METHODS FOR THE DETECTION AND TREATMENT OF PROSTATE CANCER
20230358751 · 2023-11-09
Inventors
- Samir HANASH (Houston, TX, US)
- Jody Vykoukal (Houston, TX)
- Timothy THOMPSON (Houston, TX, US)
- Johannes Francois FAHRMANN (Houston, TX, US)
Cpc classification
G01N2800/56
PHYSICS
G01N2800/52
PHYSICS
International classification
Abstract
Provided are methods and related kits for detection of early stage prostate cancer, and determination of risk of being at risk for progression of prostate cancer.
Claims
1.-3. (canceled)
4. A method of: classifying a subject with prostate cancer as being at risk of developing aggressive prostate cancer or not being at risk of developing aggressive prostate cancer, predicting a predisposition to aggressive prostate cancer in a subject, diagnosing aggressive prostate cancer in a subject with prostate cancer, determining the risk of a subject for having aggressive prostate cancer, predicting the likelihood of progression of prostate cancer in a subject with prostate cancer, providing a prognosis for a subject with prostate cancer, or selecting a subject with prostate cancer for treatment with an anticancer therapy, comprising: (a) measuring the levels of trihexosylceramide 34:1 (TriHexCer 34:1) in a biological sample from said subject using an in vitro assay and (b) comparing the levels of TriHexCer 34:1 in said sample with a reference, wherein an altered amount of TriHexCer 34:1 relative to said reference provides an indication selected from the group consisting of: an indication that the subject is at risk of developing aggressive prostate cancer or not at risk of developing aggressive prostate cancer, an indication of a predisposition of the subject to aggressive prostate cancer, an indication of the likelihood of progression of the prostate cancer in the subject, an indication of progression-free survival of the subject, an indication of the likely outcome of treatment of the prostate cancer, and an indication that the subject is a candidate for treatment with an anticancer therapy.
5. A method of: classifying a subject with prostate cancer as being at risk of developing aggressive prostate cancer or not being at risk of developing aggressive prostate cancer, predicting a predisposition to aggressive prostate cancer in a subject, diagnosing aggressive prostate cancer in a subject with prostate cancer, determining the risk of a subject for having aggressive prostate cancer, predicting the likelihood of progression of prostate cancer in a subject with prostate cancer, providing a prognosis for a subject with prostate cancer, or selecting a subject with prostate cancer for treatment with an anticancer therapy, comprising: (a) measuring the levels of sphingomyelin 40:2 (SM 40:2) in a biological sample from said subject using an in vitro assay and (b) comparing the levels of SM 40:2 in said sample with a reference, wherein an altered amount of SM 40:2 relative to said reference provides an indication selected from the group consisting of: an indication that the subject is at risk of developing aggressive prostate cancer or not at risk of developing aggressive prostate cancer, an indication of a predisposition of the subject to aggressive prostate cancer, an indication of the likelihood of progression of the prostate cancer in the subject, an indication of progression-free survival of the subject, an indication of the likely outcome of treatment of the prostate cancer, and an indication that the subject is a candidate for treatment with an anticancer therapy.
6.-49. (canceled)
50. The method claim 4, further comprising: (a) measuring the levels of: sphingomyelin 40:2 (SM 40:2), lactosylceramide 36:0 (LacCer 36:0), and TriHexCer 34:1 in a biological sample from said subject using an in vitro assay and (b) comparing the levels of SM 40:2, LacCer 36:0, and TriHexCer 34:1 in said sample with a reference, wherein an altered amount of SM 40:2, LacCer 36:0, and TriHexCer 34:1 relative to said reference provides an indication selected from the group consisting of: an indication that the subject is at risk of developing aggressive prostate cancer or not at risk of developing aggressive prostate cancer, an indication of a predisposition of the subject to aggressive prostate cancer, an indication of the likelihood of progression of the prostate cancer in the subject, an indication of progression-free survival of the subject, an indication of the likely outcome of treatment of the prostate cancer, and an indication that the subject is a candidate for treatment with an anticancer therapy.
51. The method claim 4, further comprising: (a) measuring the levels of: Caveolin-1 (CAV-1), sphingomyelin 40:2 (SM 40:2), sphingomyelin 44:2 (SM 44:2), lactosylceramide 32:0 (LacCer 32:0), lactosylceramide 36:0 (LacCer 36:0), trihexosylceramide 34:1 (TriHexCer 34:1), and hexosylceramide 40:0 (HexCer 40:0) in a biological sample from said subject using an in vitro assay and (b) comparing the levels of CAV-1, SM 40:2, SM 44:2, LacCer 32:0, LacCer 36:0, TriHexCer 34:1, and HexCer 40:0 in said sample with a reference, wherein an altered amount of CAV-1, SM 40:2, SM 44:2, LacCer 32:0, LacCer 36:0, TriHexCer 34:1, and HexCer 40:0 relative to said reference provides an indication selected from the group consisting of: an indication that the subject is at risk of developing aggressive prostate cancer or not at risk of developing aggressive prostate cancer, an indication of a predisposition of the subject to aggressive prostate cancer, an indication of the likelihood of progression of the prostate cancer in the subject, an indication of progression-free survival of the subject, an indication of the likely outcome of treatment of the prostate cancer, and an indication that the subject is a candidate for treatment with an anticancer therapy.
52. The method claim 4, further comprising: (a) measuring the levels of: sphingomyelin 40:2 (SM 40:2), sphingomyelin 44:2 (SM 44:2), lactosylceramide 32:0 (LacCer 32:0), lactosylceramide 36:0 (LacCer 36:0), and trihexosylceramide 34:1 (TriHexCer 34:1) in a biological sample from said subject using an in vitro assay and (b) comparing the levels of SM 40:2, SM 44:2, LacCer 32:0, LacCer 36:0, and TriHexCer 34:1 in said sample with a reference, wherein an altered amount of SM 40:2, SM 44:2, LacCer 32:0, LacCer 36:0, and TriHexCer 34:1 relative to said reference provides an indication selected from the group consisting of: an indication that the subject is at risk of developing aggressive prostate cancer or not at risk of developing aggressive prostate cancer, an indication of a predisposition of the subject to aggressive prostate cancer, an indication of the likelihood of progression of the prostate cancer in the subject, an indication of progression-free survival of the subject, an indication of the likely outcome of treatment of the prostate cancer, and an indication that the subject is a candidate for treatment with an anticancer therapy.
53. The method of claim 4, wherein the subject has prostate cancer.
54. The method of claim 51, wherein the levels of CAV-1, SM 40:2, SM 44:2, LacCer 32:0, LacCer 36:0, TriHexCer 34:1, and/or HexCer 40:0 are elevated in the subject relative to a healthy subject.
55. The method of claim 51, wherein the measuring of the CAV-1, SM 40:2, SM 44:2, LacCer 32:0, LacCer 36:0, TriHexCer 34:1, and/or HexCer 40:0 is carried out by UV-visible spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectrometry, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), liquid chromatography-mass spectrometry (LC-MS), correlation spectroscopy (COSy), nuclear Overhauser effect spectroscopy (NOESY), rotating frame nuclear Overhauser effect spectroscopy (ROESY), LC-TOF-MS, LC-MS/MS, or capillary electrophoresis-mass spectrometry.
56. A diagnostic panel for aggressive prostate cancer comprising trihexosylceramide 34:1 (TriHexCer 34:1) and/or sphingomyelin 40:2 (SM 40:2).
57. The diagnostic panel of claim 56, further comprising Caveolin-1 (CAV-1), sphingomyelin 40:2 (SM 40:2), sphingomyelin 44:2 (SM 44:2), lactosylceramide 32:0 (LacCer 32:0), lactosylceramide 36:0 (LacCer 36:0), trihexosylceramide 34:1 (TriHexCer 34:1) and hexosylceramide 40:0 (HexCer 40:0).
58. The diagnostic panel of claim 56, further comprising sphingomyelin 40:2 (SM 40:2), sphingomyelin 44:2 (SM 44:2), lactosylceramide 32:0 (LacCer 32:0), lactosylceramide 36:0 (LacCer 36:0), and trihexosylceramide 34:1 (TriHexCer 34:1).
59. The diagnostic panel of claim 56, further comprising sphingomyelin 40:2 (SM 40:2), lactosylceramide 36:0 (LacCer 36:0), and trihexosylceramide 34:1 (TriHexCer 34:1).
60. The diagnostic panel of claim 56, further comprising sphingomyelin 40:2 (SM 40:2), lactosylceramide 36:0 (LacCer 36:0), and trihexosylceramide 34:1.
61. A method of treatment or prevention of progression of prostate cancer in a subject in whom the levels of trihexosylceramide 34:1 and/or sphingomyelin 40:2 (SM 40:2) are elevated relative to a reference without prostate cancer, comprising one or more of: administering an anticancer drug to the subject with prostate cancer; administering therapeutic radiation to the subject with prostate cancer; and surgery for partial or complete surgical removal of cancerous tissue in the subject with prostate cancer.
62. The method of claim 61, wherein the levels of Caveolin-1 (CAV-1), sphingomyelin 40:2 (SM 40:2), sphingomyelin 44:2 (SM 44:2), lactosylceramide 32:0 (LacCer 32:0), lactosylceramide 36:0 (LacCer 36:0), trihexosylceramide 34:1 and hexosylceramide 40:0 are elevated relative to a reference without prostate cancer.
63. The method of claim 61, wherein the levels of sphingomyelin 40:2 (SM 40:2), sphingomyelin 44:2 (SM 44:2), lactosylceramide 32:0 (LacCer 32:0), lactosylceramide 36:0 (LacCer 36:0), and trihexosylceramide 34:1 are elevated relative to a reference without prostate cancer.
64. The method of claim 61, wherein the levels of sphingomyelin 40:2 (SM 40:2), lactosylceramide 36:0 (LacCer 36:0), and trihexosylceramide 34:1 are elevated relative to a reference without prostate cancer.
65. The method of claim 61, wherein the levels of sphingomyelin 40:2 (SM 40:2), lactosylceramide 36:0 (LacCer 36:0), and trihexosylceramide 34:1 are elevated relative to a reference without prostate cancer.
66. The method as recited in claim 61 comprising, as a prior step, classifying a subject with prostate cancer as being at risk of developing aggressive prostate cancer or not being at risk of developing aggressive prostate cancer, predicting a predisposition to aggressive prostate cancer in a subject, diagnosing aggressive prostate cancer in a subject with prostate cancer, determining the risk of a subject for having aggressive prostate cancer, predicting the likelihood of progression of prostate cancer in a subject with prostate cancer, providing a prognosis for a subject with prostate cancer, or selecting a subject with prostate cancer for treatment with an anticancer therapy.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0071] In one aspect, the disclosure provides a method of determining the risk for progression of prostate cancer in a subject, comprising performing the following steps on a biological sample obtained from the subject: 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, and measuring the level of HexCer 40:0 in the biological sample; wherein the amount of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 determines the risk for progression of prostate cancer in the subject.
[0072] In another aspect, the disclosure provides a method of determining the risk for progression of prostate cancer in a subject, comprising performing the following steps on a biological sample obtained from the subject: contacting the sample with a first reporter molecule that binds CAV-1, a second reporter molecule that binds SM(40:2), a third reporter molecule that binds SM(44:2), a fourth reporter molecule that binds LacCer 32:0, a fifth reporter molecule that binds LacCer 36:0, a sixth reporter molecule that binds TriHexCer 34:1, and a seventh reporter molecule that binds HexCer 40:0; wherein the amount 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 determines the risk for progression of prostate cancer in the subject.
[0073] In another aspect, the disclosure provides a method of determining the risk for progression of prostate cancer in a subject, comprising performing the following steps on a biological sample obtained from the subject: performing spectrometric analysis for CAV-1, performing spectrometric analysis for SM(40:2), performing spectrometric analysis for SM(44:2), performing spectrometric analysis for LacCer 32:0, performing spectrometric analysis for LacCer 36:0, performing spectrometric analysis for TriHexCer 34:1, performing spectrometric analysis for HexCer 40:0, wherein the spectrometric analyses determine the risk for progression of prostate cancer in the subject. In some aspects, the spectrometric analyses are quantitative analyses. In some aspects, the spectrometric analyses are mass spectrometric analyses. In some aspects, the spectrometric analyses are performed concurrently. In some aspects, the spectrometric analyses are performed sequentially. In some aspects, the method further comprises a chromatographic step. In some aspects, the method further comprises a liquid chromatographic step. In some aspects, the method further comprises a high performance liquid chromatographic (“HPLC”) step. In some aspects, the method further comprises a gas chromatographic (“GC”) step. In some aspects, the chromatographic step is coupled directly to the spectrometric step. In some aspects, the chromatographic step separates at least one analyte from at least one other analyte.
[0074] In another aspect, the disclosure provides a method of determining the risk for progression of prostate cancer in a subject, comprising performing the following steps on a biological sample obtained from the subject: providing a surface that binds CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0; incubating the surface with the biological sample; contacting the surface with a first reporter molecule that binds CAV-1, contacting the surface with a second reporter molecule that binds SM(40:2), contacting the surface with a third reporter molecule that binds SM(44:2), contacting the surface with a fourth reporter molecule that binds LacCer 32:0, contacting the surface with a fifth reporter molecule that binds LacCer 36:0, contacting the surface with a sixth reporter molecule that binds TriHexCer 34:1, and contacting the surface with a seventh reporter molecule that binds HexCer 40:0; measuring the amount of the first reporter molecule that is associated with the surface; measuring the amount of the second reporter molecule that is associated with the surface; measuring the amount of the third reporter molecule that is associated with the surface; measuring the amount of the fourth reporter molecule that is associated with the surface; measuring the amount of the fifth reporter molecule that is associated with the surface; measuring the amount of the sixth reporter molecule that is associated with the surface; measuring the amount of the seventh reporter molecule that is associated with the surface; wherein the amount 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, determines the risk for progression of prostate cancer in the subject.
[0075] In another aspect, the disclosure provides a method of determining the risk for progression of prostate cancer in a subject, comprising performing the following steps on a biological sample obtained from the subject: providing a first surface with means for binding CAV-1, providing a second surface with means for binding SM(40:2), providing a third surface with means for binding SM(44:2), providing a fourth surface with means for binding LacCer 32:0, providing a fifth surface with means for binding LacCer 36:0, providing a sixth surface with means for binding TriHexCer 34:1, and providing a seventh surface with means for binding HexCer 40:0; incubating the first surface with the biological sample; incubating the second surface with the biological sample; incubating the third surface with the biological sample; incubating the fourth surface with the biological sample; incubating the fifth surface with the biological sample; incubating the sixth surface with the biological sample; incubating the seventh surface with the biological sample; contacting the first surface with a first reporter molecule that binds CAV-1, contacting the second surface with a first reporter molecule that binds SM(40:2), contacting the third surface with a first reporter molecule that binds SM(44:2), contacting the fourth surface with a first reporter molecule that binds LacCer 32:0, contacting the fifth surface with a first reporter molecule that binds LacCer 36:0, contacting the sixth surface with a first reporter molecule that binds TriHexCer 34:1, and contacting the seventh surface with a first reporter molecule that binds HexCer 40:0; measuring the amount of the first reporter molecule associated with the first surface; measuring the amount of the second reporter molecule associated with the second surface; measuring the amount of the third reporter molecule associated with the third surface; measuring the amount of the fourth reporter molecule associated with the fourth surface; measuring the amount of the fifth reporter molecule associated with the fifth surface; measuring the amount of the sixth reporter molecule associated with the sixth surface; measuring the amount of the seventh reporter molecule associated with the seventh surface; wherein the amount 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 determines the risk for progression of prostate cancer in the subject.
[0076] In another aspect, the disclosure provides a method of determining the risk for progression of prostate cancer in a subject, comprising performing the following steps on a biological sample obtained from the subject: providing a surface with means for binding CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0; incubating the surface with the biological sample; contacting the surface with a first relay molecule that binds CAV-1, contacting the surface with a second relay molecule that binds SM(40:2), contacting the surface with a third relay molecule that binds SM(44:2); contacting the surface with a fourth relay molecule that binds LacCer 32:0; contacting the surface with a fifth relay molecule that binds LacCer 36:0; contacting the surface with a sixth relay molecule that binds TriHexCer 34:1; and contacting the surface with a seventh relay molecule that binds HexCer 40:0; contacting the surface with a first reporter molecule that binds to the first relay molecule; contacting the surface with a second reporter molecule that binds to the second relay molecule; contacting the surface with a third reporter molecule that binds to the third relay molecule; contacting the surface with a fourth reporter molecule that binds to the fourth relay molecule; contacting the surface with a fifth reporter molecule that binds to the fifth relay molecule; contacting the surface with a sixth reporter molecule that binds to the sixth relay molecule; contacting the surface with a seventh reporter molecule that binds to the seventh relay molecule; measuring the amount of the first reporter molecule associated with the first relay molecule and CAV-1; measuring the amount of the second reporter molecule associated with the second relay molecule and SM(40:2); measuring the amount of the third reporter molecule associated with the third relay molecule and SM(44:2); measuring the amount of the fourth reporter molecule associated with the fourth relay molecule and LacCer 32:0; measuring the amount of the fifth reporter molecule associated with the fifth relay molecule and LacCer 36:0; measuring the amount of the sixth reporter molecule associated with the sixth relay molecule and TriHexCer 34:1; and measuring the amount of the seventh reporter molecule associated with the seventh relay molecule and HexCer 40:0; wherein the amount 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 determines the risk for progression of prostate cancer in the subject.
[0077] In one embodiment, the amounts of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 or the reporter molecules bound thereto are elevated in the subject relative to a healthy subject. In one embodiment, the amounts of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 or the reporter molecules bound thereto are elevated in the subject relative to a subject without prostate cancer. In one embodiment, the amounts of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 or the reporter molecules bound thereto are elevated in the subject relative to a subject with indolent prostate cancer.
[0078] 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 UV-visible spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectrometry, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (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 spectrometry. In another embodiment, the spectrometric method is mass spectrometry. In another embodiment, the panel comprises biomarkers that have been identified by a method selected from UV-visible spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectrometry, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (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 spectrometry. In another embodiment, the panel comprises biomarkers that have been identified by UV-visible spectroscopy or proton NMR spectroscopy.
[0079] In another embodiment, the first reporter binds selectively to CAV-1. In another embodiment, the second reporter binds selectively to SM(40:2). In another embodiment, the third reporter binds selectively to SM(44:2). In another embodiment, the fourth reporter binds selectively to LacCer 32:0. In another embodiment, the fifth reporter binds selectively to LacCer 36:0. In another embodiment, the sixth reporter binds selectively to TriHexCer 34:1. In another embodiment, the seventh reporter binds selectively to HexCer 40:0.
[0080] In another embodiment, determination of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 levels is made at substantially the same time. In another embodiment, determination of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 levels is made in a stepwise manner. In another embodiment, such methods comprise inclusion of subject history information into the determination of risk for progression of prostate cancer. In another embodiment, such methods comprise administering at least one alternate diagnostic test for a subject assigned as being at risk for risk for progression of prostate cancer.
[0081] In another aspect, the disclosure provides a method of treating a subject suspected of being at risk for progression of prostate cancer, comprising analyzing the subject for risk for progression of prostate cancer with a method as described herein, and administering a therapeutically effective amount of a treatment for the cancer. In one embodiment, the treatment is surgery, chemotherapy, immunotherapy, radiation therapy, targeted therapy, or a combination thereof.
