Methods for determining the prognosis and stage of a disease or disorder - Patents.com

Abstract

Eleven percent of all men will develop prostate cancer (PC). In the United States, 3 million men will be living with the disease this year, with 192,000 new cases expected, and 33,000 men expected to die from the disease. PC is the second leading cause of cancer (CA) death in men worldwide, and diagnosis of PC relies on a combination of tests and procedures, including digital rectal exam (DRE), magnetic resonance imaging (MRI), ultrasound, and multiple tissue biopsies, each with increased costs and risks. In 2018, the cost of PC care in the United States was $15.3 billion, of which $5.7 billion was spent on diagnosis and $8.3 billion on management. There is a significant need for early, accurate, reliable, and cancer-specific diagnosis of PC. The technology described herein relates to compositions and methods for the diagnosis and staging of cancer in a subject. TIFF2025500074000014.tif63138

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 63 / 288,775, filed December 13, 2021, the contents of which are incorporated by reference herein in their entirety.

[0002] Technical Field The field of the invention relates to cancer detection and staging. [Background technology]

[0003] background Eleven percent of all men will develop prostate cancer (PC). In the United States, three million men will be living with the disease this year, with 192,000 new cases expected, and 33,000 men expected to die from the disease. PC is the second leading cause of cancer (CA) death in men worldwide. The prostate-specific antigen (PSA) test is currently the most representative and was developed and launched to ensure early diagnosis. However, it measures antigens derived from normal prostate epithelial cells rather than cancer-specific cells, and therefore its levels are unreliable in predicting cancer progression. In prostate hyperplasia, PSA levels increase without malignant transformation, giving a "false positive" in up to 75% of cases. Confirmatory testing carries the risk of treatment complications such as lifelong erectile dysfunction and incontinence. Nevertheless, the diagnosis of PC relies on a combination of tests and procedures, including digital rectal examination (DRE), magnetic resonance imaging (MRI), ultrasound, and multiple tissue biopsies, each with increased costs and risks. In 2018, the cost of PC care in the United States was $15.3 billion, of which $5.7 billion was spent on diagnosis and $8.3 billion on management. There is a significant need for early, accurate, reliable, and cancer-specific diagnosis of PC. Summary of the Invention

[0004] overview Provided herein are methods and compositions relating to the use of platelet-sequestered proteins for the diagnosis of cancer.

[0005] Thus, in one aspect, provided herein is a method of diagnosing cancer in a subject, the method comprising the steps of: (a) measuring in a sample of platelets from the subject the levels of a set of peptides or polypeptides comprising a subset of polypeptides that represent functions in cancer development, including each of malignant cell transformation, invasion, angiogenesis, and metastasis; (b) comparing the levels of the set of polypeptides measured in step (a) with the levels of the same set of polypeptides in a reference preparation of platelets, plasma, circulating cells, or with a reference value or range thereof for each polypeptide, wherein a difference in the levels of the polypeptides that represent one or more of the functions relative to the reference is indicative of the cancer status in the subject.

[0006] In one embodiment of this aspect and all other aspects provided herein, the set of polypeptides comprises multiple polypeptides associated with each function.

[0007] In another embodiment of this aspect and all other aspects provided herein, the polypeptides that exhibit a function in cancer development include cancer stimulating and / or cancer inhibiting polypeptides.

[0008] In another embodiment of this aspect and all other aspects provided herein, an increase in one or more cancer stimulatory polypeptides or a decrease in one or more cancer inhibitory polypeptides is indicative of cancer progression.

[0009] In another embodiment of this aspect and all other aspects provided herein, a decrease in one or more cancer stimulatory polypeptides or one or more cancer inhibitory polypeptides indicates cancer regression or therapeutic response.

[0010] In another embodiment of this aspect and all other aspects provided herein, the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in malignant cell transformation include one or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

[0011] In another embodiment of this aspect and all other aspects provided herein, the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in malignant cell transformation include two or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

[0012] In another embodiment of this aspect and all other aspects provided herein, the method is for diagnosing prostate cancer, and the polypeptides exhibiting a function in malignant cell transformation include each of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

[0013] In another embodiment of this aspect and all other aspects provided herein, the method is for diagnosing prostate cancer, and the polypeptides exhibiting a function in cancer cell invasion (e.g., local tissue cancer invasion) include one or more of MYCBP2, CD47, FKBP1A, and Tribbles homolog 2.

