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WO2025049299A1 - Procédés de détermination du pronostic et de la stadification du cancer de la prostate - Google Patents

Procédés de détermination du pronostic et de la stadification du cancer de la prostate Download PDF

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Publication number
WO2025049299A1
WO2025049299A1 PCT/US2024/043620 US2024043620W WO2025049299A1 WO 2025049299 A1 WO2025049299 A1 WO 2025049299A1 US 2024043620 W US2024043620 W US 2024043620W WO 2025049299 A1 WO2025049299 A1 WO 2025049299A1
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Prior art keywords
seq
target proteins
prostate cancer
solid support
detection
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PCT/US2024/043620
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WO2025049299A9 (fr
Inventor
Giannoula Lakka Klement
Darrol G. ROBERTS
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Hessian Labs Inc
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Hessian Labs Inc
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Publication of WO2025049299A1 publication Critical patent/WO2025049299A1/fr
Publication of WO2025049299A9 publication Critical patent/WO2025049299A9/fr
Pending legal-status Critical Current
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • XML copy created on August 13, 2024, is named 094034-000102WOPT_SL.xml and is 590,407 bytes in size.
  • TECHNICAL FIELD [0003] The field of the invention relates to the detection and staging of prostate cancer. BACKGROUND [0004] Eleven percent of all men will develop prostate cancer (PC). In the US, 3 million men are living with the disease, 268,000 new cases are expected, and 34,000 men will die from the illness this year. PC is the second leading cause of cancer (CA) deaths in men worldwide. The prostate-specific antigen (PSA) test, presently the gold standard, was developed and instituted to ensure early diagnosis.
  • PSA prostate-specific antigen
  • the ability to reliably distinguish between benign prostatic hypertrophy, prostate cancer and prostate cancer of advanced vs less advanced stage using platelet analysis permits the tailoring of treatment for the appropriate 1 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT condition, thereby avoiding or limiting the need for invasive diagnostic procedures and their risks, and avoiding the use of under- or over-aggressive therapeutic approaches.
  • the methods and compositions described herein are based, in part, upon the identification of certain proteins or sets of proteins sequestered by platelets that permit the determination not only of the presence or absence of prostate cancer, but also the relative stage or aggressiveness of such cancer if present.
  • a solid support comprising one or more reagents permitting the detection and/or measurement of the level of one or more of Heme binding protein 1 (HEBP1), Cyclin- dependent kinase 16 (CDK16), Glycosylphosphatidylinositol specific phospholipase D1 (GPLD1) and Phospholipid transfer protein (PLTP) target proteins.
  • HEBP1 Heme binding protein 1
  • CDK16 Cyclin- dependent kinase 16
  • GPLD1 Glycosylphosphatidylinositol specific phospholipase D1
  • PLTP Phospholipid transfer protein
  • the solid support comprises reagents permitting the detection of three or more of HEBP1, CDK16, GPLD1 and PLTP target proteins.
  • the solid support comprises reagents permitting the detection of HEBP1, CDK16, GPLD1 and PLTP target proteins.
  • the reagents comprise an antibody or antigen-binding fragment thereof, or an aptamer that specifically binds a target protein.
  • the solid support comprises nitrocellulose membrane, PVDF membrane, nylon membrane, polystyrene multiwell plate, polypropylene multiwell plate, polycarbonate multiwell plate, borosilicate glass multiwell plate, pyrogen- free multiwell plate a bead, a microfluidics chamber, a dipstick, an enzyme-linked immunosorbent assay (ELISA) support, or a lateral flow test strip.
  • the solid support is pretreated with poly-l-lysine or a glucosaminoglycan.
  • the solid support comprises an antibody conjugated tag.
  • the antibody conjugated tag is selected from gold, latex, and a fluorophore.
  • the solid support further comprises one or more reagents permitting the detection and/or measurement of the level of one or more of Caveolae associated protein 2 (CAVIN2), Pro-platelet basic protein (PPBP), Cortactin (CTTN), and heat shock protein family B (small) member 1 (HSPB1) target proteins.
  • CAVIN2 Caveolae associated protein 2
  • PPBP Pro-platelet basic protein
  • CTTN Cortactin
  • HSPB1 heat shock protein family B (small) member 1
  • the solid support further comprises one or more reagents permitting the detection and/or measurement of the level of two or more of CAVIN2, PPBP, CTTN and HSPB target proteins. 2 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT [0017] In another embodiment of this or any of the aspects described herein, the solid support further comprises one or more reagents permitting the detection and/or measurement of the level of three or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins.
  • the solid support further comprises one or more reagents permitting the detection and/or measurement of the level of CAVIN2, PPBP, CTTN and HSPB1 target proteins.
  • the solid support further comprises one or more reagents permitting the detection and/or measurement of the level of one or more of RAB4A, member RAS oncogene family (RAB4A), Periphilin 1 (PPHLN1), mucin 2, oligomeric mucus/gel-forming (MUC2) and SR-related CTD associated factor 8 (SCAF8) target proteins.
  • the solid support further comprises reagents permitting the detection and/or measurement of the level of two or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins. [0021] In another embodiment of this or any of the aspects described herein, the solid support further comprises reagents permitting the detection and/or measurement of the level of three or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins. [0022] In another embodiment of this or any of the aspects described herein, the solid support further comprises reagents permitting the detection and/or measurement of the level of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins.
  • a solid support comprising one or more reagents permitting the detection and/or measurement of the level of one or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins.
  • the solid support comprises one or more reagents permitting the detection and/or measurement of the level of two or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins.
  • the solid support comprises one or more reagents permitting the detection and/or measurement of the level of three or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins.
  • the solid support comprises one or more reagents permitting the detection and/or measurement of the level of CAVIN2, PPBP, CTTN and HSPB1 target proteins.
  • the solid support further comprises one or more reagents permitting the detection and/or measurement of the level of one or more of HEBP1, CDK16, GPLD1 and PLTP target proteins.
  • the solid support further comprises reagents permitting the detection of two or more of HEBP1, CDK16, GPLD1 and PLTP target proteins. [0029] In another embodiment of this or any of the aspects described herein, the solid support further comprises reagents permitting the detection of three or more of HEBP1, CDK16, GPLD1 and PLTP target proteins. [0030] In another embodiment of this or any of the aspects described herein, the solid support further comprises reagents permitting the detection of HEBP1, CDK16, GPLD1 and PLTP target proteins.
  • the solid support further comprises one or more reagents permitting the detection and/or measurement of the level of one or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins.
  • the solid support further comprises reagents permitting the detection and/or measurement of the level of two or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins.
  • the solid support further comprises reagents permitting the detection and/or measurement of the level of three or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins.
  • the solid support further comprises reagents permitting the detection and/or measurement of the level of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins.
  • a solid support comprising reagents permitting the detection and/or measurement of the level of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins.
  • the solid support further comprises reagents permitting the detection and/or measurement of the level of one or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins.
  • the solid support further comprises reagents permitting the detection and/or measurement of the level of two or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins. [0038] In another embodiment of this or any of the aspects described herein, the solid support further comprises reagents permitting the detection and/or measurement of the level of three or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins. [0039] In another embodiment of this or any of the aspects described herein, the solid support further comprises reagents permitting the detection and/or measurement of the level of CAVIN2, PPBP, CTTN and HSPB1 target proteins.
  • the reagents comprise an antibody or antigen-binding fragment thereof, or an aptamer that specifically binds a target protein. 4 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT [0041]
  • the solid support comprises nitrocellulose membrane, PVDF membrane, nylon membrane, polystyrene multiwell plate, polypropylene multiwell plate, polycarbonate multiwell plate, borosilicate glass multiwell plate, pyrogen- free multiwell plate, a bead, a microfluidics chamber, a dipstick, an enzyme-linked immunosorbent assay (ELISA) support, or a lateral flow test strip.
  • ELISA enzyme-linked immunosorbent assay
  • the solid support is pretreated with poly-l-lysine or a glucosaminoglycan.
  • the solid support comprises an antibody conjugated tag.
  • the antibody conjugated tag is selected from gold, latex, and a fluorophore.
  • the solid support comprises a lateral flow test strip.
  • kits comprising reagents for detecting in a platelet sample the presence and/or amount of a set of peptides involved in the pathogenesis of prostate cancer, at least one radioactive isotope, and instructions for processing the samples using high resolution mass spectrometry.
  • the radioactive isotope comprises 13 C, 15 N, a reagent for labeling an amino acid side chain, an isotopically coded linker, and a tag.
  • the reagent is iodoacetamide.
  • the tag is biotin.
  • the radioactive isotope is conjugated to the sequence of at least one of IPNQFQSDPPAPSDK (SEQ ID NO: 1), EADYVAQATR (SEQ ID NO: 2), AALEGTATYR (SEQ ID NO: 3), GPLSSAPEIVHEDLK (SEQ ID NO: 4), AEALLSHAPR (SEQ ID NO: 5), LDSDGADLLTK (SEQ ID NO: 6), LPDTTSIFALK (SEQ ID NO: 7), ALEFLQLHNGR (SEQ ID NO: 8), DLLGIYEK (SEQ ID NO: 9), LYPTYSTK (SEQ ID NO: 10), ILEGFQPSGR (SEQ ID NO: 11), TLLLVGSPTWK (SEQ ID NO: 12), QISLEGSVK (SEQ ID NO: 13), GIQND
  • the mass spectrometry is selected from the group consisting of matrix-assisted laser desorption/ionization-time of flight (MALDI- TOF), and Orbitrap with tandem mass spectrometry.
  • MALDI- TOF matrix-assisted laser desorption/ionization-time of flight
  • Orbitrap with tandem mass spectrometry is selected from the group consisting of matrix-assisted laser desorption/ionization-time of flight (MALDI- TOF), and Orbitrap with tandem mass spectrometry.
  • the polypeptides involved in the pathogenesis of prostate cancer include one or more of HEBP1, CDK16, GPLD1, PLTP, CAVIN2, PPBP, CTTN, and HSPB1.
  • the polypeptides involved in the pathogenesis of prostate cancer include two or more of HEBP1, CDK16, GPLD1, PLTP, CAVIN2, PPBP, CTTN, and HSPB1.
  • the polypeptides involved in the pathogenesis of prostate cancer include HEBP1, CDK16, GPLD1, PLTP, CAVIN2, PPBP, CTTN, and HSPB1.
  • kits for the staging of cancer comprising reagents for detecting in a platelet sample the presence and/or amount of sets of polypeptides in functional cancer development categories including, but not limited to proliferation, angiogenesis, invasion, and metastasis.
  • the kit further comprises a diluent that permits sample normalization.
  • the samples are normalized to platelet number or platelet volume in the sample.
  • the samples are corrected for plasma contamination using albumin (ALB) or hemopexin (HPX) concentrations in the sample.
  • the solid support comprises at least one solid support comprising reagents sufficient to detect the presence and/or amount of the sets of polypeptides.
  • the solid support comprises a lateral flow test strip, a microfluidics chamber, a dipstick, beads, or an enzyme-linked immunosorbent assay (ELISA) support.
  • the solid support is coated with a glucosaminoglycan. 6 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT
  • the polypeptides representative of function in proliferation include one or more of HEBP1, CDK16, GPLD1, and PLTP.
  • the polypeptides representative of functions in proliferation include two or more of HEBP1, CDK16, GPLD1, and PLTP.
  • the polypeptides representative of functions in proliferation include three or more of HEBP1, CDK16, GPLD1, and PLTP.
  • the polypeptides representative of functions in proliferation include each of HEBP1, CDK16, GPLD1, and PLTP.
  • the polypeptides representative of functions in proliferation include each of HEBP1, CDK16, GPLD1, and PLTP.
  • the polypeptides representative of function in cancer cell angiogenesis include RAB4A.
  • the polypeptides representative of function in invasion include PPHLN1.
  • the polypeptides representative of function in metastasis include one or more of MUC2 and SCAF8.
  • the polypeptides representative of function in metastasis include each of MUC2 and SCAF8.
  • described herein is a method of detecting or staging benign prostate hypertrophy or prostate cancer in a male subject, the method comprising contacting a solid support of any one of the embodiments with a platelet protein preparation from a male subject.
  • Another aspect described herein is a method of distinguishing benign prostate hypertrophy from prostate cancer in a male subject, the method comprising contacting a solid support of any one of the embodiments with a platelet protein preparation from a male subject.
  • Another aspect described herein is a method of determining the stage of prostate cancer in a male subject, the method comprising contacting a solid support of any one of the embodiments with a platelet protein preparation from a male subject.
  • Another aspect described herein is a method of detecting or staging benign prostate hypertrophy or prostate cancer in a male subject, the method comprising assaying a platelet protein preparation from the subject for the presence or level of: i) one or more of HEBP1, CDK16, GPLD1 and PLTP target proteins; ii) one or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins; and iii) one or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins.
  • the method further comprises assaying the platelet protein preparation for the presence or amount of one or more of CDC73, LARP1, Girdin, and SMU1 target proteins. 7 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT [0076]
  • the levels of two or more of HEBP1, CDK16, GPLD1 and PLTP target proteins, two or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins, and/or the level of two or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins are assayed.
  • the levels of three or more of HEBP1, CDK16, GPLD1 and PLTP target proteins, three or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins, and/or the level of three or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins are assayed.
  • the levels of HEBP1, CDK16, GPLD1 and PLTP target proteins, CAVIN2, PPBP, CTTN and HSPB1 target proteins, and the level of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins are assayed.
  • detection of an elevated level of one or more of HEBP1, CDK16, GPLD1 and PLTP relative to a control is indicative of benign prostate hypertrophy.
  • the detection of an elevated level of one or more of HEBP1, CDK16, GPLD1 and PLTP relative to a control, in the absence of elevation of one or more of CAVIN2, PPBP, CTTN and HSPB1, is indicative of benign prostate hypertrophy.
  • the method further comprises, when the method indicates BPH, administering terazosin, alfuzosin, doxazosin, tamsulosin, silodosin, finasteride, dutasteride, and/or tadalafil to the subject.
  • detection of an elevated level of one or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins relative to a control is indicative of prostate cancer.
  • the method further comprises administering surgery, radiation therapy, cryotherapy, hormone therapy, chemotherapy, immunotherapy, PARP inhibitors, bisphosphonates, brachytherapy, denosumab, corticosteroids, external radiation therapy or a radiopharmaceutical to the subject.
  • the surgery is selected from open or laparoscopic radical prostatectomy or transurethral resection of the prostate.
  • the radiation therapy is selected from the group consisting of external beam radiation, brachytherapy, and radiopharmaceuticals.
  • the hormone therapy is selected from the group consisting of orchiectomy, LHRH agonists, LHRH antagonists, abiraterone, ketoconazole, antiandrogens, and estrogens. 8 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT [0087]
  • the LHRH agonists are selected from the group consisting of leuprolide, goserelin, triptorelin, and leuprolide mesylate.
  • the LHRH antagonists are selected from the group consisting of degarelix and relugolix.
  • the antiandrogens are selected from the group consisting of flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, and darolutamide.
  • the hormone therapy is administered as a combined androgen blockade.
  • the chemotherapy is selected from the group consisting of docetaxel, cabazitaxel, mitoxantrone, and estramustine.
  • the immunotherapy is selected from the group consisting of sipuleucel-T and PD-1 inhibitor.
  • the PARP inhibitors are selected from the group consisting of rucaparib and olaparib.
  • the radiopharmaceuticals comprise an isotope selected from the group consisting of strontium-89, samarium-153, and radium-223.
  • detection of an elevated level of RAB4A relative to a control is indicative of stage II prostate cancer.
  • the method further comprises administering external beam radiation, brachytherapy, radical prostatectomy, or active surveillance to the subject.
  • detection of an elevated level of PPHLN1 relative to a control is indicative of stage III prostate cancer.
  • the method further comprises administering external beam radiation along with hormone therapy or external beam radiation and brachytherapy with hormone therapy to the subject.
  • the method further comprises administering radical prostatectomy to the subject.
  • detection of an elevated level of MUC2 relative to a control is indicative of stage IV indolent prostate cancer.
  • detection of an elevated level of SCAF8 relative to a control is indicative of stage IV aggressive prostate cancer.
  • the method further comprises administering hormone therapy along with chemotherapy, external beam radiation along with hormone therapy, radical prostatectomy, and active surveillance. 9 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT [00103]
  • the hormone therapy is selected from the group consisting of orchiectomy, LHRH agonists, LHRH antagonists, abiraterone, ketoconazole, antiandrogens, and estrogens.
  • the surgery is selected from the group consisting of open laparoscopic radical prostatectomy, laparoscopic radical prostatectomy and transurethral resection of the prostate.
  • the method further comprises assaying the platelet protein preparation for one or more control protein markers.
  • assaying a platelet protein preparation for the presence or level of target proteins comprises an immunoassay, microfluidic assay or mass spectrometry.
  • the microfluidic assay is an ELISPOT assay.
  • the immunoassay comprises contacting the platelet protein preparation with antibodies or antigen-binding fragments thereof that specifically bind the target proteins.
  • the mass spectrometry assay identifies the levels of one or more peptides selected from IPNQFQSDPPAPSDK (SEQ ID NO: 1), EADYVAQATR (SEQ ID NO: 2), AALEGTATYR (SEQ ID NO: 3), GPLSSAPEIVHEDLK (SEQ ID NO: 4), AEALLSHAPR (SEQ ID NO: 5), LDSDGADLLTK (SEQ ID NO: 6), LPDTTSIFALK (SEQ ID NO: 7), ALEFLQLHNGR (SEQ ID NO: 8), DLLGIYEK (SEQ ID NO: 9), LYPTYSTK (SEQ ID NO: 10), ILEGFQPSGR (SEQ ID NO: 11), TLL
  • Another aspect described herein is a method of distinguishing benign prostate hypertrophy from prostate cancer in a male subject, the method comprising assaying a platelet protein preparation from the subject for the presence or level of: i) one or more of HEBP1, CDK16, GPLD1 and PLTP target proteins; and ii) one or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins.