[0082] In another aspect, the disclosure provides a method of determining the risk for progression of prostate cancer in a subject, comprising performing the following steps on a biological sample obtained from the subject: [0083] 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 standard value, wherein the ratio is predictive of the risk for progression of prostate cancer; determining the level of SM(40:2) relative to a second standard value, wherein the ratio is predictive of the risk for progression of prostate cancer; determining the level of SM(44:2) relative to a third standard value, wherein the ratio is predictive of the risk for progression of prostate cancer; determining the level of LacCer 32:0 relative to a fourth standard value, wherein the ratio is predictive of the risk for progression of prostate cancer; determining the level of LacCer 36:0 relative to a fifth standard value, wherein the ratio is predictive of the risk for progression of prostate cancer; determining the level of TriHexCer 34:1 relative to a sixth standard value, wherein the ratio is predictive of the risk for progression of prostate cancer; determining the level of HexCer 40:0 relative to a seventh standard value, wherein the ratio is predictive of the risk for progression of prostate cancer; and assigning a risk for progression of prostate cancer or not at risk for progression of prostate cancer, as determined by statistical analysis of the ratios of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 levels.
[0084] In another aspect, the disclosure provides a method of predicting the risk for progression of prostate cancer in a subject, comprising performing the following steps on a biological sample from the subject obtained from the subject: measuring the levels of the CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 biomarkers in the biological sample; and calculating a predictive factor as determined by statistical analysis of the CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 levels.
[0085] In another aspect, the disclosure provides a method of determining the risk for progression of prostate cancer in a subject, comprising performing the following steps on a biological sample obtained from the subject: measuring the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 biomarkers in the biological sample; assigning the condition of the subject as either at risk for progression of prostate cancer or not at risk for progression of prostate cancer, as determined by statistical analysis of the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 in the biological sample, and determining, from analysis of the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0, a risk for progression of prostate cancer.
[0086] In another embodiment, the first reporter binds selectively to CAV-1. In another embodiment, the second reporter binds selectively to SM(40:2). In another embodiment, the third reporter binds selectively to SM(44:2). In another embodiment, the fourth reporter binds selectively to LacCer 32:0. In another embodiment, the fifth reporter binds selectively to LacCer 36:0. In another embodiment, the sixth reporter binds selectively to TriHexCer 34:1. In another embodiment, the seventh reporter binds selectively to HexCer 40:0. In another embodiment, determination of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 levels is made at substantially the same time. In another embodiment, determination of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 levels is made in a stepwise manner. In another embodiment, such methods further comprise inclusion of subject history information into the assignment of being at risk for progression of prostate cancer or not being at risk for progression of prostate cancer. In another embodiment, such methods comprise administering at least one alternate diagnostic test for a subject assigned as being at risk for progression of prostate cancer.
[0087] In another aspect, the disclosure provides a method of treating a subject suspected of being at risk for progression of prostate cancer, comprising analyzing the subject for risk for progression of prostate cancer with a method as described herein; and administering a therapeutically effective amount of a treatment for the cancer. In another embodiment, the treatment is surgery, chemotherapy, immunotherapy, radiation therapy, targeted therapy, or a combination thereof. In another embodiment, the classification of the subject as being at risk for progression of prostate cancer has a sensitivity of 0.76 and 0.42 at 78% and 94% specificity, respectively.
[0088] In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 are elevated in comparison to the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 in a reference subject or group that has adenocarcinoma.
[0089] In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 are elevated in comparison to the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 in a reference subject or group that has squamous cell cancer.
[0090] In another aspect, the disclosure provides a kit for the method comprising a reagent solution that comprises a first solute for detection of CAV-1; a second solute for detection of SM(40:2); a third solute for detection of SM(44:2); a fourth solute for detection of LacCer 32:0; a fifth solute for detection of LacCer 36:0; a sixth solute for detection of TriHexCer 34:1; and a seventh solute for detection of HexCer 40:0.
[0091] In another embodiment, such methods further comprise a device for contacting the reagent solutions with a biological sample. In another embodiment, such methods comprise at least one surface with 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), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0.
[0092] In another aspect, the disclosure provides a method of determining the risk for progression of prostate cancer 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), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0; wherein the method comprises: performing the following steps on obtaining a biological sample from the subject; measuring the levels of the biomarkers and the protein biomarkers in the biological sample; wherein the amount of the biomarkers and the protein biomarkers determines the risk for progression of prostate cancer in the subject.
[0093] In another aspect, the disclosure provides a method of determining the risk for progression of prostate cancer in a subject, comprising performing the following steps on obtaining a biological sample from the subject; measuring the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 in the biological sample; and determining the risk for progression of prostate cancer in the subject, as determined by statistical analysis of the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 in the biological sample. In one embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 or the reporter molecules bound thereto are elevated in the subject relative to a healthy subject. In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 are elevated in comparison to the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 in a reference subject or group that does not have prostate cancer. In another embodiment, the reference subject or group is healthy. In another embodiment, such methods comprise 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), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0. 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 amount of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 is quantified. In another embodiment, detection of the amount of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 comprises the use of a solid particle. In another embodiment, the solid particle is a bead. In another embodiment, at least one of the reporter molecules is linked to an enzyme. 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 UV-visible spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectrometry, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (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 spectrometry. In another embodiment, the concentrations of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 are measured. In another embodiment, the subject is determined to be at risk for progression of prostate cancer 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 methods further comprise the steps of: comparing the measured concentrations of each biomarker in the biological sample to the prediction of a statistical model. In another embodiment, the panel is selected from the group consisting of: a. the panel consisting of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0; or b. the panel consisting of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0. In another embodiment, the panel comprises biomarkers that have been identified by a method selected from UV-visible spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectrometry, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (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 spectrometry. In another embodiment, the panel comprises biomarkers that have been identified by UV-visible spectroscopy or proton NMR spectroscopy.
[0094] In another embodiment, the first reporter binds selectively to CAV-1. In another embodiment, the second reporter binds selectively to SM(40:2). In another embodiment, the third reporter binds selectively to SM(44:2). In another embodiment, the fourth reporter binds selectively to LacCer 32:0. In another embodiment, the fifth reporter binds selectively to LacCer 36:0. In another embodiment, the sixth reporter binds selectively to TriHexCer 34:1. In another embodiment, the seventh reporter binds selectively to HexCer 40:0.
[0095] In another embodiment, determination of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 levels is made at substantially the same time. In another embodiment, determination of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 levels is made in a stepwise manner. In another embodiment, such methods further comprise inclusion of subject history information into the assignment of being at risk for progression of prostate cancer or not being at risk for progression of prostate cancer. In another embodiment, such methods comprise administering at least one alternate diagnostic test for a subject assigned as being at risk for progression of prostate cancer.
[0096] In another embodiment, the disclosure provides a kit for the method as described herein, comprising: a reagent solution that comprises a first solute for detection of CAV-1; a second solute for detection of SM(40:2); a third solute for detection of SM(44:2); a fourth solute for detection of LacCer 32:0; a fifth solute for detection of LacCer 36:0; a sixth solute for detection of TriHexCer 34:1; and a seventh solute for detection of HexCer 40:0
[0097] In another aspect, the disclosure provides a kit for a method as described herein, comprising a first reagent solution that comprises a first solute for detection of CAV-1, a second reagent solution that comprises a second solute for detection of SM(40:2), a third reagent solution that comprises a third solute for detection of SM(44:2), a fourth reagent solution that comprises a fourth solute for detection of LacCer 32:0, a fifth reagent solution that comprises a fifth solute for detection of LacCer 36:0, a sixth reagent solution that comprises a sixth solute for detection of TriHexCer 34:1, and a seventh reagent solution that comprises a seventh solute for detection of HexCer 40:0.
[0098] In another embodiment, such a kit further comprises: a reagent solution that comprises a first solute for detection of CAV-1; a second solute for detection of SM(40:2); a third solute for detection of SM(44:2); a fourth solute for detection of LacCer 32:0; a fifth solute for detection of LacCer 36:0; a sixth solute for detection of TriHexCer 34:1; and a seventh solute for detection of HexCer 40:0.
[0099] In another aspect, the disclosure provides a method of treatment or prevention of progression of prostate cancer in a subject in whom the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 classifies the subject as having or being at risk for progression of prostate cancer comprising one or more of: administering a chemotherapeutic drug to the subject with prostate cancer; administering therapeutic radiation to the subject with prostate cancer; and surgery for partial or complete surgical removal of cancerous tissue in the subject with prostate cancer.
[0100] In another aspect, the disclosure provides a method of treatment or prevention of progression of prostate cancer in a subject in whom the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 classifies the subject as having or being at risk for progression of prostate cancer comprising one or more of: administering a chemotherapeutic drug to the subject with prostate cancer; administering therapeutic radiation to the subject with prostate cancer; and surgery for partial or complete surgical removal of cancerous tissue in the subject with prostate cancer.
[0101] In another aspect, the disclosure provides a method for treating prostate cancer in a subject, comprising: detecting CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0, in a biological sample obtained from the subject; quantifying the amounts CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 in said collected sample; determining a risk score from the amounts of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0; comparing the risk score with a cutoff value to determine whether said human is at risk for progression of prostate cancer; wherein if the levels are above the cutoff value said human is at risk for progression of prostate cancer, and administering a treatment for prostate cancer to said human being at risk for progression of prostate cancer.
[0102] In another aspect, the disclosure provides a method of determining risk of a subject for progression of prostate cancer, comprising: in biological samples from a subject in need of analysis, measuring the concentration of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0; and comparing the concentration of the biomarkers in the samples of the subject in need of diagnosis and the concentration in a normal or non-diseased subject, wherein the subject in need of diagnosis is diagnosed with prostate cancer.
[0103] In another aspect, the disclosure provides a method of determining evidence for risk of progression of prostate cancer in a biological sample, comprising measuring the concentration of a biomarker panel comprising CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 in the biological sample, and determining a risk score from the amounts of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0. In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 or the reporter molecules bound thereto are elevated in the subject relative to a healthy subject. In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 are elevated in comparison to the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 in a reference subject or group that does not have prostate cancer. In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 are elevated in comparison to the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 in a reference subject or group that has indolent prostate cancer. In another embodiment, the reference subject or group is healthy. In another embodiment, at least one of the surfaces further comprises at least one receptor molecule that selectively binds to a biomarker selected from CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0. In another embodiment, at least one of the surfaces is the surface of a solid particle. In another embodiment, the solid particle comprises a bead. In another embodiment, such methods comprising: measuring the level of the biomarkers in the biological sample; wherein the amount of the biomarkers classifies the patient as being at risk for progression of prostate cancer or not at risk for progression of prostate cancer. 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 amount of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 is quantified. In another embodiment, detection of the amount of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 comprises the use of a solid particle. In another embodiment, the solid particle is a bead. In another embodiment, at least one of the reporter molecules is linked to an enzyme. 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 UV-visible spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectrometry, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (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 spectrometry. In another embodiment, the concentrations of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 are measured. In another embodiment, the subject is determined to be at risk for progression of prostate cancer 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 methods further comprise the steps of: comparing the measured concentrations of each biomarker in the biological sample to the prediction of a statistical model. In another embodiment, the panel is selected from the group consisting of: a. the panel consisting of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0; or b. the panel consisting of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0. In another embodiment, the panel comprises biomarkers that have been identified by a method selected from UV-visible spectroscopy, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectrometry, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (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 spectrometry. In another embodiment, the panel comprises biomarkers that have been identified by UV-visible spectroscopy or proton NMR spectroscopy.
[0104] In another embodiment, the first reporter binds selectively to CAV-1. In another embodiment, the second reporter binds selectively to SM(40:2). In another embodiment, the third reporter binds selectively to SM(44:2). In another embodiment, the fourth reporter binds selectively to LacCer 32:0. In another embodiment, the fifth reporter binds selectively to LacCer 36:0. In another embodiment, the sixth reporter binds selectively to TriHexCer 34:1. In another embodiment, the seventh reporter binds selectively to HexCer 40:0.
[0105] In another embodiment, determination of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 levels is made at substantially the same time. In another embodiment, determination of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 levels is made in a stepwise manner. In another embodiment, such methods comprise inclusion of subject history information into the assignment of being at risk for progression of prostate cancer or not being at risk for progression of prostate cancer. In another embodiment, such methods comprise administering at least one alternate diagnostic test for a subject assigned as being at risk for progression of prostate cancer.
[0106] In another embodiment, the method of treating a subject suspected of being at risk for progression of prostate cancer, comprising analyzing the subject for risk for progression of prostate cancer with a method as described herein; and administering a therapeutically effective amount of a treatment for the cancer. In another embodiment, the treatment is surgery, chemotherapy, immunotherapy, radiation therapy, targeted therapy, or a combination thereof. In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 are elevated in comparison to the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 in a reference subject or group that has adenocarcinoma. In another embodiment, the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 are elevated in comparison to the levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 in a reference subject or group that has squamous cell cancer. In another embodiment, the prostate cancer is diagnosed at or before the borderline resectable stage. In another embodiment, the prostate cancer is diagnosed at the resectable stage.
[0107] In another embodiment, such methods further comprise: providing a surface that binds CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0; incubating the surface with the biological sample;
[0108] measuring the amount of the first reporter molecule that is associated with the surface; measuring the amount of the second reporter molecule that is associated with the surface; measuring the amount of the third reporter molecule that is associated with the surface; measuring the amount of the fourth reporter molecule that is associated with the surface; measuring the amount of the fifth reporter molecule that is associated with the surface; measuring the amount of the sixth reporter molecule that is associated with the surface; measuring the amount of the seventh reporter molecule that is associated with the surface; wherein the amount 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 classifies the subject as being at risk for progression of prostate cancer or not at risk for progression of prostate cancer.
[0109] In another embodiment, such a kit comprises a device for contacting the reagent solutions with a biological sample. In another embodiment, such a kit comprises at least one surface with 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), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0.
[0110] In another aspect, the disclosure provides a method comprising a) performing the following steps on obtaining a sample from a subject asymptomatic for prostate cancer; b) measuring a panel of markers in the sample, wherein the markers comprise CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0; c) determining a biomarker score for each marker; d) summing the biomarker scores for each marker to obtain a composite score for each subject, quantifying the risk for the risk for progression of prostate cancer for the subject as a risk score, wherein the composite score is matched to a risk category of a grouping of stratified subject populations, wherein each risk category comprises a multiplier indicating increased likelihood of having the prostate cancer correlated to a range of composite scores as compared to use of a single threshold value, wherein the multiplier is determined from positive predictive scores of retrospective samples; and, e) administering a computerized tomography (CT) scan or other imagine modality to the subject with a quantified risk for the the risk for progression of prostate cancer. In another embodiment, the markers consist of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0. In another embodiment, the sample is blood, blood serum, blood plasma, or some part thereof. In another embodiment, the grouping of a stratified subject population, the multiplier indicating increased likelihood of having the cancer and the range of composite scores are determined from retrospective clinical samples of a population. In another embodiment, the risk category further comprises a risk identifier. In another embodiment, the risk identifier is selected from low risk, intermediate-low risk, intermediate risk, intermediate-high risk and highest risk. In another embodiment, calculating the multiplier indicating increased likelihood of having the cancer for each risk category comprises stratifying the subject cohort based on retrospective biomarker scores and weighting a known prevalence of the cancer in the cohort by a positive predictive score for each stratified population. In another embodiment, the grouping of a stratified subject population comprises at least three risk categories wherein the multiplier indicating increased likelihood of having cancer is about 2 or greater. In another embodiment, the grouping of a stratified subject population comprises at least two risk categories wherein the multiplier indicating increased likelihood of having cancer is about 5 or greater. In another embodiment, the subject is aged 50 years or older and has a history of smoking tobacco. In another embodiment, such methods further comprise generating a risk categorization table, wherein the panel of markers is measured, a biomarker score for each marker is determined, a composite score is obtained by summing the biomarker scores; determining a threshold value used to divide the composite scores into risk groups and assigning a multiplier to each group indicating the likelihood of an asymptomatic subject having a quantified risk for the progression of cancer. In another embodiment, the groups are in a form selected from an electronic table form, a software application, a computer program, and an excel spreadsheet. In another embodiment, the panel of markers comprise proteins, polypeptides, or metabolites measured in a binding assay. In another embodiment, the panel of markers comprise proteins or polypeptides measured using a flow cytometer.
[0111] Provided are methods for identifying the risk for progression of prostate cancer in a subject, the method generally comprising: (a) applying a blood sample obtained from the subject to analysis for four biomarkers: CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0; (b) quantifying the amount of the four biomarkers present in the blood sample; and (c) applying statistical analysis based on the amount of biomarkers present to determine a biomarker score with respect to corresponding prostate cancer, thereby classifying a subject as either positive for risk of progression of prostate cancer or negative for risk of progression of prostate cancer. Alternatively, provided are methods for identifying the risk for progression of prostate cancer in a subject, the method generally comprising: (a) applying a blood sample obtained from the subject to analysis for four biomarkers: CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0; (b) quantifying the amount of the four biomarkers present in the blood sample; and (c) applying statistical analysis based on the amount of biomarkers present to determine a biomarker score with respect to corresponding prostate cancer, thereby providing a means for assessing in a subject relative risk prostate cancer progression (e.g., in a non-binary fashion).
[0112] The methods presented herein enable the screening of high-risk subjects, such as those with a family history of prostate cancer, or subjects with other risk factors such as obesity, heavy smoking, and possibly diabetes. The logistic regression model disclosed herein can incorporate these factors into its classification method.
[0113] As used herein, “prostate cancer status” refers to classification of an individual, subject, or patient as being at risk for progression of prostate cancer or as not being at risk for progression of prostate cancer. In some embodiments, an individual being at risk for progression of prostate cancer may be referred to as “prostate cancer-positive.” In other embodiments, an individual not being at risk for progression of prostate cancer may be referred to as “prostate cancer-negative.” For subjects that are classified as prostate cancer-positive, further methods can be provided to clarify prostate cancer status. Classification as prostate cancer-positive can be followed by methods including, but not limited to, computed tomography (CT).
[0114] The disclosure is not limited to the specific biomolecules that are reported herein for detection of the biomarkers. Other molecules may be chosen for use in other embodiments, including, but not limited to, biomolecules based on proteins, antibodies, nucleic acids, aptamers, and synthetic organic compounds. Other molecules may demonstrate advantages in terms of sensitivity, efficiency, speed of assay, cost, safety, or ease of manufacture or storage.
[0115] In some embodiments, levels of CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 in a biological sample are measured. In some embodiments, CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 are contacted with reporter molecules, and the levels of respective reporter molecules are measured. In some embodiments, four reporter molecules are provided which specifically bind CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0, respectively. Use of reporter molecules can provide gains in convenience and sensitivity for the assay.
[0116] In some embodiments, CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 are adsorbed onto a surface that is provided in a kit. In some embodiments, reporter molecules bind to surface-adsorbed CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0. Adsorption of biomarkers can be nonselective or selective. In some embodiments, the surface comprises a receptor functionality for increasing selectivity towards adsorption of one or more biomarkers.
[0117] In some embodiments, CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 are adsorbed onto four surfaces that are selective for one or more of the biomarkers. A reporter molecule or multiple reporter molecules can then bind to surface-adsorbed biomarkers, and the level of reporter molecule(s) associated with a particular surface can allow facile quantification of the particular biomarker that is present on that surface.
[0118] In some embodiments, CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 are adsorbed onto a surface that is provided in a kit; relay molecules that are specific for one or more of these biomarkers bind to surface-adsorbed biomarkers; and receptor molecules that are specific for one or more relay molecules bind to relay molecules. Relay molecules can provide specificity for certain biomarkers, and receptor molecules can enable detection.
[0119] In some embodiments, four relay molecules are provided which specifically bind CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0, respectively. Relay molecules can be intentionally designed for specificity towards a biomarker, or can be selected from a pool of candidates due to their binding properties.