[0014] In another embodiment of this aspect and all other aspects provided herein, the method is for diagnosing prostate cancer, and the polypeptides exhibiting a function in cancer cell invasion (e.g., localized tissue cancer invasion) include two or more of MYCBP2, CD47, FKBP1A, and tribbles homolog 2.

[0015] In another embodiment of this aspect and all other aspects provided herein, the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in angiogenesis include one or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3.

[0016] In another embodiment of this aspect and all other aspects provided herein, the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in angiogenesis include two or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3.

[0017] In another embodiment of this aspect and all other aspects provided herein, the cancer is prostate cancer and the polypeptides exhibiting a function in angiogenesis include PF4, TSP-1, and integrin beta-3, respectively, and optionally basic FGF, VEGF, and PDGF beta.

[0018] In another embodiment of this aspect and all other aspects provided herein, the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in metastasis include one or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

[0019] In another embodiment of this aspect and all other aspects provided herein, the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in metastasis include two or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

[0020] In another embodiment of this aspect and all other aspects provided herein, the method is for diagnosing prostate cancer and the polypeptides exhibiting function in metastasis include each of CAMSAP2, SRGN, and synaptobrevin homolog YKT6, and optionally one or more of RANK-L, TGF-beta, and / or IGF-1.

[0021] In another embodiment of this aspect and all other aspects provided herein, the aggressiveness or stage of the cancer is indicated by the presence of markers of particular functional classes of polypeptides (e.g., polypeptides related to malignant cell transformation, invasion, angiogenesis, and / or metastasis) that fall within a reference range for a subject having a particular stage of cancer.

[0022] In another embodiment of this aspect and all other aspects provided herein, the relative malignancy increases when markers from the functional classes are found to vary in the following order: polypeptides related to malignant cell transformation, polypeptides related to invasion, polypeptides related to angiogenesis, and polypeptides related to metastasis.

[0023] In another embodiment of this aspect and all other aspects provided herein, the method further comprises administering an anti-cancer agent if a difference in the level of the polypeptide exhibiting one or more of the functions relative to the reference is indicative of cancer in the subject.

[0024] In another embodiment of this aspect and all other aspects provided herein, cancer progression is indicated when the balance of cancer stimulating versus cancer inhibiting polypeptides is imbalanced such that cancer stimulating polypeptides are increased and / or cancer inhibiting polypeptides are decreased relative to a reference (e.g., platelets from a subject(s) without cancer).

[0025] In another aspect, also provided herein is a device for diagnosing cancer or cancer progression, the device comprising reagents sufficient to detect the presence and / or amount of a set of polypeptides in a sample of platelets or platelet proteins from a subject, the set including a subset of polypeptides that represent functions in cancer development, including each of malignant cell transformation, invasion, angiogenesis, and metastasis.

[0026] In another embodiment of this aspect and all other aspects provided herein, the set of polypeptides includes multiple polypeptides associated with each function.

[0027] In another embodiment of this aspect and all other aspects provided herein, the polypeptides that exhibit a function in cancer development include cancer stimulating polypeptides and cancer inhibiting polypeptides.

[0028] In another embodiment of this aspect and all other aspects provided herein, the polypeptide exhibiting a function in malignant cell transformation comprises one or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

[0029] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in malignant cell transformation include two or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

[0030] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in malignant cell transformation include each of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

[0031] In another embodiment of this aspect and all other aspects provided herein, the polypeptide exhibiting a function in cancer cell invasion (e.g., local tissue invasion) includes one or more of MYCBP2, CD47, FKBP1A, and tribbles homolog 2.

[0032] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in cancer cell invasion (e.g., local tissue invasion) include two or more of MYCBP2, CD47, FKBP1A, and tribbles homolog 2.