  • the levels of two or more of HEBP1, CDK16, GPLD1 and PLTP target proteins, and/or two or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins are assayed.
  • the levels of three or more of HEBP1, CDK16, GPLD1 and PLTP target proteins, and/or three or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins are assayed.
  • the levels of HEBP1, CDK16, GPLD1 and PLTP target proteins, and the levels of CAVIN2, PPBP, CTTN and HSPB1 target proteins are assayed.
  • detection of an elevated level of one or more of HEBP1, CDK16, GPLD1 and PLTP relative to a control is indicative of benign prostate hypertrophy.
  • the detection of an elevated level of one or more of HEBP1, CDK16, GPLD1 and PLTP relative to a control, in the absence of elevation of one or more of CAVIN2, PPBP, CTTN and HSPB1, is indicative of benign prostate hypertrophy.
  • the method further comprises, when the method indicates BPH, administering terazosin, alfuzosin, doxazosin, tamsulosin, silodosin, finasteride, dutasteride, and/or tadalafil to the subject.
  • the method further comprises administering surgery, radiation therapy, cryotherapy, hormone therapy, chemotherapy, immunotherapy, PARP inhibitors, bisphosphonates, brachytherapy, denosumab, corticosteroids, external radiation therapy, and a radiopharmaceutical to the subject.
  • the surgery is selected from open or laparoscopic radical prostatectomy and transurethral resection of the prostate.
  • the radiation therapy is selected from external beam radiation, brachytherapy, and a radiopharmaceutical. 11 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT
  • the hormone therapy is selected from orchiectomy, LHRH agonists, LHRH antagonists, abiraterone, ketoconazole, antiandrogens, and estrogens.
  • the LHRH agonists are selected from leuprolide, goserelin, triptorelin, and leuprolide mesylate.
  • the LHRH antagonists are selected from degarelix and relugolix.
  • the antiandrogens are selected from flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide and darolutamide.
  • the hormone therapy is administered as a combined androgen blockade.
  • the chemotherapy is selected from docetaxel, cabazitaxel, mitoxantrone, and estramustine.
  • the immunotherapy is selected from sipuleucel-T and PD-1 inhibitor.
  • the PARP inhibitors are selected from rucaparib and olaparib.
  • the radiopharmaceuticals comprise an isotope selected from strontium-89, samarium-153, and radium-223.
  • Another aspect described herein is a method of determining the stage of prostate cancer in a male subject with prostate cancer, the method comprising assaying a platelet protein preparation from the subject for the presence or level of: one or more of RAB4A, PPHLN1, MUC2 and/or SCAF8 target proteins, wherein detection of an elevated level of RAB4A relative to a control indicates stage II prostate cancer; detection of an elevated level of PPHLN1 relative to a control indicates stage III prostate cancer; detection of an elevated level of MUC2 relative to a control indicates stage IV indolent prostate cancer; and detection of an elevated level of SCAF8 relative to a control indicates stage IV aggressive prostate cancer.
  • the method further comprises, when stage II prostate cancer is determined, administering external beam radiation, brachytherapy, radical prostatectomy, or active surveillance to the subject, when stage III prostate cancer is determined, administering external beam radiation along with hormone therapy or external beam radiation and brachytherapy with hormone therapy to the subject, and when stage IV prostate cancer is determined, administering hormone therapy along with chemotherapy, external beam radiation along with hormone therapy, radical prostatectomy and active surveillance.
  • the method further comprises assaying the platelet protein preparation for one or more control protein markers.
  • the one or more control protein markers comprises FERMT3, CAPZB, YWHAG, GC, USP14, and MGST2.
  • assaying a platelet protein preparation for the presence or level of target proteins comprises an immunoassay, microfluidic assay or mass spectrometry.
  • the microfluidic assay is an ELISPOT assay.
  • the immunoassay comprises contacting the platelet protein preparation with antibodies or antigen-binding fragments thereof that specifically bind the target proteins.
  • the mass spectrometry assay identifies the levels of one or more peptides selected from IPNQFQSDPPAPSDK (SEQ ID NO: 1), EADYVAQATR (SEQ ID NO: 2), AALEGTATYR (SEQ ID NO: 3), GPLSSAPEIVHEDLK (SEQ ID NO: 4), AEALLSHAPR (SEQ ID NO: 5), LDSDGADLLTK (SEQ ID NO: 6), LPDTTSIFALK (SEQ ID NO: 7), ALEFLQLHNGR (SEQ ID NO: 8), DLLGIYEK (SEQ ID NO: 9), LYPTYSTK (SEQ ID NO: 10), ILEGFQPSGR (SEQ ID NO: 11), TLL
  • Another aspect described herein is a method of treating or staging BPH or prostate cancer, the method comprising receiving results of an assay of a platelet protein preparation from a subject for the presence or level of: i) one or more of HEBP1, CDK16, GPLD1 and PLTP target proteins; ii) one or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins; and iii) one or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins; and when one or more of HEBP1, CDK16, GPLD1 and PLTP target proteins is increased relative to a control, BPH is indicated; and when one or more of CAVIN2, PPBP, 13 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT CTTN and HSPB1 target proteins is increased relative to a control, prostate cancer is indicated, and when RAB4A is increased, stage II prostate cancer is indicated; when PPHLN1 is increased, stage
  • the assay further measures the presence or amount of one or more of CDC73, LARP1, Girdin, and SMU1 target proteins.
  • the levels of two or more of HEBP1, CDK16, GPLD1 and PLTP target proteins, two or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins, and/or the level of two or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins are assayed.
  • the levels of three or more of HEBP1, CDK16, GPLD1 and PLTP target proteins, three or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins, and/or the level of three or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins are assayed.
  • the levels of HEBP1, CDK16, GPLD1 and PLTP target proteins, CAVIN2, PPBP, CTTN and HSPB1 target proteins, and the level of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins are assayed.
  • the detection of an elevated level of one or more of HEBP1, CDK16, GPLD1 and PLTP relative to a control, in the absence of elevation of one or more of CAVIN2, PPBP, CTTN and HSPB1, is indicative of benign prostate hypertrophy.
  • the method indicates BPH, the administering comprises administering terazosin, alfuzosin, doxazosin, tamsulosin, silodosin, finasteride, dutasteride, and/or tadalafil to the subject.
  • the method indicates prostate cancer
  • the administering comprises administering surgery, radiation therapy, cryotherapy, hormone therapy, chemotherapy, immunotherapy, PARP inhibitors, bisphosphonates, brachytherapy, denosumab, corticosteroids, external radiation therapy or a radiopharmaceutical to the subject.
  • the surgery is selected from open or laparoscopic radical prostatectomy or transurethral resection of the prostate.
  • the radiation therapy is selected from external beam radiation, brachytherapy, and a radiopharmaceutical.
  • the hormone therapy is selected from orchiectomy, LHRH agonists, LHRH antagonists, abiraterone, ketoconazole, antiandrogens, and estrogens. 14 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT [00149]
  • the LHRH agonists are selected from leuprolide, goserelin, triptorelin, and leuprolide mesylate.
  • the LHRH antagonists are selected from degarelix and relugolix.
  • the antiandrogens are selected from flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, and darolutamide.
  • the hormone therapy is administered as a combined androgen blockade.
  • the chemotherapy is selected from docetaxel, cabazitaxel, mitoxantrone, and estramustine.
  • the immunotherapy is selected from sipuleucel-T and PD-1 inhibitor.
  • the PARP inhibitors are selected from rucaparib and olaparib.
  • the radiopharmaceuticals comprise an isotope are selected from strontium-89, samarium-153, and radium-223.
  • the method further comprises administering external beam radiation, brachytherapy, radical prostatectomy, or active surveillance to the subject when RAB4A is increased.
  • the method further comprises administering external beam radiation along with hormone therapy or external beam radiation and brachytherapy with hormone therapy to the subject when PPHLN1 is increased.
  • the method further comprises administering radical prostatectomy to the subject when PPHLN1 is increased.
  • the method further comprises administering hormone therapy along with chemotherapy, external beam radiation along with hormone therapy, radical prostatectomy, surgery, and active surveillance when SCAF8 and/or MUC is increased.
  • receiving results further comprises results of assaying the platelet protein preparation for one or more control protein markers.
  • the one or more control protein markers comprises FERMT3, CAPZB, YWHAG, GC, USP14, and MGST2.
  • the assay for the presence or level of target proteins comprises an immunoassay, microfluidic assay or mass spectrometry.
  • the microfluidic assay is an ELISPOT assay.
  • the immunoassay comprises contacting the platelet protein preparation with antibodies or antigen-binding fragments thereof that specifically bind the target proteins.
  • the mass spectrometry assay identifies the levels of one or more peptides selected from IPNQFQSDPPAPSDK (SEQ ID NO: 1), EADYVAQATR (SEQ ID NO: 2), AALEGTATYR (SEQ ID NO: 3), GPLSSAPEIVHEDLK (SEQ ID NO: 4), AEALLSHAPR (SEQ ID NO: 5), LDSDGADLLTK (SEQ ID NO: 6), LPDTTSIFALK (SEQ ID NO: 7), ALEFLQLHNGR (SEQ ID NO: 8), DLLGIYEK (SEQ ID NO: 9), LYPTYSTK (SEQ ID NO: 10), ILEGFQPSGR (SEQ ID NO: 11), TLLLVGSPTWK (SEQ ID NO: 12), QISLEGSVK (SEQ ID NO: 13), GIQNDLTK (SEQ ID NO: 14), YQASTSNTVSK (SEQ ID NO: 1), EADYVAQATR (SEQ ID NO:
  • the subject is human.
  • the method further comprises a step of normalizing the sample to platelet number or platelet volume in the sample.
  • the method further comprises a step of correcting the sample for plasma contamination using albumin (ALB) or hemopexin (HPX) concentrations in the sample.
  • the normalizing step or the correcting step is performed using a diluent.
  • FIG. 1 is a schematic depicting exemplary subject profiles from an exemplary method for studying the ability of platelets to determine different aspects of disease.
  • Platelets sequester prostate cancer relevant proteins.
  • Study data of healthy and cancer-bearing human subjects demonstrate the ability of 16 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT platelet protein profiles to: anticipate disease onset and evolution, distinguish disease type, distinguish disease stage, and/or distinguish between disease related inflammation and non-disease inflammation.
  • FIG. 2 examines how different analytes such as plasma/serum sample, a urine sample, and a tissue sample compare to the capacity of platelet bioinformatics to diagnose prostate cancer and prostate cancer progression.
  • Platelet samples can provide diagnostic data (e.g., to detect or confirm a specific disease or condition) and predictive data (e.g., to guide treatment decisions by indicating the probable effect of a specific therapy on a patient), whereas plasma/serum sample, a urine sample, and a tissue sample can only estimate prognosis (e.g., to inform about the likelihood of clinical outcomes, such as disease recurrence or progression).
  • FIG. 3 is a schematic depicting how platelet data fit into the omics development and clinical utility in prostate cancer carcinogenesis and current diagnostic strategies. The methods described herein (e.g., HessMapsTM) can diagnose disease by stage and monitor therapy effectiveness.
  • FIG.4A-4B shows heat maps that confirmed the ability to distinguish disease grade from platelet data.
  • the heatmaps show a clear separation of differentially expressed proteins between grades II, III, and IV.
  • the grades are histological scores for tissue samples looking at the nuclear/cytoplasmic ratio across all stages of prostate cancer.
  • FIG. 4A show a heat map distinguishing prostate cancer grade 2 and grade 4.
  • FIG.4B show a heat map distinguishing prostate cancer grade 3 and grade 4.
  • platelet proteins clearly identify prostate cancer (PC) grade.
  • FIG. 5 provides an overview of the HessMap TM assays design. It describes the framework of cancer progression from benign prostatic hyperplasia (BPH) to metastatic dissemination, and introduces the point of reference for the first diagnostic quartet of proteins – i.e., those that identify the transformation of normal prostate cells into hyperplasia (proliferation; see e.g., FIG. 6A-6D).
  • FIG.5 also examines the assay design of identifying prostate cancer through identifying and measuring proteins with linear correlation with cancer progression in prostate cancer (see e.g., FIG. 8A-8D).
  • FIG. 8A-8D shows an overview of the HessMap TM assays design. It describes the framework of cancer progression from benign prostatic hyperplasia (BPH) to metastatic dissemination, and introduces the point of reference for the first diagnostic quartet of proteins – i.e., those that identify the transformation of normal prostate cells into hyperplasia (proliferation; see e.g., FIG. 6A-6D).
  • FIG.5 also
  • FIG. 5 also examines the assay design for identifying malignant transformation of normal, terminally differentiated prostatic cells into a malignant cancer cell through the identification, measurement, and functional correlation of stage II (RAB4A), stage III (PPHLN1) and stage IV (MUC2 and SCAF8) specific proteins (see e.g., FIG. 10A- 10D).
  • FIG.5 further examines the assay design for identifying proteins capable of distinguishing prostate cancer from other types of cancer (see e.g., FIG.12A-12D).
  • FIG.6A-6D provides a closer look at the proteins upregulated in and/or specific to BPH.
  • HEBP1 FIG.6A-6D provides a closer look at the proteins upregulated in and/or specific to BPH.
  • FIG. 6A HEBP1 promotes calcium mobilization and chemotaxis in monocytes and dendritic cells
  • CDK16 FIG. 6B; CDK16 is a member of dc2/cdkx subfamily of the ser/thr protein kinases, signal transduction, exocytosis, secretory cargo
  • GPLD1 FIG. 6C; GPLD1 hydrolyzes the GPI anchor thus releasing proteins from the membrane
  • PLTP FIG.
  • FIG. 7A-7B examines protein-protein interaction and functional activity and if the functional activity is related to the interaction being studied. These diagrams focus on interactions (Fig. 7A) and functional activity related to inflammation (Fig. 7B). BPH specific platelet proteins thus activate inflammatory, metabolic and immune pathways.
  • FIG.8A-8D shows in detail the malignant transformation of prostate cancer-related proteins (e.g., prostate cancer progression specific proteins) CAVIN2 (FIG.8A; CAVIN2 is a suppressive regulator of TNF-induced Mesenchymal Stromal Cell Inflammation and Angiogenic Phenotypes), CTTN (FIG.
  • FIG.9A-9B show an example of a network of proteins and their known functions and biological pathways relating to the statistically significant upregulation of prostate cancer progression-specific protein CTTN.
  • CTTN (Cortactin) is a protein contributing to the organization of the actin cytoskeleton and cell shape , with an essential role in the formation of lamellipodia and cell migration. The pathway and hallmarks annotations confirm that CTTN activates or participates in growth, transport and angiogenesis related signaling pathways.
  • CTTN is both significantly upregulated in platelets of subjects with prostate cancer, and participates in functions important for cancer progression.
  • FIG.9A shows the protein interaction map of CTTN
  • FIG.9B shows the enrichment table of CTTN interactions.
  • growth progression specific platelet proteins activate growth signaling pathways.
  • FIGS. 10A-10D show in detail the confidence intervals of the stage-specific upregulation of proteins involved in prostate cancer including RAB4A for stage II (FIG.10A; RAB4A regulates vesicular trafficking, enhances VEGFR2 signaling by mediating VEGFR2 endosomal trafficking), PPHLN1 for stage III (FIG.10B; PPHLN1 is a component of the HUSH complex, which regulates cell cycle, mediates epigenetic repression), MUC2 for stage IV (FIG.10C; MUC2 has a role in the mucinous differentiation of prostate and is associated with more indolent growth), and SCAF8 for stage IV (FIG.
  • FIG. 11A-11B depict gene network maps and gene ontology (GO) annotations for the upregulation of MUC2 versus MUC1.
  • Mucin and SCAF8 are markers of a well differentiated vs aggressive undifferentiated cancer.
  • Mucins are glycosylated proteins essential for the formation of protective mucous barriers on epithelial surfaces. Their placement on the apical surface of epithelial cells facilitates intracellular signaling, and their over-expression, aberrant intracellular localization, or changes in glycosylation lead to formation of carcinomas. Described herein is an example of alternate splicing and different transcript variants leading to different cancer phenotypes. Unlike the primary isoform (MUC1), the inducible MUC2 is associated with quiescent phenotype. As shown in FIG.
  • FIG. 11A when MUC2 is upregulated, the prostate cancer is classified as a quiescent, well-differentiated phenotype as pathway and hallmarks annotations indicate.
  • FIG.11B when the generally constitutive SCAF8 is upregulated, the resulting network is closely associated with the constitutively active MUC1 and the EGFR growth pathway and proliferation.
  • the prostate cancer with MUC1 increased in platelets is therefore classified as aggressive, undifferentiated phenotype based on the respective functional annotations.
  • FIG.12A shows a statistically significant upregulation of prostate cancer-related proteins CDC73
  • CDC73 is a parafibromin implicated in regulation of development and maintenance of embryonic stem cell pluripotency
  • LARP1 (FIG.12B; LARP1 promotes cancer cell invasion in prostate cancer)
  • Girdin (FIG.12C; Girdin is an actin-binding protein activated by the ser/thr kinase Akt and plays a role in cytoskeleton remodeling and cell migration)
  • SMU1 FIG.12D; SMU1 is a DNA replication regulator and spliceosomal factor
  • FIG.13 depicts the current emerging molecular-based tools for prostate cancer care.
  • tests use a sample source from either plasma or urine.
  • samples use sample sources of tissue, plasma, and urine.
  • additional tests are used for risk stratification and use a tissue source for their sample.
  • FIG. 14 shows a simplified molecular-based tool for prostate cancer care.
  • the HESSMAP diagnostic test can be used at any stage of the diagnosis pathway (suspected prostate cancer, biopsy, and risk stratification) and can make the step of biopsy optional.