[0120] In some embodiments, CAV-1, SM(40:2), SM(44:2), LacCer 32:0, LacCer 36:0, TriHexCer 34:1 and HexCer 40:0 are adsorbed onto four discrete surfaces that are provided in a kit; relay molecules that are specific for one or more of these biomarkers bind to surface-adsorbed biomarkers; and receptor molecules bind to relay molecules. Analysis of the surfaces can be accomplished in a stepwise or concurrent fashion.
[0121] In some embodiments, the reporter molecule is linked to an enzyme, facilitating quantification of reporter molecule. In some embodiments, quantification can be achieved by catalytic production of a substance with desirable spectroscopic properties.
[0122] In some embodiments, the amount of biomarker is determined with spectroscopy. In some embodiments, the spectroscopy that is utilized is UV-visible spectroscopy. In some embodiments, the spectroscopy that is utilized is mass spectrometry. In other embodiments, the spectroscopy that is utilized is nuclear magnetic resonance (NMR) spectroscopy, such as including, but not limited to, proton NMR spectroscopy, nuclear magnetic resonance (NMR) spectrometry, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (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 spectrometry.
[0123] The quantity of biomarker or biomarkers that is found in a particular assay can be directly reported to an operator, or alternately it can be stored digitally and readily made available for mathematical processing. A system can be provided for performing mathematical analysis, and can further report classification as prostate cancer-positive or prostate cancer-negative to an operator.
[0124] In some embodiments, additional assays known to those of ordinary skill in the art can function with the disclosure herein. Other assays include, but are not limited to, assays utilizing mass-spectrometry, immunoaffinity LC-MS/MS, surface plasmon resonance, chromatography, electrochemistry, acoustic waves, immunohistochemistry and array technologies.
[0125] The various system components discussed herein may include one or more of the following: a computer comprising one or more processors for processing digital data; short- or long-term digital memory; an input analog-to-digital converter for providing digitized data; an application program made available to the processor for directing processing of digital data by the processor; an input device for collecting information from the subject or operator, and an output device for displaying information to the subject or operator.
[0126] Also provided herein are methods of treatment for subjects who are classified as prostate cancer-positive. Treatment for prostate cancer-positive patients can include, but is not limited to, surgery, chemotherapy, radiation therapy, targeted therapy, or a combination thereof.
[0127] With regard to the detection of the biomarkers detailed herein, the disclosure is not limited to the specific biomolecules reported herein. In some embodiments, other biomolecules can be chosen 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 demonstrate advantages in terms of sensitivity, efficiency, speed of assay, cost, safety, or ease of manufacture or storage. In this regard, those of ordinary skill in the art will appreciate that the predicative and diagnostic power of the biomarkers disclosed herein may extend to the analysis of not just the protein form of these biomarkers, but other representations of the biomarkers as well (e.g., nucleic acid). Further, those of ordinary skill in the art will appreciate that the predicative and diagnostic power of the biomarkers disclosed herein can also be used in combination with an analysis of other biomarkers associated with prostate cancer. In some embodiments, other biomarkers associated with prostate cancer can be protein-based biomarkers.
[0128] The foregoing has outlined rather broadly the features and technical benefits of the disclosure in order that the detailed description may be better understood. It should be appreciated by those skilled in the art that the specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures or processes for carrying out the same purposes of the disclosure. It is to be understood that the present disclosure is not limited to the particular embodiments described, as variations of the particular embodiments may be made and still fall within the scope of the appended claims.
Definitions
[0129] As used herein, the term “prostate cancer” refers to a malignant neoplasm of the prostate characterized by the abnormal proliferation of cells, the growth of which cells exceeds and is uncoordinated with that of the normal tissues around it.
[0130] As used herein, the term “prostate cancer-positive” refers to classification of a subject as being at risk for progression of prostate cancer.
[0131] As used herein, the term “prostate cancer-negative” refers to classification of a subject as not being at risk for progression of prostate cancer.
[0132] As used herein, the terms “subject” or “patient” refer to a mammal, preferably a human, for whom a classification as prostate cancer-positive or prostate cancer-negative is desired, and for whom further treatment can be provided.
[0133] As used herein, a “reference patient,” “reference subject,” or “reference group” refers to a group of patients or subjects to which a test sample from a patient or subject suspected of having or being at risk for progression of prostate cancer may be compared. In some embodiments, such a comparison may be used to determine whether the test subject has prostate cancer. A reference patient or group may serve as a control for testing or diagnostic purposes. As described herein, a reference patient or group may be a sample obtained from a single patient, or may represent a group of samples, such as a pooled group of samples.
[0134] As used herein, “healthy” refers to an individual in whom no evidence of prostate cancer is found, i.e., the individual does not have prostate cancer. Such an individual may be classified as “prostate cancer-negative” or as having a healthy prostate gland, or normal, non-compromised prostate function. A healthy patient or subject has no 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 to diseased or suspected diseased samples for determination of prostate cancer in a patient or a group of patients.
[0135] As used herein, the terms “treatment” or “treating” refer to the administration of medicine or the performance of medical procedures with respect to a subject, for either prophylaxis (prevention) or to cure or reduce the extent of or likelihood of occurrence or recurrence of the infirmity or malady or condition or event in the instance where the subject or patient is afflicted. As related to the present disclosure, the term may also mean the administration of pharmacological substances or formulations, or the performance of non-pharmacological methods including, but not limited to, radiation therapy and surgery. Pharmacological substances as used herein may include, but are not limited to, chemotherapeutics that are established in the art, such as abiraterone acetate (Zytiga), apalutamide (Erleada), bicalutamide (Casodex), cabazitaxel (Jevtana), darolutamide (Nubega), degarelix (Firmagon), docetaxel (Taxotere), Eligard (leuprolide acetate), enzalutamide (Xtandi), flutamide, goserelin acetate (Zoladex), leuprolide acetate (Lupron or Lupron Depot), olaparib (Lynparza), mitoxantrone hydrochloride, nilutamide (Nilandron), sipuleucel-T (Provenge), radium 223 dichloride (Xofigo), and rucaparib camsylate (Rubraca).
[0136] Pharmacological substances may include substances used in immunotherapy, such as checkpoint inhibitors. Treatment may include a multiplicity of pharmacological substances, or a multiplicity of treatment methods, including, but not limited to, surgery and chemotherapy.
[0137] As used herein, the term “ELISA” refers to enzyme-linked immunosorbent assay. This assay generally involves contacting a fluorescently tagged sample of proteins with antibodies having specific affinity for those proteins. Detection of these proteins can be accomplished with a variety of means, including, but not limited to, laser fluorimetry.
[0138] As used herein, the term “regression” refers to a statistical method that can assign a predictive value for an underlying characteristic of a sample based on an observable trait (or set of observable traits) of said sample. In some embodiments, the characteristic is not directly observable. For example, the regression methods used herein can link a qualitative or quantitative outcome of a particular biomarker test, or set of biomarker tests, on a certain subject, to a probability that said subject is for prostate cancer-positive.
[0139] As used herein, the term “logistic regression” refers to a regression method in which the assignment of a prediction from the model can have one of several allowed discrete values. For example, the logistic regression models used herein can assign a prediction, for a certain subject, of either prostate cancer-positive or prostate cancer-negative.
[0140] As used herein, the term “biomarker score” refers to a numerical score for a particular subject that is calculated by inputting the particular biomarker levels for said subject to a statistical method.
[0141] As used herein, the term “composite score” refers to a summation of the normalized values for the predetermined markers measured in the sample from the subject. In one embodiment, the normalized values are reported as a biomarker score and those biomarker score values are then summed to provide a composite score for each subjected tested. When used in the context of the risk categorization table and correlated to a stratified grouping based on a range of composite scores in the Risk Categorization Table, the “composite score” is used to determine the “risk score” for each subject tested wherein the multiplier indicating increased likelihood of having the cancer for the stratified grouping becomes the “risk score,”
[0142] As used herein, the term “risk score” refers to a single numerical value that indicates an asymptomatic human subject's risk for progression of cancer cancer as compared to the known prevalence of cancer progression in the disease cohort. In certain embodiments, the composite score as calculated for a human subject and correlated to a multiplier indicating risk for progression of prostate cancer, wherein the composite score is correlated based on the range of composite scores for each stratified grouping in the risk categorization table. In this way the composite score is converted to a risk score based on the multiplier indicating increased likelihood of having the cancer for the grouping that is the best match for the composite score.
[0143] As used herein, the term “cutoff” or “cutoff point” refers to a mathematical value associated with a specific statistical method that can be used to assign a classification of prostate cancer-positive of prostate cancer-negative to a subject, based on said subject's biomarker score.
[0144] As used herein, when a numerical value above or below a cutoff value “is characteristic of prostate cancer,” what is meant is that the subject, analysis of whose sample yielded the value, either has prostate cancer or is at risk for progression of prostate cancer.
[0145] As used herein, a subject who is “risk for progression of prostate cancer” is one who may not yet evidence overt symptoms of prostate cancer, or whose prostate cancer is currently indolent, but who is producing levels of biomarkers which indicate that the subject has prostate cancer, or may develop it in the near term. A subject who has prostate cancer or is suspected of harboring prostate cancer may be treated for the cancer or suspected cancer.
[0146] As used herein, the term “classification” refers to the assignment of a subject as being at risk for progression of prostate cancer or not being at risk for progression of prostate cancer, based on the result of the biomarker score that is obtained for said subject.
[0147] As used herein, the term “Wilcoxon rank sum test,” also known as the Mann-Whitney U test, Mann-Whitney-Wilcoxon test, or Wilcoxon-Mann-Whitney test, refers to a specific statistical method used for comparison of two populations. For example, the test can be used herein to link an observable trait, in particular a biomarker level, to the absence or the risk for progression of prostate cancer in subjects of a certain population.
[0148] As used herein, the term “true positive rate” refers to the probability that a given subject classified as positive by a certain method is truly positive.
[0149] As used herein, the term “false positive rate” refers to the probability that a given subject classified as positive by a certain method is truly negative.
[0150] As used herein, the term “sensitivity” refers to, in the context of various biochemical assays, the ability of an assay to correctly identify those with a disease (i.e., the true positive rate). By comparison, as used herein, the term “specificity” refers to, in the context of various biochemical assays, the ability of an assay to correctly identify those without the disease (i.e., the true negative rate). Sensitivity and specificity are statistical measures of the performance of a binary classification test (i.e., classification function). Sensitivity quantifies the avoiding of false negatives, and specificity does the same for false positives.
[0151] As used herein, a “sample” refers to a test substance to be tested for the presence of, and levels or concentrations thereof, of a biomarker as described herein. A sample may be any substance appropriate in accordance with the present disclosure, including, but not limited to, blood, blood serum, blood plasma, or any part thereof.
[0152] As used herein, a “metabolite” refers to small molecules that are intermediates and/or products of cellular metabolism. Metabolites may perform a variety of functions in a cell, for example, structural, signaling, stimulatory and/or inhibitory effects on enzymes. In some embodiments, a metabolite may be a non-protein, plasma-derived metabolite marker, such as including, but not limited to, acetylspermidine, diacetylspermine, lysophosphatidylcholine (18:0), lysophosphatidylcholine (20:3), and an indole-derivative.
[0153] As used herein, the term “ROC” refers to receiver operating characteristic, which is a graphical plot used herein to gauge the performance of a certain diagnostic method at various cutoff points. A ROC plot can be constructed from the fraction of true positives and false positives at various cutoff points.
[0154] As used herein, the term “AUC” refers to the area under the curve of the ROC plot. AUC can be used to estimate the predictive power of a certain diagnostic test. Generally, a larger AUC corresponds to increasing predictive power, with decreasing frequency of prediction errors. Possible values of AUC range from 0.5 to 1.0, with the latter value being characteristic of an error-free prediction method.
[0155] As used herein, the term “p-value” or “p” refers to the probability that the distributions of biomarker scores for prostate cancer-positive and prostate cancer-negative subjects are identical in the context of a Wilcoxon rank sum test. Generally, a p-value close to zero indicates that a particular statistical method will have high predictive power in classifying a subject.
[0156] As used herein, the term “CI” refers to a confidence interval, i.e., an interval in which a certain value can be predicted to lie with a certain level of confidence. As used herein, the term “95% CI” refers to an interval in which a certain value can be predicted to lie with a 95% level of confidence.
[0157] As used herein, the term “disease progression” or “early disease progression” is defined as upgrading of Gleason score and/or increased tumor volume on surveillance biopsy within 18 months after start of active surveillance.
[0158] As used herein, the term “indolent disease” or “indolent” prostate cancer is defined as the absence of progression for five or more years after start of active surveillance.
Abbreviations
[0159] AKT=RAC serine/threonine-protein kinase; AS=active surveillance; AUC=area under the curve; Cav-1=caveolin-1; CCLE=Broad Institute Cancer Cell Line Encyclopedia; CE=cholesterol ester; CM=conditioned media; DiI=1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine; DP=disease progression; FC=free cholesterol; GS=Gleason score; HexCer=hexosylceramide; HexCer 40:0=hexosylceramide(40:0); HR=hazard ratio; LacCer=lactosylceramide; LacCer 32:0=lactosylceramide(32:0); LacCer 36:0=lactosylceramide(36:0); MAPK=MAP-kinase=mitogen-activated 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 characteristic; SEM=standard error of the mean: SFM=serum-free media; sHDL=synthetic HDL-like particles; sLDL=synthetic LDL-like particles; SM=sphingomyelin; SSALP=synthetic self-assembled lipid particle; TCGA=The Cancer Genome Atlas; TO=trioleate; TriHexCer=trihexosylceramide; TriHexCer 34:1=trihexosylceramide(34:1).
EXAMPLES
[0160] The following examples are included to demonstrate embodiments of the disclosure. The following examples are presented only by way of illustration and to assist one of ordinary skill in using the disclosure. The examples are not intended in any way to otherwise limit the scope of the disclosure. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
Example 1: Isolation of Extracellular Vesicles by Density Gradient Flotation
[0161] Extracellular vesicles were isolated as previously described. Briefly, microvesicles were depleted from biospecimen samples by centrifugation at 2000×g for 20 min followed by 16,500×g for 30 min; resulting supernatant was filtered through a pre-wetted 0.22 μm vacuum filter. Microvesicle-depleted biospecimen was densified by mixing with OptiPrep iodixanol solution (Sigma D1556) to a final density of 1.16-1.30 g mL.sup.−1 and loaded into the bottom of a polycarbonate ultracentrifuge tube and overlaid with 0.5-2 mL aliquots of iodixanol/PBS solution in the 1.20-1.01 g mL.sup.−1 (35-0% wt:vol) range, proceeding from the highest to lowest density to form a single- or multi-step density fractionation gradient as needed. Ultracentrifugation was performed for at 100,000×g for 4 hrs at 8° C. Vesicles were collected from the top of the tube, proceeding downward to recover volume equal to 90% of overlaid gradient volume. Density of harvested fractions was assessed against a standard curve based on sample absorbance at 250 nm using a NanoDrop microvolume spectrophotometer (ThermoFisher Scientific, Wilmington, DE). Vesicle harvests were stored at −80° C.
Example 2: Proteomic Profiling of Extracellular Vesicles
[0162] Proteomic profiling of extracellular vesicles was performed according to the following standardized workflows. Briefly, ECV-derived protein digestion and identification by LC-MS/MS was performed using established protocols. NanoAcquity UPLC system coupled in-line with WATERS SYNAPT G2-Si mass spectrometer was used for the separation of 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 analytical column (75 μm×150 mm, 1.8 μm). The column oven temperature was set at 50° C., and the temperature of the tray compartment in the auto-sampler was set at 6° C. LC-HDMSE data were acquired in resolution mode with SYNAPT G2-Si using Waters Masslynx (version 4.1, SCN 851). The capillary voltage was set to 2.80 kV, sampling cone voltage to 30 V. source offset to 30 V and source temperature to 100° C. Mobility utilized high-purity N2 as the drift gas in the IMS TriWave cell. Pressures in the helium cell, Trap cell, IMS TriWave cell and Transfer cell were 4.50 mbar, 2.47×10.sup.−2, 2.90. and 2.53×10.sup.−3 mbar, respectively. IMS wave velocity was 600 n s.sup.−1, helium cell DC 50 V, Trap DC bias 45 V, IMS TiWave DC bias V and IMS wave delay 1000 μs. The mass spectrometer was operated in V-mode with a typical resolving power of at least 20,000. All analyses were performed using positive mode ESI using a NanoLockSpray source. The lock mass channel was sampled every 60 s. The mass spectrometer was calibrated with a [Glu1] fibrinopeptide solution (300 fmol μL.sup.−1) delivered through the reference sprayer of the NanoLockSpray source. Accurate mass LC-HDMSE data were collected in an alternating, low energy (MS) and high energy (MSE) mode of acquisition with mass scan range from m z.sup.−1 50 to 1800. The spectral acquisition time in each mode was 1.0 s with a 0.1-s inter-scan delay. In low energy HDMS mode, data were collected at constant collision energy of 2 eV in both Trap cell and Transfer cell. In high-energy HDMSE mode, the collision energy was ramped from 25 to 55 eV in the Transfer cell only. The RF applied to the quadrupole mass analyzer was adjusted such that ions from m z.sup.−1 300 to 2000 were efficiently transmitted, ensuring that any ions observed in the LC-HDMSE data<m z.sup.−1 of 300 arose from dissociations in the Transfer collision cell. The acquired LC-HDMSE data were processed and searched against protein knowledge database (Uniprot) through ProteinLynx Global Server (PLGS, Waters Company) with 4% FDR.
Example 3: Metabolomics Analyses
[0163] Sample Extraction Following transfections, cell lysates were washed 2× with pre-chilled 0.9% NaCl followed by addition of 2.5 mL of pre-chilled 3:1 isopropanol:ultrapure water. Cells were scraped using a 25 cm Cell Scraper (Sarstedt) in extraction solvent and transferred to a 15 mL conical tube (Eppendorf). Samples were briefly vortexed followed by centrifugation at 4° C. for 10 min at 2,000×g. Thereafter, 1.2 mL of metabolite extracts were transferred to 1.5 mL Eppendorf tubes and stored in −20° C. until metabolomic analysis.
[0164] Primary Metabolites and Biogenic Amines Plasma metabolites were extracted from pre-aliquoted EDTA plasma (10 μL) with 30 μL of LCMS grade methanol (ThermoFisher) in a 96-well microplate (Eppendorf). Plates were heat sealed, vortexed for 5 min at 750 rpm, and centrifuged at 2000×g for 10 minutes at room temperature. The supernatant (10 μL) was carefully transferred to a 96-well plate, leaving behind the precipitated protein. The supernatant was further diluted with 10 μL of 100 mM ammonium formate, pH 3. For Hydrophilic Interaction Liquid Chromatography (HILIC) analysis, the samples were diluted with 60 μL LCMS grade acetonitrile (ThermoFisher), whereas samples for C18 analysis were diluted with 60 μL water (GenPure ultrapure water system, ThermoFisher). Each sample solution was transferred to 384-well microplate (Eppendorf) for LCMS analysis. For conditioned media, frozen samples were thawed on ice and 30 μl transferred to a 96-well microplate (Eppendorf). Aliquots were diluted with an additional 30 μL of 100 mM ammonium formate. Microplates were heat sealed, vortexed for 5 min at 1500 rpm, and centrifuged at 2000×g for 10 minutes at room temperature. For Hydrophilic Interaction Liquid Chromatography (HILIC) analysis, the 25 μL of sample was transferred to a new 96 well microplate containing 75 μL acetonitrile, whereas samples for C18 analysis were transferred to a new 96-well microplate containing 75 μL water (GenPure ultrapure water system, ThermoFisher). Each sample solution was transferred to 384-well microplate (Eppendorf) for LCMS analysis. Cell lysate supernatant, 100 μL (3:1 isopropanol:ultrapure water) was aliquoted into two 96-well plates (Eppendorf) and evaporated to dryness under vacuum. The samples were then reconstituted as follows: for the HILIC assays, the dried samples were dissolved in 65 μL of ACN (ThermoFisher): 100 mM Ammonium Formate pH3 (9:1) whereas for the C18 reverse phase assays, the dried samples were dissolved in 65 μL of H.sub.2O: 100 mM Ammonium Formate pH3 (9:1). The samples were spun down to remove any insoluble materials and then transferred to a 384-well plate for high throughput mass analysis using LCMS.
