide (32:1) (d) Lactosylceramide (34:1) (e) NeuAc?2-3Gal?1-4Glc?-Cer(d18:1 / 16:0) (f) NeuAc?2-3Gal ?-Cer(34:1)(g)NeuAc?2-3Gal?-Cer(36:1)(h)TriHexCer34:1(i)SM(32:1)(j)SM(32:2)( k)SM(34:1)(l)SM(34:2)(m)SM(36:1)(n)SM(36:2)(o)SM(40:2)(p)SM(42:1)(q)SM(40:3). [Figure 3](a) Depict immunoblot of Cav-1 in PC-3M cells following 72 hours of treatment with SFM or lipid-containing SFM. SSALP of defined lipid composition was generated and spiked into the medium. sLDL: synthetic "LDL-like" particles; sHDL: synthetic "HDL-like" particles; PC-phosphatidylcholine; TO-trioleate; CE: cholesteryl oleate; FC: free cholesterol. (i)SFM(vehicle)(ii)sHDL(PC / TOlow)(iii)sHDL(PC / CE / TOhigh)(iv)sHDL(PC / FC / CE / TOhigh)(v)sHDL(PC / TOhigh)(vi)sHDL(PC / SM / CE / TOhigh)(vii)sHDL(PC / SM / FC / CE / TOhigh) )(viii)sHDL(PC / CE / TOhigh)(ix)sHDL(PC / SM / TOlow)(x)sHDL(PC / SM / FC / TOhigh)(xi)SFM(control)(xii)sHDL(PC / SM / FC / CE / TOlow)(xiii)sHDL(PC / SM / CE / TOlow)(xiv)SFM(vehicle). (b) Relative lipid composition of SSALP (i) LDL (PC / TOhigh) (ii) LDL (PC / CE / TOhigh) (iii) LDL (PC / SM / TOhigh) (iv) LDL (PC / SM / CE / TOhigh) (v) LDL (PC / SM / FC / TOhigh) (vi) LDL (PC / SM / FC / CE / TOhigh) (vii) HDL (PC / SM / TOlow) (viii) HDL (PC / SM / CE / TOlow) (ix) HDL (PC / SM / FC / TOlow) (x) HDL (PC / SM / FC / TOlow) (c) Representative immunoblots of Cav-1 in LNCaP (left) and PC-3M (right) prostate cancer cells following Cav-1 overexpression or knockdown, respectively. (d) Baseline evaluation of Dil-SSALP uptake by LNCaP, PC-3M, and RM-9 prostate cancer cells pretreated with Dil-SSALP. [Figure 4](a) Depicts the multiplicative changes in the lipid domain (vertical axis relative to the median of cell line-specific control) following overexpression or transient knockdown of CAV-1 in LNCaP and PC-3M, respectively. (i) Acylcarnitine (ii) Cardiolipin (iii) Ceramide (iv) Cholesterol ester (v) Diacylglycerol (vi) Sphingoglycolipid (vii) Lysophospholipid (viii) Phospholipid (ix) Sphingomyelin (x) Triacylglycerol. For the lipid domain, the aggregation strength of the individual annotated lipid species corresponding to each lipid domain was used. Statistical significance was determined by a two-sided Student's t-test. (b) Relative abundance of lactosylceramide (area unit ± StDev) following overexpression of CAV-1 in LNCaP or transient knockdown of CAV-1 in PC-3M. (i) lactosylceramide (30:1) (ii) lactosylceramide (18:1 / 20:4) (iii) lactosylceramide (18:1 / 16:0). Statistical significance was determined by a two-tailed Student's t-test. Lipid abundances were normalized based on total cell number. [Figure 5] This diagram illustrates the biochemical networks illustrating the gene expression of a core enzyme in ceramide metabolism in prostate cancer cell lines (a) and prostate adenocarcinoma (b), stratified by high or low CAV1 expression. For CCLE data (a), prostate cancer cell lines were stratified by mean CAV1 mRNA expression to either high (log2 mRNA range: 11.01–13.61) or low (log2 mRNA range: 4.16–6.88) CAV1 expression. For TCGA data (b), prostate adenocarcinoma was stratified to the highest or lowest CAV1 expression quartile. Node size reflects the magnitude of change. Edges and arrows indicate the direction of biochemical reactions. Thick black node boundaries indicate statistically significant differences. [Figure 6] This provides an overview of the ceramide biosynthesis pathway. [Figure 7](a) Schematic diagram illustrating the potential biochemical fate of sphingomyelin (18:1 / 18:1)-d9; (b) depiction of (i) sphingomyelin (18:1 / 18:1), (ii) ceramide (18:1 / 18:1), (iii) glucosylceramide (18:1 / 18:1), and their deuterated (d9) isotope substitutions (iv, v, and vi, respectively) in LNCaP, PC-3M, and RM-9 following 48 hours of treatment with sphingomyelin (18:1 / 18:1)-d9-rich SSALP. The values ​​presented above the bar graphs indicate the ratio of ceramide (18:1 / 18:1)-d9 to sphingomyelin (18:1 / 18:1)-d9. [Figure 8] This shows the relationship between CAV-1 and mitochondrial morphology. (a) Representative images of mitochondria and lysosomes in PC-3M (top) and LNCaP (bottom) cells transfected with CellLight Lysosome-GFP (lysosome-associated membrane protein 1) and CellLight Mitochondria-RFP (leader sequence of E1α-pyruvate dehydrogenase). (b) Incorporation of C11 TopFluor-SM in PC-3M cells following CAV1 knockdown. (c) Violin plot illustrating the intensity of TopFluor-SM in PC-3M cells following CAV1 knockdown. Vertical axis = RFU ± SEM. Statistical significance was determined using one-way ANOVA; pairwise comparisons were performed using Tukey HSD multiple comparison tests and adjusted p-values ​​were reported. (i) siCtrl (ii) simulated (iii) siCAV-1 (iv) siCAV-2. [Figure 9] The following images show representative images from (i) lysosomes (CellLight Lysosome-GFP (Lysosome-associated membrane protein 1)), (ii) mitochondria (CellLight Mitochondria-RFP (E1α pyruvate dehydrogenase leader sequence)) and (iii) binding images in PC-3M cells following CAV1 knockdown. [Figure 10](a) Representative images from mitochondrial clump staining (MitoTracker Green) in PC-3M cells following CAV1 knockdown are shown. Bar scales represent 20 μm. Intensity scale bars are provided next to each figure. Violin plots showing the intensities of MitoTracker Green (b) and MitoTracker CMXRos (c) in PC-3M cells following CAV1 knockdown are also illustrated. Vertical axis = RFU ± SEM. Statistical significance was determined using one-way ANOVA; pairwise comparisons were performed using Tukey HSD multiple comparison tests, and adjusted p-values ​​are reported. [Figure 11] (a)(ii)CAV1(FITC) and (iii)mitochondrial potential / reactive oxygen species (MitoTracker Red CMXRos) co-staining in PC-3M cells following CAV1 knockdown, and {and and}(i) merged images {b} are shown. (b)Intracellular levels of reactive oxygen species assessed via CellROX Deep red in PC-3M cells following CAV1 knockdown. (i)siCtrl(ii)ciCAV1-1. [Figure 12] This shows the secretion of Cav-1 containing extracellular vesicles rich in sphingomyelin and lactosylceramide when Cav1 expression is elevated. (a) Schematic diagram of the multi-fraction approach. (b) Cav-1 levels (ng / mL) in extracellular vesicles from LNCaP and PC-3M conditioned media following Cav-1 overexpression or transient CAV1 knockdown, respectively, in the presence or absence of BSA or human lipoprotein. (i) Medium + serum-lipoprotein + LDL(ii) medium + serum-lipoprotein + BSA(iii) medium + serum-lipoprotein. Vertical axis = ngCAV1 / mL. (c) Number of particles per 1 mL (vertical axis) from (i) LNCaP and (ii) PC-3M conditioned media following Cav-1 overexpression or CAV1 knockdown, respectively. Horizontal axis = size / nm. Statistical significance was determined by a two-tailed Student's t-test, comparing the area under the curve following Cav-1 overexpression or knockdown in each scrambled subject. [Figure 13] The following shows the levels of sphingomyelin (a) and lactosylceramide (b) in conditioned media for LNCaP and PC-3M following Cav-1 overexpression or transient knockdown, respectively, in the presence or absence of BSA or human-derived lipoprotein. (i) Basal medium (ii) CAV(NC1) (iii) CAV(si8) (iv) CAV- (v) CAV+ (vi) Basal medium + BSA (vii) CAV(NC1) / BSA(viii) CAV(si8) / BSA(ix) CAV- / BSA(x) CAV+ / BSA(xi) Basal medium + LDL (xii) CAV(NC1) / LDL (xiii) CAV(si8) / LDL (xiv) CAV- / LDL (xv) CAV+ / LDL. (c) Lipid composition of Ev isolated from conditioned media of LNCaP (left) and PC-3M (right) prostate cancer cells. (i) Acylcarnitine (ii) Oxylipin (iii) Lysophospholipid (iv) Phospholipid (v) Sphingomyelin (vi) Ceramide (vii) Sphingoglycolipid (viii) Cardiolipin (ix) Monoacylglycerol (x) Diacylglycerol (xi) Triacylglycerol (xii) Cholesterol ester. (d) Proteins of PC-3M-derived EV annotated to be localized to mitochondria based on COMPARTMENTS localization evidence database score. (i) Peroxisome (ii) Golgi apparatus (iii) Endosome (iv) Lysosome (v) Endoplasmic reticulum (vi) Mitochondria (vii) Cytoskeleton (viii) Extracellular region (ix) Plasma membrane (x) Nucleus (xi) Cytosol. [Figure 14] (a) Heatmaps showing the intracellular localization of protein properties identified in EVs isolated from PC-3M or (b) LNCaP prostate cancer cells in conditioned medium. Cellular localization is based on COMPARTMENTS localization evidence database scores. (i) Peroxisome (ii) Golgi apparatus (iii) Endosome (iv) Lysosome (v) Endoplasmic reticulum (vi) Mitochondria (vii) Cytoskeleton (viii) Extracellular region (ix) Plasma membrane (x) Nucleus (xi) Cytosol. [Figure 15](a) Survival curves of (left) RM-9 cells and (right) PC-3M cells following 48-hour treatment with (i) PPMP, (ii) PDMP, or (iii) eliglustat. Horizontal axis = logarithmic scale (μM); vertical axis = % survival rate compared to control. Also shown are the relative abundances of (b) ceramide and (c) sphingoglycolipids following 6-hour treatment of (left) RM-9 and (right) PC-3M with vehicle (ethanol) or inhibitor. (i) vehicle (ii) 128 μM eliglustat (iii) 64 μM PDMP (iv) 64 μM PPMP. Statistical significance was determined by a two-tailed Student's t-test comparing the aggregation strength of individual lipid species corresponding to each lipid domain. [Figure 16] (a) This shows the induction of autophagy / mitophagy in PC-3M prostate cancer cells upon exposure to eliglustat. (i) Cytotoxicity (ii) Apoptosis at 4 hours (iii) Apoptosis at 24 hours. N = 3 biologically independent replicated tests per experimental condition. Values ​​are logarithmic (relative fluorescence units) ± StDev. Also shown are (b) volcano plots showing differences in annotated lipid species stratified by lipid domain in RM-9 prostate cancer cells following a 6-hour challenge with PC-3M and (c) 128 μM eliglustat. (i) Acylcarnitine (ii) Cardiolipin (iii) Diacylglycerol (iv) Etherlysophospholipid (v) Etherlysophospholipid (vi) Lysophospholipid (vii) Phospholipid (viii) Cholesterol ester (ix) Triacylglycerol (x) Sphingomyelin. N = 3 biologically independent replicated tests per experimental condition. Statistical significance was determined by a two-sided Student's t-test. [Figure 17]Representative confocal microscopy images of PC-3M cells are shown following acute (6-hour) treatment with either (a) vehicle or (c) eliglustat (128 μM), following 24-hour pre-transfer with (i) CellLight Mitochondria-RFP (leader sequence of E1α pyruvate dehydrogenase) or (ii) CellLight Lysosome-GFP (lysosome-associated membrane protein 1). (b) and (d) correspond to functional enhancements from (a) and (c), respectively. (iii) Merged image. [Figure 18] (a) Viability of PC-3M cells treated with 128 μM eliglutat following (left) knockdown of CAV1 or (right) pretreatment with a Cav-1 monoclonal blocking antibody (MTS assay): (i) siCtrl (ii) siCAV1-1 (iii) siCAV1-2 (iv) IgG = vehicle (v) IgG + eliglutat (vi) abCAV1 + vehicle (vii) abCAV1 + eliglutat. (b) Viability of LNCaP cells treated with 64 μM eliglutat following Cav-1 overexpression (MTS assay). [Figure 19] This study demonstrates the efficacy of eliglustat in an in vivo mouse model. (a) Relative fluorescence units ± SEM of RM-9 tumors following daily intraperitoneal injection of either (i) physiological saline (n=23) or (ii) eliglustat (60 mg / kg) (n=9). (b) Tumor volume ± SEM following treatment with physiological saline (n=15) or eliglustat (60 mg / kg) (n=8). Statistical significance was determined by a two-sided Wilcoxon rank-sum test. (c) Representative IVIS images following treatment. (d) Relative abundance (area units ± StDev) of sphingoglycolipids in RM-9 tumors following treatment. Statistical significance was determined by a Wilcoxon rank-sum test comparing the aggregation strength of individual lipid species corresponding to each lipid domain. [Figure 20]Quantitative analyses obtained from immunohistochemical staining of (a) Cav-1, (b) BrdU-TUNEL, (c) PCNA, (d) HMGB1, and (e) LC3B in RM-9 tumors following treatment with either saline (n=6-8 mice) or eliglustat (n=7 mice) are shown. The vertical axis represents (a) Cav-1 staining score, (b) apoptotic bodies / fd, (c) % PCNA labeling, (d) % cytoplasmic HMGB1, and (e) LC-3B positive cells / fd. Statistical significance was determined by a two-sided Wilcoxon rank-sum test. Also shown are (f) volcano plots illustrating the multiplicative changes of individual annotated lipid species stratified by lipid domain in the plasma of RM-9-carrying C57BL / 6N mice or control mice. (i) Acylcarnitine (ii) Ceramide (iii) Cholesterol ester (iv) Diacylglycerol (v) Free fatty acid (vi) Sphingoglycolipid (vii) Lysophospholipid (viii) Oxylipin (ix) Phospholipid (x) Sphingomyelin (x) Triacylglycerol. [Figure 21] In an independent validation cohort of 248 participants (35 with progressive disease and 213 with non-progressive disease), the odds ratios (95% CI) for increase per unit of TrihexosylCer (34:1), LactosylCer (36:0), LactosylCer (32:0), SM (44:2), SM (40:2), plasma sphingolipid signature (SphingoSignature), and Simplified Signature were depicted to assess the risk of disease progression in men under active surveillance for prostate cancer. * indicates statistical significance, one-sided p<0.05. [Modes for carrying out the invention]

[0051] In one embodiment, the disclosure provides a method for determining the risk of prostate cancer progression in a subject, comprising the steps of: measuring the level of CAV-1 in a biological sample obtained from the subject; measuring the level of SM(40:2) in the biological sample; measuring the level of SM(44:2) in the biological sample; measuring the level of LacCer(32:0) in the biological sample; measuring the level of LacCer(36:0) in the biological sample; measuring the level of TriHexCer(34:1) in the biological sample; and measuring the level of HexCer40:0 in the biological sample, wherein the amounts of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 determine the risk of prostate cancer progression in the subject.

[0052] In another embodiment, the disclosure provides a method for determining the risk of prostate cancer progression in a subject, comprising the step of contacting a biological sample obtained from the subject with a first reporter molecule bound to CAV-1, a second reporter molecule bound to SM(40:2), a third reporter molecule bound to SM(44:2), a fourth reporter molecule bound to LacCer32:0, a fifth reporter molecule bound to LacCer36:0, a sixth reporter molecule bound to TriHexCer34:1, and a seventh reporter molecule bound to HexCer40:0, wherein the amounts of the first, second, third, fourth, fifth, sixth, and seventh reporter molecules determine the risk of prostate cancer progression in the subject.