  • the functional role of the selected proteins also distinguishes it from so-called 19 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT liquid biopsy in that that approach which detects cancer cells or molecules or degradation products released from them in a body fluid sample, but does not indicate which of those products are participating in the tumor’s development or evolution.
  • the detection and/or measurement of the selected proteins in platelets establishes cell functions relevant to each cancer stage , focusing on platelet-sequestered proteins that drive the evolution or progression of the cancer.
  • the identified platelet-sequestered proteins are functionally connected to cancer initiation or progression, they can provide not only an indication of the presence of a cancer, i.e., diagnosis, but also reliable information regarding the stage of that cancer. Reliable determination of the stage of cancer can be invaluable in guiding treatment decisions, e.g., by indicating the probable effect of a specific therapy on the cancer.
  • the compositions and methods described herein focus on the constellation of proteins and the relevant cell functions, including malignant transformation, local tissue invasion and metastatic escape that define each stage of a cancer. The relevance of the selected proteins is established by the evaluation of the pathways in which those proteins participate.
  • mammal is intended to encompass a singular "mammal” and plural “mammals,” and includes, but is not limited to humans; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and bears.
  • a mammal is a human.
  • a subject can be of any age including a neonate toddler, child, teen, adult or a geriatric subject.
  • Subjects that can be treated using the methods, and compositions as described herein include “healthy” individuals in respect to cancer, such as, e.g., (normal with no history of cancer; cancer not yet detected) but may have underlying health concerns, such as, e.g. (obesity, diabetes), and those suffering from, or at risk for, hyperplasia and/or cancer and/or associated inflammation.
  • the subjects include human patients (adults and children) who have or are at risk of developing cancer. Where the cancer is prostate cancer, the subject will be male.
  • a subject to be diagnosed, staged or treated according to the methods described herein can be a healthy individual or one who has been diagnosed with a disease, or condition associated with neoplasia, cancer, and/or inflammation, such as a disease or condition of the prostate, or one at risk of developing 20 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT these conditions.
  • a “healthy” subject is one that lacks detectable disease at the time of testing.
  • a healthy individual can also refer to an individual who has successfully been treated for neoplasia, and/or cancer, and is “disease free.”
  • the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g., a cancer, e.g., prostate cancer.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a cancer. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced.
  • treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable.
  • treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
  • pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a pharmaceutically acceptable carrier can be a carrier other than water.
  • a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment.
  • a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in or within nature.
  • administering refers to the placement of a composition or compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the composition, compound, or metabolite thereof at a desired site.
  • Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
  • administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and/or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and/or the subject being treated.
  • cancer refers to the presence of cells in tissue, or circulation, possessing characteristics typical of cancer cells as understood by those of skill in the art of medicine.
  • Neoplasia As used herein, the terms “neoplasia,” and “hyperplasia,” are meant as disease that is caused by, or results from, high levels of cell division, or low levels of apoptosis, or both. Neoplasia is characterized by the pathological proliferation and migration to, or invasion of, other tissues or organs.
  • cancer refers to a hyperproliferation of cells that have lost normal cellular control, resulting in unregulated growth, lack of differentiation, local tissue invasion, and metastasis. Cancers are classified based on the histological type (e.g., the tissue in which they originate) and their primary site (e.g., the location of the body the cancer first develops), and can be a carcinoma, a melanoma, a sarcoma, a myeloma, a leukemia, or a lymphoma. “Cancer” can also refer to a solid tumor.
  • tumor refers to an abnormal growth of cells or tissues, e.g., of malignant type or benign type.
  • Cancer can be metastatic, meaning the cancer cells have disseminated from its primary site of origin and migrated to a secondary site.
  • cancers wherein methylation status plays a role, include, but are not limited to, breast cancer, a melanoma, adrenal gland cancer, biliary tract cancer, bladder cancer, brain or central nervous system cancer, bronchus cancer, blastoma, carcinoma, a chondrosarcoma, cancer of the oral cavity or pharynx, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, glioblastoma, hepatic carcinoma, hepatoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreas cancer, peripheral nervous system cancer, prostate cancer, sarcoma, salivary gland cancer, small bowel or appendix cancer, small-cell lung cancer, squamous cell cancer, stomach cancer, testis cancer, thyroid cancer, urinary bladder cancer, uterine or endometrial cancer, and vulval cancer.
  • the term “grade” refers to how normal or abnormal cancer cells look under a microscope. The more normal the cells look, the less aggressive the cancer and the more slowly it grows and spreads. Tumor grade is determined by studying a sample of tissue, usually obtained by performing a biopsy, and studying the sample’s histology under the microscope. Systems for describing tumor grade can differ depending on the type of cancer. Most tumors are graded X, 1, 2, 3, or 4. In the grading of prostate cancer, a Gleason score is used to describe how different the cancer cells look from normal cells under a microscope and how likely the tumor is to spread. Grade X is a grade that cannot be assessed (undetermined grade).
  • Grade 1 is a well-differentiated sample (low grade) with a Gleason score of 6 or less.
  • Grade 2 is a moderately differentiated sample (intermediate grade) with a Gleason score of 7.
  • Grade 3 is a poorly differentiated sample (high grade) with a Gleason score of 8.
  • Grade 4 is an undifferentiated sample (high grade) with a Gleason score of 9 or 10.
  • stage refers to how large a cancer tumor is and how aggressive it is in terms of metastatic activity or potential. Tumor grade is not the same as cancer stage.
  • isolated refers to materials that are enriched or removed from the native or endogenous state from which they are found. “Isolate” denotes a degree of separation from original source or surroundings. A related term, “Purify” denotes a degree of separation that is greater than isolation, such as e.g., (isolation of platelets from whole blood, and purification of platelet associated proteins). A “purified,” or “biologically pure” protein, is sufficiently free of other materials so that any impurities do not impact biological properties, or if they do it is to a negligible degree.
  • platelet-associated protein and “platelet-sequestered polypeptide” are synonymous and used to refer to short peptides, polypeptides or proteins that are detectable in isolated platelets and can be concentrated in platelets against a concentration gradient in plasma.
  • protein or “polypeptide,” or “peptide” is meant any chain of more than two natural or unnatural amino acids, regardless of post-translational modifications, e.g., glycosylation or phosphorylation, among others, constituting all or part of a naturally-occurring or non-naturally occurring polypeptide or peptide, as is described herein.
  • isolated polypeptide refers to a polypeptide that has been separated from its natural components.
  • An isolated polypeptide can be obtained, for example, by extraction from a natural source, such as whole blood or platelets.
  • polypeptides representative of functions in cancer development refers to polypeptides that have been determined to be present or to be present in increased concentrations or occur in altered amounts during a particular phase of cancer development and their presence/absence or amount is therefore predictive or indicative of a particular disease state or stage of cancer.
  • angiogenesis refers to the developmentally abnormal production of new blood vessels (e.g., neovascularization) induced in response to oncogenic stimuli as the nutrient needs of cancer cells or tumors increase. Cancers generally attain, as part of their development, the ability to actively promote angiogenesis to satisfy supply demands for nutrients, inflammatory cells, stem cells and other cancer supporting cells.
  • cancer cell invasion refers to a process by which a cancer cell extends and penetrates into a neighboring tissue.
  • cancer cell invasion can also be referred to herein as “local tissue cancer invasion.”
  • metastasis refers to the movement of cancer cells from a primary site through the circulatory system or lymphatic system to a site distinct from the primary site, thereby permitting adhesion of the metastatic cells at the new site and establishment of a secondary cancer site (i.e. a metastasis).
  • cancer stimulatory polypeptides refers to polypeptides whose expression is associated with cancer growth and metastasis.
  • cancer inhibitory polypeptides refers to polypeptides whose lack of expression or insufficient expression is associated with cancer growth and metastasis. 23 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT [00209]
  • normalization refers to a method of modifying quantitative measures (e.g., protein concentration), to minimize and/or mitigate the impact of factors which can prevent equivalent comparison, for example, minimizing the impact of highly variable baseline of a protein in a population or removing the impact of differing amounts of starting material.
  • Exemplary methods of normalization include, but are not limited to, utilizing a ratio in platelet number, or volume, or platelet associated reference to adjust for differences in platelets in a given sample.
  • Normalization approaches commonly used in other systems e.g., normalization to the level of a relatively invariant housekeeping protein or gene can be used, but are generally not as accurate as normalization to platelet number or to a reference protein or factor that is specific to platelets, to generate a ratio or “normalization / correction factor,” to correct for equivalent comparison.
  • a diluent that permits sample normalization can be used and/or included in any of the aspects described herein (e.g., methods, kits, systems, etc.).
  • the samples are normalized to platelet number or platelet volume in the sample. In some embodiments of any of the aspects, the samples are corrected for plasma contamination using albumin (ALB) or hemopexin (HPX) concentrations in the sample [00210]
  • ALB albumin
  • HPX hemopexin
  • “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease in expression or activity of a given platelet-associated polypeptide by at least 10% as compared to a reference expression or activity level for the same platelet- associated polypeptide and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more.
  • a platelet-associated polypeptide may be “absent” or “below detectable levels” under certain conditions. For example, in conditions where cancer is not present, one or more cancer stimulatory polypeptides may be absent or below detectable levels. Alternatively, in conditions associated with cancer or cancer progression, one or more cancer inhibitory polypeptides may be absent or below detectable levels.
  • the terms “increased,” “increase” or “enhance” or “activate” are all used herein to generally mean an increase of a property, level, or other parameter (e.g., amount of a platelet-associated polypeptide) by a statistically significant amount; for the avoidance of any doubt, the terms “increased”, “increase” or “enhance” or “activate” means an increase in expression or activity of a given platelet-associated 24 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT polypeptide in the panel by at least 10% as compared to a reference level (e.g., a platelet sample from one or more healthy individuals), for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at
  • a reference level can be the amount of each member of the panel of proteins in a subject or population of subjects lacking cancer.
  • a variant amino acid or DNA sequence can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence.
  • the degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g. BLASTp or BLASTn with default settings).
  • the methods, assays, and devices described herein can be used to detect the presence of polypeptides in platelets that are associated with particular prostate cancer functions or processes, which in turn can be used to diagnose or stage prostate cancer.
  • prostate cancer functions or processes include malignant cell transformation, angiogenesis, cell invasion, metastasis, etc.
  • the methods and assays can be used to detect the presence of prostate cancer.
  • Benign prostatic hyperplasia is a condition that is characterized by an enlarged prostate gland and is common in men as they age.
  • Symptoms include frequent or urgent need to urinate, increased frequency of urination at night, difficulty starting urination, weak urine stream or a stream that stops and starts, dribbling at the end of urination, inability to completely empty the bladder, urinary tract infection, inability to urinate, and blood in the urine. These symptoms can gradually get worse over time.
  • Risk factors of BPH include aging, family history, diabetes, heart disease, and obesity.
  • diagnosis of BPH includes a digital rectal exam, urine test, blood test, and prostate-specific antigen (PSA) blood test, which measures the amount of PSA in the blood. PSA levels increase when the prostate is enlarged.
  • PSA prostate-specific antigen
  • Additional tests can include a urinary flow test, postvoid residual volume test, which measures if the bladder can be emptied completely, transrectal ultrasound, and prostate biopsy.
  • Current treatment of BPH includes, but is not limited to, alpha blockers (e.g., alfuzosin, doxazosin, tamsulosin, and silodosin), 5-alpha reductase inhibitors (e.g., finasteride, dutasteride), combination drug therapy, tadalafil, minimal invasive or surgical therapy (e.g., transurethral resection of the prostate (TURP), transurethral incision of the prostate (TUIP), transurethral microwave thermotherapy (TUMT), transurethral needle ablation (TUNA), laser therapy, prostatic urethral lift (PUL), embolization, and open or robot-assisted prostatectomy.
  • alpha blockers e.g., alfuzosin, doxazosin, tamsul
  • each stage of prostate cancer is determined by combining the tumor (T), nodes (N), and metastasis (M) categories, which is also referred to as TNM system, and is typical for all cancer types.
  • TNM system tumor
  • M metastasis
  • the Gleason score and the PSA level at the time of diagnosis also provide a additional clinical staging for estimating the potential for cancer progression.
  • the Gleason Score provides an estimate of cancer aggressiveness based on histological features characteristic of aggressive growth and metastasis. Combining these factors is called “stage grouping” and the result is the assignment of a Roman numeral stage from I to IV and a letter grouping where A characterizes low aggressiveness and B highly aggressive histological features.
  • Stage I prostate cancer is the least advanced form of prostate cancer. Cancer in this stage is small and hasn’t spread past the prostate gland. It is commonly associated with a PSA of less than 10 ng/mL and 26 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT Gleason score of less than or equal to 6.
  • Treatment of stage I prostate cancer can include active surveillance, radical prostatectomy, and radiation therapy. Transition of BPH to Stage I cancer can be characterized by proliferation and expression/upregulation of genes involved in proliferation. Proliferation can be division that outpaces cell turnover and/or active division by cells.
  • Stage II prostate cancer is still confined to the prostate and hasn’t spread to lymph nodes or other parts of the body.
  • Stage II tumors may appear on ultrasound imaging, and are commonly associated with a PSA less than 20ng/mL. Based on the histological patterns (Gleason Score) and on the level of PSA Stage II prostate cancer can be Stage IIA – low grade, or Stage IIB high grade. Treatment can include removing the prostate and some surrounding lymph nodes, hormone therapy, radiation therapy, or active surveillance. Transition of stage I prostate cancer to stage II prostate cancer can be characterized by angiogenesis, proliferation, and local invasion detectable in the expression/upregulation of genes characterizing these processes. [00224] Stage III prostate cancer is characterized by cancer cells that may or may not have spread outside the prostate to other tissues.
  • the PSA level is 20ng/mL or higher, the histology is characterized by increases in stromal elements (cribriform pattern), and the Gleason Score ranges between 8-10.
  • Stage III cancer can be Stage IIIA and Stage IIIB depending on the aggressiveness of the histology and PSA.
  • Treatment can include external beam radiation plus hormone therapy, radiation (external beam and brachytherapy) plus hormone therapy, radical prostatectomy, usually with removal of the pelvic lymph nodes; radiation therapy and/or hormone therapy may follow. Transition from stage II prostate cancer to stage III prostate cancer can be characterized by increased expression of genes/proteins involved in the process of cell adhesion, and tissue invasion. Invasion can be an intrusion of prostate cancer cells into another tissue or organ type.
  • Stage IV prostate cancer is the most advanced stage of prostate cancer, and can be of any grade, and any PSA value.
  • Stage IVA has spread to nearby lymph nodes but has not spread to other parts of the body.
  • Stage IVB has spread to distant lymph nodes, other parts of the body, or to the bones and can be any grade groups and any PSA value.
  • stage IV prostate cancer can be classified as indolent or aggressive.
  • Treatment can include hormone therapy, chemotherapy, external beam radiation, radiation targeting bones, surgical removal of the prostate or symptomatic metastases. Transition from stage III prostate cancer to stage IV prostate cancer can be characterized by increased expression of genes/proteins upregulated during the process of metastasis and tissue invasion.
  • Metastasis can be shown as the development of secondary malignant growths at a distance from a primary site of cancer, such as another tissue or organ type.
  • prostatectomy refers to the partial (“simple prostatectomy”) or complete removal (“radical prostatectomy”) of the prostate gland. It can be performed to treat prostate cancer or benign prostatic hyperplasia. Simple prostatectomy is usually carried out for benign conditions, whereas radical prostatectomy is performed for cancer. 27 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT [00220]
  • radiation therapy refers to treatment that uses ionizing radiation to kill prostate cancer cells or keep them from growing and dividing, while minimizing damage to healthy cells.
  • Radiopharmaceuticals can also be referred to as X-ray therapy.
  • radiation therapy can also be referred to as X-ray therapy.
  • radiopharmaceuticals comprise an isotope such as strontium-89, samarium-153, and radium- 223.
  • isotope such as strontium-89, samarium-153, and radium- 223.
  • hormone therapy refers to a type of treatment that slows or stops the growth of cancer that uses hormones to grow. It can also be referred to as hormonal therapy, hormone treatment, or endocrine therapy.
  • Hormone therapy falls into two broad groups: those that block the body’s ability to produce hormones and those that interfere with how hormones behave in the body. Hormone therapy can be used either alone or in conjunction with other prostate cancer treatments. Hormone therapy can be administered continuously after diagnosis of prostate cancer. Hormone therapy can be used intermittently after diagnosis of prostate cancer. Examples of hormone therapy can include, but are not limited to orchiectomy, luteinizing hormone-releasing hormone (LHRH) agonists, LHRH antagonists, abiraterone, ketoconazole, antiandrogens, and estrogens.
  • the LHRH agonists comprise of leuprolide, goserelin, triptorelin, and leuprolide mesylate.
  • LHRH antagonists comprise of degarelix and relugolix.
  • antiandrogens can include flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, and darolutamide.
  • the subject has or is at risk of developing a disease or condition associated with precancerous prostate lesions and/or prostate cancer, of various associated tissues of the prostate, e.g., prostate tissue and/or associated lymph nodes.
  • described herein is a method of differentially diagnosing presence and stage of prostate cancer, or preventing unnecessary intervention, or treatment, of a disease or condition characterized by precancerous lesions, or prostate cancer, in a subject, with the method including isolation, examination, or characterization of platelet-sequestered proteins.
  • the diagnosis and staging of hyperplasia and tumor lesions is determined by AJCC (American Joint Committee on Cancer) TNM system including but not limited to; clinical, or pathologic tumor category, such as, e.g., (cT1-4,a-c, or pT1-4,a-c,), the N category of spreading to lymph nodes, such as, e.g., (N0, or N1), the M category of metastasis, such as, e.g., (M0, or M1), and any combination of the above categories.