[0165] Complex Lipids Pre-aliquoted EDTA plasma samples (10 μL) were extracted with 30 μL of LCMS grade 2-propanol (ThermoFisher) in a 96-well microplate (Eppendorf). Plates were heat sealed, vortexed for 5 min at 750 rpm, and centrifuged at 2000×g for 10 minutes at room temperature. The supernatant (10 μL) was carefully transferred to a 96-well plate, leaving behind the precipitated protein. The supernatant was further diluted with 90 μL of 1:3:2 100 mM ammonium formate, pH 3 (ThermoFisher): acetonitrile: 2-propanol and transferred to a 384-well microplate (Eppendorf) for lipids analysis using LCMS. For cell lysates, in a 96 well plate, 10 μL (3:1 isopropanol:ultrapure water) of the cell lysates supernatant was diluted with 90 μL of 1:3:2 100 mM ammonium formate, pH 3:acetonitrile:2-propanol (ThermoFisher) and transferred to a 384-well microplate (Eppendorf) for analysis by LC-MS.
[0166] Untargeted Analysis of Primary Metabolites and Biogenic Amines
[0167] Untargeted metabolomics analysis was conducted on Waters Acquity™ UPLC system with 2D column regeneration configuration (I-class and H-class) coupled to a Xevo G2-XS quadrupole time-of-flight (qTOF) mass spectrometer. Chromatographic separation was performed using HILIC (Acquity™ UPLC BEH amide, 100 Å, 1.7 μm 2.1×100 mm, Waters Corporation, Milford, U.S.A) and C18 (Acquity™ UPLC HSS T3, 100 Å, 1.8 μm, 2.1×100 mm, Water Corporation, Milford, U.S.A) columns at 45° C. Quaternary solvent system mobile phases were (A) 0.1% formic acid in water, (B) 0.1% formic acid in acetonitrile and (D) 100 mM ammonium formate, pH 3. Samples were separated using the following gradient profile: for the HILIC separation a starting gradient of 95% B and 5% D was increase linearly to 70% A, 25% B and 5% D over a 5 min period at 0.4 mL min.sup.−1 flow rate, followed by 1 min isocratic gradient at 100% A at 0.4 mL min.sup.−1 flow rate. For C18 separation, a chromatography gradient of was as follows: starting conditions, 100% A, with linear increase to final conditions of 5% A, 95% B followed by isocratic gradient at 95% B, 5% D for 1 min. Binary pump was used for column regeneration and equilibration. The solvent system mobile phases were (A1) 100 mM ammonium formate, pH 3, (A2) 0.1% formic in 2-propanol and (B1) 0.1% formic acid in acetonitrile. The HILIC column was stripped using 90% A2 for 5 min followed by 2 min equilibration using 100% B1 at 0.3 mL min.sup.−1 flowrate. Reverse phase C18 column regeneration was performed using 95% A1, 5% B1 for 2 min followed by column equilibration using 5% A1, 95% B1 for 5 min.
[0168] Untargeted Analysis of Complex Lipids For the lipidomic assay, untargeted metabolomics analysis was conducted on a Waters Acquity™ UPLC system coupled to a Xevo G2-XS quadrupole time-of-flight (qTOF) mass spectrometer. Chromatographic separation was performed using a C18 (Acquity™ UPLC HSS T3, 100 Å, 1.8 μm, 2.1×100 mm, Water Corporation, Milford, USA) column at 55° C. The mobile phases were (A) water, (B) Acetonitrile, (C) 2-propanol and (D) 500 mM ammonium formate, pH 3. A starting elution gradient of 20% A, 30% B, 49% C and 1% D was increased linearly to 10% B, 89% C and 1% D for 5.5 min, followed by isocratic elution at 10% B, 89% C and 1% D for 1.5 min and column equilibration with initial conditions for 1 min.
Example 4: Mass Spectrometry
[0169] Data Acquisition. Mass spectrometry data was acquired using sensitivity mode in positive and negative electrospray ionization mode within 50-1200 Da range for primary metabolites and 100-2000 Da for complex lipids. For the electrospray acquisition, the capillary voltage was set at 1.5 kV (positive), 3.0 kV (negative), sample cone voltage 30 V, source temperature at 120° C., cone gas flow 50 L h.sup.−1 and desolvation gas flow rate of 800 L h.sup.−1 with scan time of 0.5 sec in continuum mode. Leucine Enkephalin; 556.2771 Da (positive) and 554.2615 Da (negative) was used for lockspray correction and scans were performed at 0.5 min. The injection volume for each sample was 3 μL, unless otherwise specified. The acquisition was carried out with instrument auto gain control to optimize instrument sensitivity over the samples acquisition time.
[0170] Data Processing LC-MS and LC-MSe data were processed using Progenesis QI (Nonlinear, Waters) and values were reported as area units. Annotations were determined by matching accurate mass and retention times using customized libraries created from authentic standards and/or by matching experimental tandem mass spectrometry data against the NIST MSMS or HMDB v3 theoretical fragmentations.
[0171] Data Normalization To correct for injection order drift, each feature was normalized using data from repeat injections of quality control samples collected every 10 injections throughout the run sequence. Measurement data were smoothed by Locally Weighted Scatterplot Smoothing (LOESS) signal correction (QC-RLSC) as previously described. Feature values between quality control samples were interpolated by a cubic spline. Metabolite values were rescaled by using the overall median of the historical quality control peak areas across all samples. Only detected features exhibiting a relative standard deviation (RSD) less than 30 in either the historical or pooled quality controls samples were considered for further statistical analysis. To reduce data matrix complexity, annotated features with multiple adducts or acquisition mode repeats were collapsed to one representative unique feature. Features were selected based on replicate precision (RSD<30), highest intensity and best isotope similarity matching to theoretical isotope distributions. Values are reports as ratios relative to the historical quality control reference samples that is included in every analytical run (plasma/conditioned media) or adjusted area units (lysates).
[0172] Statistical Analyses In order to find the cut-off point for the covariate that gives the largest difference between individuals in the two already defined groups, the previously described method was employed. Using log rank statistic-based on the groups defined by cut-off affords:
[0173] where D is the total number of distinct events (disease progression (DP)), d.sub.i is the total number of DP at each event time (t.sub.i), d.sub.i.sup.+ is the total number of DP when the Cav-1-sphingolipid signature value is bigger than the cut-off point. r.sub.i and r.sub.i.sup.+ also define as the total number at risk for all Cav-1-sphingolipid signature values and Cav-1-sphingolipid signature values larger than cut-off point, respectively. S.sub.k was calculated for all possible cut point in Cav-1-sphingolipid signature column and the estimated cut point is the value that yields the maximum S.sub.k. In this analysis, the maximum value of S.sub.k is at the top 16.4% of Cav-1-sphingolipid signature values. In another word, the top 16.4% of Cav-1-sphingolipid signature values are in the high-risk group and the other 83.6% are in the low-risk group.
[0174] In order to calculate the p-value of this test use of the following formula provided the value of 0.009. It suggests that the Cav-1-sphingolipid signature level highly relates to progression free survival.
Example 5: Prediction of AS Gleason Grade Progression by Plasma Lipid Signature
[0175] Untargeted metabolomics analyses were conducted on clinically matched baseline plasma samples (n=16 per group) prospectively collected from patients with clinically low-risk early stage prostate cancer undergoing AS who exhibited early DP or indolent disease (Table 1). A total of 269 unique annotated metabolite features were identified in baseline plasmas from the discovery cohort; 14 features exhibited statistically significant (unadjusted Wilcoxon-rank sum test p-value<0.05) ROC AUC values>0.7.
TABLE-US-00001 TABLE 1 Patient characteristics for MDACC discovery cohort. Total Cases Controls p N 32 16 16 Age, y 64.4 ± 7.9 64.3 ± 8.2 64.4 ± 7.9 0.9548 BMI, kg/m.sup.2 27.0 ± 3.9 27.3 ± 3.5 26.7 ± 4.3 0.7219 Family history of PC 1st degree 8 (25) 3 (18.8) 5 (31.3) 0.6851 2nd degree 4 (12.5) 3 (18.8) 1 (6.3) 0.5996 Hypertension 13 (40.6) 5 (31.3) 8 (50) 0.4725 Diabetes 5 (15.6) 3 (18.8) 2 (12.5) 1 Smoking (ever smoker) 14 (43.8) 8 (50) 6 (37.5) 0.4757 5ARI 3 (9.4) 0 (0) 3 (18.8) 0.2258 Statins 10 (31.3) 4 (25) 6 (37.5) 0.7043 PSA 3.8 ± 2.1 3.7 ± 1.8 3.9 ± 2.3 0.9849 Testosterone 383.7 ± 179.2 400.9 ± 197.0 366.5 ± 164.1 0.6922 TRUS (Total) 42.0 ± 18.1 41.8 ± 19.6 42.2 ± 17.3 0.771 TRUS (TZ) 18.7 ± 11.0 19.2 ± 11.3 18.3 ± 11.0 0.8264 Summation Total Tumor Length (mm) 4.6 ± 4.9 4.8 ± 3.9 4.5 ± 5.9 0.4249 Highest Gleason Score 1 3 + 3 24 (75) 12 (75) 12 (75) 3 + 4 7 (21.9) 3 (18.8) 4 (25) 4 + 3 1 (3.1) 1 (6.3) 0 (0)
[0176] Seven of the 14 features were complex lipids; in particular, sphingomyelins and glycosphingolipids (Table 2).
TABLE-US-00002 TABLE 2 Metabolite features identified in the discovery cohort Accepted Compound ID Domain AUC1 p-value# AUC2 p-value# AUC3 p-value# 1-Methylhistidine (a) 0.41 0.381 0.50 1.000 0.52 0.867 1-Methylnicotinamide (a) 0.37 0.224 0.45 0.669 0.54 0.724 2-aminobenzamide (a) 0.43 0.515 0.45 0.642 0.61 0.287 3-Methoxytyramine (a) 0.46 0.752 0.39 0.287 0.36 0.184 3-Methylhistamine (a) 0.38 0.239 0.52 0.897 0.54 0.752 3-Methylhistidine (a) 0.32 0.094 0.45 0.616 0.38 0.254 4-Acetamido-2- (a) 0.59 0.423 0.50 0.985 0.59 0.423 aminobutanoic acid 4-Hydroxy-L-proline (a) 0.57 0.491 0.37 0.210 0.48 0.897 4-phosphopantothenoyl- (a) 0.40 0.361 0.52 0.867 0.49 0.926 cysteine 5-Oxo-D-proline (a) 0.52 0.867 0.55 0.669 0.65 0.149 α-N-Phenylacetyl-L- (a) 0.49 0.956 0.42 0.445 0.48 0.867 glutamine Aniline (a) 0.45 0.616 0.42 0.468 0.48 0.867 Citrulline (a) 0.36 0.184 0.62 0.270 0.53 0.780 Creatine (a) 0.50 0.985 0.54 0.696 0.50 0.985 Creatinine (a) 0.39 0.305 0.46 0.724 0.45 0.669 D-Aspartate (a) 0.59 0.381 0.41 0.423 0.50 0.985 Diethanolamine (a) 0.56 0.564 0.46 0.752 0.48 0.838 DL-5-Hydroxylysine (a) 0.45 0.616 0.54 0.696 0.55 0.669 D-Ornithine (a) 0.31 0.073 0.47 0.809 0.48 0.897 Glutathione (a) 0.46 0.752 0.61 0.305 0.51 0.956 Homoserine (a) 0.55 0.616 0.62 0.254 0.67 0.110 L-Arginine (a) 0.45 0.642 0.52 0.838 0.49 0.956 L-Asparagine (a) 0.45 0.616 0.40 0.341 0.52 0.897 L-Cystathionine (a) 0.55 0.616 0.46 0.696 0.51 0.956 L-Cystine (a) 0.46 0.752 0.47 0.809 0.46 0.752 Leucine (a) 0.58 0.468 0.61 0.323 0.62 0.270 L-Glutamine (a) 0.52 0.897 0.60 0.341 0.58 0.468 L-Histidine (a) 0.67 0.110 0.63 0.224 0.65 0.160 L-Histidinol (a) 0.34 0.119 0.45 0.616 0.38 0.254 L-Isoleucine (a) 0.51 0.926 0.64 0.184 0.61 0.287 L-Kynurenine (a) 0.32 0.086 0.46 0.752 0.49 0.956 L,L-2.6-Diamino- (a) 0.65 0.160 0.48 0.897 0.68 0.086 heptanedioate L-Lysine (a) 0.40 0.361 0.55 0.616 0.60 0.341 L-Methionine (a) 0.43 0.515 0.61 0.305 0.61 0.323 L-N.sup.γ.-Monomethylarginine (a) 0.57 0.515 0.63 0.239 0.63 0.224 L-Phenylalanine (a) 0.55 0.669 0.46 0.752 0.67 0.110 L-Pipecolic Acid (a) 0.61 0.305 0.57 0.539 0.59 0.381 L-Proline (a) 0.52 0.897 0.66 0.138 0.65 0.149 L-Serine (a) 0.46 0.752 0.34 0.119 0.51 0.926 L-Threonine (a) 0.51 0.956 0.46 0.724 0.56 0.590 L-Tryptophan (a) 0.56 0.590 0.45 0.616 0.67 0.102 L-Tyrosine (a) 0.52 0.897 0.55 0.616 0.61 0.305 L-Valine (a) 0.56 0.590 0.51 0.926 0.56 0.564 N(Pai)-Methyl-L-Histidine (a) 0.33 0.102 0.52 0.867 0.45 0.669 N.sup.8-Acetylspermidine (a) 0.53 0.809 0.49 0.926 0.60 0.361 N.sup.6,N.sup.6,N.sup.6-Trimethyllysine (a) 0.57 0.515 0.45 0.616 0.54 0.752 N.sup.γ,N.sup.