[0053] In another embodiment, the disclosure provides a method for determining the risk of prostate cancer progression in a subject, comprising the steps of performing a CAV-1 spectroscopic analysis on a biological sample obtained from the subject, a SM(40:2) spectroscopic analysis, a SM(44:2) spectroscopic analysis, a LacCer32:0 spectroscopic analysis, a LacCer36:0 spectroscopic analysis, a TriHexCer34:1 spectroscopic analysis, and a HexCer40:0 spectroscopic analysis, wherein the spectroscopic analysis determines the risk of prostate cancer progression in the subject. In some embodiments, the spectroscopic analysis is quantitative. In some embodiments, the spectroscopic analysis is mass spectrometry. In some embodiments, the spectroscopic analysis is performed simultaneously. In some embodiments, the spectroscopic analysis is performed sequentially. In some embodiments, the method further comprises a chromatography step. In some embodiments, the method further comprises a liquid chromatography step. In some embodiments, the method further comprises a high-performance liquid chromatography (HPLC) step. In some embodiments, the method further comprises a gas chromatography ("GC") step. In some embodiments, the chromatography step is directly linked to the spectroscopy step. In some embodiments, the chromatography step separates at least one analyte from at least one other analyte.

[0054] In another aspect, the disclosure provides a surface bound to CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 for a biological sample obtained from a subject; incubates the surface with the biological sample; contacts the surface with a first reporter molecule that binds to CAV-1; contacts the surface with a second reporter molecule that binds to SM(40:2); contacts the surface with a third reporter molecule that binds to SM(44:2); contacts the surface with a fourth reporter molecule that binds to LacCer32:0; contacts the surface with a fifth reporter molecule that binds to LacCer36:0; contacts the surface with a sixth reporter molecule that binds to TriHexCer34:1; and applies Hex A method is provided for determining the risk of prostate cancer progression in a subject, comprising the steps of: contacting a seventh reporter molecule bound to Cer40:0; measuring the amount of a first reporter molecule bound to the surface; measuring the amount of a second reporter molecule bound to the surface; measuring the amount of a third reporter molecule bound to the surface; measuring the amount of a fourth reporter molecule bound to the surface; measuring the amount of a fifth reporter molecule bound to the surface; measuring the amount of a sixth reporter molecule bound to the surface; and measuring the amount of a seventh reporter molecule bound to the surface, wherein the amounts of the first, second, third, fourth, fifth, sixth, and seventh reporter molecules determine the risk of prostate cancer progression in the subject.

[0055] In another aspect, the disclosure provides a biological sample obtained from a subject by providing a means for binding CAV-1 to a first surface; a means for binding SM(40:2) to a second surface; a means for binding SM(44:2) to a third surface; a means for binding LacCer32:0 to a fourth surface; a means for binding LacCer36:0 to a fifth surface; a means for binding TriHexCer34:1 to a sixth surface; a means for binding HexCer40:0 to a seventh surface; incubation the first surface with the biological sample; incubation the second surface with the biological sample; incubation the third surface with the biological sample; incubation the fourth surface with the biological sample; incubation the fifth surface with the biological sample; incubation the sixth surface with the biological sample; and incubation the seventh surface The steps are: incubation with a physical sample; contacting the first surface with a first reporter molecule that binds to CAV-1; contacting the second surface with a first reporter molecule that binds to SM(40:2); contacting the third surface with a first reporter molecule that binds to SM(44:2); contacting the fourth surface with a first reporter molecule that binds to LacCer32:0; contacting the fifth surface with a first reporter molecule that binds to LacCer36:0; and contacting the sixth surface with TriHe The steps include: bringing the seventh surface into contact with a first reporter molecule bound to xCer34:1; bringing the seventh surface into contact with a first reporter molecule bound to HexCer40:0; measuring the amount of the first reporter molecule bound to the first surface; measuring the amount of the second reporter molecule bound to the second surface; measuring the amount of the third reporter molecule bound to the third surface; measuring the amount of the fourth reporter molecule bound to the fourth surface; and measuring the amount of the fifth reporter molecule bound to the fifth surface.A method is provided for determining the risk of prostate cancer progression in a subject, comprising the steps of: measuring the amount of a sixth reporter molecule bound to a sixth surface; and measuring the amount of a seventh reporter molecule bound to a seventh surface, wherein the amounts of the first, second, third, fourth, fifth, sixth, and seventh reporter molecules determine the risk of prostate cancer progression in the subject.

[0056] In another aspect, the disclosure provides a surface to a biological sample obtained from a subject that binds to CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0; incubates the surface with the biological sample; contacts the surface with a first relay molecule that binds to CAV-1; contacts the surface with a second relay molecule that binds to SM(40:2); and contacts the surface with a third relay molecule that binds to SM(44:2). The steps are: bringing the surface into contact with a relay molecule; bringing the surface into contact with a fourth relay molecule that binds to LacCer32:0; bringing the surface into contact with a fifth relay molecule that binds to LacCer36:0; bringing the surface into contact with a sixth relay molecule that binds to TriHexCer34:1; bringing the surface into contact with a seventh relay molecule that binds to HexCer40:0; bringing the surface into contact with a first reporter molecule that binds to the first relay molecule; and bringing the surface into contact with a second reporter molecule that binds to the second relay molecule. The steps are: to bring the surface into contact with a third reporter molecule that binds to a third relay molecule; to bring the surface into contact with a fourth reporter molecule that binds to a fourth relay molecule; to bring the surface into contact with a fifth reporter molecule that binds to a fifth relay molecule; to bring the surface into contact with a sixth reporter molecule that binds to a sixth relay molecule; to bring the surface into contact with a seventh reporter molecule that binds to a seventh relay molecule; and to measure the amount of the first relay molecule and the first reporter molecule that binds to CAV-1. The steps include: measuring the amount of the second relay molecule and the second reporter molecule bound to SM(40:2); measuring the amount of the third relay molecule and the third reporter molecule bound to SM(44:2); measuring the amount of the fourth relay molecule and the fourth reporter molecule bound to LacCer32:0; measuring the amount of the fifth relay molecule and the fifth reporter molecule bound to LacCer36:0; and measuring the amount of the sixth relay molecule and the sixth reporter molecule bound to TriHexCer34:1.A method is provided for determining the risk of prostate cancer progression in a subject, comprising the step of measuring the amount of a seventh relay molecule and a seventh reporter molecule bound to HexCer40:0, wherein the amounts of the first reporter molecule, second reporter molecule, third reporter molecule, fourth reporter molecule, fifth reporter molecule, sixth reporter molecule, and seventh reporter molecule determine the risk of prostate cancer progression in the subject.

[0057] In one embodiment, the amounts of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 or reporter molecules bound thereto are elevated in the subjects compared to healthy subjects. In one embodiment, the amounts of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 or reporter molecules bound thereto are elevated in the subjects compared to subjects without prostate cancer. In one embodiment, the amounts of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 or reporter molecules bound thereto are elevated in the subjects compared to subjects with inactive prostate cancer.

[0058] In another embodiment, at least one of the reporter molecules provides a detectable signal. In another embodiment, the detectable signal is detectable by a method selected from ultraviolet-visible spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, gas chromatography-mass spectroscopy (GC-MS), liquid chromatography-mass spectroscopy (LC-MS), correlation spectroscopy (COSy), nuclear Overhauser effect spectroscopy (NOESY), rotating-frame nuclear Overhauser effect spectroscopy (ROESY), LC-TOF-MS, LC-MS / MS, and capillary electrophoresis-mass spectroscopy. In another embodiment, the spectroscopy method is mass spectrometry. In another embodiment, the panel comprises biomarkers identified by a method selected from ultraviolet-visible spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, gas chromatography-mass spectroscopy (GC-MS), liquid chromatography-mass spectroscopy (LC-MS), correlation spectroscopy (COSy), nuclear Overhauser effect spectroscopy (NOESY), rotating-frame nuclear Overhauser effect spectroscopy (ROESY), LC-TOF-MS, LC-MS / MS, and capillary electrophoresis-mass spectroscopy. In yet another embodiment, the panel comprises biomarkers identified by ultraviolet-visible spectroscopy or proton NMR spectroscopy.

[0059] In another embodiment, the first reporter selectively binds to CAV-1. In another embodiment, the second reporter selectively binds to SM(40:2). In another embodiment, the third reporter selectively binds to SM(44:2). In another embodiment, the fourth reporter selectively binds to LacCer32:0. In another embodiment, the fifth reporter selectively binds to LacCer36:0. In another embodiment, the sixth reporter selectively binds to TriHexCer34:1. In another embodiment, the seventh reporter selectively binds to HexCer40:0.

[0060] In another embodiment, the determination of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 levels is performed substantially simultaneously. In another embodiment, the determination of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 levels is performed in a stepwise manner. In another embodiment, such a method includes including the subject's medical history information in determining the risk of progression of prostate cancer. In another embodiment, such a method includes administering at least one alternative diagnostic test to subjects assigned as at risk for progression of prostate cancer.

[0061] In another embodiment, the Disclosure provides a method for treating a subject suspected to be at risk of progression of prostate cancer, comprising the steps of: analyzing the subject for the risk of progression of prostate cancer using the method described herein; and administering a dose of treatment effective for treating the cancer. In one embodiment, the treatment is surgery, chemotherapy, immunotherapy, radiotherapy, targeted therapy, or a combination thereof.

[0062] In another aspect, the disclosure provides a biological sample obtained from a subject, comprising the steps of: measuring the level of CAV-1 in the biological sample; measuring the level of SM(40:2) in the biological sample; measuring the level of SM(44:2) in the biological sample; measuring the level of LacCer(32:0) in the biological sample; measuring the level of LacCer(36:0) in the biological sample; measuring the level of TriHexCer(34:1) in the biological sample; measuring the level of pro-SFTPB in the biological sample; determining the level of CAV-1 relative to a first reference value and predicting the risk of prostate cancer progression therein; determining the level of SM(40:2) relative to a second reference value and predicting the risk of prostate cancer progression therein; and determining the level of SM(44:2) relative to a third reference value, The present invention provides a method for determining the risk of prostate cancer progression in a subject, comprising the steps of: predicting the risk of prostate cancer progression within a given range; determining the level of LacCer32:0 relative to a fourth reference value and predicting the risk of prostate cancer progression within that range; determining the level of LacCer36:0 relative to a fifth reference value and predicting the risk of prostate cancer progression within that range; determining the level of TriHexCer34:1 relative to a sixth reference value and predicting the risk of prostate cancer progression within that range; determining the level of HexCer40:0 relative to a seventh reference value and predicting the risk of prostate cancer progression within that range; and assigning whether or not there is a risk of prostate cancer progression by statistical analysis of the ratios of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 levels.

[0063] In another aspect, the disclosure provides a method for predicting the risk of prostate cancer progression in a subject, comprising the steps of: measuring the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 biomarkers in a biological sample obtained from a subject; and calculating predictors determined by statistical analysis of the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0.

[0064] In another aspect, the disclosure provides a biological sample obtained from a subject, and measures the levels of the CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 biomarkers in the biological sample; and measures the levels of the CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1 in the biological sample. The present invention provides a method for determining the risk of prostate cancer progression in a subject, comprising the steps of: assigning to the subject's status whether there is a risk of prostate cancer progression based on a statistical analysis of the HexCer40:0 level; and determining the risk of prostate cancer progression from an analysis of the CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 levels.

[0065] In another embodiment, the first reporter selectively binds to CAV-1. In another embodiment, the second reporter selectively binds to SM(40:2). In another embodiment, the third reporter selectively binds to SM(44:2). In another embodiment, the fourth reporter selectively binds to LacCer32:0. In another embodiment, the fifth reporter selectively binds to LacCer36:0. In another embodiment, the sixth reporter selectively binds to TriHexCer34:1. In another embodiment, the seventh reporter selectively binds to HexCer40:0. In another embodiment, the determination of the CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 levels is performed substantially simultaneously. In another embodiment, the determination of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 levels is performed in a stepwise manner. In another embodiment, such a method further includes including subject medical history information in the assignment of whether or not there is a risk of progression of prostate cancer. In another embodiment, such a method includes administering at least one alternative diagnostic test to subjects assigned as being at risk of progression of prostate cancer.

[0066] In another embodiment, the Disclosure provides a method for treating a subject suspected to be at risk of progression of prostate cancer, comprising the steps of: analyzing the subject for risk of progression of prostate cancer using the method described herein; and administering a dose of treatment effective for treating the cancer. In another embodiment, the treatment is surgery, chemotherapy, immunotherapy, radiotherapy, targeted therapy, or a combination thereof. In another embodiment, the classification of subjects considered to be at risk of progression of prostate cancer has a specificity of 78% and 94% and a sensitivity of 0.76 and 0.42, respectively.

[0067] In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are elevated compared to the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a reference subject or group with adenocarcinoma.

[0068] In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are elevated compared to the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a reference subject or group having squamous cell carcinoma.

[0069] In another aspect, the present disclosure provides a kit for a method comprising: a reagent solution comprising a first solute for the detection of CAV-1; a second solute for the detection of SM(40:2); a third solute for the detection of SM(44:2); a fourth solute for the detection of LacCer32:0; a fifth solute for the detection of LacCer36:0; a sixth solute for the detection of TriHexCer34:1; and a seventh solute for the detection of HexCer40:0.

[0070] In another embodiment, such a method further comprises a device for bringing a reagent solution into contact with a biological sample. In another embodiment, such a method comprises at least one surface having means for binding at least one biomarker. In another embodiment, the at least one biomarker is selected from the group consisting of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0.

[0071] In another aspect, the Disclosure provides a method for determining the risk of prostate cancer progression in a subject, comprising a biomarker panel and a protein marker panel, wherein the biomarker panel comprises CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0; wherein the method comprises performing the step of measuring the levels of biomarkers and protein biomarkers in a biological sample when obtaining a biological sample from a subject, wherein the amounts of biomarkers and protein biomarkers determine the risk of prostate cancer progression in the subject.

[0072] In another aspect, the disclosure provides a method for determining the risk of prostate cancer progression in a subject, comprising the steps of: taking a biological sample from the subject and measuring the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in the biological sample; and determining the risk of prostate cancer progression in the subject by statistical analysis of the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in the biological sample. In one embodiment, levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 or reporter molecules bound to them are elevated in the subjects compared to healthy subjects. In another embodiment, levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are elevated compared to levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a reference subject or group without prostate cancer. In another embodiment, the reference subject or group is healthy. In another embodiment, such a method comprises at least one receptor molecule that selectively binds to a biomarker selected from the group consisting of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0. In another embodiment, the sample comprises a biological sample selected from blood, plasma, and serum. In another embodiment, the biological sample is serum. In another embodiment, the amounts of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are quantified.In another embodiment, the detection of the amounts of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 involves the use of solid particles. In another embodiment, the solid particles are beads. In another embodiment, at least one reporter molecule is linked to the enzyme. In another embodiment, at least one reporter molecule provides a detectable signal. In another embodiment, the detectable signal is detectable by a method selected from ultraviolet-visible spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, proton NMR spectroscopy, gas chromatography-mass spectroscopy (GC-MS), liquid chromatography-mass spectroscopy (LC-MS), correlation spectroscopy (COSy), nuclear Overhauser effect spectroscopy (NOESY), rotating-frame nuclear Overhauser effect spectroscopy (ROESY), LC-TOF-MS, LC-MS / MS, and capillary electrophoresis-mass spectroscopy. In another embodiment, the concentrations of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are measured. In another embodiment, the subject is determined to be at risk of prostate cancer progression based on the measured concentrations of the biomarkers. In another embodiment, the measured concentrations are used to calculate a biomarker score based on sensitivity and specificity values ​​at a given cutoff. In another embodiment, such a method further includes the step of comparing the measured concentration of each biomarker in the biological sample with the prediction of a statistical model. In another embodiment, the panel is selected from the group consisting of a. a panel comprising CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0; or b. a panel comprising CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0.In another embodiment, the panel comprises biomarkers identified by a method selected from ultraviolet-visible spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, gas chromatography-mass spectroscopy (GC-MS), liquid chromatography-mass spectroscopy (LC-MS), correlation spectroscopy (COSy), nuclear Overhauser effect spectroscopy (NOESY), rotating-frame nuclear Overhauser effect spectroscopy (ROESY), LC-TOF-MS, LC-MS / MS, and capillary electrophoresis-mass spectroscopy. In yet another embodiment, the panel comprises biomarkers identified by ultraviolet-visible spectroscopy or proton NMR spectroscopy.

[0073] In another embodiment, the first reporter selectively binds to CAV-1. In another embodiment, the second reporter selectively binds to SM(40:2). In another embodiment, the third reporter selectively binds to SM(44:2). In another embodiment, the fourth reporter selectively binds to LacCer32:0. In another embodiment, the fifth reporter selectively binds to LacCer36:0. In another embodiment, the sixth reporter selectively binds to TriHexCer34:1. In another embodiment, the seventh reporter selectively binds to HexCer40:0.