  • the subject has or is at risk for irreversible morbidity associated with prostate cancer, e.g., erectile dysfunction, or incontinence.
  • Platelets are small, irregularly shaped clear cell structures (meaning “cells” that do not have a nucleus), that are 4-7 ⁇ m in diameter, shed from pseudopodial extensions of precursor bone marrow cells termed “megakaryocytes.” In a healthy individual, platelet counts range from 150,000 to 450,000, platelets per microliter of blood; thus, an adult with 5 liters of blood has a total of 750000000, to 2250000000 platelets. Thrombocytosis is having greater than 450,000 platelets per uL of blood while 28 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT thrombocytopenia is less than 150,000 platelets per uL.
  • platelets The main function of platelets is the maintenance of hemostasis, but they also store and deliver many proteins necessary for tissue repair and regeneration.
  • the classical role of platelets is in thrombus formation when damage to the endothelium, such as a laceration leads to damage of blood vessels and release of blood. Conversely, improper thrombus formation leads to morbidity, such as in acute coronary thrombosis.
  • the ability of platelets to store and transport proteins is utilized in cancer for storage and transport of cancer regulating growth factors.
  • angiogenesis regulators are sequestered in platelets actively, and against the concentration gradient (Cervi D et al.2008, “Platelet-associated PF-4 as a biomarker of early tumor growth.” Blood.111: 1201-1207; Klement GL et al. 2009 “Platelets Actively Sequester Angiogenesis Regulators.” Blood. 113(12):2835-42, which are incorporated by reference herein in their entireties.). Furthermore, angiogenesis regulator sequestration is selective (Klement GL et al.2009). [00224] These observations indicate that platelets are “first responders” to injury and sequester proteins in a dynamic manner that permits the diagnosis of disease.
  • Platelets carry a highly responsive and continuously changing ‘cargo’ of protein stimulators and inhibitors of e.g., cancer or neoplasia.
  • platelets can carry stimulators and inhibitors of angiogenesis that play a role in new blood vessel development and tissue reconstruction (e.g., VEGF, bFGF, and PDGF).
  • tissue reconstruction advances, the amount and type of stimulators and inhibitors in the platelet cargo changes in a predictable manner.
  • the constantly changing platelet protein landscape permits early cancer diagnosis, detection of tissue response to tumor progression and invasion, detection of metastatic growth, and monitoring of response to therapeutic agents. Proteins sequestered in platelets can permit monitoring of an entire cancer-relevant protein landscape including known polypeptides.
  • platelet-sequestered proteins can be used to group platelet-associated proteins by their functions and impacts (e.g. angiogenesis modulating, inflammatory, growth promoting, etc.) and can show changes in the platelet protein landscape relevant to disease stage and progression.
  • the methods and compositions described herein allow for real time evaluation of the efficacy and patient response to therapy guiding patient treatment and clinical trial enrollment and design.
  • Prior studies indicate that cancer-relevant growth factors are actively sequestered in platelets against a concentration gradient in plasma and is more robust and more specific in regard to cancer status than protein concentrations found in plasma or serum.
  • the data described herein in the working examples demonstrate that sequestration of cancer-relevant proteins occurs early in malignant progression, but unambiguously reflects the cancer stage and response to therapy.
  • platelet rich plasma is separated 29 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT into a fresh tube and platelet concentrate obtained by centrifuging platelets at higher speed. The platelet concentrate is then resuspended in a standard lysis buffer and associated proteins are isolated. [00227] In some embodiments, isolation of the sample is non-invasive. Routine blood draw by venipuncture is considered “non-invasive” as that term is used herein.
  • isolation of platelets is by routine phlebotomy techniques (e.g., venipuncture) utilizing collection tubes containing anti- coagulants, such as, but not limited to, e.g., 3.2% Sodium citrate, Sodium or lithium heparin without gel, Potassium EDTA, Sodium fluoride, and sodium or potassium oxalate or a combination thereof, in various concentrations of the above-mentioned compounds.
  • anti- coagulants such as, but not limited to, e.g., 3.2% Sodium citrate, Sodium or lithium heparin without gel, Potassium EDTA, Sodium fluoride, and sodium or potassium oxalate or a combination thereof, in various concentrations of the above-mentioned compounds.
  • anti- coagulants such as, but not limited to, e.g., 3.2% Sodium citrate, Sodium or lithium heparin without gel, Potassium EDTA, Sodium fluoride, and sodium or potassium oxa
  • normalization of platelet-associated proteins is by the number of platelets, e.g., counts obtained by manual counting, or flow cytometry. In some embodiments, normalization of platelet-associated proteins is by volume, e.g., platelet volume or Mean platelet volume (MPV). In some embodiments, normalization of platelet-associated proteins is by an endogenous protein (e.g., tubulin or actin, the skeletal proteins of platelets), exogenous protein (e.g., a labeled heavy peptide control for quantification of a specific protein), or compound sequestered by platelets, e.g., loading with an exogenous derivative, or modification, of a platelet sequestered protein.
  • endogenous protein e.g., tubulin or actin, the skeletal proteins of platelets
  • exogenous protein e.g., a labeled heavy peptide control for quantification of a specific protein
  • compound sequestered by platelets e.g., loading with an exogenous derivative,
  • Proteins sequestered by platelets can provide dynamic information regarding the status of a disease or disorder, such as cancer.
  • Platelet-sequestered proteins provide a ubiquitous, constantly circulating, physiologically dynamic system that monitors disease development and biological events involving genetic changes not only in cancer cells, but also in the subject’s physiology. That is, such proteins can bind to and be released by platelets in a dynamic manner reflective of their expression levels and the subject’s disease status. For example, as cancer progresses, proteins sequestered by platelets will be indicative of the cancerous state, including an increase in the amount of cancer stimulators and/or a decrease in the amount of cancer inhibitors.
  • Polypeptides sequestered by platelets can be representative of any function known to be associated with cancer development or progression including, but not limited to, proliferation, evasion of growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion, malignant cell transformation, angiogenesis and/or metastasis. Isomers or cleavage/processing products or intermediates of polypeptides can also be detected. [00229] Table 1: Exemplary panel of polypeptides for cancer diagnosis and staging Gene Name Gene Symbol NCBI Gene Ascension No.
  • This protein includes a natural chemoattractant peptide of 21 amino acids at the N-terminus, which is a natural ligand for formyl peptide receptor-like receptor 2 (FPRL2) and promotes calcium mobilization and chemotaxis in monocytes and dendritic cells.
  • FPRL2 formyl peptide receptor-like receptor 2
  • Related pathways are peptide ligand-binding receptors and signaling by GPCR.
  • CDK16 Cyclin-dependent kinase 16: The protein encoded by this gene belongs to the cdc2/cdkx subfamily of the ser/thr family of protein kinases.
  • GPLD1 Glycosylphosphatidylinositol Specific Phospholipase D1; Phosphatidylinositol-glycan- specific phospholipase: Many proteins are tethered to the extracellular face of eukaryotic plasma membranes by a glycosylphosphatidylinositol (GPI) anchor.
  • GPI glycosylphosphatidylinositol
  • PLTP Phospholipid transfer protein
  • CAVIN2 (Caveolae-associated protein 2): This gene encodes a calcium-independent phospholipid-binding protein. Its low expression is associated with poor prognosis is renal cancer and with high survival in stomach cancer. It is a suppressive regulator of TNF-induced Mesenchymal Stromal Cell Inflammation and Angiogenic Phenotypes. This protein is a substrate for protein kinase C (PKC) phosphorylation and recruits polymerase I and transcript release factor (PTRF) to caveolae. Removal of this protein causes caveolae loss and its over-expression results in caveolae deformation and membrane tubulation.
  • PKC protein kinase C
  • PTRF polymerase I and transcript release factor
  • CAVIN2 plays a role in caveolar biogenesis and morphology by inducing membrane curvature within caveolae (see e.g., Hansen et al. Nat Cell Biol. 2009, 11(7): 807-814). CAVIN2 plays a role in caveola formation in a tissue-specific manner.
  • PPBP Platinum basic protein
  • LA-PF4 stimulates DNA synthesis, mitosis, glycolysis, intracellular cAMP accumulation, prostaglandin E2 secretion, and synthesis of hyaluronic acid and sulfated glycosaminoglycan. It also stimulates the formation and secretion of plasminogen activator by human synovial cells.
  • NAP-2 is a ligand for CXCR1 and CXCR2, and NAP-2, NAP-2(73), NAP-2(74), NAP-2(1-66), and most potent NAP-2(1- 63) are chemoattractants and activators for neutrophils.
  • TC-1 and TC-2 are antibacterial proteins, in vitro released from activated platelet alpha-granules.
  • CTAP-III(1-81) is more potent than CTAP-III desensitize chemokine-induced neutrophil activation.
  • CTTN Strc substrate cortactin
  • This gene has two known roles: (1) regulating the interactions between components of adherens-type junctions and (2) organizing the cytoskeleton and cell adhesion structures of epithelia and carcinoma cells.
  • the encoded protein is degraded in a caspase-dependent manner.
  • the aberrant regulation of this gene contributes to tumor cell invasion and metastasis.
  • Related pathways are cell motility, negative regulation of extrinsic apoptotic signaling pathway, and receptor-mediated endocytosis.
  • HSPB1 Heat Shock Protein Family B (Small) Member 1: Expression of this gene is correlated with poor clinical outcome in multiple human cancers, and the encoded protein promotes cancer cell proliferation and metastasis, while protecting cancer cells from apoptosis. Mutations in this gene have been identified in human patients with Charcot-Marie-Tooth disease and distal hereditary motor neuropathy.
  • RAB4A Ras-related protein Rab-4A: This gene is a member of the largest group in the Ras superfamily of small GTPases, which regulate membrane trafficking.
  • RAB4A is a small GTPase which cycles between an active GTP-bound and an inactive GDP-bound state (see e.g., Huber et al. FEBS Lett. 2005, 579(13): 2821-2829; Eathiraj et al. Nature. 2005, 436(7049): 415-419).
  • RAB4A is involved in protein transport.
  • RAB4A plays a role in vesicular traffic.
  • RAB4A mediates VEGFR2 endosomal trafficking to enhance VEGFR2 signaling (see e.g., Kofler et al.
  • PPHLN1 Periphilin 1
  • HUSH complex Component of the HUSH complex, a multiprotein complex that mediates epigenetic repression.
  • the HUSH complex is recruited to genomic loci rich in H3K9me3 and involved in maintaining transcriptional silencing by promoting recruitment of SETDB1, a histone methyltransferase that mediates further deposition of H3K9me3.
  • the HUSH complex is also involved in the silencing of unintegrated retroviral DNA: some part of the retroviral DNA formed immediately after infection remains unintegrated in the host genome and is transcriptionally repressed (see e.g., Zhu et al. Nature.2018, 564(7735): 278-282).
  • PPHLN1 can be involved in epithelial differentiation by contributing to epidermal integrity and barrier formation (see e.g., Kazerounian et al. J Biol Chem. 2003, 278(38): 36707-36717).
  • MUC2 Moc 2
  • Mucins are high molecular weight glycoproteins produced by many epithelial tissues.
  • the protein encoded by this gene is secreted and forms an insoluble mucous barrier that protects the lumen.
  • the protein polymerizes into a gel of which 80% is composed of oligosaccharide side chains by weight.
  • the protein features a central domain containing tandem repeats rich in threonine and proline that varies between 50 and 115 copies in different individuals. Downregulation of this gene has been observed in patients with Crohn disease and ulcerative colitis.
  • MUC2 has a role in the mucinous differentiation of prostate, is associated with more indolent growth.
  • SCAF8 SR-Related CTD Associated Factor 8 prevents early mRNA termination during transcription.
  • SCAF8 Childhood Ependymoma. Together with SCAF4, SCAF8 acts by suppressing the use of early, alternative poly(A) sites, thereby preventing the accumulation 35 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT of non-functional truncated proteins. Mechanistically, SCAF8 associates with the phosphorylated C- terminal heptapeptide repeat domain (CTD) of the largest RNA polymerase II subunit (POLR2A), and subsequently binds nascent RNA upstream of early polyadenylation sites to prevent premature mRNA transcript cleavage and polyadenylation.
  • CCD C- terminal heptapeptide repeat domain
  • CDC73 Cell Division Cycle 73; Parafibromin: a product of the HPRT2 tumor suppressor gene involved in transcriptional and post-transcriptional control. It is involved in cell cycle progression through regulation of cyclin D1/PRAD1 expression. CDC73 is a component of the PAF1 complex (PAF1C), which has multiple functions during transcription by RNA polymerase II and is implicated in regulation of development and maintenance of embryonic stem cell pluripotency.
  • PAF1C associates with RNA polymerase II through interaction with POLR2A CTD non-phosphorylated and 'Ser-2'- and 'Ser-5'- phosphorylated forms and is involved in transcriptional elongation, acting both independently and synergistically with TCEA1 and in cooperation with the DSIF complex and HTATSF1. PAF1C is required for transcription of Hox and Wnt target genes.
  • PAF1C is involved in hematopoiesis and stimulates transcriptional activity of KMT2A/MLL1; it promotes leukemogenesis through association with KMT2A/MLL1-rearranged oncoproteins, such as KMT2A/MLL1-MLLT3/AF9 and KMT2A/MLL1- MLLT1/ENL.
  • PAF1C is involved in histone modifications such as ubiquitination of histone H2B and methylation on histone H3 'Lys-4' (H3K4me3).
  • PAF1C recruits the RNF20/40 E3 ubiquitin-protein ligase complex and the E2 enzyme UBE2A or UBE2B to chromatin which mediate monoubiquitination of 'Lys- 120' of histone H2B (H2BK120ub1); UB2A/B-mediated H2B ubiquitination is proposed to be coupled to transcription.
  • PAF1C is involved in mRNA 3' end formation, e.g., through association with cleavage and poly(A) factors. In case of infection by influenza A strain H3N2, PAF1C associates with viral NS1 protein, thereby regulating gene transcription.
  • LARP1 (La-related protein 1): promotes cancer cell invasion in prostate cancer (see e.g., Kato et al. Int J Oncol. 2015, 47(2):710-718). LARP1 permits eukaryotic initiation factor 4E binding activity; nucleic acid binding activity; and ribosomal small subunit binding activity.
  • LARP1 is involved in several processes, including TORC1 signaling; cellular response to rapamycin; and posttranscriptional regulation of gene expression (see e.g., Burrows et al. Nucleic Acids Res.2010, 38(16):5542-5553; Aoki et al. FEBS Lett.2013, 587(14): 2173-2178; Tcherkezian et al. Genes Dev.2014, 28(4):357-371; Fonseca et al. J Biol Chem. 2015, 290(26): 15996-16020; Hong et al. Elife. 2017, 6:e25237; Gentilella et al. Mol Cell.
  • LARP1 is located in cytoplasmic stress granule. LARP1 colocalizes with TORC1 complex and polysomal ribosome.
  • GRDN Zirdin, CCDC88A, Coiled-Coil Domain Containing 88A is a protein coding gene. It is an actin-binding protein that is activated by the serine/threonine kinase Akt and plays a role in cytoskeleton remodeling and cell migration.
  • the encoded protein also enhances Akt signaling by mediating 36 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT phosphoinositide 3-kinase (PI3K)-dependent activation of Akt by growth factor receptor tyrosine kinases and G protein-coupled receptors. Increased expression of this gene and phosphorylation of the encoded protein can play a role in cancer metastasis. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene.
  • PI3K phosphoinositide 3-kinase
  • SMU1 (WD40 repeat-containing protein SMU1): Involved in pre-mRNA splicing as a component of the spliceosome (see e.g., Bertram et al. Cell.2017, 170(4):701-713.e11). SMU1 regulates alternative splicing of the HSPG2 pre-mRNA (e.g., by similarity). SMU1 is required for normal accumulation of IK (see e.g., Fournier et al. PLoS Pathog.2014, 10(6):e1004164).
  • SMU1 is required for normal mitotic spindle assembly and normal progress through mitosis (e.g., by similarity; SMU1_HUMAN,Q2TAY7, Microbial infection). SMU1 is required, together with IK, for normal splicing of influenza A virus NS1 pre-mRNA, which is required for the production of the exportin NS2 and for the production of influenza A virus particles. SMU1 is not required for the production of VSV virus particles. High expression of this protein is associated with poor survival in prostate cancer. [00246] An assay that captures or analyzes proteins that are prostate-specific can be useful in establishing that a cancer is prostate cancer.
  • polypeptides representative of function in proliferation of normal cells to hyperplasia are detected in platelets and include one or more of HEPB1, CDK16, CPLD1, and PLTP. In one embodiment, such polypeptides representative of function in proliferation of normal cells to hyperplasia can be used to detect and/or stage prostate cancer.
  • the method can be used to diagnose prostate cancer, and the polypeptides representative of function in angiogenesis include one or more of CAVIN2, PPBP, CTTN, and HSPB1.
  • the method can be used to diagnose prostate cancer, and the polypeptides representative of function in cancer cell invasion (e.g., local tissue cancer invasion) include one or more of RAB4A, PPHLN1, MUC2, and SCAF8.
  • the method can be used to diagnose prostate cancer, and the polypeptides representative of function in metastasis include one or more of CDC73, LARP1, Girdin, and SMU1.
  • the method comprises detection of at least one polypeptide (e.g., at least 2, or all 3 polypeptides) from a set of polypeptides representative of function in angiogenesis in combination with at least one polypeptide (e.g. at least 2, 3, 4, 5 or 6) from at least one functional group consisting of: polypeptides representative of function in malignant cell transformation, cancer cell invasion (e.g., local tissue cancer invasion), and/or metastasis.
  • the method comprises detection of at least 3 proteins selected from the group consisting of: HEBP1, CDK16, GPLD1, PLTP, CAVIN2, PPBP, CTTN, HSPB1, RAB4A, PPHLN1, MUC2, SCAF8, CDC73, LARP1, Girdin, and SMU1.