γ-Dimethyl-L- (a) 0.48 0.897 0.46 0.752 0.48 0.897 Arginine Nicotinamide (a) 0.49 0.956 0.68 0.086 0.54 0.724 Nicotinamide (a) 0.71 0.039 0.55 0.669 0.69 0.067 Mononucleotide Nicotinate β-D- (a) 0.50 0.985 0.56 0.590 0.46 0.752 Ribonucleotide Nicotinuric Acid (a) 0.38 0.239 0.45 0.616 0.43 0.539 N-Methyl-D-Aspartic Acid (a) 0.52 0.897 0.61 0.323 0.54 0.752 N-Methyl-L-Glutamate (a) 0.55 0.616 0.51 0.956 0.55 0.616 N-Methylnicotinamide (a) 0.39 0.323 0.52 0.867 0.49 0.926 N-Undecanoylglycine (a) 0.32 0.086 0.42 0.468 0.36 0.196 O-Acetyl-L-Serine (a) 0.38 0.270 0.36 0.184 0.34 0.119 O-Phospho-L-Serine (a) 0.49 0.956 0.25 0.014 0.49 0.956 Picolinic Acid (a) 0.36 0.184 0.52 0.897 0.48 0.838 Pyroglutamic Acid (a) 0.66 0.119 0.62 0.254 0.67 0.110 Quinoline (a) 0.52 0.838 0.46 0.752 0.60 0.361 Thyroxine (a) 0.69 0.067 0.63 0.210 0.76 0.011 Tyramine (a) 0.45 0.616 0.51 0.926 0.51 0.926 Urocanate (a) 0.36 0.184 0.36 0.184 0.36 0.171 6-Phosphogluconic Acid (b) 0.38 0.270 0.54 0.724 0.34 0.128 α-D-Glucose (b) 0.61 0.305 0.53 0.809 0.57 0.539 D-Galactose (b) 0.73 0.026 0.57 0.539 0.72 0.035 D-Glucose-6-Phosphate (b) 0.35 0.160 0.49 0.956 0.29 0.047 Galactitol (b) 0.63 0.239 0.44 0.590 0.48 0.897 Maltose (b) 0.48 0.867 0.41 0.381 0.51 0.956 Melibiose (b) 0.53 0.809 0.58 0.468 0.60 0.341 N-Acetyllactosamine (b) 0.41 0.423 0.39 0.323 0.50 0.985 N-Acetylneuraminate (b) 0.36 0.184 0.39 0.323 0.39 0.287 Acetylcarnitine (c) 0.53 0.780 0.47 0.809 0.43 0.515 Acylcarnitine(C10:0) (c) 0.50 0.985 0.41 0.402 0.44 0.590 Acylcarnitine(C14:0) (c) 0.50 0.985 0.41 0.402 0.53 0.809 Acylcarnitine(C16:0) (c) 0.46 0.752 0.58 0.468 0.54 0.696 Acylcarnitine(C18:0) (c) 0.48 0.838 0.53 0.809 0.48 0.897 Acylcarnitine(C18:1) (c) 0.53 0.809 0.60 0.341 0.49 0.956 Deoxycarnitine (c) 0.63 0.239 0.55 0.616 0.58 0.468 Dodecanedioylcarnitine (c) 0.45 0.616 0.45 0.616 0.44 0.590 Glutarylcarnitine (c) 0.47 0.809 0.47 0.780 0.44 0.590 Hexanoylcarnitine (c) 0.63 0.224 0.51 0.956 0.53 0.809 Hydroxybutyrylcarnitine (c) 0.39 0.287 0.44 0.564 0.34 0.119 L-Carnitine (c) 0.51 0.926 0.41 0.423 0.45 0.642 O-Acetyl-L-Carnitine (c) 0.57 0.491 0.47 0.809 0.48 0.897 Octanoylcarnitine (c) 0.55 0.642 0.44 0.590 0.50 1.000 Propionylcarnitine (c) 0.61 0.287 0.54 0.724 0.57 0.539 Tiglylcarnitine (c) 0.49 0.926 0.51 0.956 0.47 0.809 Cer(41:1) (d) 0.55 0.616 0.52 0.867 0.59 0.402 Aspartyl-threonine (e) 0.55 0.642 0.43 0.515 0.52 0.867 Glycyl-threonine (e) 0.42 0.468 0.64 0.184 0.58 0.468 Histidinyl-proline (e) 0.54 0.724 0.56 0.564 0.75 0.015 1-(Hydroxymethyl)-5,5- (f) 0.54 0.696 0.63 0.239 0.65 0.160 dimethyl-2,4- imidazolidinedione Exogenous 13-Nor-6-eremophilene- (f) 0.40 0.341 0.44 0.590 0.39 0.287 8,11-dione Exogenous Benzofuran (f) 0.52 0.867 0.56 0.590 0.62 0.254 cis-Quinceoxepane (f) 0.58 0.445 0.50 0.985 0.50 0.985 Metformin (f) 0.53 0.780 0.52 0.897 0.47 0.809 Trazodone (f) 0.55 0.669 0.30 0.051 0.39 0.323 4,7-Dioxo-octanoic Acid (g) 0.59 0.423 0.35 0.149 0.61 0.323 cis-9, cis-12- (g) 0.65 0.149 0.46 0.696 0.59 0.423 Octadecadienoic Acid Dodecanoic acid (g) 0.64 0.184 0.54 0.696 0.57 0.515 Heptanoic acid (g) 0.54 0.724 0.51 0.956 0.53 0.780 Linoleate (g) 0.62 0.254 0.48 0.838 0.52 0.838 Myristoleic acid (g) 0.54 0.752 0.38 0.270 0.64 0.184 GlcCer(39:2) (h) 0.74 0.019 0.77 0.008 0.82 0.001 LacCer(32:0) (h) 0.74 0.019 0.76 0.012 0.82 0.001 LacCer(32:1) (h) 0.69 0.067 0.69 0.073 0.78 0.007 LacCer(34:1) (h) 0.64 0.171 0.67 0.102 0.68 0.094 Neuac?2-3Gal?1-4Glc?- (h) 0.49 0.956 0.53 0.809 0.51 0.926 Cer(D18:1/16:0) [Trihexosylcer- (D18:1/16:0)] Neuac?2-3Gal?-Cer(34:1) (h) 0.62 0.254 0.60 0.361 0.66 0.138 [Dihexosylcer(34:1)] Neuac?2-3Gal?-Cer(36:1) (h) 0.62 0.270 0.60 0.341 0.66 0.119 [Dihexosylcer(36:1)] Trihexosylceramide(34:1) (h) 0.64 0.171 0.55 0.642 0.55 0.642 Corticosterone (i) 0.65 0.149 0.48 0.897 0.64 0.196 Cortisol (i) 0.54 0.724 0.39 0.305 0.74 0.021 Cortisol 21-Acetate (i) 0.54 0.724 0.37 0.224 0.57 0.539 Cortisone (i) 0.55 0.669 0.48 0.867 0.64 0.184 Noradrenaline (i) 0.35 0.160 0.43 0.539 0.50 0.985 Putative_Progesterone 3- (i) 0.71 0.043 0.73 0.023 0.79 0.005 Biotin Serotonin (i) 0.52 0.838 0.47 0.809 0.66 0.138 Glycerophosphocholine (j) 0.57 0.491 0.58 0.445 0.59 0.402 Phosphocholine (j) 0.50 1.000 0.64 0.196 0.52 0.838 Sn-Glycerol 3-Phosphate (j) 0.50 0.985 0.57 0.515 0.52 0.897 LPC(14:0) (k) 0.58 0.468 0.50 0.985 0.59 0.423 LPC(15:0) (k) 0.54 0.752 0.56 0.590 0.58 0.468 LPC(16:0) (k) 0.55 0.616 0.51 0.926 0.61 0.323 LPC(16:1) (k) 0.54 0.696 0.52 0.838 0.54 0.696 LPC(17:0) (k) 0.61 0.287 0.48 0.867 0.62 0.254 LPC(17:1) (k) 0.54 0.752 0.52 0.897 0.54 0.724 LPC(18:0) (k) 0.60 0.341 0.55 0.642 0.63 0.224 LPC(18:2) (k) 0.50 1.000 0.51 0.956 0.53 0.780 LPC(18:3) (k) 0.47 0.780 0.44 0.564 0.52 0.897 LPC(20:1) (k) 0.54 0.724 0.57 0.491 0.49 0.926 LPC(20:2) (k) 0.56 0.564 0.57 0.539 0.52 0.838 LPC(20:3) (k) 0.50 1.000 0.54 0.752 0.47 0.809 LPC(20:4) (k) 0.44 0.590 0.52 0.838 0.42 0.468 LPC(20:5) (k) 0.55 0.642 0.44 0.564 0.54 0.724 LPC(22:5) (k) 0.57 0.515 0.58 0.445 0.53 0.809 LPC(22:6) (k) 0.51 0.926 0.50 1.000 0.52 0.897 LPC(26:0) (k) 0.61 0.323 0.61 0.287 0.74 0.019 LPC(P-16:0) (k) 0.52 0.838 0.63 0.224 0.55 0.669 LPC(P-18:0) (k) 0.68 0.086 0.59 0.381 0.62 0.254 LPC(P-18:0/0:0) or LPC(O- (k) 0.62 0.254 0.61 0.305 0.58 0.468 18:1) LPE(18:1) (l) 0.43 0.539 0.48 0.867 0.46 0.696 LPE(18:2) (l) 0.45 0.669 0.54 0.696 0.45 0.669 LPE(19:0) (l) 0.66 0.128 0.61 0.287 0.61 0.305 LPE(22:0) (l) 0.65 0.149 0.63 0.239 0.68 0.080 LPE(22:6) (l) 0.62 0.254 0.49 0.956 0.54 0.696 1-Linoleoylglycerol (m) 0.67 0.102 0.60 0.361 0.69 0.067 Monoelaidin (m) 0.57 0.515 0.55 0.642 0.55 0.642 1-Methyladenosine (n) 0.53 0.780 0.44 0.590 0.54 0.752 5,6-Dihydrouridine (n) 0.43 0.491 0.44 0.590 0.41 0.402 Adenosine 3′.5′-diphosphate (n) 0.49 0.956 0.56 0.564 0.43 0.515 Adenosine 5′- (n) 0.52 0.897 0.57 0.539 0.50 1.000 monophosphate Alloxan (n) 0.57 0.491 0.66 0.119 0.69 0.067 Guanosine 3′.5′-cyclic (n) 0.68 0.080 0.59 0.423 0.64 0.171 Monophosphate Hypoxanthine (n) 0.63 0.210 0.55 0.642 0.71 0.043 Paraxanthine (n) 0.66 0.119 0.62 0.254 0.79 0.005 UDP-GlcNAc (n) 0.41 0.423 0.52 0.838 0.43 0.539 Uridine (n) 0.71 0.043 0.60 0.361 0.68 0.086 Xanthine (n) 0.48 0.867 0.52 0.897 0.57 0.515 Xanthurenic Acid (n) 0.60 0.361 0.63 0.239 0.66 0.119 1-Hydroxy-2-Naphthoate (o) 0.43 0.539 0.47 0.780 0.48 0.867 2-Hydroxy-4-(methylthio)- (o) 0.50 1.000 0.41 0.381 0.43 0.515 butyric acid 3-(4-Hydroxyphenyl)- (o) 0.57 0.491 0.50 0.985 0.53 0.780 propionic acid 4-Hydroxy-2-quinoline- (o) 0.73 0.026 0.57 0.515 0.62 0.270 carboxylic acid Citrate (o) 0.57 0.491 0.67 0.102 0.59 0.402 Coumaric acid (o) 0.50 0.985 0.55 0.616 0.58 0.445 Hydroxyphenanthrene (o) 0.71 0.043 0.54 0.752 0.73 0.029 Indole-3-lactic acid (o) 0.56 0.590 0.54 0.696 0.51 0.926 Indoleacrylic acid (o) 0.51 0.926 0.47 0.809 0.68 0.094 Pyrrole-2-carboxylic acid (o) 0.44 0.590 0.45 0.642 0.45 0.642 Urate (o) 0.48 0.838 0.51 0.956 0.49 0.926 Betaine (p) 0.54 0.752 0.49 0.956 0.45 0.669 Biliverdin (p) 0.71 0.047 0.67 0.102 0.66 0.119 Caffeine (p) 0.57 0.539 0.61 0.305 0.65 0.160 Dethiobiotin (p) 0.57 0.491 0.53 0.780 0.66 0.128 Indole-3-acetaldehyde (p) 0.57 0.515 0.47 0.780 0.66 0.128 Indoleacetaldehyde (p) 0.52 0.838 0.57 0.491 0.56 0.590 Piperine (p) 0.45 0.669 0.51 0.926 0.51 0.956 PC(30:0) (q) 0.43 0.515 0.47 0.809 0.53 0.780 PC(31:0) (q) 0.50 0.985 0.46 0.724 0.49 0.926 PC(32:1) (q) 0.39 0.305 0.45 0.642 0.46 0.752 PC(32:2) (q) 0.56 0.590 0.68 0.094 0.61 0.323 PC(34:1) (q) 0.62 0.270 0.35 0.149 0.48 0.867 PC(34:2) (q) 0.70 0.061 0.43 0.539 0.54 0.724 PC(35:1) (q) 0.66 0.119 0.45 0.669 0.61 0.287 PC(36:2) (q) 0.73 0.026 0.47 0.780 0.73 0.023 PC(36:3) (q) 0.56 0.564 0.52 0.838 0.65 0.149 PC(36:4) (q) 0.57 0.491 0.38 0.270 0.57 0.539 PC(36:5) (q) 0.55 0.642 0.42 0.468 0.56 0.564 PC(38:5) (q) 0.55 0.669 0.46 0.724 0.55 0.616 PC(38:6) (q) 0.54 0.752 0.58 0.445 0.62 0.254 PC(38:7) (q) 0.54 0.724 0.49 0.926 0.56 0.590 PC(40:7) (q) 0.61 0.323 0.60 0.341 0.61 0.287 PC(o-30:1) or PC(p-30:0) (q) 0.56 0.564 0.46 0.724 0.59 0.381 PC(o-32:1) or PC(p-32:0) (q) 0.41 0.423 0.53 0.809 0.53 0.809 PC(o-34:4) or PC(p-34:3) (q) 0.50 0.985 0.47 0.809 0.52 0.867 PC(o-38:4) or PC(p-38:3) (q) 0.54 0.724 0.57 0.515 0.63 0.239 PC(o-38:6) or PC(p-38:6) (q) 0.56 0.590 0.59 0.423 0.55 0.616 PC(o-40:7) or PC(p-40:6) (q) 0.56 0.590 0.51 0.956 0.60 0.361 PE(36:1) (r) 0.65 0.149 0.58 0.445 0.70 0.056 PE(36:2) (r) 0.57 0.539 0.64 0.196 0.68 0.086 PE(36:4) (r) 0.44 0.590 0.45 0.669 0.48 0.867 PE(37:4) (r) 0.65 0.149 0.45 0.669 0.56 0.590 PE(38:3) (r) 0.69 0.073 0.60 0.361 0.73 0.023 PE(38:3) (r) 0.58 0.445 0.58 0.445 0.66 0.119 PE(40:6) (r) 0.56 0.590 0.41 0.423 0.52 0.867 PE(o-36:5) or PE(p-36:4) (r) 0.64 0.171 0.64 0.184 0.70 0.051 PE(p-18:0_20:4) (r) 0.60 0.361 0.65 0.160 0.71 0.039 PI(38:4) (s) 0.66 0.119 0.67 0.110 0.74 0.021 Putative_PI(36:2) (s) 0.72 0.032 0.76 0.012 0.76 0.012 Putative_PI(40:3) (s) 0.65 0.149 0.70 0.056 0.78 0.006 Leukotriene B4 (t) 0.46 0.696 0.46 0.752 0.39 0.305 Prostaglandin A1 (t) 0.75 0.014 0.55 0.642 0.64 0.184 Prostaglandin E2 (t) 0.55 0.616 0.36 0.184 0.52 0.867 SM(32:1) (u) 0.63 0.224 0.57 0.539 0.62 0.270 SM(32:2) (u) 0.65 0.149 0.64 0.196 0.63 0.239 SM(34:1) (u) 0.50 0.985 0.59 0.423 0.55 0.616 SM(34:2) (u) 0.56 0.590 0.61 0.287 0.58 0.468 SM(36:1) (u) 0.63 0.239 0.61 0.323 0.65 0.149 SM(36:2) (u) 0.48 0.867 0.53 0.780 0.55 0.642 SM(40:2) (u) 0.73 0.029 0.65 0.160 0.72 0.032 SM(42:1) (u) 0.52 0.838 0.52 0.867 0.53 0.809 SM(42:3) (u) 0.74 0.019 0.64 0.171 0.71 0.039 7-oxo-cholesterol (v) 0.61 0.305 0.48 0.897 0.50 0.985 8-Deoxy-11,13- (v) 0.44 0.564 0.53 0.809 0.35 0.149 dihydroxygrosheimin Cholate (v) 0.66 0.119 0.51 0.926 0.58 0.445 Deoxycholate (v) 0.55 0.642 0.53 0.780 0.54 0.724 Glycocholate (v) 0.55 0.616 0.50 0.985 0.52 0.838 Sterol (v) 0.62 0.254 0.55 0.669 0.60 0.361 TG(16:0_18:1_18:2) (w) 0.50 0.985 0.51 0.956 0.47 0.809 TG(44:0) (w) 0.58 0.468 0.47 0.809 0.54 0.696 TG(45:0) (w) 0.61 0.323 0.47 0.780 0.59 0.423 TG(46:0) (w) 0.62 0.254 0.48 0.897 0.64 0.184 TG(46:1) (w) 0.58 0.468 0.53 0.809 0.55 0.669 TG(48:1) (w) 0.55 0.642 0.49 0.926 0.56 0.564 TG(48:2) (w) 0.57 0.539 0.53 0.780 0.55 0.642 TG(49:0) (w) 0.59 0.381 0.44 0.590 0.55 0.616 TG(49:1) (w) 0.54 0.696 0.41 0.381 0.55 0.616 TG(50:2) (w) 0.47 0.780 0.51 0.926 0.50 1.000 TG(50:3) (w) 0.55 0.669 0.54 0.752 0.53 0.809 TG(51:3) (w) 0.56 0.590 0.55 0.669 0.54 0.696 TG(51:4) (w) 0.57 0.515 0.54 0.724 0.57 0.491 TG(52:0) (w) 0.54 0.752 0.50 1.000 0.53 0.780 TG(52:1) (w) 0.46 0.724 0.46 0.752 0.50 1.000 TG(52:4) (w) 0.50 0.985 0.54 0.752 0.52 0.897 TG(52:7) (w) 0.61 0.323 0.47 0.780 0.59 0.423 TG(53:1) (w) 0.57 0.515 0.43 0.539 0.59 0.423 TG(53:2) (w) 0.54 0.724 0.46 0.696 0.51 0.926 TG(54:1) (w) 0.59 0.381 0.51 0.956 0.54 0.724 TG(54:2) (w) 0.43 0.515 0.52 0.897 0.44 0.564 TG(54:3) (w) 0.40 0.361 0.49 0.956 0.46 0.724 TG(54:5) (w) 0.43 0.539 0.52 0.838 0.50 0.985 TG(54:6) (w) 0.47 0.809 0.48 0.897 0.52 0.897 TG(55:2) (w) 0.48 0.867 0.43 0.539 0.52 0.897 TG(56:7) (w) 0.54 0.752 0.52 0.867 0.57 0.515 TG(57:1) (w) 0.44 0.564 0.48 0.838 0.50 1.000 TG(58:9) (w) 0.54 0.724 0.50 0.985 0.59 0.423 TG(61:13) (w) 0.55 0.642 0.32 0.086 0.54 0.752 TG(65:11) (w) 0.43 0.515 0.44 0.590 0.57 0.491 TG(65:11)_iso (w) 0.52 0.838 0.45 0.669 0.59 0.402 3-cis-Hydroxy-b,e-caroten- (x) 0.68 0.080 0.49 0.956 0.66 0.128 3′-one 4-Pyridoxate (x) 0.35 0.149 0.48 0.838 0.49 0.926 Choline (x) 0.51 0.956 0.46 0.724 0.49 0.956 D-Pantothenic acid (x) 0.38 0.270 0.43 0.515 0.50 0.985 Folic acid (x) 0.53 0.780 0.42 0.445 0.49 0.956 Riboflavin (x) 0.62 0.254 0.54 0.724 0.55 0.669 (a) amino acid or amide (b) carbohydrate (c) carnitine (d) ceramide (e) dipeptide (f) exogenous (g) fatty acid (h) glycosphingolipid (i) hormone (j) lipid (k) lysophosphatidylcholine (l) lysophosphatidylethanolamine (m) monoacylglycerol (n) purine/pyrimidine (o) organic acid (p) other (q) phosphatidylcholine (r) phosphatidylethanolamine (s) phosphatidylinositol (t) prostanoid (u) sphingomyelin (v) stereol (w) triacylglycerol (x) vitamin
[0177] Although none of the 14 features remained individually significant after adjusting for multiple hypothesis testing, the significantly elevated lipids are biochemically linked to ceramide metabolism, suggesting a coordinated signal. Further, sphingomyelins and glycosphingolipids, in general, were elevated in baseline plasma samples of progressor cases (early DP) as compared to controls (no disease progression for a minimum of 5 years after start of AS) (
TABLE-US-00003 TABLE 3 Patient characteristics for MDACC validation cohort Case Control P Subjects, N 98 361 age, mean ± stdev 65 ± 7.4 63 ± 8.4 0.035{circumflex over ( )} BMI, mean ± stdev 29.2 ± 4.53 28.9 ± 4.51 0.748{circumflex over ( )} PSA Density, mean ± stdev 0.13 ± 0.083 0.11 ± 0.103 0.002{circumflex over ( )} Testosterone, mean ± stdev 396 ± 161.8 396 ± 162.5 0.922{circumflex over ( )} Smoking, N (%) Yes 61 (62) 206 (57) 0.419{circumflex over ( )}{circumflex over ( )} No 37 (38) 155 (43) 5-ARI Treatment, N (%) Yes 7 (7) 42 (12) 0.268{circumflex over ( )}{circumflex over ( )} No 91 (93) 319 (88) Statin Use, N (%) Yes 52 (53) 165 (46) 0.211{circumflex over ( )}{circumflex over ( )} No 46 (47) 196 (54) Follow-up time (months), 25 (12-96) 41.7 (6-120) mean (range) {circumflex over ( )}2-sided Wilcoxon-rank Sum Test {circumflex over ( )}{circumflex over ( )}2-sided Fisher's Exact Test
Consistent with findings in the initial discovery cohort, multiple SMs and glycosphingolipids were positively associated (Hazard Ratio>1.5) with DP based on GS (