[0074] In another embodiment, the determination of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 levels is performed substantially simultaneously. In another embodiment, the determination of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 levels is performed in a stepwise manner. In another embodiment, such a method further includes including the subject's medical history information in the assignment of whether or not they are at risk of progression of prostate cancer. In another embodiment, such a method includes administering at least one alternative diagnostic test to subjects assigned as being at risk of progression of prostate cancer.

[0075] In another embodiment, the Disclosure provides a kit for the method described herein, comprising a reagent solution comprising a first solute for the detection of CAV-1; a second solute for the detection of SM(40:2); a third solute for the detection of SM(44:2); a fourth solute for the detection of LacCer32:0; a fifth solute for the detection of LacCer36:0; a sixth solute for the detection of TriHexCer34:1; and a seventh solute for the detection of HexCer40:0.

[0076] In another aspect, the Disclosure provides a kit for the method described herein, comprising: a first reagent solution comprising a first solute for the detection of CAV-1; a second reagent solution comprising a second solute for the detection of SM(40:2); a third reagent solution comprising a third solute for the detection of SM(44:2); a fourth reagent solution comprising a fourth solute for the detection of LacCer32:0; a fifth reagent solution comprising a fifth solute for the detection of LacCer36:0; a sixth reagent solution comprising a sixth solute for the detection of TriHexCer34:1; and a seventh reagent solution comprising a seventh solute for the detection of HexCer40:0.

[0077] In another embodiment, such a kit further comprises a reagent solution comprising a first solute for the detection of CAV-1; a second solute for the detection of SM(40:2); a third solute for the detection of SM(44:2); a fourth solute for the detection of LacCer32:0; a fifth solute for the detection of LacCer36:0; a sixth solute for the detection of TriHexCer34:1; and a seventh solute for the detection of HexCer40:0.

[0078] In another aspect, the Disclosure provides a method for treating or preventing the progression of prostate cancer in a subject having prostate cancer, comprising the steps of: administering a chemotherapeutic agent to the subject having prostate cancer; administering therapeutic radiation to the subject having prostate cancer; and performing one or more surgeries for partial or complete surgical removal of cancerous tissue in the patient having prostate cancer, wherein the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 classify the subject as having or being at risk of progression of prostate cancer.

[0079] In another aspect, the Disclosure provides a method for treating or preventing the progression of prostate cancer in a subject having prostate cancer, comprising the steps of: administering a chemotherapeutic agent to the subject having prostate cancer; administering therapeutic radiation to the subject having prostate cancer; and performing one or more surgeries for partial or complete surgical removal of cancerous tissue in the patient having prostate cancer, wherein the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 classify the subject as having or being at risk of progression of prostate cancer.

[0080] In another embodiment, the disclosure includes the steps of: detecting CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a biological sample obtained from a subject; quantifying the amounts of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in the collected sample; and detecting CAV-1, SM(40:2), SM(44:2), A method for treating prostate cancer in a subject is provided, comprising the steps of: determining a risk score from the amounts of LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0; and comparing the risk score to a cutoff value to determine whether the person is at risk of prostate cancer progression, wherein if the level exceeds the cutoff value, the person is at risk of prostate cancer progression, and the person at risk of prostate cancer progression is given treatment for prostate cancer.

[0081] In another aspect, the disclosure provides a method for determining the risk of progression of prostate cancer in a subject, comprising the steps of: measuring the concentrations of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a biological sample from a subject to be analyzed; and comparing the concentrations of the biomarkers in a sample from a subject to be diagnosed with prostate cancer to the concentrations in a normal or non-disease subject, wherein the subject to be diagnosed with prostate cancer.

[0082] In another embodiment, the disclosure provides a method for determining evidence of risk of prostate cancer progression in a biological sample, comprising the steps of: measuring the concentration of a biomarker panel comprising CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in the biological sample; and determining a risk score from the amounts of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0. In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 or reporter molecules bound thereto are elevated in the subject compared to a healthy subject. In another embodiment, levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are elevated compared to levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a reference group or group without prostate cancer. In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are elevated compared to the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a reference subject or group having inactive prostate cancer. In another embodiment, the reference subject or group is healthy. In another embodiment, at least one surface further comprises at least one receptor molecule that selectively binds to a biomarker selected from CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0. In another embodiment, at least one surface is the surface of a solid particle.In another embodiment, the solid particles comprise beads. In another embodiment, such a method comprises the step of measuring the level of a biomarker in a biological sample; in which the amount of the biomarker classifies whether the patient is at risk of progression of prostate cancer or not. In another embodiment, the sample comprises a biological sample selected from blood, plasma, and serum. In another embodiment, the biological sample is serum. In another embodiment, the amounts of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are quantified. In another embodiment, the detection of the amounts of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 comprises the use of solid particles. In another embodiment, the solid particles are beads. In another embodiment, at least one reporter molecule is linked to the enzyme. In another embodiment, at least one reporter molecule provides a detectable signal. In another embodiment, the detectable signal is detectable by a method selected from ultraviolet-visible spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, gas chromatography-mass spectroscopy (GC-MS), liquid chromatography-mass spectroscopy (LC-MS), correlation spectroscopy (COSy), nuclear Overhauser effect spectroscopy (NOESY), rotating-frame nuclear Overhauser effect spectroscopy (ROESY), LC-TOF-MS, LC-MS / MS, and capillary electrophoresis-mass spectroscopy. In another embodiment, the concentrations of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are measured. In another embodiment, a subject is determined to be at risk of prostate cancer progression based on the measured concentration of a biomarker. In yet another embodiment, a biomarker score is calculated using the measured concentration based on sensitivity and specificity values ​​at a given cutoff.In another embodiment, such a method further includes the step of comparing the measured concentration of each biomarker in the biological sample with the prediction of a statistical model. In another embodiment, the panel is selected from the group consisting of a. a panel comprising CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0; or b. a panel comprising CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0. In another embodiment, the panel comprises biomarkers identified by a method selected from ultraviolet-visible spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, gas chromatography-mass spectroscopy (GC-MS), liquid chromatography-mass spectroscopy (LC-MS), correlation spectroscopy (COSy), nuclear Overhauser effect spectroscopy (NOESY), rotating-frame nuclear Overhauser effect spectroscopy (ROESY), LC-TOF-MS, LC-MS / MS, and capillary electrophoresis-mass spectroscopy. In yet another embodiment, the panel comprises biomarkers identified by ultraviolet-visible spectroscopy or proton NMR spectroscopy.

[0083] In another embodiment, the first reporter selectively binds to CAV-1. In another embodiment, the second reporter selectively binds to SM(40:2). In another embodiment, the third reporter selectively binds to SM(44:2). In another embodiment, the fourth reporter selectively binds to LacCer32:0. In another embodiment, the fifth reporter selectively binds to LacCer36:0. In another embodiment, the sixth reporter selectively binds to TriHexCer34:1. In another embodiment, the seventh reporter selectively binds to HexCer40:0.

[0084] In another embodiment, the determination of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 levels is performed substantially simultaneously. In another embodiment, the determination of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 levels is performed in a stepwise manner. In another embodiment, such a method includes including the subject's medical history information in assigning whether or not they are at risk of progression of prostate cancer. In another embodiment, such a method includes administering at least one alternative diagnostic test to subjects assigned as being at risk of progression of prostate cancer.

[0085] In another embodiment, a method for treating a subject suspected to be at risk of progression of prostate cancer comprises the steps of: analyzing the subject for the risk of progression of prostate cancer using the method described herein; and administering a dose of treatment effective for treating the cancer. In another embodiment, the treatment is surgery, chemotherapy, immunotherapy, radiotherapy, targeted therapy, or a combination thereof. In another embodiment, levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are elevated compared to levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a reference subject or group having adenocarcinoma. In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are elevated compared to the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a reference subject or group with squamous cell carcinoma. In another embodiment, prostate cancer is diagnosed at or before the borderline resectable stage. Prostate cancer is diagnosed at the resectable stage.

[0086] In another embodiment, such a method includes the steps of: providing a surface to which CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 can be bound; incubating the surface with a biological sample; measuring the amount of a first reporter molecule bound to the surface; measuring the amount of a second reporter molecule bound to the surface; measuring the amount of a third reporter molecule bound to the surface; and measuring the amount of a fourth reporter molecule bound to the surface. The method further comprises the steps of measuring the amount of a molecule; measuring the amount of a fifth reporter molecule bound to the surface; measuring the amount of a sixth reporter molecule bound to the surface; and measuring the amount of a seventh reporter molecule bound to the surface, wherein the amounts of the first reporter molecule, the second reporter molecule, the third reporter molecule, the fourth reporter molecule, the fifth reporter molecule, the sixth reporter molecule, and the seventh reporter molecule are used to classify whether the subject is at risk of prostate cancer progression or not.

[0087] In another embodiment, such a kit comprises a device for bringing a reagent solution into contact with a biological sample. In another embodiment, such a kit comprises at least one surface having means for binding at least one biomarker. In another embodiment, the at least one biomarker is selected from the group consisting of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0.

[0088] In another aspect, the disclosure provides a step of a) taking a sample from an asymptomatic subject with prostate cancer; b) measuring a panel of markers in the sample comprising CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0; c) determining the biomarker score for each marker; d) summing the biomarker scores for each marker to obtain a composite score for each subject; and determining the risk of progression of prostate cancer for the subject. The present invention provides a method comprising the steps of: e) quantifying {for} as a risk score (wherein the composite score corresponds to a risk category of a group of stratified target populations, wherein each risk category includes a multiplier indicating an increased likelihood of having prostate cancer that correlates to a range of composite scores compared to the use of a single threshold, wherein the multiplier is determined from positive predictive scores of retrospective samples); and e) performing computed tomography (CT) scans or other imaginary {imagine} forms on subjects who have a quantified risk for the progression of prostate cancer. In another embodiment, the markers consist of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0. In another embodiment, the sample is blood, serum, plasma, or a portion thereof. In another embodiment, the grouping of the stratified target population, the multiplier indicating an increased likelihood of having cancer, and the range of composite scores are determined from retrospective clinical samples of the population. In another embodiment, the risk category further includes a risk identifier. In another embodiment, the risk identifier is selected from low risk, medium-low risk, medium risk, medium-high risk, and highest risk. In another embodiment, calculating a multiplier indicating the increased likelihood of having cancer for each risk category includes the steps of stratifying the target cohort based on retrospective biomarker scores and weighting the known cancer prevalence in the cohort by the positive predictive score of each stratified population.In another embodiment, the grouping of the stratified population comprises at least three risk categories, among which the multiplier indicating a high probability of cancer is approximately 2 or greater. In another embodiment, the grouping of the stratified population comprises at least two risk categories, among which the multiplier indicating a high probability of cancer is approximately 5 or greater. In another embodiment, the subjects are 50 years of age or older and have a history of smoking tobacco. In another embodiment, such a method further comprises the steps of: generating a risk classification table (in which a panel of markers is measured and a biomarker score is determined for each marker, and a composite score is obtained by summing the biomarker scores); determining thresholds used to divide the composite score into risk groups and assigning a multiplier to each group indicating that asymptomatic subjects have a quantified risk of cancer progression. In another embodiment, the groups are in a format selected from electronic tabular format, software application, computer program, and Excel spreadsheet. In another embodiment, the panel of markers comprises proteins, polypeptides, or metabolites measured by binding assays. In another embodiment, the panel of markers comprises proteins or polypeptides measured using a flow cytometer.

[0089] Generally, a method is provided for identifying the risk of prostate cancer progression in a subject, comprising the steps of: (a) applying a blood sample obtained from the subject to the analysis of four biomarkers CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0; (b) quantifying the amounts of the four biomarkers present in the blood sample; and (c) applying statistical manipulation based on the amounts of the present biomarkers to determine the corresponding biomarker score for prostate cancer, thereby classifying the subject as either positive or negative for the risk of prostate cancer progression. As an alternative, a method is provided for identifying the risk of prostate cancer progression in a subject, which generally includes the steps of: (a) applying a blood sample obtained from the subject to the analysis of four biomarkers CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0; (b) quantifying the amounts of the four biomarkers present in the blood sample; and (c) applying a statistical analysis based on the amounts of the present biomarkers to determine a corresponding biomarker score for prostate cancer, thereby providing a means (e.g., a non-binary method) for evaluating the relative risk of prostate cancer progression in the subject.

[0090] The methods presented herein enable screening of high-risk individuals, such as those with a family history of prostate cancer or those with other risk factors such as obesity, heavy smoking, and possibly diabetes. The logistic regression models disclosed herein may incorporate these factors into their classification methods.

[0091] As used herein, “prostate cancer status” refers to a classification of individuals, subjects, or patients who are at risk of or not at risk of progression of prostate cancer. In some embodiments, individuals at risk of progression of prostate cancer may be referred to as “prostate cancer positive.” In other embodiments, individuals not at risk of progression of prostate cancer may be referred to as “prostate cancer negative.” For subjects classified as prostate cancer positive, further methods may be provided to clarify the prostate cancer status. Classification as prostate cancer positive may be followed by methods including, but not limited to, computed tomography (CT).

[0092] This disclosure is not limited to the specific biomolecules reported herein for the detection of biomarkers. Other molecules, including but not limited to proteins, antibodies, nucleic acids, aptamers, and biomolecules based on synthetic organic compounds, may be selected for use in other embodiments. Other molecules may also offer advantages in terms of sensitivity, efficiency, assay speed, cost, safety, or ease of manufacture or storage.

[0093] In some embodiments, the levels of CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in a biological sample are measured. In some embodiments, CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are contacted with a reporter molecule, and the level of each reporter molecule is measured. In some embodiments, reporter molecules are provided that specifically bind to CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0, respectively. The use of reporter molecules can provide increased convenience and sensitivity of the assay.

[0094] In some embodiments, CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are adsorbed onto a surface provided in the kit. In some embodiments, a reporter molecule binds to the surface-adsorbed CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0. The adsorption of the biomarkers may be non-selective or selective. In some embodiments, the surface includes receptor functionality to enhance the selectivity for the adsorption of one or more biomarkers.

[0095] In some embodiments, CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are adsorbed onto four surfaces selectively for one or more biomarkers. The reporter molecule or a group of reporter molecules can then bind to the surface-adsorbed biomarker, and the level of reporter molecules binding to a particular surface allows for easy quantification of the specific biomarker present on that surface.

[0096] In some embodiments, CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are adsorbed onto a surface provided in the kit; relay molecules specific to one or more of these biomarkers bind to the surface-adsorbed biomarkers; and receptor molecules specific to one or more relay molecules bind to the relay molecules. The relay molecules may provide specificity to specific biomarkers, and the receptor molecules may enable detection.

[0097] In some embodiments, relay molecules are provided that specifically bind to CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0, respectively. The relay molecules may be intentionally designed for specificity to biomarkers or selected from a pool of candidates based on their binding properties.

[0098] In some embodiments, CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 are adsorbed onto four discontinuous surfaces provided in the kit; relay molecules specific to one or more of these biomarkers bind to the surface-adsorbed biomarkers; and receptor molecules bind to the relay molecules. Surface analysis can be achieved in a stepwise or parallel manner.

[0099] In some embodiments, the reporter molecule is linked to an enzyme to facilitate its quantification. In some embodiments, quantification can be achieved by catalytic production of a substance having desired spectroscopic properties.

[0100] In some embodiments, the amount of biomarker is determined using spectroscopy. In some embodiments, the spectroscopy method used is ultraviolet-visible spectroscopy. In some embodiments, the spectroscopy method used is mass spectrometry. In other embodiments, the spectroscopy method used is nuclear magnetic resonance (NMR) spectroscopy, including but not limited to proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, gas chromatography-mass spectroscopy (GC-MS), liquid chromatography-mass spectroscopy (LC-MS), correlation spectroscopy (COSy), nuclear Overhauser effect spectroscopy (NOESY), rotating-frame nuclear Overhauser effect spectroscopy (ROESY), LC-TOF-MS, LC-MS / MS, and capillary electrophoresis-mass spectroscopy.

[0101] The amounts of biomarkers or biomarker groups detected in a particular assay may be reported directly to the operator, or alternatively, stored digitally for easy use in mathematical processing. A system for performing mathematical analysis may be provided, and the classification as prostate cancer positive or negative may be further reported to the operator.

[0102] In some embodiments, additional assays known to those skilled in the art may function in conjunction with the disclosure herein. Other assays include, but are not limited to, those utilizing mass spectrometry, immunoaffinity LC-MS / MS, surface plasmon resonance, chromatography, electrochemistry, acoustic wave, immunohistochemistry, and array techniques.