[0033] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in angiogenesis include one or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3.

[0034] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in angiogenesis include two or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3.

[0035] In another embodiment of this aspect and all other aspects provided herein, the polypeptides that exhibit a function in angiogenesis include each of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3.

[0036] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in metastasis include one or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

[0037] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in metastasis include two or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

[0038] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in metastasis include each of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and the synaptobrevin homolog YKT6.

[0039] In another embodiment of this aspect and all other aspects provided herein, the device comprises a solid substrate comprising reagents that allow for detection of the presence and / or amount of said set of polypeptides.

[0040] In another embodiment of this aspect and all other aspects provided herein, the solid substrate comprises a lateral flow test strip, a microfluidic chamber, a dipstick, a bead, or an enzyme-linked immunosorbent assay (ELISA).

[0041] In another embodiment of this aspect and all other aspects provided herein, the solid substrate is coated with a glycosaminoglycan.

[0042] Another aspect provided herein relates to a kit for detecting the presence and / or amount of a set of polypeptides comprising a subset of polypeptides that represent functions in cancer development, including each of transformation, invasion, angiogenesis, and metastasis, in a sample of platelets or platelet protein from a subject, the kit comprising reagents sufficient to detect the presence and / or amount of the set of polypeptides comprising a subset of polypeptides that represent functions in cancer development, including each of transformation, invasion, angiogenesis, and metastasis, in a sample of platelets or platelet protein from a subject, and packaging materials therefor.

[0043] In another aspect, a kit for staging cancer is also provided herein, the kit comprising reagents necessary for detecting the presence and / or amount of a set of polypeptides in functional cancer development categories, including each of malignant cell transformation, invasion, angiogenesis, and metastasis, in a platelet sample.

[0044] In another embodiment of this aspect and all other aspects provided herein, the kit comprises at least one solid support comprising sufficient reagents to detect the presence and / or amount of said sets of polypeptides.

[0045] In another embodiment of this aspect and all other aspects provided herein, the solid support comprises a lateral flow test strip, a microfluidic chamber, a dipstick, a bead, or an enzyme-linked immunosorbent assay (ELISA).

[0046] In another embodiment of this aspect and all other aspects provided herein, the solid substrate is coated with a glycosaminoglycan.

[0047] In another embodiment of this aspect and all other aspects provided herein, the kit comprises reagents for the detection of actin.

[0048] In another embodiment of this aspect and all other aspects provided herein, the polypeptide exhibiting a function in malignant cell transformation comprises one or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

[0049] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in malignant cell transformation include two or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

[0050] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in malignant cell transformation include each of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

[0051] In another embodiment of this aspect and all other aspects provided herein, the polypeptide exhibiting a function in cancer cell invasion includes one or more of MYCBP2, CD47, FKBP1A, and tribbles homolog 2.

[0052] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in cancer cell invasion include two or more of MYCBP2, CD47, FKBP1A, and tribbles homolog 2.

[0053] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in angiogenesis include one or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3.

[0054] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in angiogenesis include two or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3.

[0055] In another embodiment of this aspect and all other aspects provided herein, the polypeptides that exhibit a function in angiogenesis include each of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3.

[0056] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in metastasis include one or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

[0057] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in metastasis include two or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

[0058] In another embodiment of this aspect and all other aspects provided herein, the polypeptides exhibiting a function in metastasis include each of CAMSAP2, SRGN, and the synaptobrevin homolog YKT6, and optionally one or more of RANK-L, TGF-beta, and IGF-1.

[0059] In another aspect, also provided herein is a solid support comprising reagents sufficient to detect polypeptides in each of a set of functional cancer development categories, including malignant cell transformation, invasion, angiogenesis, and metastasis, in a sample of platelet proteins from a subject.

[0060] In one embodiment of this aspect and all other aspects provided herein, reagents sufficient to detect the presence and / or amount of a plurality of sets of polypeptides are disposed on the support in four regions, one region each for reagents sufficient to detect polypeptides in functional cancer development categories including malignant cell transformation, invasion, angiogenesis, and metastasis, each region containing reagents sufficient to detect the presence and / or amount of one or more polypeptides in one of the respective functional cancer development categories.