TABLE-US-00004 TABLE 4 Hazard ratios of individual metabolites for progression- free survival in the MDACC validation cohort 2-sided 1-sided Lower Upper Metabolite Domain HR# p-value.sup.‡ p-value 95 CI 95 CI Acetylcarnitine (a) 1.4 0.123 0.062 0.92 2.09 Acylcarnitine(C14:0) (a) 1.1 0.616 0.308 0.72 1.73 Acylcarnitine(C16:0) (a) 1.1 0.693 0.346 0.61 2.09 Acylcarnitine(C18:0) (a) 1.3 0.510 0.255 0.64 2.44 Acylcarnitine(C18:1) (a) 1.2 0.499 0.249 0.70 2.11 Acylcarnitine(C18:2n-6) (a) 1.1 0.639 0.320 0.69 1.85 Acylcarnitine(C8:0) (a) 1.0 0.696 0.348 0.93 1.12 Dodecanedioylcarnitine (a) 1.2 0.506 0.253 0.72 1.97 Lauroylcarnitine (a) 1.1 0.298 0.149 0.89 1.46 O-butanoyl-R-carnitine (a) 1.2 0.599 0.299 0.68 1.95 O-decanoylcarnitine (a) 1.0 0.487 0.243 0.93 1.17 O-hexanoyl-R-carnitine (a) 1.2 0.412 0.206 0.82 1.63 O-octanoyl-R-carnitine (a) 1.0 0.620 0.310 0.91 1.17 Ceramide(18:1_24:1) (b) 0.8 0.474 0.237 0.43 1.48 Ceramide(18:2_16:0) (b) 1.3 0.550 0.275 0.55 3.07 Ceramide(30:1) (b) 1.7 0.396 0.198 0.52 5.21 Ceramide(32:1) (b) 0.9 0.745 0.373 0.34 2.16 Ceramide(34:1) (b) 1.8 0.286 0.143 0.62 5.18 Ceramide(39:1) (b) 0.9 0.819 0.410 0.52 1.68 Ceramide(40:0) (b) 0.9 0.628 0.314 0.56 1.43 Ceramide(40:1) (b) 1.0 0.880 0.440 0.61 1.77 Ceramide(40:2) (b) 0.9 0.622 0.311 0.51 1.50 Ceramide(41:1) (b) 1.1 0.850 0.425 0.56 2.02 Ceramide(42:0) (b) 0.9 0.592 0.296 0.54 1.42 Ceramide(42:1) (b) 1.2 0.523 0.262 0.68 2.14 Ceramide(42:2) (b) 0.8 0.526 0.263 0.33 1.77 Ceramide(43:1) (b) 0.9 0.827 0.414 0.46 1.86 Cholesterol Ester(16:1) (c) 1.0 0.867 0.434 0.67 1.41 Cholesterol Ester(20:4) (c) 2.6 0.004 0.002 1.36 4.83 Cholesterol Ester(20:5) (c) 1.1 0.406 0.203 0.85 1.51 Cholesterol Ester(22:6) (c) 1.1 0.805 0.403 0.72 1.54 Diacylglycerol(32:0) (d) 0.9 0.298 0.149 0.74 1.10 Diacylglycerol(34:0) (d) 0.9 0.258 0.129 0.67 1.12 Diacylglycerol(34:1) (d) 0.9 0.384 0.192 0.66 1.18 Diacylglycerol(34:2) (d) 0.9 0.524 0.262 0.67 1.22 Diacylglycerol(36:3) (d) 0.9 0.420 0.210 0.58 1.26 Diacylglycerol(36:4) (d) 1.0 0.981 0.490 0.74 1.36 Diacylglycerol(37:7) (d) 0.9 0.333 0.166 0.76 1.10 Leukotriene B4 (e) 0.3 0.166 0.083 0.07 1.57 Prostaglandin D2; Prostaglandin E2 (e) 4.4 0.047 0.024 1.02 18.99 Prostaglandin D2; Prostaglandin E2 (e) 1.5 0.463 0.232 0.52 4.23 4,7-dioxo-octanoic acid (f) 0.8 0.564 0.282 0.31 1.89 Free Fatty Acid (18:1) (f) 1.1 0.429 0.214 0.92 1.23 Free Fatty Acid (18:2) (linoleic acid) (f) 1.1 0.162 0.081 0.96 1.32 Free Fatty Acid (20:4) (arachidonic acid) (f) 1.2 0.145 0.073 0.93 1.64 Free Fatty Acid (22:6) (f) 1.0 0.953 0.477 0.80 1.27 (docosahexaenoic acid) Glucosyl/GalactosylCer(40:0) (g) 3.3 0.030 0.015 1.12 9.40 GlucosylCeramide(42:1) (g) 1.3 0.501 0.251 0.63 2.57 LactosylCeramide(18:1/16:0) (g) 1.6 0.247 0.123 0.72 3.53 Lactosylceramide(18:1/20:4) (g) 1.0 0.909 0.455 0.46 2.01 LactosylCeramide(32:0) (g) 2.0 0.016 0.008 1.14 3.51 Lactosylceramide(32:0) (g) 1.4 0.388 0.194 0.68 2.75 LactosylCeramide(34:1) (g) 1.8 0.199 0.100 0.75 4.09 LactosylCeramide(36:0) (g) 1.9 0.039 0.019 1.03 3.52 LactosylCeramide(36:0) (g) 2.9 0.055 0.027 0.98 8.36 Trihexosylceramide(34:1) (g) 2.6 0.047 0.023 1.01 6.54 Trihexosylceramide(40:1) (g) 2.2 0.069 0.034 0.94 5.16 Lysophosphatidylcholine(14:0) (h) 0.9 0.727 0.363 0.55 1.52 Lysophosphatidylcholine(15:0) (h) 1.1 0.887 0.444 0.46 2.44 Lysophosphatidylcholine(15:1) (h) 1.0 0.979 0.489 0.56 1.77 Lysophosphatidylcholine(16:0) (h) 1.0 0.948 0.474 0.45 2.34 Lysophosphatidylcholine(16:1) (h) 1.0 0.919 0.460 0.59 1.81 Lysophosphatidylcholine(17:0) (h) 1.3 0.373 0.187 0.71 2.46 Lysophosphatidylcholine(17:1) (h) 1.3 0.390 0.195 0.70 2.54 Lysophosphatidylcholine(17:2) (h) 1.4 0.219 0.109 0.81 2.53 Lysophosphatidylcholine(18:0) (h) 1.3 0.475 0.237 0.63 2.69 Lysophosphatidylcholine(18:1) (h) 1.5 0.201 0.100 0.82 2.60 Lysophosphatidylcholine(18:2) (h) 1.4 0.225 0.113 0.81 2.45 Lysophosphatidylcholine(18:3) (h) 1.4 0.125 0.062 0.92 2.05 Lysophosphatidylcholine(20:0) (h) 1.2 0.258 0.129 0.86 1.75 Lysophosphatidylcholine(20:1) (h) 1.2 0.535 0.268 0.68 2.11 Lysophosphatidylcholine(20:2) (h) 1.4 0.420 0.210 0.63 3.09 Lysophosphatidylcholine(20:3) (h) 1.1 0.728 0.364 0.69 1.69 Lysophosphatidylcholine(20:4) (h) 1.1 0.402 0.201 0.84 1.57 Lysophosphatidylcholine(20:5) (h) 1.1 0.597 0.299 0.85 1.34 Lysophosphatidylcholine(22:4) (h) 1.2 0.456 0.228 0.76 1.85 Lysophosphatidylcholine(22:5) (h) 1.1 0.489 0.244 0.80 1.59 Lysophosphatidylcholine(22:6) (h) 1.2 0.357 0.179 0.83 1.66 Lysophosphatidylcholine(24:0) (h) 1.4 0.365 0.183 0.67 3.00 Lysophosphatidylcholine(26:0) (h) 1.8 0.280 0.140 0.62 5.29 Lysophosphatidylethanolamine(16:0) (h) 1.2 0.543 0.271 0.62 2.49 Lysophosphatidylethanolamine(18:0) (h) 1.4 0.395 0.197 0.67 2.76 Lysophosphatidylethanolamine(18:1) (h) 1.4 0.102 0.051 0.94 2.01 Lysophosphatidylethanolamine(18:2) (h) 1.3 0.330 0.165 0.78 2.11 Lysophosphatidylethanolamine(20:3) (h) 1.9 0.124 0.062 0.84 4.27 Lysophosphatidylethanolamine(20:4) (h) 1.3 0.374 0.187 0.74 2.22 Lysophosphatidylethanolamine(22:0) (h) 1.6 0.336 0.168 0.62 4.14 Lysophosphatidylethanolamine(22:6) (h) 1.1 0.634 0.317 0.68 1.88 Plas_Lysophosphatidylcholine(P-18:0/0:0) (h) 1.1 0.822 0.411 0.57 2.04 or Plas_Lysophosphatidylcholine(O-18:1) PlasLysophosphatidylcholine(P-16:0) (h) 1.7 0.087 0.043 0.93 3.07 PlasLysophosphatidylethanolamine(p-22:0) (h) 1.5 0.268 0.134 0.75 2.84 1-Linoleoylglycerol (i) 0.5 0.641 0.320 0.03 8.13 2-Arachidonylglycerol (i) 1.3 0.112 0.056 0.95 1.69 Monoacylglycerol(20:0/0:0/0:0) (i) 1.2 0.676 0.338 0.51 2.83 Phosphatidylcholine(30:1) (j) 1.1 0.666 0.333 0.75 1.58 Phosphatidylcholine(32:0) (j) 1.2 0.559 0.279 0.60 2.58 Phosphatidylcholine(32:1) (j) 0.9 0.450 0.225 0.60 1.25 Phosphatidylcholine(32:2) (j) 0.8 0.494 0.247 0.52 1.37 Phosphatidylcholine(33:5) (j) 0.9 0.815 0.407 0.53 1.66 Phosphatidylcholine(34:1) (j) 0.9 0.799 0.399 0.33 2.33 Phosphatidylcholine(34:4) (j) 0.9 0.420 0.210 0.59 1.25 Phosphatidylcholine(35:1); (j) 1.2 0.650 0.325 0.56 2.53 Phosphatidylethanolamine(38:1) Phosphatidylcholine(35:2) (j) 1.0 0.965 0.482 0.47 2.19 Phosphatidylcholine(36:1) (j) 1.3 0.492 0.246 0.63 2.64 Phosphatidylcholine(36:2) (j) 1.2 0.818 0.409 0.34 3.93 Phosphatidylcholine(36:4) (j) 1.6 0.279 0.140 0.68 3.79 Phosphatidylcholine(36:5) (j) 1.1 0.333 0.166 0.88 1.45 Phosphatidylcholine(38:5) (j) 2.4 0.022 0.011 1.13 4.96 Phosphatidylcholine(38:6) (j) 1.0 0.906 0.453 0.55 1.97 Phosphatidylcholine(38:7) (j) 1.1 0.769 0.384 0.62 1.90 Phosphatidylcholine(40:5) (j) 1.3 0.237 0.118 0.85 1.98 Phosphatidylcholine(40:7) (j) 1.3 0.397 0.199 0.74 2.12 Phosphatidylcholine(40:8) (j) 1.4 0.352 0.176 0.70 2.73 Phosphatidylethanolamine(36:1) (j) 1.2 0.476 0.238 0.72 2.02 Phosphatidylethanolamine(36:1) (j) 1.0 0.998 0.499 0.61 1.65 Phosphatidylethanolamine(38:4) (j) 1.1 0.589 0.295 0.73 1.75 Phosphatidylglycerol(43:0) (j) 1.6 0.294 0.147 0.68 3.64 Phosphatidylserine(41:1) (j) 1.3 0.562 0.281 0.57 2.84 Plas_Phosphatidylcholine(o-30:1) or (j) 0.8 0.615 0.307 0.28 2.13 Plas_Phosphatidylcholine(p-30:0) Plas_Phosphatidylcholine(o-32:1) or (j) 1.2 0.628 0.314 0.57 2.56 Plas_Phosphatidylcholine(p-32:0) Plas_Phosphatidylcholine(o-34:1) or (j) 1.2 0.620 0.310 0.53 2.92 Plas_Phosphatidylcholine(p-34:0) Plas_Phosphatidylcholine(o-34:3) or (j) 1.1 0.220 0.110 0.94 1.32 Plas_Phosphatidylcholine(p-34:2) Plas_Phosphatidylcholine(o-38:6) or (j) 1.9 0.084 0.042 0.92 4.03 Plas_Phosphatidylcholine(p-38:5) Plas_Phosphatidylcholine(o-38:7) or (j) 1.7 0.066 0.033 0.97 3.01 Plas_Phosphatidylcholine(p-38:6) Plas_Phosphatidylcholine(o-40:6) or (j) 1.5 0.230 0.115 0.78 2.88 Plas_Phosphatidylcholine(p-40:5) Plas_Phosphatidylcholine(o-40:7) or (j) 1.9 0.026 0.013 1.08 3.29 Plas_Phosphatidylcholine(p-40:6) Plas_Phosphatidylethanolamine(o-38:5) or (j) 1.4 0.198 0.099 0.85 2.24 Plas_Phosphatidylethanolamine(p-38:4) Plas_Phosphatidylethanolamine(o-40:5) or (j) 1.9 0.023 0.011 1.09 3.37 Plas_Phosphatidylethanolamine(p-40:4) Plas_Phosphatidylethanolamine(o-40:5) or (j) 1.2 0.432 0.216 0.74 2.03 Plas_Phosphatidylethanolamine(p-40:4) Plas_Phosphatidylethanolamine(o-40:6) or (j) 1.5 0.179 0.089 0.84 2.49 Plas_Phosphatidylethanolamine(p-40:5) Plas_Phosphatidylserine(p-39:1) (j) 1.2 0.680 0.340 0.45 3.38 Plas_Phosphatidylserine(p-40:1) (j) 1.0 0.916 0.458 0.53 2.02 PlasPhosphatidylcholine(o-32:1) or (j) 2.1 0.059 0.029 0.97 4.51 PlasPhosphatidylcholine(p-32:0) PlasPhosphatidylcholine(o-34:1) or (j) 1.4 0.420 0.210 0.61 3.25 PlasPhosphatidylcholine(p-34:0) PlasPhosphatidylcholine(o-40:2) or (j) 2.4 0.036 0.018 1.06 5.56 PlasPhosphatidylcholine(p-40:1) PlasPhosphatidylcholine(o-40:7) or (j) 1.4 0.348 0.174 0.72 2.59 PlasPhosphatidylcholine(p-40:6) PlasPhosphatidylcholine(o-42:5) or (j) 1.3 0.554 0.277 0.60 2.58 PlasPhosphatidylcholine(p-42:4) PlasPhosphatidylethanolamine(o-38:5) or (j) 1.5 0.152 0.076 0.87 2.44 PlasPhosphatidylethanolamine(p-38:4) Sphingomyelin(32:1) (k) 1.2 0.534 0.267 0.70 2.01 Sphingomyelin(32:2) (k) 1.2 0.503 0.252 0.69 2.15 Sphingomyelin(33:1) (k) 2.4 0.091 0.046 0.87 6.34 Sphingomyelin(33:2) (k) 1.4 0.447 0.224 0.57 3.59 Sphingomyelin(34:0) (k) 1.7 0.167 0.084 0.79 3.78 Sphingomyelin(34:1) (k) 2.8 0.091 0.045 0.85 8.90 Sphingomyelin(34:2) (k) 1.5 0.337 0.168 0.67 3.29 Sphingomyelin(36:1) (k) 1.5 0.277 0.138 0.73 2.95 Sphingomyelin(36:2) (k) 1.5 0.233 0.117 0.76 3.11 Sphingomyelin(36:3) (k) 1.2 0.441 0.221 0.72 2.14 Sphingomyelin(38:1) (k) 1.5 0.274 0.137 0.74 2.89 Sphingomyelin(39:2) (k) 1.9 0.161 0.081 0.77 4.71 Sphingomyelin(40:1) (k) 1.7 0.119 0.060 0.88 3.09 Sphingomyelin(40:2) (k) 2.2 0.070 0.035 0.94 5.19 Sphingomyelin(40:3) (k) 1.4 0.285 0.143 0.76 2.52 Sphingomyelin(41:1) (k) 2.0 0.087 0.044 0.90 4.42 Sphingomyelin(42:1) (k) 1.8 0.089 0.045 0.91 3.51 Sphingomyelin(42:2) (k) 1.7 0.140 0.070 0.83 3.61 Sphingomyelin(42:3) (k) 1.6 0.242 0.121 0.72 3.62 Sphingomyelin(43:2) (k) 1.4 0.350 0.175 0.70 2.71 Sphingomyelin(44:2) (k) 3.9 0.007 0.004 1.45 10.60 Triacylglycerol(16:0_18:1_18:2) (l) 0.4 0.459 0.229 0.05 4.04 Triacylglycerol(40:0) (l) 1.0 0.115 0.057 0.99 1.06 Triacylglycerol(42:2) (l) 1.1 0.123 0.062 0.99 1.11 Triacylglycerol(44:0) (l) 1.0 0.450 0.225 0.94 1.16 Triacylglycerol(44:1) (l) 1.0 0.795 0.397 0.87 1.12 Triacylglycerol(44:2) (l) 1.0 0.967 0.483 0.87 1.16 Triacylglycerol(45:0) (l) 1.0 0.992 0.496 0.71 1.41 Triacylglycerol(46:0) (l) 1.3 0.107 0.053 0.94 1.87 Triacylglycerol(46:1) (l) 1.0 0.636 0.318 0.85 1.10 Triacylglycerol(46:2) (l) 0.9 0.503 0.252 0.74 1.16 Triacylglycerol(46:3) (l) 1.0 0.935 0.468 0.80 1.23 Triacylglycerol(47:0) (l) 1.2 0.567 0.284 0.59 2.63 Triacylglycerol(47:3) (l) 1.0 0.981 0.490 0.72 1.40 Triacylglycerol(48:0) (l) 1.1 0.787 0.393 0.67 1.71 Triacylglycerol(48:1) (l) 1.0 0.949 0.474 0.70 1.41 Triacylglycerol(48:2) (l) 1.0 0.934 0.467 0.63 1.53 Triacylglycerol(48:3) (l) 0.9 0.660 0.330 0.66 1.30 Triacylglycerol(48:3) (l) 1.0 0.849 0.425 0.83 1.26 Triacylglycerol(48:4) (l) 1.0 0.792 0.396 0.82 1.31 Triacylglycerol(49:1) (l) 0.9 0.601 0.300 0.71 1.22 Triacylglycerol(49:2) (l) 0.8 0.275 0.138 0.62 1.15 Triacylglycerol(49:3) (l) 0.9 0.614 0.307 0.57 1.39 Triacylglycerol(50:1) (l) 1.1 0.706 0.353 0.59 2.19 Triacylglycerol(50:3) (l) 0.8 0.461 0.231 0.38 1.56 Triacylglycerol(50:5) (l) 1.1 0.617 0.308 0.74 1.67 Triacylglycerol(51:1) (l) 1.1 0.570 0.285 0.78 1.57 Triacylglycerol(51:2) (l) 0.9 0.648 0.324 0.54 1.48 Triacylglycerol(51:3) (l) 0.8 0.360 0.180 0.43 1.36 Triacylglycerol(51:4) (l) 0.9 0.685 0.342 0.63 1.36 Triacylglycerol(52:2) (l) 1.0 0.943 0.471 0.22 4.06 Triacylglycerol(52:4) (l) 0.7 0.575 0.288 0.16 2.79 Triacylglycerol(52:7) (l) 1.5 0.092 0.046 0.94 2.34 Triacylglycerol(53:1) (l) 0.9 0.624 0.312 0.66 1.29 Triacylglycerol(53:2) (l) 1.1 0.657 0.328 0.72 1.70 Triacylglycerol(53:3) (l) 0.8 0.580 0.290 0.44 1.59 Triacylglycerol(53:4) (l) 0.9 0.755 0.378 0.56 1.53 Triacylglycerol(54:3) (l) 1.0 0.879 0.440 0.60 1.56 Triacylglycerol(54:4) (l) 1.7 0.320 0.160 0.61 4.54 Triacylglycerol(54:6) (l) 1.3 0.322 0.161 0.77 2.18 Triacylglycerol(55:1) (l) 1.2 0.602 0.301 0.66 2.06 Triacylglycerol(55:2) (l) 0.5 0.246 0.123 0.19 1.52 Triacylglycerol(55:2) (l) 1.0 0.887 0.444 0.52 1.77 Triacylglycerol(56:7) (l) 1.1 0.732 0.366 0.80 1.37 Triacylglycerol(56:9) (l) 1.4 0.032 0.016 1.03 1.97 Triacylglycerol(57:3) (l) 1.2 0.736 0.368 0.36 4.33 Triacylglycerol(57:8) (l) 1.1 0.652 0.326 0.75 1.58 Triacylglycerol(58:9) (l) 1.3 0.063 0.031 0.99 1.68 Triacylglycerol(59:4) (l) 1.3 0.419 0.210 0.67 2.60 Triacylglycerol(59:5) (l) 1.3 0.330 0.165 0.77 2.19 Triacylglycerol(60:3) (l) 1.1 0.892 0.446 0.46 2.46 Triacylglycerol(61:3) (l) 1.0 0.955 0.477 0.45 2.32 Triacylglycerol(61:6) (l) 1.4 0.347 0.174 0.68 2.98 Triacylglycerol(61:7) (l) 1.4 0.239 0.119 0.79 2.59 Triacylglycerol(65:11) (l) 1.2 0.177 0.088 0.91 1.64 # treated as continuous variables .sup.‡p-values (a) acylcarnitine (b) ceramide (c) cholesterol ester (d) diacylglycerol (e) eicosanoid (f) free fatty acid (g) glycosphingolipid (h) lysophospholipid (i) monoacylglycerol (j) phospholipid (k) sphingolipid (l) triacylglycerol
[0178] Next, from the set of sphingolipids that exhibited statistically significant (p<0.05) HRs and, using a logistic regression model, a signature panel was developed which comprised plasma Cav-1 and six sphingolipids that exhibited positive β-estimates in the logistic regression model: SM(40:2), SM(44:2), lactosylceramide(32:0), lactosylceramide(36:0), trihexosylceramide(34:1) and hexosylceramide(40:0). Using log rank test statistics from the Cox model, an optimal cut-off point was calculated for the plasma Cav-1-sphingolipid signature that would yield the greatest difference between subjects on AS that exhibited disease progression (defined as upgrading of GS and/or increased tumor volume) from those that did not. This resulted in a cut-off value of 4.33. Assessment of the association of the signature with progression free survival was achieved using Cox-proportional hazard models. In a multivariate analyses, adjusted for age, 5-alpha reductase treatment, and baseline tumor volume, AS subjects with a plasma Cav-1-sphingolipid signature score>4.33 exhibited statistically significantly worse DP free survival as compared to those with a plasma Cav-1-sphingolipid signature score≤4.33 (HR: 2.70, 95% CI: 1.75-4.16, p-value: <0.001) (Table 5). Notably, non-proportionality hazard model tests yielded non-significant p-values. Of relevance, in this analyses BMI was not associated with increased risk of DP (HR: 1.02, 95% CI: 0.40-2.64, p-value: 0.965) indicating that this lipid signature is unlikely to be biased by obesity.