[0103] The various system components considered herein include a computer comprising one or more processors for processing digital data; short-term or long-term digital memory; an input analog-to-digital converter for providing digitized data; and application programs available to the processor for directing the processor's processing of digital data. These may also include one or more input devices for collecting information from a subject or operator, and one or more output devices for presenting information to the subject or operator.

[0104] Furthermore, this specification also provides treatment methods for individuals classified as prostate cancer positive. Treatment options for prostate cancer positive patients include, but are not limited to, surgery, chemotherapy, radiation therapy, targeted therapy, or a combination thereof.

[0105] With regard to the detection of biomarkers detailed herein, this disclosure is not limited to the specific biomolecules reported herein. In some embodiments, other biomolecules may be selected for the detection and analysis of the disclosed biomarkers, including but not limited to biomolecules based on proteins, antibodies, nucleic acids, aptamers, and synthetic organic compounds. Other molecules may offer advantages in terms of sensitivity, efficiency, assay speed, cost, safety, or ease of manufacture or storage. In this regard, those skilled in the art will understand that the predictive and diagnostic power of the biomarkers disclosed herein may extend not only to the protein forms of these biomarkers but also to the analysis of other representations of the biomarkers (e.g., nucleic acids). Furthermore, those skilled in the art will understand that the predictive and diagnostic power of the biomarkers disclosed herein may be used in combination with the analysis of other biomarkers associated with prostate cancer. In some embodiments, the other biomarkers associated with prostate cancer may be protein-based biomarkers.

[0106] The above provides a fairly broad overview of the features and technical benefits of this disclosure in order to better understand the detailed explanation. It should be understood by those skilled in the art that the particular embodiments disclosed may readily be used as a basis for modifying or designing other structures or processes to accomplish the same objectives of the disclosure. It should be understood that this disclosure is not limited to the particular embodiments described, as variations of the particular embodiments may be made and still be included within the scope of the appended claims.

[0107] definition As used herein, the term “prostate cancer” refers to a malignant neoplasm of the prostate gland characterized by the abnormal proliferation of cells, the proliferation of which exceeds and does not coordinate with the proliferation of surrounding normal tissue.

[0108] As used herein, the term “prostate cancer positive” refers to a classification of individuals considered to be at risk of progression of prostate cancer.

[0109] As used herein, the term “prostate cancer negative” refers to a classification of individuals who are considered to be at no risk of progression of prostate cancer.

[0110] In the use of this specification, the terms “subject” or “patient” refer to a mammal, preferably human, that is preferably classified as prostate cancer-positive or prostate cancer-negative and for which further treatment may be offered.

[0111] In the context of this specification, “reference patient,” “reference subject,” or “reference group” refers to a group of patients or subjects from whom test samples may be compared to those from patients or subjects suspected to have prostate cancer or to be at risk of progression. In some embodiments, such comparisons may be used to determine whether a test subject has prostate cancer. The reference patient or group may serve as a control for the test or diagnostic purposes. As described herein, the reference patient or group may be a sample obtained from a single patient or may represent a group of samples, such as a pooled sample group.

[0112] As used herein, “healthy” means an individual in which no evidence of prostate cancer is found, i.e., an individual who does not have prostate cancer. Such an individual may be classified as “prostate cancer negative,” or as having a healthy prostate or normal, unimpaired prostate function. A healthy patient or subject is free from symptoms of prostate cancer or other prostate disease. In some embodiments, a healthy patient or subject may be used as a reference patient for comparison with a sample of disease or suspected disease in order to determine prostate cancer in a patient or group of patients.

[0113] In the use of this specification, the terms “treatment” or “to treat” mean the administration of a drug or the implementation of a medical procedure to a subject or patient for the purpose of preventing or curing the occurrence or recurrence of weakness, disease, condition or event, or reducing its severity or likelihood, if the subject or patient is ill. In connection with this disclosure, the terms may also mean the administration of a pharmacological substance or preparation, or the implementation of a non-pharmacological method, including but not limited to radiotherapy and surgery. In the use described herein, pharmacological substances may include, but are not limited to, abiraterone acetate (Zytiga), apalutamide (Erleada), bicalutamide (Casodex), cabazitaxe (Jevtana), darolutamide (Nubeqa), degarelix (Firmagon), docetaxel (Taxotere), Eligard (leuprolide acetate), enzalutamide (Xtandi), flutamide, goserelin acetate (Zoladex), leuprolide acetate (Lupron or Lupron reservoir), olaparib (Lynparza), mitoxantrone hydrochloride, nilutamide (Nilandron), ciproisel T (Provenge), radium-223 dichloride (Xofigo), and rucaparibu cansylate (Rubraca), as well as other established chemotherapeutic agents in the art.

[0114] Examples of pharmacological substances may include substances used in immunotherapy, such as checkpoint inhibitors. Examples of treatment may include a number of pharmacological substances, or a number of treatment methods, including but not limited to surgery and chemotherapy.

[0115] In the use of this specification, the term "ELISA" refers to an enzyme-linked immunosorbent assay. This assay generally involves contacting a sample of fluorescently labeled proteins with antibodies that have a specific affinity for those proteins. Detection of these proteins can be achieved by a variety of means, including but not limited to laser fluorescence assays.

[0116] As used herein, the term “regression” refers to a statistical method that can assign predictive values ​​to fundamental characteristics of a sample based on observable traits (or sets of observable traits) of the sample. In some embodiments, the characteristics are not directly observable. For example, the regression methods used herein may associate qualitative or quantitative results of a particular biomarker test or set of biomarker tests for a particular subject with the probability that the subject is positive for prostate cancer.

[0117] As used herein, the term “logistic regression” refers to a regression method in which the assignment of a prediction from a model may have one of several allowable discrete values. For example, a logistic regression model used herein may assign a prediction of either prostate cancer positive or prostate cancer negative for a particular subject.

[0118] As used herein, the term “biomarker score” refers to a numerical score for a particular subject calculated by inputting specific biomarker levels for that subject into a statistical method.

[0119] As used herein, the term “composite score” refers to the sum of normalized values ​​of a given marker measured in a sample from a subject. In one embodiment, the normalized values ​​are reported as biomarker scores, and these biomarker score values ​​are then summed to provide a composite score for each subject tested. When used in the context of a risk classification table and associated with groups stratified based on a range of composite scores in the risk classification table, the “composite score” is used to determine the “risk score” for each subject tested, where the “risk score” is a multiplier indicating an increased likelihood that the stratified group has cancer.

[0120] As used herein, the term “risk score” refers to a single numerical value representing the risk of cancer progression in an asymptomatic human subject compared to the known prevalence of cancer progression in a disease cohort. In certain embodiments, a composite score is calculated for a human subject and associated with a multiplier representing the risk of prostate cancer progression, where the composite score is associated based on the range of composite scores for each stratified group in a risk classification table. In this way, the composite score is converted into a risk score based on a multiplier representing the increased likelihood of having cancer for the grouping that best matches the composite score.

[0121] As used herein, the terms “cutoff” or “cutoff point” refer to a mathematical value related to a specific statistical method that may be used to assign a classification of prostate cancer positive or prostate cancer negative based on the biomarker score of the subject.

[0122] In the context of this specification, if a value above or below a cutoff value is considered "characteristic of prostate cancer," it means that the subject from whom the analysis of the sample yielded that value has prostate cancer or is at risk of prostate cancer progression.

[0123] In the context of this specification, a subject at “risk of progression of prostate cancer” means a subject who may not yet be showing obvious symptoms of prostate cancer, or whose prostate cancer is currently inactive, but who is producing levels of biomarkers indicating that the subject has prostate cancer or is likely to develop it in the near future. A subject who has prostate cancer or is suspected of having prostate cancer may be treated for cancer or suspected cancer.

[0124] As used herein, the term “classification” refers to the assignment of subjects to either be at risk of progression of prostate cancer or not at risk of progression of prostate cancer, based on the results of biomarker scores obtained for the subject.

[0125] As used herein, the term “Wilcoxon rank-sum test” refers to a specific statistical method used for comparing two populations, also known as the Mann-Whitney U test, the Mann-Whitney-Wilcoxon test, or the Wilcoxon-Mann-Whitney test. For example, the test may be used herein to associate an observable trait, particularly a biomarker level, with the risk of the absence or progression of prostate cancer in a particular population.

[0126] As used herein, "true positive rate" refers to the probability that a subject classified as positive by a specific method is truly positive.

[0127] As used herein, “false positive rate” refers to the probability that a subject classified as positive by a particular method is actually negative.

[0128] As used herein, the term “sensitivity” refers to the ability of an assay to correctly identify individuals with a disease (i.e., the true positive rate) in the context of various biochemical assays. In contrast, as used herein, the term “specificity” refers to the ability of an assay to correctly identify individuals without a disease (i.e., the true negative rate) in the context of various biochemical assays. Sensitivity and specificity are statistical measures of the performance (i.e., classification function) of a binary classification test. Sensitivity quantifies the avoidance of false negatives, while specificity quantifies the avoidance of false positives.

[0129] In the use of this specification, “sample” means a test substance to be tested for the presence, and the level or concentration thereof of a biomarker described herein. The sample may include, but is not limited to, blood, serum, plasma, or any part thereof, any suitable substance in accordance with this disclosure.

[0130] In the use of this specification, “metabolites” refer to small molecules that are intermediates and / or products of cellular metabolism. Metabolites may perform a variety of intracellular functions, such as structural, signaling, stimulating, and / or inhibitory effects on enzymes. In some embodiments, metabolites may be non-protein plasma-derived metabolite markers, including but not limited to acetylspermidine, diacetylspermine, lysophosphatidylcholine (18:0), lysophosphatidylcholine (20:3), and indole derivatives.

[0131] As used herein, the term "ROC" refers to receiver operating characteristics, which are graph plots used herein to measure the performance of a particular diagnostic method at various cutoff points. ROC plots may be constructed from the ratio of true positives to false positives at various cutoff points.

[0132] In the context of this specification, the term "AUC" refers to the area under the curve of a ROC plot. AUC can be used to estimate the predictive power of a particular diagnostic test. Generally, a larger AUC indicates higher predictive power and a lower frequency of prediction errors. Possible AUC values ​​range from 0.5 to 1.0, with the latter being characteristic of an error-free prediction method.

[0133] In the context of this specification, the terms “p-value” or “p” refer to the probability that the distributions of biomarker scores for prostate cancer-positive and prostate cancer-negative subjects are identical in the context of the Wilcoxon rank-sum test. Generally, a p-value close to zero indicates that a particular statistical method has high predictive power in classifying subjects.

[0134] As used herein, the term "CI" refers to a confidence interval, that is, an interval within which a particular value is expected to exist with a certain level of confidence. As used herein, the term "95% CI" refers to an interval within which a particular value is expected to exist with a 95% confidence level.

[0135] In the context of this specification, the terms “disease progression” or “early disease progression” are defined as an increase in the Gleason score and / or tumor volume in a surveillance biopsy within 18 months of the commencement of active surveillance.

[0136] In the context of this specification, the terms “inactive disease” or “inactive” prostate cancer are defined as a disease that has not progressed for more than five years after the commencement of active monitoring.

[0137] Abbreviation AKT = RAC serine / threonine protein kinase; AS = active monitoring; AUC = area under the curve; Cav-1 = caveolin-1; CCLE = Broad Institute cancer cell line encyclopedia; CE = cholesterol ester; CM = conditioned medium; DiI = 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine; DP = disease progression; FC = free cholesterol; GS = Gleason score; HexCer = hexosylceramide; HexCer40:0 = hexosylceramide (40:0); HR = hazard ratio; LacCer = lactosylceramide; LacCer32:0 = lactosylceramide (32:0); LacCer36:0 = lactosylceramide (36:0); MAPK = MAP kinase = mitogen-activated protein kinase Protein kinase; PC = phosphatidylcholine; PDMP = 1-phenyl-2-decanoylamino-3-morpholino-1-propanol; PPMP = D-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol; PI3K = phosphoinositide 3-kinase = phosphatidylinositol-3-kinase; RFU = relative fluorescence unit; ROC = receiver operating characteristics; SEM = mean standard error; SFM = serum-free medium; sHDL = synthetic HDL-like particles; sLDL = synthetic LDL-like particles; SM = sphingomyelin; SSALP = synthetic self-assembling lipid particles; TCGA = cancer genome atlas; TO = trioleate; TriHexCer = trihexosylceramide; TriHexCer34:1 = trihexosylceramide (34:1). [Examples]

[0138] The following examples are included to demonstrate embodiments of the present disclosure. These examples are presented solely for illustrative purposes and to assist those skilled in the art in using the present disclosure. The examples are not intended to limit the scope of the disclosure in any sense. Those skilled in the art should understand that many modifications are possible in the particular embodiments disclosed, and that similar or comparable results can still be obtained without departing from the spirit and scope of the invention.

[0139] Example 1: Separation of extracellular vesicles by density gradient suspension. Extracellular vesicles were separated as previously described. Briefly, microvesicles were depleted from the biological specimen samples by centrifugation at 2000 × g for 20 minutes, followed by 16,500 × g for 30 minutes; the resulting supernatant was filtered through a pre-moistened 0.22 μm vacuum filter. The microvesicle-depleted biological specimens were mixed with OptiPrep iodixanol solution (Sigma D1556), compacted to a final density of 1.16–1.30 g / mL, loaded into the bottom of a polycarbonate ultracentrifuge tube, and topped with 0.5–2 mL aliquots of iodixanol / PBS solution in the range of 1.20–1.01 g / mL (35–0% wt:vol), progressing from highest to lowest density as needed, forming a single or multi-step density fractionation gradient. Ultracentrifugation was performed at 8°C and 100,000 × g for 4 hours. Vesicles were collected from the top of the tube and moved downwards until a volume equal to 90% of the superimposed gradient volume was recovered. The density of the recovered fraction was evaluated against a standard curve based on the absorbance of the sample at 250 nm using a NanoDrop microvolume spectrophotometer (ThermoFisher Scientific, Wilmington, Delaware). The harvested vesicles were stored at -80°C.

[0140] Example 2: Proteomic profiling of extracellular vesicles Proteomics profiling of extracellular vesicles was performed according to the following standardized workflow. Briefly, digestion and identification of ECV-derived proteins were performed by LC-MS / MS using an established protocol. A NanoAcquity UPLC system, inline connected to a Waters SYNAPT G2-Si mass spectrometer, was used to separate pooled digested protein fractions. The system was equipped with a Waters Symmetry C18 nanoAcquity trap column (180 μm × 20 mm, 5 μm) and a Waters HSS-T3 C18 nanoAcquity analysis column (75 μm × 150 mm, 1.8 μm). The column oven temperature was set to 50°C, and the tray compartment temperature in the automated sampler was set to 6°C. LC-HDMSE data were acquired in decomposition mode using a Waters Masslynx (version 4.1, SCN 851) with a SYNAPT G2-Si. The capillary voltage was set to 2.80kV, the sampling cone voltage to 30V, the source offset to 30V, and the source temperature to 100°C. For mobility, high-purity N2 was used as the drift gas for the IMS TriWave cell. The pressures of the helium cell, trap cell, IMS TriWave cell, and transfer cell were 4.50 millibars and 2.47 × 10⁻⁶, respectively. -2 , 2.90, and 2.53 × 10 -3The pressure was in millibars. The IMS wave velocity was 600 m / s, the helium cell DC was 50 V, the trap DC bias was 45 V, and the IMS TriWave DC bias V and IMS wave delay were 1000 μs. The mass spectrometer was operated in V mode, and the typical resolution was at least 20,000. All analyses were performed in positive mode ESI using a NanoLockSpray source. The lock mass channel was sampled every 60 seconds. The mass spectrometer was calibrated with [Glu1] fibrinopeptide solution (300 fmol / μL) supplied from the reference sprayer of the NanoLockSpray source. Accurate mass LC-HDMSE data were collected in alternating low-energy (MS) and high-energy (MSE) acquisition modes using a mass scan range of 50–1800 m / z. The spectral acquisition time in each mode was 1.0 sec, and the inter-scan delay was 0.1 sec. In low-energy HDMS mode, data was collected at a constant collision energy of 2 eV in both the trap and transfer cells. In high-energy HDMSE mode, the collision energy was increased from 25 to 55 eV in the transfer cell only. The RF of the quadrupole mass spectrometer was adjusted to efficiently transmit ions in the 300-2000 m / z range, and it was confirmed that all ions observed in LC-HDMSE data below 300 m / z originated from dissociation in the transfer collision cell. The acquired LC-HDMSE data were processed and searched against the protein knowledge database (Uniprot) via the ProteinLynx Global Server (PLGS, Waters Company) at 4% FDR.