  • the method comprises detection of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or all 16 proteins selected from the group consisting of: HEBP1, CDK16, GPLD1, PLTP, CAVIN2, PPBP, CTTN, HSPB1, RAB4A, PPHLN1, MUC2, SCAF8, CDC73, LARP1, Girdin, and SMU1.
  • the method comprises detection of proteins consisting essentially of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or all 16 proteins from the group consisting of: HEBP1, CDK16, GPLD1, PLTP, CAVIN2, PPBP, CTTN, HSPB1, RAB4A, PPHLN1, MUC2, SCAF8, CDC73, LARP1, Girdin, and SMU1.
  • proteins consisting essentially of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or all 16 proteins from the group consisting of: HEBP1, CDK16, GPLD1, PLTP, CAVIN2, PPBP, CTTN, HSPB1, RAB4A, PPHLN1, MUC2, SCAF8, CDC73, LARP1, Girdin, and SMU1.
  • the polypeptides detected in platelets consist of: HEBP1, CDK16, GPLD1, PLTP, CAVIN2, PPBP, CTTN, HSPB1, RAB4A, PPHLN1, MUC2, SCAF8, CDC73, LARP1, Girdin, and SMU1.
  • Table 2 Peptide sequences of proteins selected for mass spectrometry P urpose Protein Peptide Sequence 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT GPLSSAPEIVHEDLK (SEQ ID NO: 4) NP_001163931.1 (SEQ ID NO: 57) platelet AEALLSHAPR (SEQ ID NO: 5) N r ker CDK P_006192.1 (SEQ ID NO: 58) m a 16 LDSDGADLLTK (SEQ ID NO: 6) NP 148978.2 (SEQ ID NO: 59) 2 ) 3 ) 4) 5) 6 ) 7 ) 8 ) 5) 6) 7) 5) 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT beta' VLPTIPK (SEQ ID NO: 237) isoform 2) LPEAAFLAR (SEQ ID NO: 238) FLEQELETITIPDLR (SEQ ID NO: 30)
  • Examining platelet-sequestered proteins in the context of activation and/or repression of biological function pathways provides a context as to whether the protein(s) being studied are related to the pathogenesis of prostate cancer. That is, such proteins can bind to and be released by platelets in a dynamic manner reflective of the biological functional pathways that are being activated and/or repressed.
  • Representative biological functional pathways being activated can include, but are not limited to, any function known to be associated with cancer development or progression including, but not limited to, proliferation, evasion of growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion, malignant cell transformation, angiogenesis and/or metastasis.
  • Representative biological functional pathways that can be affected in prostate cancer pathogenesis can include, but are not limited to, any function known to be associated with cancer cell restriction, including, but not limited to, nuclear factor kappa B (NF- ⁇ B) pathway, Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway, toll-like receptor (TLR) pathway, cGAS-STING pathway, mitogen-activated protein kinase (MAPK) pathway, vascular endothelial growth factor (VEGF) signaling pathway, transforming growth factor (TGF)- ⁇ pathway, and phosphatidylinositol (3,4,5)-triphosphate (PI3K)/AKT pathway.
  • NF- ⁇ B nuclear factor kappa B
  • JAKT Janus kinase/signal transducers and activators of transcription
  • TLR toll-like receptor
  • cGAS-STING pathway cGAS-STING pathway
  • MAPK mitogen-activated protein
  • Methods for evaluating physical protein-protein interactions include, but are not limited to yeast two-hybrid analyses, protein microarrays, mass spectrometry, immunoprecipitation, dual polarization interferometry (DPI), and tandem affinity purification. These physical assays have served to create academic databases of these networks of protein-protein interactions, such as, Reactome.
  • the assays, systems, and methods described herein can comprise a lateral flow immunoassay (LFIA), also known as the immunochromatographic assay, or strip test to measure or determine the level of HEBP1, CDK16, GPLD1, PLTP, RAB4A, PPHL1, MUC2, SCAF8, CAVIN2, PPBP, CTTN, and/or HSPB1 polypeptide in a sample.
  • LFIA lateral flow immunoassay
  • LFIAs provide a simple device intended to detect the presence (or absence) of HEBP1, CDK16, GPLD1, PLTP, RAB4A, PPHL1, MUC2, SCAF8, CAVIN2, PPBP, CTTN, and/or HSPB1 in a sample.
  • LFIA tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action.
  • LFIAs are essentially immunoassays adapted to operate along a single axis to suit the test strip format or a dipstick format.
  • Strip tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of antigens from fluid samples such as urine, blood, water samples etc. Strip tests are also known as dip stick test, the name bearing from the literal action of “dipping” the test strip into a fluid sample to be tested.
  • LFIA strip test are easy to use, require minimum training and can easily be included as components of point-of-care test (POCT) diagnostics to be used on site in the field. LFIA tests can be operated as either competitive or sandwich assays. Sandwich LFIAs are similar to sandwich ELISA.
  • test results area comprising a reaction membrane - typically a hydrophobic nitrocellulose or cellulose acetate membrane onto which antibody 43 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT reagents are immobilized in a line across the membrane as a capture zone or test line (a control zone may also be present, containing antibodies specific for the antibody reagents conjugated to the particles or microspheres); and (4) optional wick or waste reservoir - a further absorbent pad designed to draw the sample across the reaction membrane by capillary action and collect it.
  • the components of the strip are usually fixed to an inert backing material and may be presented in a simple dipstick format or within a plastic casing with a sample port and reaction window showing the capture and control zones. While not strictly necessary, most tests will incorporate a second line which contains an antibody that picks up free latex/gold in order to confirm the test has operated correctly.
  • the use of “dip sticks” or LFIA test strips and other solid supports has been described in the art in the context of an immunoassay for a number of antigens. U.S. Pat. Nos.4,943,522; 6,485,982; 6,187,598; 5,770,460; 5,622,871; 6,565,808, U. S. patent applications Ser.
  • the apparatuses and methods of these three patents broadly describe a first component fixed to a solid surface on a “dip stick” which is exposed to a solution containing a soluble antigen that binds to the component fixed upon the “dip stick,” prior to detection of the component-antigen complex upon the stick. It is within the skill of one in the art to modify the teaching of these “dip stick” technologies as necessary for the detection of HEBP1, CDK16, GPLD1, PLTP, RAB4A, PPHL1, MUC2, SCAF8, CAVIN2, PPBP, CTTN, and/or HSPB1 polypeptides.
  • any method for detecting a plurality (e.g., at least 2) of polypeptides sequestered by platelets and associated with one or more cancer functions as described herein can be used with the methods described herein. While microfluidic devices, lateral flow assays or test strips and mass spectrometry can each be used to detect the presence or amount of platelet proteins associated with cancer functions, isolation and detection of such platelet-sequestered proteins by other means is also specifically contemplated herein.
  • High-resolution mass spectrometry is a mass spectrometry technique for trace level analysis. HRMS can achieve mass measurement accuracy of 0.001 Da and detect trace quantities (mDa) of elements.
  • the underlying mass spectrometry principle involves ionization of analyte compounds in vapor phase, accelerating the ionized fragments in an electric field and separating them by mass analyzer, based on the mass to charge ratios (m/z). The separated ions are detected using electron multiplier detectors. The intensities of the detected ions, when plotted against mass to charge ratios, constitute the characteristic mass spectra.
  • the mass spectrometry is selected from the group including matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and Orbitrap with tandem mass spectrometry.
  • MALDI-TOF matrix-assisted laser desorption/ionization-time of flight
  • Orbitrap with tandem mass spectrometry One who is skilled in the art will be able to prepare samples and operate the HRMS in order to identify and classify patient samples.
  • stable isotopic labeled peptides also known as heavy peptides, are chemically synthesized peptides with the native sequence, but some of the constituent amino acids are replaced by stable isotope-labeled amino acids, such as amino acids in which 12 C is replaced by 13 C, 14 N by 15 N, and 1 H by 2 H.
  • Stable isotope-labeled peptides are chemically and physically indistinguishable from their endogenous counterparts with respect to retention time, ionization efficiency and fragmentation mechanism. They only differ by mass. These properties make the stable isotope-labeled peptides widely applicable in quantification analysis (e.g. ICAT, iTRAQ and AQUA), nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) and as reference materials for pharmacokinetic analyses and metabolite identification. [00269] HRMS can also utilize at least one radioactive isotope. Radioactive labeling in mass spectrometry is used for quantification of compounds, especially those that are not readily quantified by reliable extraction methods.
  • the radioactive isotope is conjugated to the sequence of at least one of IPNQFQSDPPAPSDK (SEQ ID NO: 1), EADYVAQATR (SEQ ID NO: 2), AALEGTATYR (SEQ ID NO: 3), GPLSSAPEIVHEDLK (SEQ ID NO: 4), AEALLSHAPR (SEQ ID NO: 5), LDSDGADLLTK (SEQ ID NO: 6), LPDTTSIFALK (SEQ ID NO: 7), ALEFLQLHNGR (SEQ ID NO: 8), DLLGIYEK (SEQ ID NO: 9), LYPTYSTK (SEQ ID NO: 10), ILEGFQPSGR (SEQ ID NO: 11), TLLLVGSPTWK (SEQ ID NO: 12), QISLEGSVK (SEQ ID NO: 13), GIQNDLTK (SEQ ID NO: 14), YQASTSNTVSK (SEQ ID NO: 15), YEGSYALTSEEAER (SEQ ID NO: 15
  • multiple reaction monitoring is a method used in tandem mass spectrometry in which an ion of a particular mass is selected in the first stage of a tandem mass spectrometer and an ion product of a fragmentation reaction of the precursor ions is selected in the second mass spectrometer stage for detection.
  • SRM has been developed to be a highly sensitive and reproducible mass spectrometry-based protein targeted detection platform (entitled "SAFE-SRM"), and it has been demonstrated that the SRM-based new pipeline has advantages in clinical proteomics applications over traditional SRM pipelines, 46 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT Capillary action lateral flow test strips etc.
  • SAFE-SRM mass spectrometry-based protein targeted detection platform
  • the testing device will comprise a sample receiving zone to which a platelet or blood sample from a subject is added, and (ii) a conjugate zone comprising a plurality of labelled binding reagents, each of which specifically binds to one of the polypeptides selected from HEBP1, CDK16, GPLD1, PLTP, CAVIN2, PPBP, CTTN, HSPB1, RAB4A, PPHLN1, MUC2, SCAF8, CDC73, LARP1, Girdin, and SMU1.
  • a conjugate zone comprising a plurality of labelled binding reagents, each of which specifically binds to one of the polypeptides selected from HEBP1, CDK16, GPLD1, PLTP, CAVIN2, PPBP, CTTN, HSPB1, RAB4A, PPHLN1, MUC2, SCAF8, CDC73, LARP1, Girdin, and SMU1.
  • a testing device can comprise a conjugate zone comprising a plurality of labelled binding reagents, each of which specifically binds to one of the polypeptides HEBP1, CDK16, GPLD1, PLTP, RAB4A, PPHL1, MUC2, SCAF8, CAVIN2, PPBP, CTTN, and/or HSPB1.
  • test lines for each polypeptide are spatially separated to permit levels of each polypeptide to be measured and discriminated from levels of the other polypeptide(s).
  • the boundary of the sample receiving zone can be marked for the user's convenience; for instance, using one or more symbols such as arrows. The user should dip the sample receiving zone portion of the strip into the sample up to the one or more symbols. This ensures that the sample receiving zone is sufficiently brought into contact with the sample to be tested but that the downstream components (e.g. test lines) are not.
  • the solid support comprises a chromatographic medium or a capillary flow device.
  • the device can be provided in a test strip format in some embodiments.
  • a region is provided downstream of the test lines (and control line and/or absorbent material if present) which can be held by hand by the user. Thus, the user can easily manipulate the testing device without compromising the sample and subsequent testing thereof.
  • the region 47 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT can be called a “hold region” and can be made of any suitable material, such as plastic.
  • the region can be visibly marked “hold region” or simply “hold” or similar for the user's convenience.
  • the testing device comprises a dipstick or test strip with a plurality of testing zones, wherein each testing zone can detect a given polypeptide.
  • detecting a color change in the dipstick or test strip can indicate the measurement of specific analytes or polypeptides in each test zone of the panel.
  • Each test zone can change the amount of colored light reflected from one of the components of the dipstick or can change color based on a chemical reaction. For a negative result (i.e. the presence of a given polypeptide is not detected), the strip can remain its original color, or it can change to a specific color. For a positive result (i.e.
  • the amount of each polypeptide present can be determined to provide further information as to the health of the subject.
  • lower or higher levels of polypeptides, and not just their presence can be relevant to e.g., the stage of cancer.
  • a quantification device is included in the panel itself and is not a separate device.
  • the quantification device can include or be coupled to a computer with software that is capable of performing analysis using the data thus obtained with an analyzing mechanism.
  • the analyzing mechanism can compute values of each of the polypeptides in the tests, perform normalization, as well as compute relationships of the test results with each other, the test results can be calculated to determine the presence and/or stage of a cancer.
  • the results can be incorporated into a report on an individual's wellness or cancer risk assessment that includes, but is not limited to, the results of the tests, comparison to the values and ratios computed to normal ranges that have previously been established for normal healthy men and women of different ages, ethnicities (if relevant) and/or other relevant parameters.
  • a report can also incorporate historical data for an individual subject that was obtained using the same method(s), which can be used to monitor effectiveness of treatment.
  • a desired panel of platelet-sequestered polypeptides to be tested can including the use of a single, easy-to-use, and disposable test strip that comprises multiple test zones and remove the need for individual assays for each of the various polypeptides discussed herein.
  • a desired panel of platelet-sequestered polypeptides to be tested can including the use of a single, easy-to-use, and disposable test strip that comprises multiple test zones and remove the need for individual assays for each of the various polypeptides discussed herein.
  • [00283] Furthermore, with the integration of all of these tests onto a single platform, it is possible to aggregate the data from all results and to compile it in a complementary way so that the data from individual test zone enhances the interpretation of other test zones on the strip.
  • the presence or amount of a single platelet-sequestered polypeptide as described herein may or may not be indicative of cancer presence or stage when viewed alone, but when combined with information detailing the amount or presence of multiple other platelet-sequestered proteins, it is now possible to identify the subject as having cancer or a particular stage of cancer.
  • the testing device can further comprise a visual aid such as a printed document (e.g. a printed card) displaying different line intensity patterns from which the user (e.g., clinician) can interpret the results of the completed assay(s).
  • the lines can be graded (Grade lines 1-10) wherein Grade line 1 is the lightest colored line followed by Grade line 2 which is more intense in color and so on to Grade line 10 which is the darkest (i.e. most intensely colored) line; Grade lines 1 and 2 being calibrated at or below a pre-determined threshold level and indicating that the specific polypeptide is present but within normal parameters and therefore a cancer or function thereof is not detected whilst Grade lines 3-10, calibrated above the pre-determined threshold level, indicate that the specific polypeptide is present in abnormally high levels and therefore a cancer is detected and/or can be staged.
  • Grade lines 1 is the lightest colored line followed by Grade line 2 which is more intense in color and so on to Grade line 10 which is the darkest (i.e. most intensely colored) line
  • Grade lines 1 and 2 being calibrated at or below a pre-determined threshold level and indicating that the specific polypeptide is present but within normal parameters and therefore a cancer or function thereof is not detected whilst Grade lines 3-10, calibrated above the pre-
  • Grade lines 3-10 enables the user, particularly when analyzing multiple samples taken over time using the monitoring methods described herein, to understand whether the polypeptide levels are continuing to abnormally increase and therefore whether the cancer is progressing and/or current treatment is ineffective.
  • a null grade line (Grade line 0) can also be provided for which no colored line is displayed on the visual aid indicating that the polypeptide is absent (or present at negligible levels) from the platelet or blood sample.
  • detection of a given polypeptide or panel thereof is by way of enzymatic activity, which can be measured directly in the platelet or platelet-protein sample or using a lateral flow assay or liquid bioassay.
  • Enzymatic activity can be measured for example by detecting processing of a substrate, which can be labelled, in the sample.
  • the assay can be a fluorogenic substrate assay.
  • suitable assay formats include the assays set forth in International Patent Applications WO2009/024805, WO2009/063208, WO2007/128980, WO2007/096642, WO2007/096637, WO2013/156794, WO2015/059487 and WO2013/156795 (the content of each of which is hereby incorporated by reference). 49 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT [00286]
  • the determined levels of the polypeptides are compared with a corresponding threshold level.
  • Threshold levels of a given polypeptide can be defined from population studies or be specific to the individual (i.e. personalized, or from a prior cancer risk or staging assessment). Personalized levels can be more relevant to monitoring applications, although monitoring is preferably also achieved by comparison with pre-determined threshold levels. Threshold levels can be set with reference to a training data set comprising samples defined in relation to a particular cancer status or stage. Thus, threshold levels do not need to be measured each time an assay according to the methods as performed. They can be pre- programmed into a reader device or provided for comparative purposes when performing the such methods.
  • the earlier time points can comprise at least two, and possibly 3, 4, 5, 6, 7, 8, 9, 10 etc., earlier measurements immediately preceding the determination of the level of the polypeptide in the current sample.
  • Isolation and Detection of proteins sequestered by platelets [00289] If desired, proteins can be isolated from platelets as described in “Current Protocols in Immunology by F. M. Ausubel, R. Brent, R. E. Scientific, D. D. Moore, J. G. Seidman, K. Struhl and V. B. Chanda (Editors), John Wiley & Sons, 2004.”, incorporated herein by reference.
  • the procedure generally involves the extraction of proteins in one solubilizing step.
  • the results of this procedure are intact proteins, substantially free of cross-contamination.
  • the isolated proteins maintain activity, allowing analysis through any number of assays. 50 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT [00290]
  • the buffers for the protein isolation step can include one or more of buffer components, salt (s), detergents, protease inhibitors, and phosphatase inhibitors.