[0061] In another embodiment of this aspect and all other aspects provided herein, each region contains sufficient pooled reagents to detect the presence of multiple polypeptides in each functional oncogenesis category.

[0062] In another embodiment of this aspect and all other aspects provided herein, the amount of detectable signal in each region upon detection of the sets of polypeptides in a platelet or platelet protein sample provides an indication of the presence and / or stage of cancer in the subject from which the platelets were obtained.

[0063] Another aspect provided herein relates to a method of diagnosing or staging cancer in a subject, the method comprising contacting a solid support as described herein with a sample of platelets or platelet proteins obtained from the subject, wherein the contacting allows detection of the presence and / or amount of platelet proteins in the sample in one or more of the functional cancer occurrence categories.

[0064] Another aspect provided herein pertains to a kit comprising the solid support described herein and packaging materials therefor.

[0065] Another aspect provided herein relates to a method of monitoring a cancer treatment for effectiveness in a subject, the method comprising the steps of: (a) measuring in a first sample of platelets from the subject the levels of a set of polypeptides comprising a subset of polypeptides that represent functions in cancer development comprising each of malignant cell transformation, invasion, angiogenesis, and metastasis; (b) measuring in a second sample of platelets from the subject, the second sample being taken after administration of a cancer therapy to the subject following step (a); and (c) comparing the levels of the polypeptides measured in steps (a) and (b), wherein a change in the level of polypeptides in one or more of the subset of polypeptides measured in steps (a) and (b) provides an indication of the effectiveness of the cancer therapy.

[0066] In one embodiment of this aspect and all other aspects provided herein, polypeptides that exhibit a function in cancer development include cancer stimulating polypeptides and cancer inhibiting polypeptides.

[0067] In another embodiment of this aspect and all other aspects provided herein, a decrease in the level of one or more cancer stimulatory polypeptides or an increase in the level of one or more cancer inhibitory polypeptides measured in step (b) relative to that measured in step (a) indicates that the cancer therapy is efficacious.

[0068] In another embodiment of this aspect and all other aspects provided herein, a positive prognostic change in the level of a polypeptide indicative of malignant cell transformation, invasion, angiogenesis, or metastasis indicates that the therapy is efficacious, and a negative prognostic change in the level of a polypeptide indicative of malignant transformation, invasion, angiogenesis, or metastasis indicates that the therapy is not efficacious.

[0069] In another embodiment of this aspect and all other aspects provided herein, the method further comprises continuing to administer the therapy at the current dosage and frequency or reducing the dosage and / or frequency of administration of the therapy if the comparing step indicates that the therapy is effective, or administering another therapy alone or together with the therapy or increasing the dosage and / or frequency of the therapy if the comparing step indicates that the therapy is not effective. [Brief description of the drawings]