[0141] Example 3: Metabolomics Analysis Sample Extraction Following translocation, the cell lysates were washed twice with pre-cooled 0.9% NaCl, followed by the addition of 2.5 mL of pre-cooled 3:1 isopropanol:ulopure water. Cells were scraped off in the extraction solvent using a 25 cm cell scraper (Sarstedt) and transferred to a 15 mL conical tube (Eppendorf). The samples were briefly vortexed and then centrifuged at 4°C and 2,000 × g for 10 minutes. Subsequently, 1.2 mL of metabolite extract was transferred to a 1.5 mL Eppendorf tube and stored at -20°C until metabolomic analysis.

[0142] Primary metabolites and biological amines: Plasma metabolites were extracted from pre-dispensed EDTA plasma (10 μL) using 30 μL of LCMS-grade methanol (ThermoFisher) in a 96-well microplate (Eppendorf). The plate was heat-sealed, vortexed at 750 rpm for 5 minutes, and centrifuged at 2000 × g for 10 minutes at room temperature. The supernatant (10 μL) was carefully transferred to a 96-well plate, leaving the precipitated proteins. The supernatant was further diluted with 10 μL of 100 mM ammonium formate, pH 3. For hydrophilic interaction liquid chromatography (HILIC) analysis, samples were diluted with 60 μL of LCMS-grade acetonitrile (ThermoFisher), while samples for C18 analysis were diluted with 60 μL of water (GenPure ultrapure water system, ThermoFisher). For LCMS analysis, each sample solution was transferred to a 384-well microplate (Eppendorf). For acclimatization, frozen samples were thawed on ice, and 30 μl was transferred to a 96-well microplate (Eppendorf). Aliquots were diluted with an additional 30 μL of 100 mM ammonium formate. The microplates were heat-sealed, vortexed at 1500 rpm for 5 minutes, and centrifuged at 2000 × g for 10 minutes at room temperature. For hydrophilic interaction liquid chromatography (HILIC) analysis, 25 μL of the sample was transferred to a new 96-well microplate containing 75 μL of acetonitrile, while the sample for C18 analysis was transferred to a new 96-well microplate containing 75 μL of water (GenPure ultrapure water system, ThermoFisher). For LCMS analysis, each sample solution was transferred to a 384-well microplate (Eppendorf). 100 μL of cell lysate supernatant (3:1 isopropanol:ultrapure water) was dispensed into two 96-well plates (Eppendorf) and evaporated to dryness under vacuum. Next, the samples were reconstituted as follows: For the HILIC assay, the dried sample was dissolved in 65 μL of ACN (ThermoFisher):100 mM ammonium formate, pH 3 (9:1), while for the C18 reverse-phase assay, the dried sample was dissolved in 65 μL of H2O:100 mM ammonium formate, pH 3 (9:1).The sample was centrifuged to remove insoluble substances and transferred to a 384-well plate for high-throughput mass spectrometry using LC-MS.

[0143] Complex Lipids: A pre-aliquoted EDTA plasma sample (10 μL) was extracted in a 96-well microplate (Eppendorf) using 30 μL of LC-MS grade 2-propanol (Thermo Fisher). The plate was heat-sealed, vortexed at 750 rpm for 5 minutes, and centrifuged at 2000 × g for 10 minutes at room temperature. Leaving the precipitated proteins behind, the supernatant (10 μL) was carefully transferred to a 96-well plate. The supernatant was further diluted with 90 μL of 1:3:2 100 mM ammonium formate, pH 3 (Thermo Fisher):acetonitrile:2-propanol and transferred to a 384-well microplate (Eppendorf) for lipid analysis using LC-MS. For the cell lysates, 10 μL of cell lysate supernatant (3:1 isopropanol:ultrapure water) was diluted in 96-well plates with 90 μL of 1:3:2 100 mM ammonium formate, pH 3:acetonitrile:2-propanol (ThermoFisher), transferred to a 384-well microplate (Eppendorf), and analyzed by LC-MS.

[0144] Non-targeted analysis of primary metabolites and biological amines Non-targeted metabolomics analysis was performed on a Waters Acquity® UPLC system with a 2D column regeneration configuration (Class I and Class H) connected to a Xevo G2-XS quadrupole time-of-flight (qTOF) mass spectrometer. Chromatographic separation was performed using HILIC (Acquity® UPLC BEH amide, 100A, 1.7 μm, 2.1 × 100 mm, Waters Corporation, Milford, USA) and C18 (Acquity® UPLCHSS T3, 100A, 1.8 μm, 2.1 × 100 mm, Waters Corporation, Milford, USA) columns at 45°C. The mobile phases of the quaternary solvent system were (A) 0.1% formic acid in water, (B) 0.1% formic acid in acetonitrile, and (D) 100 mM ammonium formate, pH 3. The samples were separated using the following gradient profiles: For HILIC separation, the initial gradients of 95% B and 5% D were linearly increased over 5 minutes at a flow rate of 0.4 mL / min to 70% A, 25% B, and 5% D, followed by a homogeneous solvent gradient of 100% A at a flow rate of 0.4 mL / min for 1 minute. The chromatographic gradient for C18 separation was as follows: initial condition of 100% A, linear increase to final condition of 5% A and 95% B, followed by a homogeneous solvent gradient of 95% B and 5% D for 1 minute. A binary pump was used for column regeneration and equilibration. The solvent mobile phases were (A1) 100 mM ammonium formate, pH 3, (A2) 0.1% formic acid in 2-propanol, and (B1) 0.1% formic acid in acetonitrile. The HILIC column was stripped with 90% A2 for 5 minutes and equilibrated with 100% B1 at a flow rate of 0.3 mL / min for 2 minutes. Reverse-phase C18 column regeneration was performed using 95% A1 and 5% B1 for 2 minutes, followed by column equilibration using 5% A1 and 95% B1 for 5 minutes.

[0145] Non-targeted analysis of complex lipids: In the lipidomics assay, targeted metabolomics analysis was performed on a Waters Acquity® ULC system connected to a Xevo G2-XS quadrupole time-of-flight (qTOF) mass spectrometer. Chromatographic separation was performed at 55°C using a C18 column (Acquity® ULC HSS T3, 100A, 1.8 μm, 2.1 × 100 mm, Water Corporation, Milford, USA). The mobile phases were (a) water, (b) acetonitrile, (c) 2-propanol, and (d) 500 mM ammonium formate, pH 3. The initial elution gradients of 20%A, 30%B, 49%C, and 1%D were linearly increased over 5.5 minutes to 10%B, 89%C, and 1%D, followed by homogeneous solvent elution at 10%B, 89%C, and 1%D for 1.5 minutes, and equilibration to the initial conditions for 1 minute.

[0146] Example 4: Mass Spectrometry Data acquisition. Mass spectrometry data were acquired using positive and negative electrospray ionization sensitivity modes, within the range of 50–1200 Da for primary metabolites and 100–2000 Da for complex lipids. For electrospray acquisition, the capillary voltage was set to 1.5 kV (positive) and 3.0 kV (negative), the sample cone voltage to 30 V, the source temperature to 120 °C, the cone gas flow rate to 50 L / h, the desolvation gas flow rate to 800 L / h, the scan time to 0.5 seconds, and continuous mode. For rock-spray correction, leucine enkephalin; 556.2771 Da (positive) and 554.2615 Da (negative) were used, and the scan was performed at 0.5 minutes. The injection volume of each sample was 3 μL unless otherwise specified. Acquisition was performed using the instrument's automatic gain control to optimize the instrument's sensitivity over the sample acquisition time.

[0147] Data Processing: LC-MS and LC-MSe data were processed using Progenesis QI (nonlinear, Waters), and values ​​were reported in area units. Annotations were determined by matching accurate mass and retention times using a customized library created from genuine standards, and / or by matching experimental tandem mass spectrometry data against theoretical fragmentation of NIST MSMS or HMDB v3.

[0148] Data Normalization: To compensate for variations in injection order, each characteristic was normalized using data from repeated injections of quality control samples collected every 10 injections throughout the entire run order. Measurement data were smoothed by Locally Weighted Scatter Smoothing (LOESS) signal correction (QC-RLSC), as previously described. Feature values ​​between quality control samples were interpolated using cubic splines. Metabolite values ​​were rescaled using the overall median of historical quality control peak areas across all samples. Only detected characteristics exhibiting a relative standard deviation (RSD) of less than 30 in either historical or pooled quality control samples were considered for further statistical analysis. To reduce the complexity of the data matrix, annotated characteristics with multiple adducts or acquisition mode repetitions were combined into a single representative intrinsic characteristic. Characteristics were selected based on reproducibility (RSD < 30), the highest intensity matching the theoretical isotopic distribution, and the highest isotopic similarity. The values ​​are reported as a percentage of the historical quality control reference sample included in all analytical runs (plasma / conditioned medium) or adjusted area units (lysates).

[0149] Statistical Analysis: The method described above was used to find the cutoff point of the covariate that gives the maximum difference between individuals in two already defined groups. Using log-rank statistics based on the groups defined by the cutoff, the following becomes possible:

number

Number

Number

[0150] To calculate the p-value of this test, the following equation was used to obtain a value of 0.009. This suggests that the Cav-1 sphingolipid signature level is highly associated with the progression-free survival period. p-value ≈ 2exp(-2Q 2 ) where

Number

Number

[0151] Example 5: Prediction of AS Gleason grade progression by plasma lipid signature Non-targeted metabolomics analysis was performed on clinically matched baseline plasma samples (n=16 per group) prospectively collected from clinically low-risk early prostate cancer patients undergoing AS who presented with early DP or inactive disease (Table 1). A total of 269 unique annotated metabolite characteristics were identified in the baseline plasma of the discovery cohort; 14 characteristics showed statistically significant ROC AUC values ​​of <0.7 (unadjusted Wilcoxon rank-sum test, p<0.05).

[0152] [Table 1]

[0153] Seven of the 14 characteristics were complex lipids; in particular, sphingomyelin and sphingolipids (Table 2).

[0154] [Table 2]

[0155] [Table 3]

[0156] [Table 4]

[0157] [Table 5]

[0158] [Table 6]

[0159] [Table 7]

[0160] [Table 8]

[0161] [Table 9]

[0162] [Table 10]

[0163] After adjusting for multiple hypothesis tests, none of the 14 characteristics remained individually significant, however, the significantly elevated lipids were biochemically related to ceramide metabolism, suggesting a regulated signal. Furthermore, sphingomyelin and sphingoglycolipids were generally elevated in baseline plasma samples from progressive patient cases (early DP) compared to controls (no disease progression for at least 5 years after AS initiation) (Figure 1(a)). Importantly, detected SM and sphingoglycolipids remained elevated in DP compared to tracking time-matched subjects. No statistically significant differences were observed in within-case comparisons of SM and sphingoglycolipids at baseline versus 12 months (Figure 2). In light of the known lipid transport function of Cav-1 (Cheng 2016), the observed elevation of plasma sphingolipid signatures suggested a biological association with previous findings of elevated plasma Cav-1 in association with disease progression. To investigate this, we extended the analysis to include non-targeted metabolomics profiling of 459 baseline plasma samples prospectively collected from patients with early-stage prostate cancer undergoing AS (Table 3).

[0164] [Table 11]

[0165] Consistent with findings in the initial discovery cohort, several SMs and sphingoglycolipids were positively associated with GS-based DP (hazard ratio greater than 1.5) (Figure 1(b); Table 4).

[0166] [Table 12]

[0167] [Table 13]

[0168] [Table 14]

[0169] [Table 15]

[0170] [Table 16]

[0171] [Table 17]

[0172] [Table 18]

[0173] Next, using a logistic regression model, we developed a signature panel consisting of six sphingoglycolipids—SM(40:2), SM(44:2), lactosylceramide(32:0), lactosylceramide(36:0), trihexosylceramide(34:1), and hexosylceramide(40:0)—that showed positive β estimates in both plasma Cav-1 and the logistic regression model. Using the log-rank test statistic of the Cox model, we calculated the optimal cutoff point for the plasma Cav-1-sphingolipid signature where the difference between subjects who experienced disease progression (defined as increased GS and / or increased tumor volume) and those who did not was maximized. This yielded a cutoff value of 4.33. The association with signature progression-free survival was evaluated using the Cox proportional hazards model. In a multivariate analysis adjusted for age, 5α-reductase therapy, and baseline tumor volume, AS subjects with a plasma Cav-1-sphingolipid signature score greater than 4.33 showed statistically significantly worse DP-free survival compared to patients with a plasma Cav-1-sphingolipid signature score of 4.33 or less (HR: 2.70, 95% CI: 1.75–4.16, p: <0.001) (Table 5). In particular, the non-proportional hazards model test yielded a non-significant p-value. Relatedly, in this analysis, BMI was not associated with an increased risk of DP (HR: 1.02, 95% CI: 0.40–2.64, p: 0.965), suggesting that this lipid index is not biased by obesity.

[0174] [Table 19]

[0175] Kaplan-Meier survival curves illustrating progression-free survival for participants with plasma Cav-1-sphingoglycolipid signature scores below the cutoff (<4.33) or above the cutoff (≧4.33) are shown in Table 6 and Figure 1(c).

[0176] [Table 20]

[0177] To further clarify the relationship between lipid metabolism and Cav-1 in the context of the Cav-1 sphingolipid signature, we first compared gene expression profiles reflecting lipid management machinery and CAV1 mRNA expression in 333 prostate tumors using the Cancer Genome Atlas (TCGA). High CAV1 mRNA expression was found to be positively associated with genes annotated with ontology related to lipid cleavage and metabolism, glycosylceramide metabolic processes, and the ceramide pathway. Next, we investigated the association between elevated CAV1 and associated lipid management machinery and previously defined molecular subtypes (iClusters) of primary prostate cancer. The results showed that high CAV1 mRNA expression was mainly associated with iCluster 3, which is characterized by elevated PI3K / AKT, MAP-kinase, and receptor tyrosine kinase activity.

[0178] Example 6: Relationship between Cav-1 and high-lipid deficiency phenotype Next, we evaluated the response of Cav-1 to the availability of extracellular lipids. Comparing lipid-containing serum-free medium with lipid-free serum-free medium, lipid deficiency reduced Cav-1 protein expression in RM-9 and PC-3M prostate cancer cell lines (Figure 3(a) and Figure 3(b)). In particular, the presence of apolipoproteins, cholesterol, or cholesterol esters was not necessary to maintain elevated Cav-1 protein levels, suggesting that Cav-1 responds specifically to the phospholipid components of extracellular lipid complexes (Figure 3(a) and Figure 3(b)).

[0179] Next, we investigated the involvement of Cav-1 in lipid uptake. To achieve this objective, we then evaluated the ability of Cav-1-low (Figure 3(b)) LNCaP and Cav-1-positive (Figure 3(a)) PC-3M and RM-9 prostate cancer cell lines to remove extracellular fluorescent DiI-coupled SSALP. PC-3M and RM-9 prostate cancer cells showed substantially higher fluorescence accumulation compared to LNCaP (Figure 3(d)).

[0180] Example 7: Regulation of sphingoglycolipid biosynthesis by Cav-1 Next, baseline lipid profiles of LNCaP and PC-3M cells were compared, as well as lipid profiles following Cav-1 overexpression or transient knockdown of Cav-1, respectively. Immunoblots comparing total cell lysate Cav-1 levels in LNCaP and PC-3M cells following Cav-1 overexpression or transient knockdown of Cav-1 are provided in Figure 3(b). Compared to baseline LNCaP, baseline PC-3M cells showed elevated levels of triacylglycerols, cholesterol esters, and lysophospholipids, but decreased levels of sphingolipids. Cav-1 overexpression resulted in a significant overall increase in levels of major lipid classes, while Cav-1 knockdown resulted in a significant decrease in phospholipids, diacylglycerols, sphingomyelin, and sphingoglycolipids, particularly lactosylceramide (Figures 4(a) and 4(b)). Next, the relationship between Cav-1 and key enzymes in ceramide metabolism was evaluated using CCLE and TCGA gene expression datasets. For CCLE data, prostate cancer cell lines were stratified based on mean CAV1 gene expression into either high-CAV1-expressing cell lines (log2 value > 11 (range 11-01 to 13.61); HPrEC, DU145, PC-3) or low-CAV1-expressing cell lines (log2 value < 7 (range 4.16 to 6.88); NCIH660, MDAPCa2B, LNCaP, VCaP, CWR22Rv1). TCGA data for prostate adenocarcinoma were stratified to the highest or lowest CAV1 expression quartile, and the association between CAV1 mRNA expression and mRNA expression of genes involved in sphingolipid metabolism in the most different populations was evaluated. Table 7 shows Spearman correlation analysis based on the entire TCGA prostate adenocarcinoma dataset using continuous values ​​of CAV1 mRNA expression and genes involved in sphingolipid metabolism. Compared to prostate cancer cell lines and prostate tumors with low CAV1 mRNA levels, those exhibiting high CAV1 mRNA levels also tend to show reduced mRNA expression levels for genes involved in ceramide biosynthesis, including dihydroceramide desaturase (DEGS), ceramide synthase (CERS), and sphingomyelinase (SPMD) (Figures 5(a) and 5(b)).In contrast, mRNA expression of enzymes involved in sphingoglycolipid biosynthesis, including glucosylceramide synthase (UCGC) and lactosylceramide synthases B4GALT5 and B4GALT6, was elevated in CAV1 hyperprostate cancer cell lines and prostate tumors.