TABLE-US-00005 TABLE 5 Hazard models for the Cav-1-sphingolipid signature and disease progression free-survival Univariable Multivariable† Variable HR 95% CI 2-sided P HR 95% CI 2-sided P Age <64 Reference Reference ≥64 1.66 1.11-2.48 0.014 1.68 1.12-2.53 0.013 BMI{circumflex over ( )} 1.02 0.40-2.64 0.965 — PSA Density‡ 3.77 0.99-14.44 0.053 — 5-ARI treatment No Reference Reference Yes 0.50 0.23-1.07 0.074 0.37 0.17-0.82 0.014 Risk Group I Reference Reference II 2.94 1.75-4.96 <0.001 2.60 1.54-4.38 <0.001 Sphingolipid Signature Below Cutoff (≤4.33) Reference Reference Above Cutoff (>4.33) 2.62 1.71-4.01 <0.001 2.70 1.75-4.16 <0.001
[0179] Kaplan-Meier survival curves depicting progression free survival for participants with plasma Cav-1—sphingolipid signature scores below (<4.33) or equal to or above the cutoff (>4.33) are provided in Table 6 and
TABLE-US-00006 TABLE 6 Progression-free survival signature time(months) cutoff 0 10 20 30 40 50 60 70 80 90 100 110 120 ≤4.33 383 378 269 229 165 109 92 49 27 18 8 2 1 >4.33 76 75 52 41 27 16 13 3 1 1 1 0 0 Multivariate Cox-proportional hazard models for the Cav-1-sphingolipid signature and its association with disease progression free-survival. Cox proportional hazard models using a plasma Cav-1-sphingolipid signature cut-off value of 4.33. Optimal cut-off values for plasma Cav-1-sphingolipid signature were derived using log rank statistic based methods as previously described. Age, 5-α reductase treatment and baseline tumor volume (Risk Group 1: 1 positive biopsy core with tumor focus of <3.0 mm in Gleason 3 + 3 = 6 patients or <2.0 mm in Gleason 3 + 4 = 7 patients; Risk Group 2: >1 core or baseline tumor length greater than those of Risk Group 1) were included as co-variables based on a backward stepwise selection method (likelihood ratio). † Variables included into the equation after selection using a backward stepwise method (likelihood ratio) {circumflex over ( )} per unit log 2 increase ‡ per unit increase
[0180] To better define the relationship between lipid metabolism and Cav-1 within the context of the Cav-1—sphingolipid signature, gene expression profiles reflective of lipid managing apparati and mRNA expression of CAV1 in 333 prostate tumors using The Cancer Genome Atlas (TCGA) were first compared. High CAV1 mRNA expression was found to be positively associated with genes annotated to ontologies related to lipid scavenging and metabolism, glycosylceramide metabolic process as well as the ceramide pathway. Association of elevated CAV1 and associated lipid managing apparti with previously defined molecular subtypes (iClusters) of primary prostate cancers was next probed. The results indicated that high CAV1 mRNA expression was predominately associated with iCluster 3, which is characterized by elevated PI3K/AKT, MAP-Kinase and receptor tyrosine kinase activity.
Example 6: Association of Cav-1 with a High-Lipid Scavenging Phenotype
[0181] The response of Cav-1 to extracellular lipid availability was next assessed. In comparing serum-free media with and without lipids, lipid-deprivation reduced Cav-1 protein expression in RM-9 and PC-3M prostate cancer cell lines (
[0182] Participation of Cav-1 in lipid uptake was next explored. To this end, the ability of Cav-1 low
Example 7: Regulation of Glycosphingolipid Biosynthesis by Cav-1
[0183] Comparison of baseline lipid profiles of LNCaP and PC-3M cells was next performed, as well as lipid profiles following respective overexpression of Cav-1, or transient knockdown of CAV1. Immunoblots comparing whole cell lysate Cav-1 levels in LNCaP and PC-3M cells following respective overexpression of Cav-1 or transient knockdown of CAV1 are provided in
TABLE-US-00007 TABLE 7 Spearman correlation analysis Pathway Gene Spearman Rho 2-sided P (a) ASAH1 0.07 0.1629 (a) DEGS1 −0.2 0.0001 (a) DEGS2 0.07 0.1684 (a) KDSR 0.4 <0.0001 (a) SPTLC1 0.03 0.5933 (a) SPTLC2 0.03 0.5189 (a) SPTLC3 0.37 <0.0001 (b) ACER1 0.38 <0.0001 (b) ACER2 0 0.9855 (b) ACER3 −0.12 0.0186 (b) CERS1 −0.31 <0.0001 (b) CERS2 −0.32 <0.0001 (b) CERS3 0.38 <0.0001 (b) CERS4 −0.41 <0.0001 (b) CERS5 0.09 0.1009 (b) CERS6 0.01 0.8473 (b) PLPP1 (PPAP2A) −0.2 0.0001 (b) PLPP2 (PPAP2C) −0.07 0.1786 (b) PLPP3 (PPAP2B) 0.73 <0.0001 (b) SGPL1 −0.11 0.0336 (b) SGPP1 −0.11 0.0353 (b) SGPP2 0.03 0.5302 (b) SPHK1 0.14 0.0076 (b) SPHK2 −0.27 <0.0001 (c) SGMS1 −0.19 0.0004 (c) SGMS2 0.09 0.0941 (c) SMPD1 −0.16 0.0028 (c) SMPD2 −0.29 <0.0001 (c) SMPD3 −0.11 0.0336 (c) SMPDL3A 0.02 0.7576 (c) SMPDL3A 0.02 0.7576 (d) GBA2 −0.29 <0.0001 (d) GLB1 −0.14 0.0074 (d) GALC 0.01 0.8319 (d) GLB1L2 0.09 0.0961 (d) GLB1L 0.1 0.0576 (d) UGCG 0.1 0.0566 (d) GLB1L3 0.18 0.0008 (d) UGT8 0.28 <0.0001 (d) B4GALT5 0.45 <0.0001 (d) B4GALT6 0.45 <0.0001 (a) de novo pathway (b) sphingosine recycling (c) sphingomyelinase pathway (d) glycosphingolipid metabolism
Example 8: Sphingomyelins as a Source of Ceramides and Glycosphingolipids
[0184] Investigation of the uptake of extracellular sphingomyelins as a potential source of ceramides and (via glycosylation), their glycosphingolipid derivatives. Ceramides are principally derived through three metabolic pathways: de novo, recycling or salvaging (
[0185] PC-3M, RM-9 and LNCaP prostate cancer cells were treated for 48 hours with SSALPs containing sphingomyelin(d18:1/18:1)-deuterium(d).sub.9. The biochemical fate of this compound was traced using liquid chromatography mass spectrometry (
Example 9: Facilitation of Mitochondrial Components by Cav-1
[0186] The findings above suggest a Cav-1-associated mechanistic framework that directs ceramide pools into glycosphingolipids, consistent with the observation that glycosphingolipids, particularly lactosylceramides, are key features of the plasma sphingolipid signature (
Example 10: Release of Cav-1-Sphingomyelin/Lactosylceramide-Enriched EVs
[0187] It was previously demonstrated that secretion of membrane associated Cav-1 by prostate cancer cells has been previously demonstrated. Consistent with this previous report, assessment of CM confirmed detectable levels of Cav-1 in CM from PC-3M and RM-9 prostate cancer cell lines but not LNCaP. Isolation of extracellular lipid vesicles (EVs) from CM of LNCaP and PC-3M following respective overexpression of Cav-1, or knockdown of CAV1, confirmed that Cav-1 is present on EVs (
[0188] To determine the lipid composition and protein cargo of EVs, lipidomic and proteomic analyses were performed using mass spectrometry on EVs derived from LNCaP and PC-3M, respectively. Consistent with findings by others, analyses of the EV-lipidome identified sphingolipids as well as phosphatidylcholines to be particularly enriched. Interestingly cardiolipins, important lipid constituents of the inner mitochondrial membrane, were found to be present in prostate cancer cell line-derived EVs. Evaluation of the EV-proteome identified 237 and 341 (>5 spectral abundance) high confidence proteins in LNCaP and PC-3M derived EVs, respectively. To investigate for functional aspects of protein features, performed subcellular localization analysis, based on the COMPARTMENTS localization evidence database scores [doi.org/10.1093/database/bauOl2], were performed, filtering for genes confidently assigned to at least one ofthe 11 subcellular localizations (confidence score c2): nucleus, cytosol, cytoskeleton, peroxisome, lysosome, endoplasmic reticulum, Golgi apparatus, plasma membrane, endosome, extracellular spaces and mitochondrion (
TABLE-US-00008 TABLE 8 Disease Function adjusted P- Predicted Disease function value activation state # molecules Cell movement 2.09E−30 Increased 125 Apoptosis 2.8E−22 Decreased 119 Necrosis 1.06E−27 Decreased 134 Degranulation of cells 2.51E−31 Increased 69 Cell proliferation of 8.87E−20 Increased 112 tumor cell lines
TABLE-US-00009 TABLE 9 Ingenuity canonical pathways −log(p- # Ingenuity canonical pathways value) Ratio molecules Virus entry via endocytic pathways 27.6 0.28 30/107 Caveolar-mediated endocytosis signaling 21.1 0.301 22/73 Phagosome maturation 20.2 0.193 27/140 Remodeling of epithelial adherens 19.3 0.303 20/66 junctions Germ cell-sertoli cell junction signaling 18.2 0.163 27/166 14-3-3-mediated signaling 17.9 0.190 24/126 Ephrin receptor signaling 17.4 0.152 27/178 Axonal guidance signaling 17.2 0.0878 41/467 Epithelial adherens junction signaling 17.1 0.167 25/150 Clathrin-mediated endocytosis signaling 16.5 0.141 27/192
[0189] Collectively, these findings indicate that prostate cancer cells robustly secrete Cav-1-containing sphingolipid-enriched EVs that are enriched in a diverse repertoire of proteins including mitochondrial-associated proteins and lipids. On the basis of these findings, Cav-1 mediated uptake of sphingomyelin (
Example 11: Targeting of the Shunt from Ceramides to Glycosphingolipids
[0190] These findings of an adaptive Cav-1-mediated glycosphingolipid mechanism suggest that that targeting of the ceramide to glycosphingolipid conversion may represent an actionable metabolic vulnerability in prostate cancer. To test this hypothesis, the efficacy of PDMP, PPMP and eliglustat three different inhibitors of glucosylceramide synthase (also known as UGCG; UDP-glucose:ceramide glucosyltransferase), a rate-limiting enzyme in glycosphingolipid metabolism, for reducing viability of RM-9 and PC-3M prostate cancer cells in vitro was then evaluated. Treatment of RM-9 and PC-3M prostate cancer cells with PDMP, PPMP and eliglustat resulted in dose-dependent cytotoxicity (
Example 12: Inhibition of RM-9 Tumor Growth by Eliglustat
[0191] Inhibition of tumor growth by eliglustat was next examined. RM-9 cells harbor driver oncogenic RAS and MYC genes which model RAS-MAPK pathway activation and MYC-driven transcriptional activities which are associated with aggressive primary prostate cancer. RM-9-luciferase cells were subcutaneously implanted into C57BL/6N mice. RM-9 tumor growth was suppressed by eliglustat (
Example 13: Calculation of Biomarker Score
[0192] Measured concentrations of plasma Cav-1-sphingolipid signature features were used to calculate a biomarker score based on a logistic regression model. In this model for progression of prostate cancer, the values for the plasma analyte signature features are combined into a model that is applied to predict risk of disease progression.
[0193] The table below illustrates the ratio between the actual hazard and the baseline hazard rate as well as the hazard rate at different cutoff points of the biomarker panel score. Here, the hazard is defined as the risk of an event (i.e., disease progression) as a function of time where a hazard rate>than 1 implies that the time-to-disease progression is shorter.
[0194] The table shows two things as a function of model score. The first is the risk of progression relative to the calculated “baseline hazard rate” for a given signature, and the second considers the use of the signature score as a classifier cut point; the last column then gives the relative risk of disease progression between the high and low scoring groups, ie the “hazard ratio.”
[0195] The first column of the table shows the biomarker panel score in the Active Surveillance cohort, the second column describes the change in the actual hazard relative to the baseline hazard, and the third column illustrates the hazard ratio as well as the corresponding 95% confidence interval, based on a dichotomization of the population using different cutoff points for the biomarker panel score.