[0070] [Figure 1] FIG. 1 depicts an exemplary method for isolation of platelets and platelet-poor plasma. [Diagram 2] FIG. 1 depicts an exemplary method for generating a profile of isolated platelet proteins (eg, a HessMap™) indicative of cancer or cancer stage. [Diagram 3]1 is a table showing relative levels of cancer progression indicators in platelets. T2a = tumor has invaded one half (or less than half) of the prostate. T3a = tumor has developed outside the prostate but has not spread to the seminal vesicles. Stimulatory factor levels increase from healthy to stage T2a and further increase in stage T3a, while inhibitory factor levels decrease from healthy to stage T2a and further decrease in stage T3a. [Figure 4] Cancer stage specific proteins are shown in both box plot formats. [Diagram 5] 1 is a table showing normalized levels of cancer progression indicators in platelets. These proteins represent stimulators or inhibitors of carcinogenesis, cancer progression, and metastasis. These data show that the levels of stimulators increase from healthy to stage T2a and further increase in stage T3a, while the levels of inhibitors decrease from healthy to stage T2a and further decrease in stage T3a. These values ​​are normalized to healthy platelet protein levels. These proteins can stage prostate cancer as stage T2a or T3a. [Figure 6] Normalized levels of cancer progression indicators in platelets are shown. [Figure 7] 1 is a table showing prostate cancer-related biomarkers, i.e., proteins or peptides that are not present in the platelets of healthy subjects but are significantly elevated in the platelets of subjects with prostate cancer. Mean raw data values ​​are peak areas x 106. Redundancies in proteins such as HSP90 are due to differential expression of receptor subunits. [Figure 8] Graph showing prostate cancer polypeptide.Comparing platelets isolated from 5 healthy subjects and 5 subjects with T2a prostate cancer, some proteins are not detected in the platelets from healthy controls, but are detected in subjects with stage T2a prostate cancer.Some proteins are not detectable or are present in very small amounts in healthy platelets. [Figure 9]An exemplary HessMap™ reveals proteins associated with identifying "escape from dormancy". These data show how sequestration of basic fibroblast growth factor (bFGF) in platelets can be an early marker of tumor aggressiveness (i.e., signaling escape from dormancy). This human liposarcoma dormant clone is known to take an average of 131 days to become palpable, i.e., to reach a tumor size of 200-300 mm3. In this study, platelet-associated bFGF detected escape from dormancy in 2 / 5 mice at day 32 and in all mice by day 120. In contrast, plasma bFGF was undetectable in any of the mice throughout the 130 days of the study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] Detailed Description Provided herein are methods, assays, and devices that utilize the presence of polypeptides in platelets or platelet preparations that are associated with and therefore predict or indicate disease states such as cancer. Such platelet-sequestered polypeptides represent the entire cancer-associated protein profile (e.g., detect the presence of cancer or allow cancer staging), allowing real-time evaluation of efficacy and patient response to treatment. Real-time evaluation of proteins in platelets can be used to guide patient treatment and clinical trial enrollment and design. Polypeptides sequestered by platelets can be classified based on their function and impact on cancer (e.g., angiogenesis, cell invasion / local tissue cancer invasion, metastasis, etc.).

[0072] definition For convenience, certain terms used throughout this application (including the specification, examples, and appended claims) are collected here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0073] As used herein, the term "subject" includes humans and mammals. The term "mammal" is intended to include the single "mammal" and the plural "mammals", including, but not limited to, humans; primates such as apes, monkeys, orangutans, and chimpanzees; canines such as dogs and wolves; felines such as cats, lions, and tigers; equines 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. In some embodiments, the mammal is a human. The subject can be of any age, including newborn infants, children, teenagers, adults, or elderly subjects. Subjects that can be treated using the methods and compositions described herein include individuals who are "healthy" with respect to cancer, e.g., (no history of cancer, normal; cancer not yet detected), but may have underlying health concerns, e.g., (obesity, diabodies), as well as those suffering from or at risk of hyperplasia and / or cancer and / or associated inflammation.Subjects include human patients (adults and children) who have cancer or are at risk of developing cancer.

[0074] The subject to be diagnosed, staged or treated according to the methods described herein may be a healthy individual, or a person who has been diagnosed with a disease or condition associated with "neoplasm", "cancer" and / or inflammation, such as a disease or condition of the prostate, or a person who is at risk of developing such a condition.A "healthy" subject is a person who does not have detectable disease at the time of examination.A healthy individual may also refer to an individual who has been successfully treated for neoplasm and / or cancer, and is "disease-free".

[0075] As used herein, the term "cancer" refers to the presence in tissue or circulation of cells that possess the typical characteristics of cancer-inducing cells as understood by those skilled in the art of medicine.Exemplary characteristics of cancerous or tumor cells include, but are not limited to, uncontrolled cell proliferation, loss of normal function, immortality, metastatic potential, lack of apoptotic activity or increased anti-apoptotic activity, high growth and proliferation rate, recalcitrance to regulatory mechanisms, and certain characteristic morphology and cell markers.

[0076] As used herein, the terms "neoplasia" and "hyperplasia" are intended as diseases caused or resulting from high levels of cell division or low levels of apoptosis, or both. Neoplasia is characterized by pathological growth and migration or invasion into other tissues or organs.