[0181] [Table 21]

[0182] [Table 22]

[0183] Example 8: Sphingomyelin as a source of ceramides and sphingoglycolipids Investigation of extracellular sphingomyelin uptake as a potential source of ceramides and their sphingoglycolipid derivatives (via glycosylation). Ceramides are induced primarily through three metabolic pathways: de novo, recycling, or salvage (Figure 6). Ceramide biosynthesis via the salvage pathway is mediated by hydrolysis of sphingomyelin via sphingomyelinase.

[0184] PC-3M, RM-9, and LNCaP prostate cancer cells were treated with SSALP containing sphingomyelin (d18:1 / 18:1)-deuterium(d)9 for 48 hours. The biochemical fate of this compound was tracked using liquid chromatography-mass spectrometry (Figure 7(a)). The ceramide (18:1 / 18:1)-d9 isotope substitution was detected in all three cell lines, while the glucosylceramide (18:1 / 18:1)-d9 isotope substitution was detected only in the PC-3M and RM-9 prostate cancer cell lines. In particular, based on peak area, the ratio of ceramide (18:1 / 18:1)-d9 to sphingomyelin (18:1 / 18:1)-d9 was considerably higher in PC-3M (ratio: 0.14) and RM-9 (ratio: 0.23) compared to LNCaP (ratio: 0.02), indicating a higher overall metabolic flow to ceramide biosynthesis via sphingomyelin salvage (Figure 7(b)). Neither oleic acid-d9 nor ceramide (18:1 / 18:1-d9)-1-phosphate was observed, suggesting that ceramide derived from sphingomyelin is preferentially allocated to the sphingoglycolipid pathway rather than hydrolysis or phosphorylation by ceramidase. These findings provide direct biochemical evidence that sphingomyelin is indeed a source of ceramide and their glycosylated derivatives (Figure 4(b)).

[0185] Example 9: Promotion of mitochondrial components by Cav-1. The above findings suggest a Cav-1-related mechanistic framework that induces ceramide pools into sphingoglycolipids, consistent with the observation that sphingoglycolipids, particularly lactosylceramide, are important features of the plasma sphingolipid signature (Figure 1(b)). Ceramides are bioactive sphingolipids that are actively involved in mediating cell death, including inducing apoptosis via cytochrome c release from mitochondria and inducing lethal mitophagy by targeting tochondria to autophagosomes. Next, we investigated the relationship between Cav-1 and mitochondrial morphology in both PC-3M (high Cav-1) and LNCaP (low Cav-1) cell lines. PC-3M cells showed a more branched, fusion-like mitochondrial structure, and lysosome staining was also diffuse, while mitochondrial and lysosome morphology was more scattered in LNCaP cells (Figure 8(a)). The differential transport of sphingomyelin in PC-3M cells following CAV1 knockdown was evaluated using fluorescently labeled SSALP containing C11 TopFluor-SM. CAV1 knockdown resulted in a statistically significant (Tukey multiple comparison test, two-sided adjusted p<0.001) decrease in the uptake of C11 TopFluor-SM-containing SSALP (Figure 8(b) and Figure 8(d)). Notably, CAV1 knockdown in PC-3M also resulted in punctate mitochondria accumulation (Figure 9; Figures 10(a) and 10(b)) and decreased lysosome proliferation (Figure 9); these changes were accompanied by an increase in reactive oxygen species (Tukey multiple comparison test, two-sided adjusted p<0.001), as assessed by MitoTracker Red CMXRos (Poot 1996) and CellROX Deep red (Figure 11, Figure 10(c)).

[0186] Example 10: Release of EVs rich in Cav-1-sphingomyelin / lactosylceramide. It was previously demonstrated that secretion of membrane-bound Cav-1 by prostate cancer cells has been previously demonstrated. Consistent with this report, evaluation of cell culture medium (CM) confirmed detectable levels of Cav-1 in PC-3M and RM-9 prostate cancer cell lines, but not in LNCaP. Isolation of extracellular lipid vesicles (EVs) from LNCaP and PC-3M CM following overexpression of Cav-1 or knockdown of Cav-1 confirmed the presence of Cav-1 on EVs (Figure 8(d), Figure 12(a), and Figure 12(b)). In particular, the amount of Cav-1-containing EVs was considerably higher when the culture medium was supplemented with exogenous low-density lipoprotein (Figure 12(b)), consistent with previous observations that lysate Cav-1 protein expression is responsive to the availability of extracellular lipids. (Figure 3(a). Consistently, in LNCaP following Cav-1 overexpression, the concentration of EV particles in CM was statistically significantly higher compared to the respective control groups (comparison of areas under the curve with a two-sided Student t-test p:0.02), while the number of EVs in CM from PC-3M following CAV1 knockdown was statistically significantly reduced (comparison of areas under the curve with a two-sided Student t-test p<0.001) (Figures 12(c) and 12(d))). Furthermore, using density gradient fractionation, it was determined that Cav-1-containing EVs were most concentrated in the fraction of plasma high-density lipoprotein suspension densities in the range of 1.06–1.15 g / mL, suggesting that secreted Cav-1 is present in HDL-like lipid protein particles. Analysis of the CM lipidomes of LNCaP and PC-3M following Cav-1 overexpression or Cav1 knockdown, respectively, also showed increases in the relative abundances of sphingomyelin and lactosylceramide, which are dependent on Cav-1 and extracellular lipid bioavailability (Figures 13(a) and 13(b)).

[0187] To determine the lipid composition and protein load of extracellular proteins (EVs), lipidomics and proteomics analyses were performed using mass spectrometry on EVs derived from LNCaP and PC-3M, respectively. Consistent with the findings of other researchers, analysis of the EV lipidome confirmed particularly high concentrations of sphingolipids and phosphatidylcholine. Interestingly, cardiolipin, an important lipid component of the inner mitochondrial membrane, was found to be present in EVs derived from prostate cancer cell lines. Evaluation of the EV proteome identified 237 and 341 (abundances of 5 spectra or more) highly reliable proteins in EVs derived from LNCaP and PC-3M, respectively. To investigate the functional aspects of protein properties, we performed intracellular localization analysis by filtering genes (with a confidence score of 2 or more) that were confidently assigned to at least one of 11 intracellular localizations: nucleus, cytosol, cytoskeleton, peroxisomes, lysosomes, endoplasmic reticulum, Golgi apparatus, plasma membrane, endosomes, extracellular space, and mitochondria, based on COMPARTMENTS localization evidence database scores [doi.org / 10.1093 / database / bau012] (Figure 14). Intracellular localization analysis of EV-derived protein properties demonstrated the presentation of proteins annotated as localizing to mitochondria (Figure 13(c); Figure 14). IPA analysis of 341 protein properties detected in PC-3M-derived EVs revealed caveolae-mediated endocytosis and phagocytic maturation as top networks, while apoptosis and necrosis were reduced and cell motility, degranulation, and cell proliferation were activated as top disease functions (Tables 8 and 9).

[0188] [Table 23]

[0189] [Table 24]

[0190] These findings suggest that prostate cancer cells vigorously secrete extracellular proteins (EVs) rich in Cav-1-containing sphingolipids, which have a diverse repertoire of proteins, including mitochondrial-related proteins and lipids. Based on these findings, Cav-1-mediated uptake of sphingomyelin (Figures 8(b) and 8(c)), conversion of sphingomyelin to ceramide and subsequent sphingoglycolipid derivatives (Figure 7(b)), and inclusion of mitochondrial proteins in the EV cargo (Figures 12(c), 12(d), and 14) appear to be related to the clearance of mitochondrial components.

[0191] Example 11: Targeting of a shunt from ceramide to sphingoglycolipid These findings regarding the adaptive Cav-1-mediated sphingoglycolipid mechanism suggest that targeting of ceramide-to-sphingoglycolipid conversion may represent a practical metabolic vulnerability in prostate cancer. To test this hypothesis, we evaluated the efficacy of three different inhibitors of glucosylceramide synthase (UGCG; also known as UDP-glucose:ceramideglucosyltransferase), the rate-limiting enzyme in sphingoglycolipid metabolism, PDMP, PPMP, and eliglustat, against reduced viability in vivo for RM-9 and PC-3M prostate cancer cells. Treatment of RM-9 and PC-3M prostate cancer cells with PDMP, PPMP, and eliglustat resulted in dose-dependent cytotoxicity (Figure 15). Next, we evaluated the changes in lipidome following pharmacological inhibition of UGCG in RM-9 and PC-3M prostate cancer cells. RM-9 and PC-3M cells were challenged with PDMP, PPMP, eliglustat, or a vehicle and evaluated after 6 hours of treatment to capture initial metabolic changes, particularly in sphingolipid metabolism, and to mitigate the effects of secondary events that may lead to an increase in the ceramide pool and the decrease in GLS expression that may result from reduced cell viability. Short-term (6-hour) challenge of RM-9 and PC-3M prostate cells with PDMP, PPMP, or eliglustat resulted in the accumulation of intracellular ceramides, acylcarnitines, lysophospholipids, and diacylglycerols, as well as a decrease in sphingoglycolipids, phospholipids, and triacylglycerols (Figures 15 and 16). In particular, the acute cytotoxic effect of eliglustat was mediated via a non-apoptotic mechanism (Figure 16(a)). The decrease in phospholipids and triacylglycerols, coupled with an increase in downstream catabolisms, suggested mitophagy. Consistent with this, treatment of PC-3M cells with eliglustat increased the protein expression of the mitophagy-related marker LC3B-II (Figure 14).Evaluation of mitochondrial morphology in PC-3M cells following acute (6-hour) treatment with eliglustat revealed loss of branched, fusion-like mitochondrial structures and accumulation of punctate mitochondria co-localized with lysosomes (Figure 17); these changes, accompanied by increased Parkin and PINK1 protein expression, further suggest increased mitophagy. Previous reports have demonstrated that ceramides induce lethal mitophagy by targeting autophagosomes to mitochondria. In particular, knockdown of CAV1 or pretreatment with a Cav-1 specific monoclonal antibody (Cav-1 mAb) further sensitized PC-3M cells to eliglustat, while overexpression of Cav-1 in LNCaP reduced the anticancer effect of eliglustat (Figure 18).

[0192] Example 12: Inhibition of RM-9 tumor growth by eliglustat Next, we investigated the inhibition of tumor growth by eliglustat. RM-9 cells possess the driving mechanisms of oncogenic RAS and MYC genes and model RAS-MAPK pathway activation and MYC-driven transcriptional activity associated with invasive primary prostate cancer. RM-9-luciferase cells were transplanted subcutaneously into C57BL / 6N mice. RM-9 tumor growth was suppressed by eliglustat (Figure 19(a), Figure 19(b), and Figure 19(c)). Metabolomics analysis of tumor tissue from all treatment groups showed that eliglustat was associated with a decrease in sphingoglycolipids in RM-9 tumor-carrying mice (Figure 19(d)). Immunohistochemical analysis of tumor tissue showed that treatment with eliglustat statistically significantly reduced Cav-1 and PCNA staining (two-sided Wilcoxon rank-sum test, p:0.008 and 0.001, respectively), while BrdU-TUNEL and mitophagy-related marker (LC3B and HMGB1) staining were statistically significantly increased (two-sided Wilcoxon rank-sum test, p:0.008 for all three markers) (Figure 20). In particular, these results indicated that RM-9 tumors were associated with a plasma lipid signature similar to that observed in prostate cancer patients, including elevated levels of several sphingomyelins and sphingoglycolipids (Figure 20). Furthermore, RM-9 tumor-carrying mice tended to have elevated plasma Cav-1 concentrations compared to control mice. Interestingly, plasma levels of Cav-1 and identified lipid species that are part of the Cav-1 sphingolipid signature increased in RM-9 tumor-carrying mice upon treatment with eliglustat. Treatment of RM-9 tumor-carrying mice with eliglustat also resulted in a statistically significant increase in plasma ceramide (two-sided Wilcoxon rank-sum test p: 0.004), suggesting that the elevation of plasma Cav-1 and sphingolipids may be a consequence of cell death (Figure 19(a), Figure 19(b), and Figure 19(c)).

[0193] Example 13: Calculation of Biomarker Score Using the measured concentrations of plasma Cav-1 sphingolipid signature properties, biomarker scores were calculated based on a logistic regression model. In this model of prostate cancer progression, the values ​​of plasma analytes signature properties were combined into the model and applied to predict the risk of disease progression.

[0194] The table below shows the ratio between the actual hazard and the baseline hazard rate, as well as the hazard rates at various cutoff points of the biomarker panel score. Here, hazard is defined as the risk of an event (i.e., disease progression) as a function of time, and a hazard rate greater than 1 means a short time to disease progression.

[0195] The table shows two things as functions of the model score: firstly, the progression risk relative to the “baseline hazard rate” calculated for a particular signature; secondly, considering the signature score as a classifier cutpoint; and finally, the last column shows the relative risk of disease progression between the high-scoring group and the low-scoring group, i.e., the “hazard ratio”.

[0196] The first column of the table shows the biomarker panel scores for the actively monitored cohort; the second column shows the change in actual hazards relative to baseline hazards; and the third column shows the hazard ratio and corresponding 95% confidence interval based on a population dichotomy using different cutoff points for the biomarker panel scores.

[0197] [Table 25]

[0198] [Table 26]

[0199] [Table 27]

[0200] [Table 28]

[0201] [Table 29]

[0202] [Table 30]

[0203] [Table 31]

[0204] [Table 32]

[0205] [Table 33]

[0206] [Table 34]

[0207] [Table 35]

[0208] Example 14: Verification Test To assess the risk of prostate cancer progression in men under active surveillance for prostate cancer, an independent validation study was conducted using a cohort of 248 participants (35 with advanced disease and 213 with non-advanced disease). Levels of TrihexosylCer(34:1), LactosylCer(36:0), LactosylCer(32:0), SM(44:2), SM(40:2), plasma sphingolipid signature (SphingoSignature), and simplified sphingolipid signature [consisting of simplified signature, TrihexosylCer(34:1), LactosylCer(36:0), and SM(40:2)] were examined in each participant. Cav-1 was not included in this analysis. Models were derived using fixed coefficients from logistic regression as previously described.

[0209] TrihexosylCer(34:1) and SM(40:2) were found to have statistically significant odds ratios per unit increase, along with a simplified sphingolipid signature, while hexosylceramide(40:0) could not be quantified in the cohort sample (see Figure 21).