  • one effective buffer for extracting proteins to be analyzed by immunohistochemistry includes the buffer Tris-HCl, NaCl, the detergents Nonidet (g) P-40, EDTA, and sodium pyrophosphate, the protease inhibitors aprotinin and leupeptin, and the phosphatase inhibitors sodium deoxycholate, sodium orthovanadate, and 4-(2- aminoethyl)benzenesulfonyl fluoride (AEBSF).
  • Another salt that could be used is LiCl, while glycerol is a suitable emulsifying agent that can be added to the fraction buffer.
  • Additional optional protease inhibitors include soybean trypsin inhibitor and pepstatin.
  • Suitable phosphatase inhibitors include phenylmethylsulfonyl fluoride, sodium molybdate, sodium fluoride, and beta-glycerol phosphate.
  • phenylmethylsulfonyl fluoride sodium molybdate, sodium fluoride, and beta-glycerol phosphate.
  • SELDI® simple lysis with a 1% SDS solution is effective, while ultimate analysis using the SELDI® process requires Triton-X-100, a non-ionic detergent (Sigma, St. Louis, Mo.), MEGA109 (ICN, Aurora, Ohio), and octyl B-glucopyranoside (ESA, Chelmsford, Mass.) in a standard PBS base.
  • the proteins sequestered by platelets can be used to predict, indicate, diagnose, or monitor progression or regression of cancer and can be measured using any process known to those of skill in the art including, but not limited to, enzyme linked immunosorbent assay (ELISA), fluorescence polarization immunoassay (FPIA) and homogeneous immunoassays, point of care tests using conventional lateral flow immunochromatography (LFA), quantitative point of care tests using determination of chemiluminescence, fluorescence, and magnetic particles, as well as latex agglutination, biosensors, gel electrophoresis, mass spectrometry (MS), gas chromatograph-mass spectrometry (GC-MS), nanotechnology based methods, proximity extension assays (e.g., the use of DNA oligonucleotides linked to antibodies against a target molecule that can be quantified with real-time polymerase chain reaction), slow-off-rate-modified-aptamer reagent (SOMAmer) as
  • Such technologies can include immunofluorescent assays, enzyme immunoassays, radioimmunoassays, chemiluminescent assays, sandwich-format assays, techniques using microfluidic or MEMS technologies, re-engineering technologies (e.g. instruments utilizing sensors for polypeptides used for telemedicine purposes), epitope- based technologies, other fluorescence technologies, microarrays, lab-on-a-chip, and rapid point-of-care screening technologies.
  • Exemplary proximity extension assays are available commercially from OLINK (Uppsalla, Sweden), while exemplary slow-off-rate-modified-aptamer reagent (SOMAmer) assays include SOMAscanTM assays available commercially from SOMAlogicTM (Boulder, CO).
  • Exemplary assays using nanoscale needles that function as label free biosensors, functionalized with capture antibodies that change color once bound to its target (which is then quantified) is available commercially from companies such as NanoMosaicTM (Woburn, MA).
  • Antibodies directed to the markers can be found commercially.
  • LF-MA0156 Invitrogen, Carlsbad, CA
  • PLTP Cat. No. MA5-42930, Invitrogen, Carlsbad, CA; Cat. No. H00005360-M01, ABNOVA, Taipei, TW; Cat. No. ab282456, AbCam, Cambridge, UK
  • CAVIN2 Cat. No. ab79397, AbCam, Cambridge, UK; Cat. No. ab133484, AbCam, Cambridge, UK; Cat. No. CABT-LM000181, Creative Diagnostics, Shirley, NY
  • PPBP Cat. No. MA5-43005, Invitrogen, Carlsbad, CA; Cat. No. DCABY-4086, Creative Diagnostics, Shirley, NY; Cat. No.
  • sc-73636 Santa Cruz Biotechnologies, Santa Cruz, CA
  • CTTN Cat. No. MA5- 15831, Invitrogen, Carlsbad, CA; Cat. No. CBMAB-C3169-CN, Creative Biolabs, Shirley NY; Cat. No. H00002017-M01, ABNOVA, Taipei, TW
  • HSPB1 Cat. No. NBP2-32972, Novus Biologicals, Littleton, CO; Cat. No. MA3-015, Invitrogen, Carlsbad, CA; Cat. No. MA5-33170, Invitrogen, Carlsbad, CA); RAB4A (Cat. No. ab109009, AbCam, Cambridge, UK; Cat No.
  • Illustrative of optical methods in addition to microscopy, both confocal and non-confocal, are detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry).
  • 52 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT
  • a sample can be analyzed by means of a biochip.
  • Biochips generally comprise solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Frequently, the surface of a biochip comprises a plurality of addressable locations, each of which has the capture reagent bound there.
  • a capture reagent also called an adsorbent or affinity reagent
  • the surface of a biochip comprises a plurality of addressable locations, each of which has the capture reagent bound there.
  • Protein biochip refers to a biochip adapted for the capture of polypeptides. Many protein biochips are described in the art. These include, for example, protein biochips produced by CIPHERGEN BIOSYSTEMS, INC.
  • the polypeptides described herein are detected by mass spectrometry, a method that employs a mass spectrometer to detect gas phase ions.
  • mass spectrometers are time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these.
  • the mass spectrometer is a laser desorption/ionization mass spectrometer.
  • the analytes are placed on the surface of a mass spectrometry probe, a device adapted to engage a probe interface of the mass spectrometer and to present an analyte to ionizing energy for ionization and introduction into a mass spectrometer.
  • a laser desorption mass spectrometer employs laser energy, typically from an ultraviolet laser, but also from an infrared laser, to desorb analytes from a surface, to volatilize and ionize them and make them available to the ion optics of the mass spectrometer.
  • the mass spectrometric technique for use in detecting the polypeptides described herein is “Surface Enhanced Laser Desorption and Ionization” or “SELDI.”
  • SELDI surface Enhanced Laser Desorption and Ionization
  • SELDI surface Enhanced Laser Desorption and Ionization
  • SELDI surface Enhanced Laser Desorption and Ionization
  • SELDI surface Enhanced Laser Desorption and Ionization
  • a mass-spectrometry method can be used to capture a polypeptide on an appropriate chromatographic resin.
  • a cation exchange resin such as CM Ceramic HYPERD F resin
  • wash the resin elute the polypeptides and detect by MALDI.
  • this method can be preceded by fractionating the sample on an anion exchange resin before application to the cation exchange resin.
  • polypeptides described herein can be detected and/or measured by immunoassay, where specific capture reagents, such as antibodies or binding fragments thereof, to bind each of the polypeptides in the panel.
  • Antibodies can be produced by methods well known in the art, e.g., by separately immunizing animals with each of the polypeptides. Polypeptides can be isolated from samples based on their binding characteristics. Alternatively, if the amino acid sequence of a polypeptide is known, the polypeptide can be synthesized and used to generate antibodies by methods well known in the art. [00303] Also specifically contemplated herein are traditional immunoassays for detecting or measuring the level of a polypeptide in the panel including, but not limited to, sandwich immunoassays including ELISA or fluorescence-based immunoassays, as well as other enzyme immunoassays.
  • an antibody or other binding reagent for the polypeptide is attached to the surface of an MS probe, such as a pre-activated PROTEINCHIP array.
  • the polypeptide can then be specifically captured on the biochip and detected by mass spectrometry.
  • Detection of a given polypeptide can require the use of a label or detectable moiety.
  • detectable moieties can be isotopic labels; magnetic, electrical or thermal labels; colored or luminescent dye; and enzymes, all of which enable detection of the polypeptide(s).
  • a secondary detectable label is used.
  • a secondary label is one that is indirectly detected including, but not limited to, one of a binding partner pair; chemically modifiable moieties; nuclease inhibitors; enzymes such as horseradish peroxidase, alkaline phosphatases, luciferases etc.
  • an enzyme can serve as the secondary label, bound to the soluble capture ligand.
  • the system relies on detecting the precipitation of a reaction product or on a change on the properties of the label, for example the color for detection.
  • a detection system for colorimetric methods can include a spectrophotometer, a colorimeter, or other device that measures absorbance or transmission of light on one or more wavelengths.
  • assessment of results can be qualitative or quantitative depending upon the specific method of detection employed.
  • the methods described herein are exemplified using analysis at the protein level, it is specifically contemplated herein that the step of determining expression of each member in a panel can be detected at the mRNA level can also be used with the methods described herein.
  • Reference Levels [00307] The results of a polypeptide or polypeptide panel from a given biological sample (e.g., a platelet sample or a sample of proteins isolated from platelets) can be compared to those of a control biological sample tested using substantially the same methods or to a visual reference card for dipstick or lateral flow style assays.
  • Attorney Docket No.094034-000102WOPT member in the panel of polypeptides in a control biological sample can permit diagnosis or staging of cancer in the subject.
  • Control biological samples can also be a reference sample taken from the subject at an earlier time point (e.g., during initial diagnosis of a given cancer or as a baseline prior to initiation of cancer treatment) to permit monitoring of disease progress in the subject.
  • control biological sample can be a sample taken from the subject one month, two months, three months, six months, or one year prior to the sample to be tested.
  • control biological sample or reference sample is obtained from the subject during, or following the administration of a given anti-cancer therapy.
  • a reference sample can be a sample from a patient or a population of patients having no detectable cancer (i.e., a negative control).
  • a reference sample can be a sample from a patient or population of patients with a known stage of cancer (e.g., prostate cancer), for example, mild, moderate or advanced cancer (e.g., prostate cancer) or specific stages such as T2a or T3a prostate cancer.
  • a reference is from a subject known to have cancer.
  • the cancer is the same cancer that is being monitored using the methods described herein.
  • the reference is from a different cancer from that being tested using the methods described herein.
  • the reference is from a subject having a secondary cancer or a plurality of different cancers.
  • one or more controls can comprise a known concentration (or range of concentrations) of each of the polypeptides in the panel in order to quantitatively detect the level of each polypeptide in the subject being tested.
  • a positive or negative control sample is a sample that is obtained or derived from a corresponding tissue or biological fluid or tumor as the sample to be analyzed in accordance with the methods as described herein. This sample will typically be from the same patient at the same or different time points.
  • the level of one or more of the polypeptides described herein can be compared to a reference value or the level the polypeptide in a control or reference sample in order to assess the “risk” of a subject for developing cancer.
  • the level of polypeptides in the panel described herein can be compared to a reference value in order to determine the prognosis of a subject (i.e., potential for survival).
  • the reference levels for one or more polypeptides are established based on polypeptide levels in a sample taken from an individual at an earlier point in time, for example, prior to onset of treatment with a therapeutic agent. Such methods permit one of skill in the art to monitor the efficacy of a given therapeutic.
  • the individual is determined to be responding to treatment for cancer if the relative amounts of the polypeptides in the biological sample have altered favorably from the polypeptide levels in a biological sample taken at an earlier first time point from the same individual; i.e.
  • the disease state of the individual may be progressing if the polypeptide levels in a biological sample are changing relative to the levels in the individual taken at an earlier time point or in 55 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT reference to the control levels.
  • the methods provided herein permit one of skill in the art to shift the subject to treatment with a more aggressive chemotherapeutic.
  • the final detection or staging of cancer can require that the measured levels are integrated, ideally to provide a simply binary result that is readily interpreted.
  • a suitable algorithm can be employed in order to interpret the data from the levels of the plurality of polypeptides (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 polypeptides in Table 2) and apply it in order to detect or stage cancer (e.g., prostate cancer).
  • the polypeptides levels can be inter-dependent and thus the algorithm is based on this predicted relationship.
  • the determined levels of the plurality of polypeptides are analyzed in a pre-determined sequence to monitor the subject. This may give rise to a decision tree to detect or stage a cancer.
  • the levels of the first of the multiple polypeptides can influence the subsequent thresholds required for the other polypeptides in order to detect or stage a cancer, as would readily be appreciated by one skilled in the art.
  • the output of the methods can also guide future sampling and treatment of the subject.
  • the determined levels of the plurality of polypeptides are weighted.
  • Weighting is a well-known method of applying a degree of relative significance to each polypeptide in the plurality of polypeptides.
  • the algorithm can be a threshold-based algorithm as discussed herein.
  • the levels of the measured polypeptides can be combined using logistic regression, decision tree analysis, neural networks and/or machine learning.
  • each node is a summation of the input (marker) multiplied by a weighting (beta coefficient).
  • summation is performed a number of times; there are a number of nodes and the input to these nodes can be nodes themselves rather than the measured levels.
  • the nodes are first entered at random with random weights, 56 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT the difference between the expected output and the observed output is then calculated. If it is not 0 (which is likely to be the case) the weightings are altered in the preceding layer and then in the layer before that until the input variables are reached.
  • Anti-Prostate Cancer Therapies [00315] Any anti-prostate cancer therapy which is useful, has been used, is currently being used, or can be used for the prevention, treatment and/or management of cancer can be used to prevent, treat, and/or manage cancer in accordance with the methods and assays described herein.
  • Exemplary anti-prostate cancer agents include, but are not limited to, peptides, polypeptides, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules.
  • Non-limiting examples of prostate cancer therapies include chemotherapies, radiation therapies, hormonal therapies, anti- angiogenesis therapies, targeted therapies, and/or biological therapies including immunotherapies and surgery.
  • administration includes surgery, radiation therapy, cryotherapy, hormone therapy, chemotherapy, immunotherapy, PARP inhibitors, bisphosphonates, brachytherapy, denosumab, corticosteroids, external radiation therapy or a radiopharmaceutical to the subject.
  • treatment approaches can advantageously be tailored to correspond to the patient's diagnosed level of disease. Such tailored treatments are adjusted to be aggressive enough to effective, while minimizing side effects caused by overly aggressive treatments.
  • stage I prostate cancer While therapy effective for stage IV prostate cancer would likely be expected to treat stage I prostate cancer, a stage I patient treated with stage IV therapy will very likely experience more numerous, and more severe side effects than if they had been treated with therapy appropriate for stage I prostate cancer. At the same time, treating a patient with stage III prostate cancer with therapy better suited to stage I prostate cancer will not likely be effective to eradicate the cancer. While the therapies for two different stages of prostate cancer (e.g., stage I and stage II as non- limiting examples) can have overlapping elements, those indicated for stage II, alone or in aggregate, will generally be more aggressive than those indicated for stage I.
  • Therapies that are suitable for benign prostate hypertrophy diagnosis include, but are not limited to administration of terazosin, alfuzosin, doxazosin, tamsulosin, silodosin, finasteride, dutasteride, and/or tadalafil to the subject.
  • Therapies that are suitable for stage II prostate cancer diagnosis include, but are not limited to administering external beam radiation, brachytherapy, radical prostatectomy, or active surveillance to the subject.
  • Therapies that are suitable for stage III prostate cancer diagnosis include, but are not limited to administering external beam radiation, brachytherapy, radical prostatectomy, or active surveillance to the subject.
  • Therapies that are suitable for stage IV indolent or aggressive prostate cancer diagnosis include, but are not limited to administering hormone therapy along with chemotherapy, external beam radiation along with hormone therapy, prostatectomy, and active surveillance.
  • Hormone therapy can include orchiectomy, LHRH agonists, LHRH antagonists, abiraterone, ketoconazole, antiandrogens, and estrogens.
  • the prostatectomy includes open or laparoscopic radical prostatectomy, or transurethral resection of the prostate.
  • Pharmaceutical compositions, Administration and Efficacy [00321] Certain embodiments described herein include administration of therapy appropriate for the treatment of BPH or prostate cancer.
  • a pharmaceutical composition can be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers with, optionally, an added preservative.
  • Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension can also contain suitable stabilizers or agents that increase the solubility of the active ingredients, to allow for the preparation of highly concentrated solutions.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use.
  • a therapeutic agent can be delivered in an immediate release form. In other embodiments, the therapeutic agent can be delivered in a controlled-release system or sustained-release system.
  • a pump can be used (Langer, Science 249:1527-1533 (1990); Sefton, CRC Crit. Ref Biomed. Eng.14:201 (1987); Buchwald et al., Surgery 88:507 (1980); and Saudek et al., N. Engl. J. Med 321:574 (1989)).
  • polymeric materials can be used (see Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol.
  • Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compositions.
  • fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture.
  • delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations.
  • a time-delay material such as glycerol monostearate or glycerol stearate can also be used.
  • Oral compositions can include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In one embodiment, the excipients are of pharmaceutical grade.
  • compositions can also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.
  • rectal compositions such as suppositories or retention enemas
  • conventional suppository bases such as cocoa butter or other glycerides.
  • the appropriate dosage range for a given therapeutic agent depends upon the potency, and includes amounts large enough to produce the desired effect, e.g., reduction in at least one symptom of cancer.
  • the dosage of the therapeutic agent should not be so large as to cause unacceptable or life- threatening adverse side effects or should be used under close supervision by a medical professional.
  • the dosage will vary with the type of anti-cancer agent, and with the age and condition of the patient.
  • a therapeutically effective agent is administered to a subject concurrently with a combination therapy.
  • the term “concurrently” is not limited to the administration of the two or more agents at exactly the same time, but rather, it is meant that they are administered to a subject in a sequence and within a time interval such that they can act together (e.g., synergistically to provide an increased benefit than if they were administered otherwise).
  • the combination of therapeutics can be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic effect, preferably in a synergistic fashion.
  • the agents can be administered separately, in any appropriate form and by any suitable route.
  • each of the therapeutic agents in a combination are not administered in the same pharmaceutical composition, it is understood that they can be administered in any order to a subject in need thereof.
  • the first therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of the second therapeutic agent, to a subject in need thereof (or vice versa).
  • the delivery of either therapeutic agent ends before the delivery of the other agent/treatment begins.
  • the treatment is more effective because of combined administration.