[0077] As used herein, the term "tumor" refers to any neoplastic cell proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues, including tissue-resident, circulating, and metastatic tumors.

[0078] As used herein, the term "isolated" refers to a substance that has been concentrated or removed from the natural or endogenous state in which it is found. "Isolate" refers to some degree of separation from the original source or surrounding substances. The related term "purify" refers to a higher degree of separation than isolation, such as, for example, (isolation of platelets from whole blood and purification of platelet-associated proteins). A "purified" or "biologically pure" protein is sufficiently free of other substances such that any impurities do not affect the biological properties, or if they do, they do so to a negligible extent.

[0079] As used herein, the terms "platelet-associated protein" and "platelet-sequestered polypeptide" are synonymous and are 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.

[0080] As used herein, the term "protein" or "polypeptide" or "peptide" as used herein refers to any chain of more than two natural or non-natural amino acids, without post-translational modifications such as, for example, glycosylation or phosphorylation, that constitutes all or part of a naturally occurring or non-naturally occurring polypeptide or peptide.

[0081] Unless otherwise defined, the term "isolated polypeptide" as used herein refers to a polypeptide that is separated from its natural associated components. An isolated polypeptide can be obtained, for example, by extraction from a natural source such as whole blood or platelets.

[0082] As used herein, the term "polypeptide exhibiting a function in cancer development" refers to a polypeptide that has been determined to be present during a particular phase of cancer development, or to be present at an increased concentration, or to occur with an altered amount, and therefore, its presence / absence or amount is predictive or indicative of a particular condition or stage of cancer.

[0083] As used herein, the term "function in cancer development" refers to the multi-step progression during cancer development and provides an organizing principle for assigning platelet-associated polypeptides to cancers with certain characteristics. Exemplary functions include malignant cell transformation, promotion of angiogenesis, cancer cell invasion (e.g., local tissue invasion), metastasis, and the like.

[0084] The term "malignant cell transformation", as used herein, refers to a process in which a cell is no longer responsive to mechanisms that regulate normal proliferation or cell death (e.g., via apoptosis). Such a cell(s) may undergo uncontrolled proliferation and growth processes that may ultimately lead to the formation of a cancer mass or tumor. In some aspects, malignant cell transformation is associated with changes in cell morphology, such as, for example, an increase in the nuclear to cytoplasmic ratio, metaplasia, chromatin reorganization, accumulation of chromosomal alterations and mutations, and loss of contact inhibition.

[0085] The term "angiogenesis" refers to the developmentally abnormal generation of new blood vessels (e.g., neovascularization) induced in response to oncogenic stimuli as the nutritional needs of cancer cells or tumors increase. Cancers generally achieve the ability to actively promote angiogenesis as part of their development to meet the demands and supply of nutrients, inflammatory cells, stem cells, and other cancer-supporting cells.

[0086] As used herein, the term "cancer cell invasion" refers to the process by which cancer cells spread and invade adjacent tissues. The term "cancer cell invasion" may also be referred to herein as "local tissue cancer invasion."

[0087] As used herein, the term "metastasis" refers to the migration of cancer cells from a primary site via the circulatory or lymphatic system to a site different from the primary site, thereby allowing the attachment of metastatic cells and the establishment of a secondary cancer site at the new site (i.e., metastasis).

[0088] As used herein, the term "cancer-stimulating polypeptide" refers to a polypeptide whose expression is associated with cancer growth and metastasis.

[0089] As used herein, the term "cancer-inhibiting polypeptide" refers to a polypeptide whose lack or insufficient expression is associated with cancer growth and metastasis.