[0210] Other embodiments The above detailed description is provided to assist those skilled in the art in implementing this disclosure. However, the disclosure described and claimed herein is not limited in scope by any particular embodiment disclosed herein, as these embodiments are intended to be illustrative of some aspects of the disclosure. Any equivalent embodiment is intended to be within the scope of this disclosure. In fact, in addition to those shown and described herein, various modifications of the disclosure will be apparent to those skilled in the art from the foregoing description, which does not deviate from the spirit or scope of the discovery of the invention. Such modifications are also intended to be within the scope of the appended claims. This specification includes the following disclosures: (Note 1) (a) A step of measuring the levels of caveolin-1 (CAV-1), sphingomyelin 40:2 (SM40:2), sphingomyelin 44:2 (SM44:2), lactosylceramide 32:0 (LacCer32:0), lactosylceramide 36:0 (LacCer36:0), trihexosylceramide 34:1 (TriHexCer34:1), and hexosylceramide 40:0 (HexCer40:0) in a biological sample from the subject using an in vitro assay; (b) A step of comparing the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in the sample with a reference. Consists of, - Classify individuals with prostate cancer as either at risk of developing invasive prostate cancer or not at risk of developing invasive prostate cancer. - Predicting the predisposition to invasive prostate cancer in the target patient. - To diagnose invasive prostate cancer in subjects with prostate cancer, - Determining the risk of the subject having invasive prostate cancer. - Predicting the likelihood of prostate cancer progression in individuals with prostate cancer. - To provide a prognosis to subjects with prostate cancer, or - Select patients with prostate cancer for treatment with anticancer therapy. It is a method, The amounts of change in CAV-1, SM(40:2), SM(44:2), LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 compared to the aforementioned reference are: - An indicator indicating whether the subject is at risk of developing invasive prostate cancer or not, - Indicators of predisposition to the aforementioned target for invasive prostate cancer, - An indicator of the likelihood of prostate cancer progression in the aforementioned subjects, - An indicator of progression-free survival for the aforementioned subjects, - An indicator of the probable outcome of the treatment of the aforementioned prostate cancer, - An indicator that the subject is a candidate for treatment with anti-cancer therapy. A method that provides an index selected from a group consisting of the following. (Note 2) (a) A step of measuring the levels of sphingomyelin 40:2 (SM40:2), sphingomyelin 44:2 (SM44:2), lactosylceramide 32:0 (LacCer32:0), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 (TriHexCer34:1) in a biological sample from the subject using an in vitro assay, (b) The step of comparing the levels of SM40:2, SM44:2, LacCer32:0, LacCer36:0, and TriHexCer34:1 in the sample with a reference, - Classify individuals with prostate cancer as either at risk of developing invasive prostate cancer or not at risk of developing invasive prostate cancer. - Predicting the predisposition to invasive prostate cancer in the target patient. - To diagnose invasive prostate cancer in subjects with prostate cancer, - Determining the risk of the subject having invasive prostate cancer. - Predicting the likelihood of prostate cancer progression in individuals with prostate cancer. - To provide a prognosis to subjects with prostate cancer, or - Select patients with prostate cancer for treatment with anticancer therapy. It is a method, The change in the quantities of SM40:2, SM44:2, LacCer32:0, LacCer36:0, and TriHexCer34:1 relative to the aforementioned reference is - An indicator indicating whether the subject is at risk of developing invasive prostate cancer or not, - Indicators of predisposition to the aforementioned target for invasive prostate cancer, - An indicator of the likelihood of prostate cancer progression in the aforementioned subjects, - An indicator of progression-free survival for the aforementioned subjects, - An indicator of the probable outcome of the treatment of the aforementioned prostate cancer, - An indicator that the subject is a candidate for treatment with anti-cancer therapy. A method that provides an index selected from a group consisting of the following. (Note 3) (a) A step of measuring the levels of sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 (TriHexCer34:1) in a biological sample from the subject using an in vitro assay, (b) A step of comparing the levels of SM40:2, LacCer36:0, and TriHexCer34:1 in the sample with a reference. Consists of, - Classify individuals with prostate cancer as either at risk of developing invasive prostate cancer or not at risk of developing invasive prostate cancer. - Predicting the predisposition to invasive prostate cancer in the target patient. - To diagnose invasive prostate cancer in subjects with prostate cancer, - Determining the risk of the subject having invasive prostate cancer. - Predicting the likelihood of prostate cancer progression in individuals with prostate cancer. - To provide a prognosis to subjects with prostate cancer, or - A method for selecting subjects with prostate cancer for treatment with anticancer therapy, The changes in the amounts of SM40:2, LacCer36:0, and TriHexCer34:1 relative to the aforementioned reference are: - An indicator indicating whether the subject is at risk of developing invasive prostate cancer or not, - Indicators of predisposition to the aforementioned target for invasive prostate cancer, - An indicator of the likelihood of prostate cancer progression in the aforementioned subjects, - An indicator of progression-free survival for the aforementioned subjects, - An indicator of the probable outcome of the treatment of the aforementioned prostate cancer, - An indicator that the subject is a candidate for treatment with anti-cancer therapy. A method that provides an index selected from a group consisting of the following. (Note 4) (a) A step of measuring the level of trihexosylceramide 34:1 (TriHexCer34:1) in a biological sample from the subject using an in vitro assay, (b) A step of comparing the level of TriHexCer34:1 in the sample with a reference. Consists of, - Classify individuals with prostate cancer as either at risk of developing invasive prostate cancer or not at risk of developing invasive prostate cancer. - Predicting the predisposition to invasive prostate cancer in the target patient. - To diagnose invasive prostate cancer in subjects with prostate cancer, - Determining the risk of the subject having invasive prostate cancer. - Predicting the likelihood of prostate cancer progression in individuals with prostate cancer. - To provide a prognosis to subjects with prostate cancer, or - Select patients with prostate cancer for treatment with anticancer therapy. It is a method, The change in the amount of TriHexCer34:1 relative to the aforementioned reference is, - An indicator indicating whether the subject is at risk of developing invasive prostate cancer or not. - Indicators of predisposition to the aforementioned target for invasive prostate cancer, - An indicator of the likelihood of prostate cancer progression in the aforementioned subjects, - An indicator of progression-free survival for the aforementioned subjects, - An indicator of the probable outcome of the treatment of the aforementioned prostate cancer, - An indicator that the subject is a candidate for treatment with anti-cancer therapy. A method that provides an index selected from a group consisting of the following. (Note 5) (a) A step of measuring the level of sphingomyelin 40:2 (SM40:2) in a biological sample from the subject using an in vitro assay, (b) A step of comparing the level of SM40:2 in the sample with a reference; Consists of, - Classify individuals with prostate cancer as either at risk of developing invasive prostate cancer or not at risk of developing invasive prostate cancer. - Predicting the predisposition to invasive prostate cancer in the target patient. - To diagnose invasive prostate cancer in subjects with prostate cancer, - Determining the risk of the subject having invasive prostate cancer. - Predicting the likelihood of prostate cancer progression in individuals with prostate cancer. - To provide a prognosis to subjects with prostate cancer, or - Select patients with prostate cancer for treatment with anticancer therapy. It is a method, The change in the amount of SM40:2 relative to the aforementioned reference is, - An indicator indicating whether the subject is at risk of developing invasive prostate cancer or not, - Indicators of predisposition to the aforementioned target for invasive prostate cancer, - An indicator of the likelihood of prostate cancer progression in the aforementioned subjects, - An indicator of progression-free survival for the aforementioned subjects, - An indicator of the probable outcome of the treatment of the aforementioned prostate cancer, - An indicator that the subject is a candidate for treatment with anti-cancer therapy. A method that provides an index selected from a group consisting of the following. (Note 6) (a) Using an in vitro assay, the biological sample from the subject - Sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1; and / or - Trihexosylceramide 34:1; and / or - A step to measure the level of sphingomyelin 40:2 (SM40:2), (b) A step of comparing the level of SM40:2 in the sample with a reference; Consists of, - Classify individuals with prostate cancer as either at risk of developing invasive prostate cancer or not at risk of developing invasive prostate cancer. - Predicting the predisposition to invasive prostate cancer in the target patient. - To diagnose invasive prostate cancer in subjects with prostate cancer, - Determining the risk of the subject having invasive prostate cancer. - Predicting the likelihood of prostate cancer progression in individuals with prostate cancer. - To provide a prognosis to subjects with prostate cancer, or - Select patients with prostate cancer for treatment with anticancer therapy. It is a method, The change in the amount of SM40:2 relative to the aforementioned reference is, - An indicator indicating whether the subject is at risk of developing invasive prostate cancer or not, - Indicators of predisposition to the aforementioned target for invasive prostate cancer, - An indicator of the likelihood of prostate cancer progression in the aforementioned subjects, - An indicator of progression-free survival for the aforementioned subjects, - An indicator of the probable outcome of the treatment of the aforementioned prostate cancer, - An indicator that the subject is a candidate for treatment with anti-cancer therapy. A method that provides an index selected from a group consisting of the following. (Note 7) The method according to any one of the appendices 1 to 6, wherein the subject has prostate cancer. (Note 8) The method according to any one of the appendices 1 to 6, wherein the subject is determined to be positive for the level of prostate cancer antigen (PCA), which indicates the presence of prostate cancer. (Note 9) The method described in any one of the appendices 1 to 6, wherein the subject is under active monitoring (AS) for disease progression. (Note 10) The method according to any one of the appendices 1 to 6, wherein the subject is classified as having clinically low-risk early-stage prostate cancer under active surveillance, exhibiting early disease progression (DP) or inactive disease. (Note 11) The method described in any one of the appendices 1 to 6, wherein the subject is not receiving treatment for prostate cancer. (Note 12) The method according to any one of the appendices 1 to 11, wherein the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are elevated in the subject compared to healthy individuals. (Note 13) The method described in any one of the appendices 1 to 11, wherein the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are elevated compared to the levels of a reference subject or group without invasive prostate cancer. (Note 14) The method according to any one of the appendices 1 to 11, wherein the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are elevated compared to the levels of a reference subject or group having inactive prostate cancer. (Note 15) The method according to any one of the appendices 1 to 14, wherein the measurement of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 is performed by ultraviolet-visible spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, gas chromatography-mass spectroscopy (GC-MS), liquid chromatography-mass spectroscopy (LC-MS), high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UPLC), liquid chromatography-mass spectroscopy (LC-MS), correlation spectroscopy (COSy), nuclear Overhauser effect spectroscopy (NOESY), rotating-frame nuclear Overhauser effect spectroscopy (ROESY), LC-TOF-MS, LC-MS / MS, and capillary electrophoresis-mass spectroscopy. (Note 16) The method according to Appendix 15, wherein the measurement of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 is performed by high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UPLC), or liquid chromatography-mass spectrometry (LC-MS). (Note 17) The method according to any one of the appendices 1 to 16, wherein the biological sample is selected from serum and plasma. (Note 18) The method according to Appendix 17, wherein the biological sample comprises a fraction of the sample rich in extracellular vesicles (EVs). (Note 19) The method according to any one of the appendices 1 to 18, wherein the measurements of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are performed at substantially the same time. (Note 20) The method according to any one of the appendices 1 to 19, wherein the measurement of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 is performed in a stepwise manner. (Note 21) a) If the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are greater than or equal to a cutoff value of 4.33 calculated by multivariate analysis, such as a multivariate Cox proportional hazards model, then the subject is: - Classified as or should be classified as being at risk of developing invasive prostate cancer. - Having a predisposition to invasive prostate cancer, -The aforementioned prostate cancer will progress, - They will not experience progression-free survival. -Probably not responding to the aforementioned prostate cancer treatment, or - Candidate for treatment with anti-cancer therapy; or b) If the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are less than the cutoff value of 4.33 calculated by the multivariate Cox proportional hazards model, then the subject is - Not classified as being at risk of developing invasive prostate cancer, or should not be classified as such. - Not having a predisposition to invasive prostate cancer, -The aforementioned prostate cancer will not progress, -They will likely experience progression-free survival. - Likely to respond to the aforementioned prostate cancer treatment, - Not a candidate for treatment with anti-cancer therapy, The method described in any one of the appendices 1 to 20. (Note 22) If the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are greater than or equal to the cutoff value of 4.33 calculated by the multivariate Cox proportional hazards model, then the subject is - Classified as or should be classified as being at risk of developing invasive prostate cancer. - Having a predisposition to invasive prostate cancer, -The aforementioned prostate cancer will progress, - They will not experience progression-free survival. -Probably not responding to the aforementioned prostate cancer treatment, -Candidates for treatment with anti-cancer therapy, The method described in any one of the appendices 1 to 20. (Note 23) If the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are less than the cutoff value of 4.33 calculated by the multivariate Cox proportional hazards model, then the subject is - Not classified as being at risk of developing invasive prostate cancer, or should not be classified as such. - Not having a predisposition to invasive prostate cancer, -The aforementioned prostate cancer will not progress, -They will likely experience progression-free survival. - Likely to respond to the aforementioned prostate cancer treatment, - Not a candidate for treatment with anti-cancer therapy, The method described in any one of the appendices 1 to 20. (Note 24) The method according to any one of the appendices 21 to 23, wherein the multivariate analysis is adjusted for age, 5-α-reductase treatment, and baseline tumor volume. (Note 25) The method as described in Appendix 24, wherein age, 5-alpha reductase treatment, and baseline tumor volume are adjusted using a retrospective stepwise selection method (likelihood ratio). (Note 26) A method of classifying individuals with prostate cancer as either at risk of developing invasive prostate cancer or not at risk of developing invasive prostate cancer, as described in any one of the appendices 1 to 25. (Note 27) The method according to any one of the appendices 1 to 25, wherein the method described above is a method for predicting the predisposition to invasive prostate cancer. (Note 28) The method according to any one of the appendices 1 to 25, wherein the method is a method for diagnosing invasive prostate cancer in a subject having prostate cancer. (Note 29) The method according to any one of the appendices 1 to 25, wherein the method is a method for predicting the likelihood of progression of prostate cancer in a subject having prostate cancer. (Note 30) The method according to any one of the appendices 1 to 25, wherein the method provides a prognosis to a subject having prostate cancer. (Note 31) The method according to any one of the appendices 1 to 30, wherein the method is a method for selecting a subject having prostate cancer for treatment with anticancer therapy. (Note 32) A diagnostic panel for invasive prostate cancer comprising caveolin-1 (CAV-1), sphingomyelin 40:2 (SM40:2), sphingomyelin 44:2 (SM44:2), lactosylceramide 32:0 (LacCer32:0), lactosylceramide 36:0 (LacCer36:0), trihexosylceramide 34:1 (TriHexCer34:1), and hexosylceramide 40:0 (HexCer40:0). (Note 33) A diagnostic panel for invasive prostate cancer comprising sphingomyelin 40:2 (SM40:2), sphingomyelin 44:2 (SM44:2), lactosylceramide 32:0 (LacCer32:0), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 (TriHexCer34:1). (Note 34) A diagnostic panel for invasive prostate cancer comprising sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 (TriHexCer34:1). (Note 35) A diagnostic panel for invasive prostate cancer comprising trihexosylceramide 34:1 (TriHexCer34:1). (Note 36) A diagnostic panel for invasive prostate cancer, comprising sphingomyelin 40:2 (SM40:2). (Note 37) ● Sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1; and / or ● Trihexosylceramide 34:1; and / or ● Sphingomyelin 40:2 (SM40:2) A diagnostic panel for invasive prostate cancer comprising the following: (Note 38) - The step of administering an anticancer drug to the subject having prostate cancer; - The step of administering therapeutic radiation to the subject having prostate cancer; - Surgery for partial or complete surgical removal of the cancerous tissue of the subject having prostate cancer. A method for treating or preventing the progression of prostate cancer in a subject in whom the levels of caveolin-1 (CAV-1), sphingomyelin 40:2 (SM40:2), sphingomyelin 44:2 (SM44:2), lactosylceramide 32:0 (LacCer32:0), lactosylceramide 36:0 (LacCer36:0), trihexosylceramide 34:1, and hexosylceramide 40:0 are elevated compared to a reference without prostate cancer. (Note 39) - The step of administering an anticancer drug to the subject having prostate cancer; - The step of administering therapeutic radiation to the subject having prostate cancer; - Surgery for partial or complete surgical removal of the cancerous tissue of the subject having prostate cancer. A method for treating or preventing the progression of prostate cancer in a subject in whom the levels of sphingomyelin 40:2 (SM40:2), sphingomyelin 44:2 (SM44:2), lactosylceramide 32:0 (LacCer32:0), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 are elevated compared to a reference without prostate cancer. (Note 40) - The step of administering an anticancer drug to the subject having prostate cancer; - The step of administering therapeutic radiation to the subject having prostate cancer; - Surgery for partial or complete surgical removal of the cancerous tissue of the subject having prostate cancer. A method for treating or preventing the progression of prostate cancer in a subject in which the levels of sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 are elevated compared to a reference without prostate cancer. (Note 41) - The step of administering an anticancer drug to the subject having prostate cancer; - The step of administering therapeutic radiation to the subject having prostate cancer; - Surgery for partial or complete surgical removal of the cancerous tissue of the subject having prostate cancer. A method for treating or preventing the progression of prostate cancer in a subject in which the level of trihexosylceramide 34:1 is elevated compared to a reference without prostate cancer, comprising one or more of the above. (Note 42) - The step of administering an anticancer drug to the subject having prostate cancer; - The step of administering therapeutic radiation to the subject having prostate cancer; - Surgery for partial or complete surgical removal of the cancerous tissue of the subject having prostate cancer. A method for treating or preventing the progression of prostate cancer in a subject in whom the level of sphingomyelin 40:2 (SM40:2)0 is elevated compared to a reference without prostate cancer, comprising one or more of the above. (Note 43) - The step of administering an anticancer drug to the subject having prostate cancer; - The step of administering therapeutic radiation to the subject having prostate cancer; - Surgery for partial or complete surgical removal of the cancerous tissue of the subject having prostate cancer. Consists of one or more of the following: among them (a) Sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1; and / or (b) Trihexosylceramide 34:1; and / or (c) A method for treating or preventing the progression of prostate cancer in subjects whose sphingomyelin 40:2 (SM40:2) levels are elevated compared to a reference without prostate cancer. (Note 44) The method according to any one of the appendices 38-43, wherein caveolin-1 (CAV-1), sphingomyelin 40:2 (SM40:2), sphingomyelin 44:2 (SM44:2), lactosylceramide 32:0 (LacCer32:0), lactosylceramide 36:0 (LacCer36:0), trihexosylceramide 34:1, and hexosylceramide 40:0 are elevated compared to a reference without prostate cancer. (Note 45) The method according to any one of the appendices 38 to 43, comprising the step of administering at least one anticancer drug to a subject having prostate cancer. (Note 46) The method according to any one of the appendices 38 to 43, wherein the anticancer agent is selected from a glucosylceramide synthase inhibitor, a Cav-1 inhibitor, or a combination of the two. (Note 47) The method according to Appendix 46, wherein the Cav-1 inhibitor is an anti-Cav-1 monoclonal antibody. (Note 48) The method according to Appendix 46, wherein the glucosylceramide synthase inhibitor is eliglustat. (Note 49) Using the method described in any of the appendices 1 to 26, or the diagnostic panel described in appendice 27, as a preliminary step, - A step of classifying individuals with prostate cancer as either at risk of developing invasive prostate cancer or not at risk of developing invasive prostate cancer. - A step to predict the predisposition to invasive prostate cancer in the target patient. - Steps for diagnosing invasive prostate cancer in subjects with prostate cancer, - A risk assessment step for subjects with invasive prostate cancer. - A step to predict the likelihood of prostate cancer progression in a subject with prostate cancer. - A step of providing a prognosis to a subject with prostate cancer, or - A step to select patients with prostate cancer for treatment with anticancer therapy. The method described in any one of the appendices 38 to 43, comprising:

Claims

1. - Classify individuals with prostate cancer as either at risk of developing invasive prostate cancer or not at risk of developing invasive prostate cancer. - Predicting the predisposition to invasive prostate cancer in the target group. - To diagnose invasive prostate cancer in subjects with prostate cancer, - Determining the risk of the subject having invasive prostate cancer, - Predicting the likelihood of prostate cancer progression in individuals with prostate cancer. - To provide prognosis to subjects with prostate cancer, - Selecting patients with prostate cancer for treatment with anticancer therapy. A method to assist in that, (a) A step of measuring the levels of caveolin-1 (CAV-1), sphingomyelin 40:2 (SM40:2), sphingomyelin 44:2 (SM44:2), lactosylceramide 32:0 (LacCer32:0), lactosylceramide 36:0 (LacCer36:0), trihexosylceramide 34:1 (TriHexCer34:1), and hexosylceramide 40:0 (HexCer40:0) in a biological sample from the subject using an in vitro assay; (b) A step of comparing the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 in the biological sample with a reference. It includes, The amounts of change in CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and HexCer40:0 compared to the above reference are: - An indicator indicating whether the subject is at risk of developing invasive prostate cancer or not, - Indicators of predisposition to the aforementioned target for invasive prostate cancer, - An indicator of the likelihood of prostate cancer progression in the aforementioned subjects, - An indicator of progression-free survival for the aforementioned subjects, - An indicator of the probable outcome of the aforementioned treatment for prostate cancer, - An indicator that the subject is a candidate for treatment with anti-cancer therapy. A method that provides an index selected from a group consisting of the following.

2. - Classify individuals with prostate cancer as either at risk of developing invasive prostate cancer or not at risk of developing invasive prostate cancer. - Predicting the predisposition to invasive prostate cancer in the target group. - To diagnose invasive prostate cancer in subjects with prostate cancer, - Determining the risk of the subject having invasive prostate cancer, - Predicting the likelihood of prostate cancer progression in individuals with prostate cancer. - To provide prognosis to subjects with prostate cancer, - Selecting patients with prostate cancer for treatment with anticancer therapy. A method to assist in that, (a) A step of measuring the levels of sphingomyelin 40:2 (SM40:2), sphingomyelin 44:2 (SM44:2), lactosylceramide 32:0 (LacCer32:0), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 (TriHexCer34:1) in a biological sample from the subject using an in vitro assay, (b) A step of comparing the levels of SM40:2, SM44:2, LacCer32:0, LacCer36:0, and TriHexCer34:1 in the biological sample with a reference. It includes, The change in the quantities of SM40:2, SM44:2, LacCer32:0, LacCer36:0, and TriHexCer34:1 relative to the aforementioned reference is - An indicator indicating whether the subject is at risk of developing invasive prostate cancer or not, - Indicators of predisposition to the aforementioned target for invasive prostate cancer, - An indicator of the likelihood of prostate cancer progression in the aforementioned subjects, - An indicator of progression-free survival for the aforementioned subjects, - An indicator of the probable outcome of the aforementioned treatment for prostate cancer, - An indicator that the subject is a candidate for treatment with anti-cancer therapy. A method that provides an index selected from a group consisting of the following.

3. - Classify individuals with prostate cancer as either at risk of developing invasive prostate cancer or not at risk of developing invasive prostate cancer. - Predicting the predisposition to invasive prostate cancer in the target group. - To diagnose invasive prostate cancer in subjects with prostate cancer, - Determining the risk of the subject having invasive prostate cancer, - Predicting the likelihood of prostate cancer progression in individuals with prostate cancer. - To provide prognosis to subjects with prostate cancer, - Selecting patients with prostate cancer for treatment with anticancer therapy. A method to assist in that, (a) A step of measuring the levels of sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 (TriHexCer34:1) in a biological sample from the subject using an in vitro assay, (b) A step of comparing the levels of SM40:2, LacCer36:0, and TriHexCer34:1 in the biological sample with a reference. It includes, The changes in the amounts of SM40:2, LacCer36:0, and TriHexCer34:1 relative to the aforementioned reference are, - An indicator indicating whether the subject is at risk of developing invasive prostate cancer or not, - Indicators of predisposition to the aforementioned target for invasive prostate cancer, - An indicator of the likelihood of prostate cancer progression in the aforementioned subjects, - An indicator of progression-free survival for the aforementioned subjects, - An indicator of the probable outcome of the aforementioned treatment for prostate cancer, - An indicator that the subject is a candidate for treatment with anti-cancer therapy. A method that provides an index selected from a group consisting of the following.

4. - Classify individuals with prostate cancer as either at risk of developing invasive prostate cancer or not at risk of developing invasive prostate cancer. - Predicting the predisposition to invasive prostate cancer in the target group. - To diagnose invasive prostate cancer in subjects with prostate cancer, - Determining the risk of the subject having invasive prostate cancer, - Predicting the likelihood of prostate cancer progression in individuals with prostate cancer. - To provide prognosis to subjects with prostate cancer, - Selecting patients with prostate cancer for treatment with anticancer therapy. A method to assist in that, (a) A step of measuring the level of trihexosylceramide 34:1 (TriHexCer34:1) in a biological sample from the subject using an in vitro assay, (b) A step of comparing the level of TriHexCer34:1 in the biological sample with a reference; It includes, The change in the amount of TriHexCer34:1 relative to the aforementioned reference is, - An indicator indicating whether the subject is at risk of developing invasive prostate cancer or not. - Indicators of predisposition to the aforementioned target for invasive prostate cancer, - An indicator of the likelihood of prostate cancer progression in the aforementioned subjects, - An indicator of progression-free survival for the aforementioned subjects, - An indicator of the probable outcome of the aforementioned treatment for prostate cancer, - An indicator that the subject is a candidate for treatment with anti-cancer therapy. A method that provides an index selected from a group consisting of the following.

5. - Classify individuals with prostate cancer as either at risk of developing invasive prostate cancer or not at risk of developing invasive prostate cancer. - Predicting the predisposition to invasive prostate cancer in the target group. - To diagnose invasive prostate cancer in subjects with prostate cancer, - Determining the risk of the subject having invasive prostate cancer, - Predicting the likelihood of prostate cancer progression in individuals with prostate cancer. - To provide prognosis to subjects with prostate cancer, - Selecting patients with prostate cancer for treatment with anticancer therapy. A method to assist in that, (a) A step of measuring the level of sphingomyelin 40:2 (SM40:2) in a biological sample from the subject using an in vitro assay, (b) The step of comparing the level of SM40:2 in the biological sample with a reference; It includes, The change in the amount of SM40:2 relative to the above reference is, - An indicator indicating whether the subject is at risk of developing invasive prostate cancer or not, - Indicators of predisposition to the aforementioned target for invasive prostate cancer, - An indicator of the likelihood of prostate cancer progression in the aforementioned subjects, - An indicator of progression-free survival for the aforementioned subjects, - An indicator of the probable outcome of the aforementioned treatment for prostate cancer, - An indicator that the subject is a candidate for treatment with anti-cancer therapy. A method that provides an index selected from a group consisting of the following.

6. - Classify individuals with prostate cancer as either at risk of developing invasive prostate cancer or not at risk of developing invasive prostate cancer. - Predicting the predisposition to invasive prostate cancer in the target group. - To diagnose invasive prostate cancer in subjects with prostate cancer, - Determining the risk of the subject having invasive prostate cancer, - Predicting the likelihood of prostate cancer progression in individuals with prostate cancer. - To provide prognosis to subjects with prostate cancer, - Selecting patients with prostate cancer for treatment with anticancer therapy. A method to assist in that, (a) Using an in vitro assay, the biological sample from the subject - Sphingomyelin 40:2 (SM40:2), lactosylceramide 36:0 (LacCer36:0), and trihexosylceramide 34:1 (TriHexCer34:1); and / or - Trihexosylceramide 34:1 (TriHexCer34:1); and / or - Sphingomyelin 40:2 (SM40:2) A step to measure the level, If (a) above includes measuring the levels of SM40:2, LacCer36:0, and TriHexCer34:1, (b1) A step of comparing the levels of SM40:2, LacCer36:0, and TriHexCer34:1 in the biological sample with a reference, If (a) above includes measuring the level of TriHexCer34:1, (b) A step of comparing the level of TriHexCer34:1 in the biological sample with a reference, If (a) above includes measuring the level of SM40:2, (b3) A step of comparing the level of SM40:2 in the biological sample with a reference, It includes and In (b1) above, The changes in the amounts of SM40:2, LacCer36:0, and TriHexCer34:1 relative to the aforementioned reference are, - An indicator indicating whether the subject is at risk of developing invasive prostate cancer or not, - Indicators of predisposition to the aforementioned target for invasive prostate cancer, - An indicator of the likelihood of prostate cancer progression in the aforementioned subjects, - An indicator of progression-free survival for the aforementioned subjects, - An indicator of the probable outcome of the aforementioned treatment for prostate cancer, - An indicator that the subject is a candidate for treatment with anti-cancer therapy. We provide an index selected from a group consisting of the following: In (b2) above, The change in the amount of TriHexCer34:1 relative to the aforementioned reference is, - An indicator indicating whether the subject is at risk of developing invasive prostate cancer or not. - Indicators of predisposition to the aforementioned target for invasive prostate cancer, - An indicator of the likelihood of prostate cancer progression in the aforementioned subjects, - An indicator of progression-free survival for the aforementioned subjects, - An indicator of the probable outcome of the aforementioned treatment for prostate cancer, - An indicator that the subject is a candidate for treatment with anti-cancer therapy. We provide an index selected from a group consisting of the following: In (b3) above, The change in the amount of SM40:2 relative to the above reference is, - An indicator indicating whether the subject is at risk of developing invasive prostate cancer or not, - Indicators of predisposition to the aforementioned target for invasive prostate cancer, - An indicator of the likelihood of prostate cancer progression in the aforementioned subjects, - An indicator of progression-free survival for the aforementioned subjects, - An indicator of the probable outcome of the aforementioned treatment for prostate cancer, - An indicator that the subject is a candidate for treatment with anti-cancer therapy. A method that provides an index selected from a group consisting of the following.

7. The method according to any one of claims 1 to 6, wherein the subject has prostate cancer.

8. The method according to any one of claims 1 to 6, wherein the subject is determined to be positive for the level of prostate cancer antigen (PSA) indicating the presence of prostate cancer.

9. The method according to any one of claims 1 to 6, wherein the subject is under active monitoring (AS) for disease progression.

10. The method according to any one of claims 1 to 6, wherein the subject is classified as having clinically low-risk early prostate cancer under active surveillance, exhibiting early disease progression (DP) or inactive disease.

11. The method according to any one of claims 1 to 6, wherein the subject is not receiving treatment for prostate cancer.

12. The method according to any one of claims 1 to 11, wherein the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are elevated in the subject compared to healthy individuals.

13. The method according to any one of claims 1 to 11, wherein the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are elevated compared to the levels of a reference subject or group that does not have invasive prostate cancer.

14. The method according to any one of claims 1 to 11, wherein the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are elevated compared to the levels of a reference subject or group having inactive prostate cancer.

15. The method according to any one of claims 1 to 14, wherein the measurement of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 is performed by ultraviolet-visible spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, gas chromatography-mass spectroscopy (GC-MS), liquid chromatography-mass spectroscopy (LC-MS), high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UPLC), liquid chromatography-mass spectroscopy (LC-MS), correlation spectroscopy (COSy), nuclear Overhauser effect spectroscopy (NOESY), rotating-frame nuclear Overhauser effect spectroscopy (ROESY), LC-TOF-MS, LC-MS / MS, and capillary electrophoresis-mass spectroscopy.

16. The method according to claim 15, wherein the measurement of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 is performed by high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UPLC), or liquid chromatography-mass spectrometry (LC-MS).

17. The method according to any one of claims 1 to 16, wherein the biological sample is selected from serum and plasma.

18. The method according to claim 17, wherein the biological sample comprises a fraction of the sample containing isolated extracellular vesicles (EVs).

19. The method according to any one of claims 1 to 18, wherein the measurements of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are performed at the same time.

20. The method according to any one of claims 1 to 18, wherein the measurement of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 is performed in a stepwise manner.

21. a) If the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are greater than or equal to a cutoff value of 4.33 calculated by multivariate analysis using a multivariate Cox proportional hazards model, then the subject is, - An indicator that will not experience progression-free survival, Provide; or b) If the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are less than the cutoff value of 4.33 calculated by multivariate analysis using a multivariate Cox proportional hazards model, then the subject is, - An indicator of the likelihood of experiencing progression-free survival, The method according to any one of claims 1 to 20, which provides the following:

22. If the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are greater than or equal to the cutoff value of 4.33 calculated by multivariate analysis using a multivariate Cox proportional hazards model, then the subject is, - An indicator that will not experience progression-free survival, The method according to any one of claims 1 to 20, which provides the following:

23. If the levels of CAV-1, SM40:2, SM44:2, LacCer32:0, LacCer36:0, TriHexCer34:1, and / or HexCer40:0 are less than the cutoff value of 4.33 calculated by multivariate analysis using a multivariate Cox proportional hazards model, then the subject is, - An indicator of the likelihood of experiencing progression-free survival, The method according to any one of claims 1 to 20, which provides the following:

24. The method according to any one of claims 21 to 23, wherein the multivariate analysis is adjusted for age, 5-α-reductase treatment, and baseline tumor volume.

25. The method according to claim 24, wherein age, 5-alpha reductase treatment, and baseline tumor volume are adjusted using a retrospective stepwise selection method (likelihood ratio).

26. The method according to any one of claims 1 to 25, which is a method for assisting in classifying subjects with prostate cancer as either at risk of developing invasive prostate cancer or not at risk of developing invasive prostate cancer.

27. The method according to any one of claims 1 to 25, wherein the method is a method for assisting in predicting the predisposition to invasive prostate cancer in a target.

28. The method according to any one of claims 1 to 25, wherein the method is a method for assisting in the diagnosis of invasive prostate cancer in a subject having prostate cancer.

29. The method according to any one of claims 1 to 25, wherein the method is a method for assisting in predicting the likelihood of progression of prostate cancer in a subject having prostate cancer.

30. The method according to any one of claims 1 to 25, wherein the method is a method that helps to provide a prognosis to a subject having prostate cancer.

31. The method according to any one of claims 1 to 30, wherein the method is a method for assisting in the selection of a subject having prostate cancer for treatment with anticancer therapy.

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