TABLE-US-00010 Ratio between the actual hazard rate Model and the baseline hazard rate Hazard Ratio (binary Score (h(t)/h0(t)) cutoff) 2.524 8.684 2.718 10.252 2.840 11.381 3.017 13.251 3.019 13.273 3.056 13.700 3.081 13.990 3.110 14.347 3.133 14.631 3.157 14.934 3.161 14.986 2.64 [0.369-18.969] 3.162 15.004 2.76 [0.385-19.807] 3.164 15.019 2.89 [0.402-20.694] 3.166 15.048 3 [0.419-21.541] 3.170 15.106 3.35 [0.467-23.999] 3.171 15.114 3.47 [0.483-24.854] 3.171 15.121 1.79 [0.441-7.266] 3.175 15.163 1.25 [0.397-3.958] 3.178 15.206 1.34 [0.423-4.223] 3.182 15.261 1.47 [0.464-4.626] 3.183 15.274 1.56 [0.493-4.918] 3.188 15.336 1.6 [0.506-5.044] 3.189 15.345 1.64 [0.519-5.178] 3.202 15.520 1.68 [0.532-5.305] 3.205 15.567 1.74 [0.551-5.498] 3.208 15.604 1.86 [0.589-5.867] 3.209 15.610 1.96 [0.621-6.187] 3.227 15.863 2.06 [0.654-6.518] 3.237 15.992 2.2 [0.696-6.939] 3.247 16.127 2.26 [0.717-7.142] 3.251 16.192 2.38 [0.755-7.524] 3.262 16.338 2.43 [0.768-7.656] 3.263 16.358 2.56 [0.812-8.088] 3.264 16.364 2.63 [0.832-8.295] 3.271 16.468 2.72 [0.861-8.582] 3.279 16.581 2.83 [0.895-8.918] 3.283 16.630 2.94 [0.93-9.267] 3.287 16.689 2.98 [0.944-9.411] 3.295 16.806 3.11 [0.984-9.81] 3.298 16.855 3.22 [1.02-10.165] 3.299 16.865 3.36 [1.066-10.617] 3.304 16.942 2.58 [0.947-7.009] 3.305 16.945 2.67 [0.982-7.272] 3.312 17.051 2.13 [0.865-5.235] 3.317 17.130 2.19 [0.89-5.387] 3.324 17.235 2.22 [0.902-5.456] 3.328 17.295 1.87 [0.818-4.27] 3.333 17.361 1.59 [0.739-3.439] 3.333 17.366 1.64 [0.76-3.54] 3.333 17.371 1.7 [0.786-3.659] 3.338 17.432 1.49 [0.722-3.069] 3.338 17.440 1.51 [0.73-3.104] 3.357 17.720 1.52 [0.738-3.139] 3.358 17.745 1.55 [0.751-3.191] 3.366 17.856 1.61 [0.783-3.325] 3.376 18.009 1.63 [0.791-3.359] 3.380 18.082 1.65 [0.799-3.394] 3.391 18.248 1.68 [0.817-3.469] 3.394 18.293 1.74 [0.844-3.583] 3.406 18.482 1.78 [0.861-3.659] 3.407 18.504 1.8 [0.874-3.712] 3.409 18.535 1.83 [0.887-3.768] 3.426 18.810 1.83 [0.887-3.768] 3.429 18.851 1.88 [0.915-3.885] 3.431 18.888 1.69 [0.849-3.346] 3.434 18.931 1.71 [0.861-3.392] 3.436 18.971 1.74 [0.878-3.458] 3.438 18.993 1.78 [0.894-3.525] 3.440 19.027 1.81 [0.913-3.598] 3.441 19.052 1.86 [0.939-3.7] 3.443 19.078 1.69 [0.879-3.252] 3.443 19.083 1.71 [0.89-3.294] 3.446 19.131 1.75 [0.912-3.375] 3.450 19.193 1.79 [0.928-3.434] 3.462 19.390 1.82 [0.944-3.495] 3.464 19.431 1.83 [0.952-3.523] 3.469 19.511 1.85 [0.964-3.566] 3.477 19.645 1.87 [0.971-3.595] 3.482 19.728 1.89 [0.983-3.639] 3.483 19.741 1.92 [1-3.701] 3.487 19.818 1.96 [1.019-3.772] 3.488 19.828 1.77 [0.945-3.313] 3.488 19.830 1.8 [0.961-3.372] 3.490 19.859 1.83 [0.98-3.436] 3.494 19.933 1.89 [1.008-3.536] 3.494 19.933 1.9 [1.016-3.562] 3.500 20.034 1.75 [0.955-3.197] 3.501 20.057 1.6 [0.892-2.868] 3.504 20.106 1.61 [0.899-2.889] 3.512 20.235 1.62 [0.905-2.91] 3.513 20.256 1.63 [0.912-2.931] 3.525 20.473 1.66 [0.927-2.98] 3.526 20.493 1.7 [0.946-3.041] 3.528 20.515 1.73 [0.963-3.096] 3.530 20.553 1.6 [0.908-2.815] 3.531 20.576 1.63 [0.926-2.872] 3.532 20.583 1.66 [0.942-2.922] 3.534 20.622 1.69 [0.958-2.971] 3.535 20.651 1.72 [0.979-3.036] 3.536 20.662 1.76 [0.998-3.094] 3.538 20.697 1.77 [1.007-3.124] 3.539 20.722 1.81 [1.027-3.184] 3.543 20.784 1.82 [1.033-3.204] 3.545 20.816 1.86 [1.055-3.271] 3.549 20.886 1.9 [1.077-3.339] 3.556 21.012 1.93 [1.099-3.407] 3.558 21.047 1.97 [1.121-3.477] 3.558 21.054 2 [1.138-3.53] 3.559 21.069 2.02 [1.145-3.552] 3.569 21.249 2.04 [1.156-3.584] 3.577 21.402 2.05 [1.163-3.606] 3.577 21.404 2.06 [1.17-3.628] 3.586 21.562 2.1 [1.19-3.691] 3.589 21.617 2.11 [1.197-3.713] 3.589 21.624 2.14 [1.214-3.765] 3.594 21.704 2.16 [1.225-3.798] 3.597 21.771 2.19 [1.246-3.864] 3.598 21.788 2.03 [1.171-3.517] 3.600 21.831 2.05 [1.181-3.548] 3.600 21.834 2.09 [1.203-3.616] 3.601 21.846 2.12 [1.224-3.676] 3.608 21.967 2.15 [1.24-3.725] 3.608 21.967 2.18 [1.257-3.778] 3.614 22.079 2.18 [1.257-3.778] 3.619 22.181 2.04 [1.194-3.486] 3.620 22.196 2.07 [1.21-3.535] 3.626 22.307 2.11 [1.233-3.6] 3.626 22.308 2.12 [1.243-3.63] 3.629 22.365 2.16 [1.266-3.697] 3.632 22.430 2.19 [1.283-3.747] 3.633 22.446 2.22 [1.301-3.8] 3.635 22.485 2.26 [1.324-3.868] 3.637 22.530 2.3 [1.345-3.93] 3.638 22.542 2.32 [1.356-3.962] 3.640 22.586 2.33 [1.364-3.984] 3.641 22.597 2.37 [1.385-4.046] 3.644 22.664 2.21 [1.308-3.723] 3.650 22.783 2.06 [1.237-3.442] 3.651 22.790 2.09 [1.253-3.487] 3.659 22.952 2.12 [1.269-3.53] 3.659 22.957 1.98 [1.203-3.276] 3.664 23.053 2.02 [1.223-3.333] 3.665 23.076 2.03 [1.23-3.351] 3.667 23.110 2.04 [1.236-3.368] 3.667 23.117 2.06 [1.25-3.406] 3.667 23.117 2.09 [1.269-3.458] 3.669 23.144 2.13 [1.288-3.51] 3.672 23.206 2.15 [1.302-3.548] 3.672 23.212 2.16 [1.309-3.566] 3.672 23.216 2.03 [1.242-3.321] 3.673 23.235 2.04 [1.249-3.338] 3.675 23.274 2.07 [1.267-3.388] 3.680 23.368 2.08 [1.274-3.405] 3.683 23.438 1.96 [1.211-3.181] 3.683 23.442 1.85 [1.153-2.98] 3.685 23.468 1.87 [1.161-3.002] 3.685 23.471 1.78 [1.115-2.839] 3.688 23.531 1.79 [1.12-2.854] 3.697 23.715 1.8 [1.126-2.868] 3.697 23.722 1.81 [1.131-2.882] 3.707 23.917 1.71 [1.08-2.713] 3.708 23.937 1.74 [1.097-2.758] 3.711 23.995 1.74 [1.097-2.758] 3.714 24.059 1.66 [1.052-2.612] 3.716 24.100 1.67 [1.058-2.626] 3.716 24.110 1.69 [1.071-2.658] 3.717 24.117 1.6 [1.024-2.513] 3.725 24.298 1.55 [0.996-2.418] 3.729 24.377 1.56 [1.004-2.437] 3.731 24.424 1.57 [1.009-2.449] 3.733 24.447 1.59 [1.021-2.479] 3.734 24.484 1.61 [1.032-2.505] 3.735 24.502 1.63 [1.047-2.541] 3.736 24.523 1.55 [1.002-2.409] 3.737 24.540 1.48 [0.961-2.288] 3.737 24.546 1.49 [0.965-2.299] 3.738 24.565 1.51 [0.981-2.336] 3.742 24.656 1.53 [0.991-2.36] 3.744 24.687 1.55 [1.005-2.393] 3.748 24.778 1.48 [0.963-2.276] 3.750 24.818 1.49 [0.97-2.292] 3.751 24.836 1.52 [0.988-2.335] 3.751 24.840 1.54 [1.001-2.364] 3.756 24.944 1.57 [1.019-2.408] 3.761 25.052 1.57 [1.019-2.408] 3.764 25.116 1.59 [1.034-2.442] 3.766 25.154 1.61 [1.046-2.47] 3.767 25.188 1.63 [1.062-2.509] 3.772 25.281 1.64 [1.07-2.527] 3.774 25.334 1.66 [1.077-2.545] 3.775 25.351 1.68 [1.09-2.575] 3.776 25.365 1.69 [1.098-2.594] 3.777 25.388 1.71 [1.111-2.625] 3.779 25.442 1.65 [1.076-2.523] 3.780 25.457 1.66 [1.087-2.548] 3.782 25.509 1.68 [1.095-2.566] 3.785 25.578 1.6 [1.051-2.445] 3.787 25.611 1.53 [1.009-2.333] 3.788 25.630 1.47 [0.969-2.228] 3.790 25.686 1.41 [0.932-2.13] 3.790 25.692 1.42 [0.938-2.144] 3.792 25.729 1.44 [0.955-2.184] 3.792 25.736 1.45 [0.96-2.194] 3.797 25.841 1.47 [0.969-2.216] 3.798 25.850 1.48 [0.981-2.241] 3.799 25.880 1.49 [0.987-2.257] 3.799 25.880 1.43 [0.949-2.159] 3.801 25.928 1.45 [0.964-2.192] 3.803 25.968 1.46 [0.97-2.207] 3.806 26.025 1.47 [0.975-2.218] 3.811 26.147 1.48 [0.982-2.233] 3.811 26.154 1.49 [0.986-2.244] 3.812 26.163 1.51 [1-2.274] 3.814 26.215 1.52 [1.005-2.285] 3.815 26.231 1.54 [1.02-2.32] 3.816 26.251 1.56 [1.031-2.346] 3.819 26.319 1.5 [0.994-2.252] 3.827 26.512 1.5 [0.999-2.262] 3.828 26.542 1.52 [1.01-2.288] 3.829 26.563 1.54 [1.022-2.314] 3.831 26.597 1.56 [1.036-2.345] 3.832 26.617 1.57 [1.046-2.368] 3.833 26.644 1.59 [1.06-2.4] 3.834 26.675 1.6 [1.065-2.411] 3.836 26.709 1.61 [1.072-2.427] 3.836 26.717 1.55 [1.035-2.333] 3.838 26.752 1.57 [1.049-2.365] 3.838 26.767 1.6 [1.063-2.397] 3.839 26.777 1.6 [1.068-2.408] 3.839 26.779 1.62 [1.08-2.436] 3.839 26.785 1.65 [1.1-2.48] 3.840 26.800 1.59 [1.058-2.377] 3.844 26.893 1.6 [1.066-2.394] 3.844 26.898 1.61 [1.076-2.417] 3.846 26.951 1.63 [1.089-2.446] 3.850 27.042 1.65 [1.101-2.475] 3.852 27.070 1.67 [1.113-2.501] 3.853 27.113 1.69 [1.128-2.535] 3.855 27.147 1.62 [1.086-2.431] 3.856 27.172 1.64 [1.098-2.459] 3.857 27.189 1.67 [1.115-2.496] 3.864 27.363 1.68 [1.12-2.508] 3.866 27.409 1.7 [1.133-2.537] 3.866 27.414 1.72 [1.146-2.567] 3.868 27.447 1.65 [1.103-2.463] 3.868 27.459 1.67 [1.115-2.489] 3.869 27.486 1.68 [1.126-2.514] 3.870 27.514 1.7 [1.137-2.538] 3.871 27.531 1.72 [1.149-2.566] 3.874 27.586 1.75 [1.171-2.614] 3.875 27.631 1.76 [1.176-2.626] 3.876 27.646 1.69 [1.132-2.521] 3.879 27.722 1.72 [1.149-2.56] 3.879 27.723 1.73 [1.161-2.586] 3.881 27.766 1.75 [1.174-2.616] 3.883 27.806 1.77 [1.188-2.646] 3.889 27.947 1.8 [1.204-2.683] 3.891 28.012 1.83 [1.223-2.725] 3.893 28.039 1.84 [1.235-2.752] 3.900 28.208 1.8 [1.204-2.678] 3.902 28.255 1.81 [1.213-2.696] 3.904 28.310 1.75 [1.172-2.6] 3.912 28.504 1.76 [1.185-2.629] 3.921 28.739 1.7 [1.14-2.526] 3.923 28.782 1.71 [1.146-2.538] 3.923 28.785 1.73 [1.164-2.578] 3.925 28.823 1.74 [1.172-2.597] 3.927 28.874 1.76 [1.18-2.615] 3.927 28.875 1.78 [1.197-2.652] 3.928 28.902 1.8 [1.209-2.679] 3.928 28.903 1.81 [1.215-2.692] 3.933 29.021 1.83 [1.229-2.722] 3.934 29.043 1.85 [1.244-2.756] 3.938 29.149 1.87 [1.258-2.787] 3.947 29.370 1.89 [1.267-2.807] 3.947 29.372 1.9 [1.276-2.827] 3.948 29.394 1.85 [1.242-2.749] 3.952 29.498 1.79 [1.206-2.666] 3.952 29.513 1.74 [1.168-2.582] 3.953 29.518 1.76 [1.185-2.619] 3.954 29.564 1.78 [1.199-2.649] 3.955 29.582 1.78 [1.199-2.649] 3.958 29.645 1.81 [1.216-2.688] 3.960 29.702 1.83 [1.23-2.719] 3.963 29.791 1.86 [1.25-2.764] 3.970 29.970 1.87 [1.26-2.784] 3.972 30.004 1.9 [1.278-2.826] 3.974 30.052 1.84 [1.239-2.737] 3.975 30.101 1.78 [1.196-2.644] 3.979 30.189 1.8 [1.209-2.673] 3.980 30.223 1.82 [1.227-2.713] 3.984 30.335 1.83 [1.234-2.726] 3.990 30.483 1.86 [1.248-2.759] 3.990 30.490 1.87 [1.258-2.78] 3.991 30.508 1.89 [1.274-2.816] 3.993 30.544 1.93 [1.296-2.864] 3.993 30.553 1.86 [1.253-2.772] 3.994 30.579 1.89 [1.272-2.814] 3.996 30.643 1.9 [1.279-2.829] 4.001 30.774 1.92 [1.289-2.851] 4.003 30.817 1.93 [1.296-2.865] 4.010 30.996 1.87 [1.255-2.778] 4.013 31.084 1.9 [1.275-2.821] 4.015 31.148 1.91 [1.285-2.844] 4.025 31.411 1.94 [1.301-2.879] 4.030 31.531 1.96 [1.318-2.918] 4.031 31.572 1.89 [1.268-2.809] 4.033 31.633 1.9 [1.274-2.824] 4.042 31.853 1.91 [1.281-2.838] 4.043 31.882 1.92 [1.292-2.861] 4.046 31.966 1.93 [1.298-2.876] 4.053 32.175 1.86 [1.248-2.769] 4.054 32.190 1.88 [1.263-2.801] 4.054 32.198 1.81 [1.214-2.697] 4.056 32.239 1.83 [1.228-2.729] 4.071 32.667 1.85 [1.238-2.751] 4.076 32.820 1.88 [1.261-2.802] 4.080 32.914 1.92 [1.288-2.864] 4.082 32.985 1.93 [1.296-2.88] 4.084 33.031 1.98 [1.325-2.946] 4.090 33.214 2.01 [1.347-2.996] 4.091 33.230 1.93 [1.294-2.885] 4.092 33.250 1.87 [1.249-2.79] 4.092 33.267 1.89 [1.264-2.824] 4.093 33.284 1.92 [1.282-2.865] 4.094 33.316 1.93 [1.29-2.881] 4.095 33.349 1.94 [1.297-2.898] 4.097 33.417 1.88 [1.255-2.812] 4.099 33.449 1.89 [1.263-2.828] 4.101 33.529 1.94 [1.293-2.897] 4.112 33.833 1.97 [1.313-2.942] 4.117 33.990 1.89 [1.26-2.832] 4.118 34.006 1.81 [1.208-2.726] 4.121 34.102 1.85 [1.232-2.781] 4.122 34.138 1.86 [1.239-2.798] 4.126 34.234 1.87 [1.247-2.814] 4.129 34.329 1.8 [1.195-2.708] 4.130 34.375 1.83 [1.213-2.75] 4.133 34.440 1.84 [1.221-2.766] 4.133 34.447 1.86 [1.237-2.802] 4.140 34.648 1.88 [1.248-2.828] 4.144 34.790 1.92 [1.274-2.888] 4.146 34.839 1.96 [1.301-2.95] 4.153 35.039 1.97 [1.309-2.968] 4.162 35.310 1.98 [1.317-2.987] 4.162 35.316 2.02 [1.339-3.036] 4.168 35.511 1.94 [1.283-2.922] 4.173 35.659 1.96 [1.301-2.962] 4.180 35.864 1.99 [1.319-3.003] 4.187 36.076 2.02 [1.337-3.045] 4.189 36.128 2.05 [1.356-3.088] 4.191 36.215 2.06 [1.365-3.108] 4.194 36.287 2.1 [1.392-3.17] 4.196 36.347 2.15 [1.424-3.243] 4.199 36.463 2.1 [1.385-3.171] 4.202 36.557 2.15 [1.418-3.247] 4.203 36.587 2.17 [1.432-3.281] 4.208 36.750 2.18 [1.442-3.303] 4.209 36.763 2.2 [1.453-3.326] 4.209 36.770 2.21 [1.463-3.349] 4.211 36.823 2.14 [1.413-3.253] 4.213 36.887 2.16 [1.423-3.276] 4.213 36.891 2.18 [1.439-3.312] 4.220 37.130 2.22 [1.462-3.364] 4.222 37.163 2.16 [1.421-3.291] 4.224 37.238 2.2 [1.446-3.348] 4.228 37.368 2.22 [1.457-3.373] 4.236 37.642 2.29 [1.503-3.484] 4.237 37.645 2.19 [1.436-3.349] 4.241 37.779 2.25 [1.474-3.44] 4.242 37.818 2.27 [1.486-3.468] 4.245 37.925 2.3 [1.504-3.509] 4.257 38.311 2.33 [1.522-3.552] 4.279 39.042 2.37 [1.553-3.624] 4.284 39.213 2.42 [1.585-3.698] 4.284 39.218 2.48 [1.624-3.79] 4.292 39.490 2.5 [1.637-3.821] 4.299 39.702 2.53 [1.658-3.869] 4.305 39.902 2.46 [1.606-3.775] 4.308 40.011 2.49 [1.628-3.824] 4.318 40.353 2.52 [1.642-3.857] 4.322 40.487 2.56 [1.673-3.929] 4.322 40.497 2.62 [1.707-4.01] 4.331 40.802 2.54 [1.648-3.901] 4.331 40.806 2.6 [1.687-3.993] 4.332 40.865 2.48 [1.608-3.836] 4.333 40.896 2.39 [1.541-3.71] 4.357 41.732 2.49 [1.603-3.868] 4.360 41.837 2.55 [1.642-3.962] 4.361 41.880 2.6 [1.676-4.045] 4.364 41.977 2.63 [1.694-4.087] 4.369 42.178 2.71 [1.745-4.21] 4.373 42.311 2.74 [1.762-4.252] 4.375 42.385 2.67 [1.709-4.164] 4.376 42.413 2.54 [1.622-3.993] 4.380 42.551 2.6 [1.659-4.084] 4.388 42.857 2.5 [1.585-3.946] 4.389 42.894 2.38 [1.499-3.779] 4.390 42.924 2.28 [1.428-3.646] 4.395 43.113 2.35 [1.467-3.748] 4.396 43.135 2.24 [1.395-3.613] 4.400 43.306 2.32 [1.442-3.737] 4.404 43.452 2.2 [1.355-3.567] 4.410 43.677 2.11 [1.29-3.456] 4.413 43.793 2.03 [1.231-3.36] 4.414 43.839 1.96 [1.174-3.272] 4.416 43.889 1.84 [1.089-3.105] 4.434 44.575 1.86 [1.102-3.144] 4.445 45.005 1.92 [1.139-3.248] 4.450 45.186 1.96 [1.162-3.313] 4.451 45.224 1.84 [1.073-3.139] 4.462 45.645 1.89 [1.107-3.238] 4.464 45.738 1.76 [1.017-3.061] 4.471 46.007 1.66 [0.939-2.918] 4.481 46.394 1.72 [0.978-3.039] 4.481 46.416 1.76 [1-3.107] 4.481 46.423 1.79 [1.015-3.152] 4.487 46.634 1.79 [1.015-3.152] 4.488 46.683 1.86 [1.053-3.272] 4.509 47.533 1.94 [1.102-3.422] 4.509 47.540 2.01 [1.14-3.54] 4.517 47.877 2.09 [1.189-3.692] 4.518 47.916 1.98 [1.105-3.558] 4.525 48.202 2.02 [1.126-3.623] 4.548 49.131 2.13 [1.186-3.817] 4.552 49.304 2.17 [1.207-3.887] 4.570 50.100 1.99 [1.089-3.651] 4.573 50.230 1.84 [0.983-3.452] 4.588 50.855 1.9 [1.012-3.553] 4.603 51.541 1.93 [1.032-3.624] 4.608 51.765 1.85 [0.963-3.57] 4.609 51.769 1.96 [1.016-3.766] 4.614 52.010 2.05 [1.066-3.951] 4.628 52.631 2.2 [1.142-4.234] 4.642 53.286 1.98 [0.997-3.935] 4.645 53.400 2.03 [1.022-4.034] 4.650 53.632 2.19 [1.103-4.354] 4.652 53.732 2.34 [1.177-4.646] 4.655 53.889 2.08 [1.011-4.3] 4.658 54.006 2.18 [1.058-4.5] 4.661 54.136 1.96 [0.91-4.24] 4.691 55.559 1.69 [0.74-3.866] 4.735 57.713 1.75 [0.765-3.994] 4.776 59.768 1.89 [0.828-4.324] 4.780 59.974 2.05 [0.896-4.676] 4.839 63.081 2.17 [0.95-4.961] 4.854 63.856 1.93 [0.786-4.758] 4.861 64.239 2.21 [0.899-5.44] 4.916 67.337 2.58 [1.049-6.348] 4.968 70.427 2.94 [1.194-7.226] 4.971 70.616 3.41 [1.385-8.385] 4.973 70.702 4.19 [1.701-10.31] 5.005 72.702 4.44 [1.628-12.111] 5.019 73.550 7.42 [2.696-20.415] 5.089 78.092 7.35 [2.306-23.435] 5.408 102.698 5.55 [1.359-22.693] 5.456 106.995 10.28 [2.509-42.081] 5.534 114.388 18.3 [2.51-133.473] 5.800 143.552
Example 14: Validation Study
[0196] In order to assess the risk of prostate cancer disease progression among men on active surveillance for prostate cancer, an independent validation study was conducted using a cohort of 248 participants (35 progressors, 213 non progressors). The levels of (TrihexosylCer(34:1), LactosylCer(36:0), LactosylCer(32:0), SM(44:2), SM(40:2), the plasma sphingolipid signature (SphingoSignature), and a simplified sphingolipid signature [Simplified Signature, consisting of TrihexosylCer(34:1), LactosylCer(36:0), and SM(40:2)] were tested in each participant. Cav-1 was not included in this analysis. The model was derived using fixed coefficients from the logistic regression as previously described.
[0197] TrihexosylCer(34:1) and SM(40:2), along with the simplified sphingolipid signature, were found to have statistically significant odds ratios per unit increase, while hexosylceramide(40:0) was not quantifiable in the cohort samples (see
Other Embodiments
[0198] The detailed description set-forth above is provided to aid those skilled in the art in practicing the present disclosure. However, the disclosure described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of several aspects of the disclosure. Any equivalent embodiments are intended to be within the scope of this disclosure. Indeed, various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description, which do not depart from the spirit or scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended claims.