  • the therapeutic agents used in combination are more effective than would be seen with either agent alone.
  • delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with either therapeutic agent alone.
  • the 61 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT effect of such a combination can be partially additive, wholly additive, or greater than additive.
  • the agent and/or other therapeutic agents, procedures or modalities can be administered during periods of active disease, or during a period of persistence or less active disease.
  • one or more of the therapeutic agents can be administered in an amount or dose that is higher, lower or the same as the amount or dosage of the given agent used individually, e.g., as a monotherapy.
  • the administered amount or dosage of a first therapeutic agent when administered in combination with a second therapeutic agent is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of the first agent when used individually.
  • the amount or dosage of a first therapeutic agent, when administered in combination with a second therapeutic agent, results in a desired effect is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of the first (or second) agent required to achieve the same therapeutic effect when administered alone.
  • a desired effect e.g., improved cognitive functioning
  • the efficacy of a given treatment for cancer can be determined by the skilled clinician.
  • a treatment is considered “effective treatment,” as the term is used herein, if any one or all of the signs or symptoms of cancer is/are altered in a beneficial manner, or other clinically accepted symptoms or markers of disease are improved, or ameliorated, e.g., by at least 10% following treatment with a therapeutic agent for cancer. Efficacy can also be measured by failure of an individual to worsen as assessed by stabilization of the disease, or the need for medical interventions (i.e., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein.
  • Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing progression of the cancer; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of the disease, or preventing secondary diseases/disorders associated with the infection (e.g., anemia).
  • An effective amount for the treatment of a disease means that amount which, when administered to a mammal in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease.
  • Efficacy of an agent can be determined by assessing physical indicators of the disease, such as e.g., anemia, white blood cell levels or identity, pain, fatigue, fever, etc.
  • the treatment according to the methods provided herein can reduce or eliminate one or more symptoms associated with cancer such as fatigue, pain, tumor size, tumor growth, etc.
  • the cancer is prostate cancer and the one or more symptoms associated with prostate cancer include trouble urinating, increased frequency of urination, pelvic pain or discomfort, decreased force of urination, difficulty starting or stopping urine stream, blood in semen, and bone pain.
  • kits that permit quantification of individual platelet-associated polypeptides or a subset of platelet-sequestered proteins using, e.g. an antibody test from whole blood targeting proteins as described herein sequestered by platelets.
  • the kits can optionally include an agent or device for quantification of the platelet proteins.
  • the kits can optionally include agents or devices for collection of whole blood, lysing platelets, such as, e.g., (0.01% Triton X), and quantification such as, e.g., (by flow cytometry or chemical assay).
  • kits can include one or more sterile applicators, such as syringes or needles.
  • the kits can optionally include other agents, for example, anesthetics or antibiotics.
  • the kit can also include a package insert that instructs a user of the kit, such as, e.g., (a physician, laboratory technician) to perform the methods disclosed herein.
  • the testing device comprises disposable single use devices to which a platelet protein sample is applied. Where the testing device is a lateral flow test strip, such a testing device can comprise a sample receiving zone to which the sample is added. These devices typically also incorporate a solid support which defines a liquid/capillary flow path for the sample once applied to the sample receiving zone.
  • the sample receiving zone can be an integral part of the solid support.
  • the solid support can comprise a chromatographic medium, such as a membrane material in some embodiments (e.g. nitrocellulose).
  • a sample applied to the sample application zone will typically rehydrate the necessary reagents to detect the marker.
  • the reagents include binding reagents which specifically interact with the polypeptides or a substrate for effector molecules where activity is measured. Further reagents immobilized further along the flow path bind to the complex of polypeptide and binding reagent.
  • the binding reagent is labelled to provide a signal at the site of immobilization of the complex of polypeptide and binding reagent (through binding to the further reagent).
  • Suitable labels include fluorescent labels, magnetic labels, latex or gold as would be readily understood by one skilled in the art.
  • the binding reagent and/or further binding reagent can bind with a substrate, for example, only after it has been modified by the enzymatic activity, or can only bind if the substrate has not been modified by the enzymatic activity.
  • the binding reagent and further reagent for such devices and detection/quantitation methods are typically antibodies.
  • the one or more testing devices, testing kits or testing compositions of matter can comprise a lateral flow test strip.
  • a single lateral flow test strip is employed to permit detection of all polypeptides that are to be determined in the test sample.
  • a separate lateral flow test strip is provided for each polypeptide that is determined.
  • the devices, kits or compositions of matter can also include a control zone to confirm sample has passed through the device satisfactorily. In the event this is not the case the system or test kit or reader of the testing device can indicate an invalid result to the user, for example via the display.
  • the devices, kits 63 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT or compositions of matter can act as competitive or sandwich assays.
  • ELISA enzyme linked immunosorbent assay
  • the systems, test kits, testing devices and testing compositions of matter can incorporate a suitable reader to provide a quantitative output (in conjunction with the processor and storage medium); this output can be an absolute or a relative output.
  • Suitable readers can incorporate an illuminator to expose the device to a specific wavelength or wavelengths of light and a suitable detector for the reflected or emitted light.
  • the systems, test kits, testing devices and testing compositions of matter can also incorporate a suitable processor and computer application to output the results based upon the detected signal.
  • the processor running the computer application will be in operable connection with the reader.
  • operable connection is meant a functional connection that permits the exchange of a signal or information between the elements.
  • the system or test kit can incorporate the appropriate number of testing devices to permit each polypeptide to be determined. This is particularly the case where the polypeptides are detecting using different platforms.
  • the one or more testing devices comprise one or more lateral flow activity assays, ELISAs, fluorogenic substrate assays etc.
  • the one or more testing devices comprise one or more lateral flow activity assays, ELISAs or competition assays. In some embodiments, the one or more testing devices comprise one or more lateral flow assays and ELISAs.
  • a test kit can include labeled peptides suitable for use in a mass spectrometry assay. As but one example, a kit can comprise labeled peptides of one or more, or any combination of SEQ ID Nos 1-51. Such peptides can permit quantitation of target peptides in a sample.
  • Computer Systems [00354] Provided herein are testing devices and kits for performing the methods described herein. Also provided herein are computer applications for use with the systems and test kits.
  • the computer applications can also be used in or in conjunction with the testing devices or testing kits described herein, for example, by incorporation of a reader.
  • the computer-implemented method, system, and computer program product can be embodied in a computer application, for example, that operates and executes on a processor, such as in the context of a computing machine.
  • the processor can be comprised within any computer, server, embedded system, or computing system.
  • the computer can include various internal or attached components such as a system bus, system memory, storage media, input/output interface, and a network interface for communicating with a network, for example.
  • the computer can be implemented as a conventional computer system, an embedded controller, a laptop, a server, a customized machine, any other hardware platform, such as a laboratory computer or device, for example, or any combination thereof.
  • the computing machine can be a distributed system configured to function using multiple computing machines interconnected via a data network or bus system, for example.
  • the processor can be configured to execute code or instructions to perform the operations and functionality described herein, manage request flow and address mappings, and to perform calculations and generate commands.
  • the processor can be configured to monitor and control the operation of the components in the computing machine.
  • the processor can be a general purpose processor, a processor core, a multiprocessor, a reconfigurable processor, a microcontroller, a digital signal processor (“DSP”), an application specific integrated circuit (“ASIC”), a graphics processing unit (“GPU”), a field programmable gate array (“FPGA”), a programmable logic device (“PLD”), a controller, a state machine, gated logic, discrete hardware components, any other processing unit, or any combination or multiplicity thereof.
  • the processor can be a single processing unit, multiple processing units, a single processing core, multiple processing cores, special purpose processing cores, co-processors, or any combination thereof.
  • the processor along with other components of the computing machine, can be a virtualized computing machine executing within one or more other computing machines.
  • the storage medium can be selected from a flash memory, other non-volatile memory device, a solid-state drive (“SSD”), any magnetic storage device, any optical storage device, any electrical storage device, any semiconductor storage device, any physical-based storage device, any other data storage device, or any combination or multiplicity thereof.
  • SSD solid-state drive
  • the storage media can store one or more operating systems, application programs and program modules such as module, data, or any other information.
  • the storage media can be part of, or connected to, the computing machine.
  • the I/O interface can include both electrical and physical connections for operably coupling the various peripheral devices to the computing machine or the processor.
  • the I/O interface can be configured to communicate data, addresses, and control signals between the peripheral devices, the computing machine, or the processor.
  • the I/O interface can be configured to implement any standard interface, such as small 65 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT computer system interface (“SCSI”), serial-attached SCSI (“SAS”), fiber channel, peripheral component interconnect (“PCI”), PCI express (PCIe), serial bus, parallel bus, advanced technology attached (“ATA”), serial ATA (“SATA”), universal serial bus (“USB”), Thunderbolt, FireWire, various video buses, and the like.
  • the I/O interface can couple the computing machine to various output devices including video displays, speakers, printers, projectors, tactile feedback devices, automation control, robotic components, actuators, motors, fans, solenoids, valves, pumps, transmitters, signal emitters, lights, and so forth.
  • the computing machine can operate in a networked environment using logical connections through the network interface to one or more other systems or computing machines across the network.
  • the network can include wide area networks (WAN), local area networks (LAN), intranets, the Internet, wireless access networks, wired networks, mobile networks, telephone networks, optical networks, or combinations thereof.
  • the network can be packet switched, circuit switched, of any topology, and can use any communication protocol.
  • Embodiments can comprise a computer program that embodies the functions described and illustrated herein, wherein the computer program is implemented in a computer system that comprises instructions stored in a machine-readable medium and a processor that executes the instructions.
  • the embodiments should not be construed as limited to any one set of computer program instructions.
  • a skilled programmer would be able to write such a computer program to implement one or more of the disclosed embodiments described herein. Therefore, disclosure of a particular set of program code instructions is not considered necessary for an adequate understanding of how to make and use the embodiments.
  • a method of simultaneously detecting and staging BPH or prostate cancer in a subject comprising: analyzing a platelet sample from the subject; identifying irregular protein activity in the platelet sample, wherein at least one irregular protein activity is functionally involved in, and indicative of BPH or a stage of prostate cancer selected from stage II, stage III and stage IV prostate cancer, whereby the presence of irregular protein activity indicates BPH or a given stage of prostate cancer in the subject; and administering a treatment appropriate for BPH or the stage of cancer indicated by the at least one irregular protein activity.
  • the method of paragraph 1 wherein the method is non-invasive.
  • the irregular protein activity comprises: i) one or more of HEBP1, CDK16, GPLD1 and PLTP target proteins; ii) one or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins; and 67 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT iii) one or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins; and when one or more of HEBP1, CDK16, GPLD1 and PLTP target proteins is increased relative to a control, BPH is indicated; and when one or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins is increased relative to a control, prostate cancer is indicated, and when RAB4A is increased, stage II prostate cancer is indicated; when PPHLN1 is increased, stage III prostate cancer is indicated; when MUC2 is increased, stage IV indolent prostate cancer is indicated, and when SCAF8 is increased, stage IV aggressive prostate cancer
  • a method of simultaneously detecting and staging BPH or prostate cancer in a subject comprising: analyzing a platelet sample from the subject; identifying at least one irregular protein activity in the platelet sample, wherein the at least one irregular protein activity is functionally involved in and indicative of BPH or a stage of prostate cancer selected from stage II, stage III and stage IV prostate cancer, whereby the presence of the at least one irregular protein activity indicates BPH or a given stage of prostate cancer in the subject; and classifying the sample as BPH or a stage of prostate cancer selected from stage II, stage III and stage IV prostate cancer. 5.
  • the method of paragraph 4 further comprising administering a treatment appropriate for BPH or the stage of cancer indicated by the at least one irregular protein activity. 6.
  • a solid support comprising one or more reagents permitting the detection and/or measurement of the level of one or more of Heme binding protein 1 (HEBP1), Cyclin-dependent kinase 16 (CDK16), Glycosylphosphatidylinositol specific phospholipase D1 (GPLD1) and Phospholipid transfer protein (PLTP) target proteins.
  • HEBP1 Heme binding protein 1
  • CDK16 Cyclin-dependent kinase 16
  • GPLD1 Glycosylphosphatidylinositol specific phospholipase D1
  • PLTP Phospholipid transfer protein
  • the solid support of paragraph 6 comprising reagents permitting the detection of HEBP1, CDK16, GPLD1 and PLTP target proteins. 10.
  • the solid support of paragraph 11, wherein the solid support comprises an antibody conjugated tag.
  • the antibody conjugated tag is selected from gold, latex, and a fluorophore.
  • CAVIN2 Caveolae associated protein 2
  • PPBP Pro-platelet basic protein
  • CTTN Cortactin
  • HSPB1 heat shock protein family B
  • the solid support of any one of paragraphs 6-18 further comprising one or more reagents permitting the detection and/or measurement of the level of one or more of RAB4A, member RAS oncogene family (RAB4A), Periphilin 1 (PPHLN1), mucin 2, oligomeric mucus/gel-forming (MUC2) and SR-related CTD associated factor 8 (SCAF8) target proteins.
  • RAB4A member RAS oncogene family
  • PPHLN1 Periphilin 1
  • MUC2 oligomeric mucus/gel-forming
  • SCAF8 SR-related CTD associated factor 8
  • the solid support of any one of paragraphs 23-26 further comprising one or more reagents permitting the detection and/or measurement of the level of one or more of HEBP1, CDK16, GPLD1 and PLTP target proteins.
  • 28. The solid support of any one of paragraphs 23-26, further comprising reagents permitting the detection of two or more of HEBP1, CDK16, GPLD1 and PLTP target proteins.
  • 29. The solid support of any one of paragraphs 23-26, further comprising reagents permitting the detection of three or more of HEBP1, CDK16, GPLD1 and PLTP target proteins.
  • the solid support of paragraph 35 further comprising reagents permitting the detection and/or measurement of the level of one or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins. 37. The solid support of paragraph 35, further comprising reagents permitting the detection and/or measurement of the level of two or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins. 38. The solid support of paragraph 35, further comprising reagents permitting the detection and/or measurement of the level of three or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins. 39. The solid support of paragraph 35, further comprising reagents permitting the detection and/or measurement of the level of CAVIN2, PPBP, CTTN and HSPB1 target proteins. 40.
  • a kit comprising reagents for detecting in a platelet sample the presence and/or amount of a set of peptides involved in the pathogenesis of prostate cancer, at least one radioactive isotope, and instructions for processing the samples using high resolution mass spectrometry.
  • the radioactive isotope comprises 13 C, 15 N, a reagent for labeling an amino acid side chain, an isotopically coded linker, and a tag.
  • the reagent is iodoacetamide.
  • the tag is biotin. 50.
  • the radioactive isotope is conjugated to the sequence of at least one of IPNQFQSDPPAPSDK (SEQ ID NO: 1), EADYVAQATR (SEQ ID NO: 2), AALEGTATYR (SEQ ID NO: 3), GPLSSAPEIVHEDLK (SEQ ID NO: 4), AEALLSHAPR (SEQ ID NO: 5), LDSDGADLLTK (SEQ ID NO: 6), LPDTTSIFALK (SEQ ID NO: 7), ALEFLQLHNGR (SEQ ID NO: 8), DLLGIYEK (SEQ ID NO: 9), LYPTYSTK (SEQ ID NO: 10), ILEGFQPSGR (SEQ ID NO: 11), TLLLVGSPTWK (SEQ ID NO: 12), QISLEGSVK (SEQ ID NO: 13), GIQNDLTK (SEQ ID NO: 14), YQASTSNTVSK (SEQ ID NO: 15), YEGSYALTSEEAER
  • the kit of paragraph 46 wherein the mass spectrometry is selected from the group consisting of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF), and Orbitrap with tandem mass spectrometry.
  • MALDI-TOF matrix-assisted laser desorption/ionization-time of flight
  • the polypeptides involved in the pathogenesis of prostate cancer include one or more of HEBP1, CDK16, GPLD1, PLTP, CAVIN2, PPBP, CTTN, and HSPB1. 53.
  • kits of any one of paragraphs 46-52, wherein the polypeptides involved in the pathogenesis of prostate cancer include two or more of HEBP1, CDK16, GPLD1, PLTP, CAVIN2, PPBP, CTTN, and HSPB1.
  • the kit of any one of paragraphs 46-53, wherein the polypeptides involved in the pathogenesis of prostate cancer include HEBP1, CDK16, GPLD1, PLTP, CAVIN2, PPBP, CTTN, and HSPB1.
  • a kit for the staging of cancer the kit comprising reagents for detecting in a platelet sample the presence and/or amount of sets of polypeptides in functional cancer development categories including each of proliferation, angiogenesis, invasion, and metastasis. 56.
  • the kit of paragraph 55 the kit further comprising a diluent that permits sample normalization.
  • the kit of paragraph 55 or 56 wherein the samples are normalized to platelet number or platelet volume in the sample.
  • the kit of paragraph 55 which comprises at least one solid support comprising reagents sufficient to detect the presence and/or amount of the sets of polypeptides.
  • the solid support comprises a lateral flow test strip, a microfluidics chamber, a dipstick, beads, or an enzyme-linked immunosorbent assay (ELISA) support.
  • ELISA enzyme-linked immunosorbent assay
  • kit of any one of paragraphs 46-60 wherein the kit further comprises reagents for the detection of one or more of FERMT3, CAPZB, YWHAG, GC, USP14, and MGST2.
  • the polypeptides representative of function in proliferation include one or more of HEBP1, CDK16, GPLD1, and PLTP.
  • the polypeptides representative of functions in proliferation include two or more of HEBP1, CDK16, GPLD1, and PLTP. 64.
  • the kit of any one of paragraphs 46-60, wherein the polypeptides representative of functions in proliferation include three or more of HEBP1, CDK16, GPLD1, and PLTP.
  • the kit of any one of paragraphs 46-60, wherein the polypeptides representative of functions in proliferation include each of HEBP1, CDK16, GPLD1, and PLTP.
  • the polypeptides representative of functions in proliferation include each of HEBP1, CDK16, GPLD1, and PLTP.
  • the polypeptides representative of function in cancer cell angiogenesis include RAB4A.
  • a method of distinguishing benign prostate hypertrophy from prostate cancer in a male subject comprising contacting a solid support of any one of paragraphs 6-45 with a platelet protein preparation from a male subject.
  • 73. A method of determining the stage of prostate cancer in a male subject, the method comprising contacting a solid support of any one of paragraphs 16-23 or 28-39 with a platelet protein preparation from a male subject. 74.
  • a method of detecting or staging benign prostate hypertrophy or prostate cancer in a male subject comprising assaying a platelet protein preparation from the subject for the presence or level of: i) one or more of HEBP1, CDK16, GPLD1 and PLTP target proteins; ii) one or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins; and iii) one or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins.
  • the method of paragraph 74 further comprising assaying the platelet protein preparation for the presence or amount of one or more of CDC73, LARP1, Girdin, and SMU1 target proteins.
  • the method of paragraph 74 wherein the levels of two or more of HEBP1, CDK16, GPLD1 and PLTP target proteins, two or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins, and/or the level of two or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins are assayed.
  • the levels of three or more of HEBP1, CDK16, GPLD1 and PLTP target proteins, three or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins, and/or the level of three or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins are assayed.
  • the radiation therapy is selected from the group consisting of external beam radiation, brachytherapy, and radiopharmaceuticals.
  • the hormone therapy is selected from the group consisting of orchiectomy, LHRH agonists, LHRH antagonists, abiraterone, ketoconazole, antiandrogens, and estrogens.
  • the LHRH agonists are selected from the group consisting of leuprolide, goserelin, triptorelin, and leuprolide mesylate.
  • the LHRH antagonists are selected from the group consisting of degarelix and relugolix. 89.
  • the antiandrogens are selected from the group consisting of flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, and darolutamide.
  • the hormone therapy is administered as a combined androgen blockade.
  • the chemotherapy is selected from the group consisting of docetaxel, cabazitaxel, mitoxantrone, and estramustine.
  • the immunotherapy is selected from the group consisting of sipuleucel-T and PD-1 inhibitor.
  • the PARP inhibitors are selected from the group consisting of rucaparib and olaparib.
  • the radiopharmaceuticals comprise an isotope selected from the group consisting of strontium-89, samarium-153, and radium-223.
  • detection of an elevated level of RAB4A relative to a control is indicative of stage II prostate cancer.
  • the method of paragraph 95 further comprising administering external beam radiation, brachytherapy, radical prostatectomy, or active surveillance to the subject. 74 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT 97.
  • the method of paragraphs 100 or 101 further comprising administering hormone therapy along with chemotherapy, external beam radiation along with hormone therapy, radical prostatectomy, and active surveillance.
  • the hormone therapy is selected from the group consisting of orchiectomy, LHRH agonists, LHRH antagonists, abiraterone, ketoconazole, antiandrogens, and estrogens.
  • the surgery is selected from the group consisting of open radical prostatectomy, laparoscopic radical prostatectomy, and transurethral resection of the prostate.
  • the method of any one of paragraphs 74-104 further comprising assaying the platelet protein preparation for one or more control protein markers.
  • control protein markers comprises FERM domain containing kindlin 3 (FERMT3), capping actin protein of muscle Z-line subunit beta (CAPZB), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG), GC vitamin D binding protein (GC), ubiquitin specific peptidase 14 (USP14), and microsomal glutathione S- transferase 2 (MGST2).
  • FERMT3 FERM domain containing kindlin 3
  • CAPZB capping actin protein of muscle Z-line subunit beta
  • YWHAG tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma
  • GC GC vitamin D binding protein
  • USP14 ubiquitin specific peptidase 14
  • MST2 microsomal glutathione S- transferase 2
  • microfluidic assay is an ELISPOT assay.
  • immunoassay comprises contacting the platelet protein preparation with antibodies or antigen-binding fragments thereof that specifically bind the target proteins.
  • the mass spectrometry assay identifies the levels of one or more peptides selected from IPNQFQSDPPAPSDK (SEQ ID NO: 1), EADYVAQATR (SEQ ID NO: 2), AALEGTATYR (SEQ ID NO: 3), GPLSSAPEIVHEDLK (SEQ ID NO: 4), AEALLSHAPR (SEQ ID NO: 5), LDSDGADLLTK (SEQ ID NO: 6), LPDTTSIFALK (SEQ ID NO: 7), ALEFLQLHNGR (SEQ ID NO: 8), DLLGIYEK (SEQ ID NO: 9), LYPTYSTK (SEQ ID NO: 10), ILEGFQPSGR (SEQ ID NO: 11), TLLLVGSPTWK (SEQ ID NO: 12), QISLEGSVK (SEQ ID NO: 13), GIQNDLTK (SEQ ID NO: 75 4874-8934-9591.7 Attorney Docket No.094034-000102
  • a method of distinguishing benign prostate hypertrophy from prostate cancer in a male subject comprising assaying a platelet protein preparation from the subject for the presence or level of: i) one or more of HEBP1, CDK16, GPLD1 and PLTP target proteins; and ii) one or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins.
  • a platelet protein preparation from the subject for the presence or level of: i) one or more of HEBP1, CDK16, GPLD1 and PLTP target proteins; and ii) one or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins.
  • the radiation therapy is selected from external beam radiation, brachytherapy, and a radiopharmaceutical.
  • the hormone therapy is selected from orchiectomy, LHRH agonists, LHRH antagonists, abiraterone, ketoconazole, antiandrogens, and estrogens.
  • the LHRH agonists are selected from leuprolide, goserelin, triptorelin, and leuprolide mesylate.
  • the LHRH antagonists are selected from degarelix and relugolix. 125.
  • the method of paragraph 122 wherein the antiandrogens are selected from flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide and darolutamide.
  • the hormone therapy is administered as a combined androgen blockade.
  • the chemotherapy is selected from docetaxel, cabazitaxel, mitoxantrone, and estramustine. 128.
  • the immunotherapy is selected from sipuleucel-T and PD-1 inhibitor.
  • the PARP inhibitors are selected from rucaparib and olaparib. 130.
  • a method of determining the stage of prostate cancer in a male subject with prostate cancer comprising assaying a platelet protein preparation from the subject for the presence or level of: one or more of RAB4A, PPHLN1, MUC2 and/or SCAF8 target proteins, wherein detection of an elevated level of RAB4A relative to a control indicates stage II prostate cancer; detection of an elevated level of PPHLN1 relative to a control indicates stage III prostate cancer; detection of an elevated level of MUC2 relative to a control indicates stage IV indolent prostate cancer; and 77 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT detection of an elevated level of SCAF8 relative to a control indicates stage IV aggressive prostate cancer.
  • assaying a platelet protein preparation for the presence or level of target proteins comprises an immunoassay, microfluidic assay or mass spectrometry.
  • the microfluidic assay is an ELISPOT assay.
  • the immunoassay comprises contacting the platelet protein preparation with antibodies or antigen-binding fragments thereof that specifically bind the target proteins.
  • the mass spectrometry assay identifies the levels of one or more peptides selected from IPNQFQSDPPAPSDK (SEQ ID NO: 1), EADYVAQATR (SEQ ID NO: 2), AALEGTATYR (SEQ ID NO: 3), GPLSSAPEIVHEDLK (SEQ ID NO: 4), AEALLSHAPR (SEQ ID NO: 5), LDSDGADLLTK (SEQ ID NO: 6), LPDTTSIFALK (SEQ ID NO: 7), ALEFLQLHNGR (SEQ ID NO: 8), DLLGIYEK (SEQ ID NO: 9), LYPTYSTK (SEQ ID NO: 10), ILEGFQPSGR (SEQ ID NO: 11), TLLLVGSPTWK (SEQ ID NO: 12), QISLEGSVK (SEQ ID NO: 13), GIQNDLTK (SEQ ID NO: 14), YQASTSNTVSK (SEQ ID NO: 15), YEGSY
  • the assay further measures the presence or amount of one or more of CDC73, LARP1, Girdin, and SMU1 target proteins.
  • the levels of two or more of HEBP1, CDK16, GPLD1 and PLTP target proteins, two or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins, and/or the level of two or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins are assayed. 142.
  • the method of paragraph 139 wherein the levels of three or more of HEBP1, CDK16, GPLD1 and PLTP target proteins, three or more of CAVIN2, PPBP, CTTN and HSPB1 target proteins, and/or the level of three or more of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins are assayed.
  • the levels of HEBP1, CDK16, GPLD1 and PLTP target proteins, CAVIN2, PPBP, CTTN and HSPB1 target proteins, and the level of RAB4A, PPHLN1, MUC2 and SCAF8 target proteins are assayed.
  • any one of paragraphs 139-143 wherein the detection of an elevated level of one or more of HEBP1, CDK16, GPLD1 and PLTP relative to a control, in the absence of elevation of one or more of CAVIN2, PPBP, CTTN and HSPB1, is indicative of benign prostate hypertrophy.
  • the administering comprises administering terazosin, alfuzosin, doxazosin, tamsulosin, silodosin, finasteride, dutasteride, and/or tadalafil to the subject. 146.
  • the chemotherapy is selected from docetaxel, cabazitaxel, mitoxantrone, and estramustine.
  • the immunotherapy is selected from sipuleucel-T and PD-1 inhibitor.
  • the PARP inhibitors are selected from rucaparib and olaparib.
  • the radiopharmaceuticals comprise an isotope are selected from strontium-89, samarium-153, and radium-223.
  • the method of paragraph 139 further comprising administering external beam radiation, brachytherapy, radical prostatectomy, or active surveillance to the subject when RAB4A is increased. 159. The method of paragraph 139, further comprising administering external beam radiation along with hormone therapy or external beam radiation and brachytherapy with hormone therapy to the subject when PPHLN1 is increased. 160. The method of paragraph 139, further comprising administering radical prostatectomy to the subject when PPHLN1 is increased. 161. The method of paragraph 139, further comprising administering hormone therapy along with chemotherapy, external beam radiation along with hormone therapy, radical prostatectomy, surgery, and active surveillance when SCAF8 and/or MUC is increased. 162.
  • receiving results further comprises results of assaying the platelet protein preparation for one or more control protein markers.
  • 80 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT 163.
  • the one or more control protein markers comprises FERMT3, CAPZB, YWHAG, GC, USP14, and MGST2.
  • the assay for the presence or level of target proteins comprises an immunoassay, microfluidic assay or mass spectrometry.
  • the microfluidic assay is an ELISPOT assay.
  • the immunoassay comprises contacting the platelet protein preparation with antibodies or antigen-binding fragments thereof that specifically bind the target proteins.
  • the mass spectrometry assay identifies the levels of one or more peptides selected from IPNQFQSDPPAPSDK (SEQ ID NO: 1), EADYVAQATR (SEQ ID NO: 2), AALEGTATYR (SEQ ID NO: 3), GPLSSAPEIVHEDLK (SEQ ID NO: 4), AEALLSHAPR (SEQ ID NO: 5), LDSDGADLLTK (SEQ ID NO: 6), LPDTTSIFALK (SEQ ID NO: 7), ALEFLQLHNGR (SEQ ID NO: 8), DLLGIYEK (SEQ ID NO: 9), LYPTYSTK (SEQ ID NO: 10), ILEGFQPSGR (SEQ ID NO: 11), TLLLVGSPTWK (SEQ ID NO:
  • EXAMPLE 1 Development of MS assay panels [00372] Phase 1: Analytical method development for round 2 proteins [00373] In this phase, the instrument parameters are optimized for the detection of the round 2 proteins/peptides including two major steps: [00374] Stable isotopic labeled peptides (SIL peptides) evaluation [00375] After the isotopic labelled peptides (heavy peptides) are delivered, the synthetic peptides from round 2 and round 1 are pooled in equimolar amount to create a standard mix.
  • SIL peptides Stable isotopic labeled peptides
  • the investigation of the round 2 peptides can be performed using Parallel Reaction Monitoring approach (PRM) on a high- resolution mass spectrometer (HRMS) coupled with a capillary flow liquid chromatography system (cap- LC). Approximately 20 measurements (1 hour each) of only the SIL peptides are required for this part.
  • PRM Parallel Reaction Monitoring approach
  • HRMS high- resolution mass spectrometer
  • cap- LC capillary flow liquid chromatography system
  • MRM method development Compared to PRM, multiple reaction monitoring (MRM) on a high flow LC coupled with a triple quadrupole MS is a more robust analytical technique. Therefore, it is frequently used in the clinical environment. In this phase, the reference spectral information derived from the previous part can be adopted. The three most abundant fragment ions of each peptide can be selected for MRM method development. Two main MRM instrument parameters, the collision energy and the declustering potential, can be optimized for each individual peptide on round 2 list. For the MRM method development, 30 measurements of SIL samples can be used.
  • Phase 2 verification of round 1 and round 2 proteins in platelet samples [00378]
  • the optimized MRM instrument parameters for round 2 peptides are combined with the round 1 values from the previous work to create one draft MRM method for all proteins.
  • EXAMPLE 2 Elevated abundance in platelets, relative to a reference or control, of any one, or any combination of HEBP1, CDK16, GPLD1 and PLTP is indicative of BPH. The data indicate that these markers are not usually elevated in platelets in prostate cancer. Thus, it would be unusual to detect an increase in one or more of cancer markers CAVIN2, PPBP, CTTN and HSPB1 in the same sample in which an increased level of one or more of HEBP1, CDK16, GPLD1 and PLTP is elevated.
  • Elevated abundance in platelets, relative to a reference or control, of any one, or any combination of CAVIN2, PPBP, CTTN and HSPB1 is indicative of prostate cancer. If elevated platelet levels of one or more of these is detected in the absence of elevated platelet levels of RAB4A, PPHLN1, MUC2 or SCAF8, stage I prostate cancer is indicated. Elevated platelet levels of any one or more of CAVIN2, PPBP, CTTN and HSPB1, along with an elevated platelet level of RAB4A is indicative of stage II prostate cancer. Elevated platelet levels of any one or more of CAVIN2, PPBP, CTTN and HSPB1, along with an elevated platelet level of PPHLN1 is indicative of stage III prostate cancer.
  • HEBP1-diagnosis of BPH [00391] CDK16-diagnosis of BPH [00392] GPLD1-diagnosis of BPH [00393] PLTP-diagnosis of BPH [00394] HEBP1, CDK16-diagnosis of BPH [00395] HEBP1, GPLD1-diagnosis of BPH 83 4874-8934-9591.7 Attorney Docket No.094034-000102WOPT [00396] HEBP1, PLTP-diagnosis of BPH [00397] HEBP1, CDK16, GPLD1-diagnosis of BPH [00398] HEBP1, CDK16, PLTP-diagnosis of BPH [00399] HEBP1, GPLD1, PLTP-diagnosis of BPH [00400] HEBP1, CDK16, GPLD1, PLTP-diagnosis of BPH [00400] HEBP1, CDK16, GPLD1, PLTP
  • CDK16, GPLD1, PLTP -diagnosis of prostate cancer [00620] CAVIN2, CTTN, CDK16, GPLD1, PLTP -diagnosis of prostate cancer [00621] CAVIN2, HSPB1, CDK16, GPLD1, PLTP -diagnosis of prostate cancer [00622] CAVIN2, PPBP, CTTN, CDK16, GPLD1, PLTP -diagnosis of prostate cancer [00623] CAVIN2, PPBP, HSPB1, CDK16, GPLD1, PLTP -diagnosis of prostate cancer [00624] CAVIN2, CTTN, HSPB1, CDK16, GPLD1, PLTP -diagnosis of prostate cancer [00625] CAVIN2, PPBP, CTTN, HSPB1, CDK16, GPLD1, PLTP -diagnosis of prostate cancer [00626] PPBP, HSPB1, CDK16, GPLD1, PLTP -diagnos

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Abstract

La technologie décrite dans la présente invention concerne des compositions et des méthodes de diagnostic, de stadification et de traitement du cancer de la prostate chez un sujet.
PCT/US2024/043620 2023-08-25 2024-08-23 Procédés de détermination du pronostic et de la stadification du cancer de la prostate Pending WO2025049299A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060041274A1 (en) * 2004-08-20 2006-02-23 Li-Ming Su Modified laparoscopic instruments and methods of use
US20160041153A1 (en) * 2008-11-12 2016-02-11 Kirk Brown Biomarker compositions and markers
US20200200753A1 (en) * 2006-11-13 2020-06-25 Life Technologies Corporation Methods and kits for detecting prostate cancer biomarkers
WO2023108166A2 (fr) * 2021-12-10 2023-06-15 The Johns Hopkins University Biomarqueurs pour détecter un cancer de la prostate agressif à partir de formes indolentes et leur traitement
WO2023114169A1 (fr) * 2021-12-13 2023-06-22 Hessian Labs, Inc. Méthodes de détermination du pronostic et du stade d'une maladie ou d'une affection

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060041274A1 (en) * 2004-08-20 2006-02-23 Li-Ming Su Modified laparoscopic instruments and methods of use
US20200200753A1 (en) * 2006-11-13 2020-06-25 Life Technologies Corporation Methods and kits for detecting prostate cancer biomarkers
US20160041153A1 (en) * 2008-11-12 2016-02-11 Kirk Brown Biomarker compositions and markers
WO2023108166A2 (fr) * 2021-12-10 2023-06-15 The Johns Hopkins University Biomarqueurs pour détecter un cancer de la prostate agressif à partir de formes indolentes et leur traitement
WO2023114169A1 (fr) * 2021-12-13 2023-06-22 Hessian Labs, Inc. Méthodes de détermination du pronostic et du stade d'une maladie ou d'une affection

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