[0090] As used herein, the term "normalization" refers to a method of modifying a quantitative measure (e.g., protein concentration) to minimize and / or reduce the effects of factors that may prevent equivalent comparison, for example, by minimizing the effects of highly variable baselines of proteins in a population or by removing the effects of different amounts of starting material. Exemplary methods of normalization include, but are not limited to, using the ratio of platelet number or volume or a platelet-related reference or set of references to a reference protein such as actin, albumin, or another other platelet-associated protein to obtain a ratio or "normalization / correction factor" to correct for equivalent comparison. For example, if the platelet protein concentration is 10X in sample A and 1X in sample B, and the platelet count is 10X in sample A and 1X in sample B, a correction factor of 10 is applied to sample B. (Platelet count of A (10X)÷Platelet count of B (1C)=Normalization factor of 10) corrects for the difference in starting numbers, indicating that there is no difference in protein concentration in the comparison of sample A and sample B.

[0091] The terms "reduce," "reduced," "reduction," or "inhibit" are all used herein to mean a decrease or alleviation of a characteristic, level, or other parameter (such as the amount of a platelet associated polypeptide or a disease symptom) by a statistically significant amount. In some embodiments, "reduce," "reduction," or "reduce" or "inhibit" typically means at least a 10% decrease in the expression or activity of a given platelet associated polypeptide as compared to a reference expression or activity level for the same platelet associated polypeptide, and can include, for example, a decrease of 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. As used herein, "reduction" or "inhibition" does not include complete inhibition or reduction when compared to a reference level. Rather, the term "complete inhibition" is used to refer to 100% inhibition when compared to an appropriate reference level. The reduction of a given cancer symptom can preferably be down to a level that is accepted as being within the normal range for individuals without cancer.

[0092] In some embodiments, 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-stimulating polypeptides may be absent or below detectable levels. Alternatively, in conditions associated with cancer or cancer progression, one or more cancer-inhibiting polypeptides may be absent or below detectable levels.

[0093] The terms "increased," "increase," or "enhance" or "activate" are all used herein to generally mean an increase in a property, level, or other parameter (e.g., the 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" mean an increase of at least a 10% in the expression or activity of a given platelet-associated polypeptide in a panel compared to a reference level (e.g., a platelet sample from one or more healthy individuals), e.g., 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 100% compared to the reference level. By "reference level" is meant an increase (including 100%), or any increase between 10 and 100%, or at least about a 2-fold increase, or at least about a 3-fold increase, or at least about a 4-fold increase, or at least about a 5-fold increase, or at least about a 10-fold increase, or at least about a 20-fold increase, or at least about a 50-fold increase, or at least about a 100-fold increase, or at least about a 1000-fold increase, or more, as compared to a reference level. In one embodiment, the reference level can be the amount of each member of the panel of proteins in a subject or population of subjects without cancer.

[0094] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective component(s) essential to the invention, but leave room for the inclusion of unspecified elements, whether or not essential to the invention.

[0095] As used herein, the term "consisting essentially of" refers to an element that is required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0096] The term "consisting of" refers to the compositions, methods, and their respective components described herein excluding any element not recited in that description of an embodiment.

[0097] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and / or steps of the type described herein and / or that will become apparent to those of skill in the art upon reading this disclosure, and so forth. It will be understood that the foregoing detailed description and the examples set forth below are merely illustrative and should not be construed as limitations on the scope of the invention. Various changes and modifications to the aspects of the present disclosure that will become apparent to those of skill in the art can be made without departing from the spirit and scope of the invention. Furthermore, all identified patents, patent applications, and publications are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodology described in such publications that may be used in connection with the present invention. Such publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by reason of prior invention or for any other reason. All statements as to the dates or phraseology of the contents of such documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of such documents.

[0098] The disclosure described herein, in preferred aspects, does not relate to processes for cloning humans, processes for modifying the genetic identity of the germ line of humans, the use of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals that may cause suffering to mankind or animals without providing any substantial medical benefit to them, nor to the animals resulting from such processes.

[0099] cancer The methods, assays, and devices used herein can be used to detect the presence of polypeptides in platelets that are associated with specific cancer functions or processes, and can therefore be used to diagnose or stage specific cancers. Such cancer functions or processes include malignant cell transformation, angiogenesis, cell invasion, metastasis, and the like. The methods and assays can be used to detect the presence of any cancer and / or determine the specific stage of that cancer.

[0100] Some non-limiting examples of cancers that can be diagnosed, staged, and / or treated using the methods and compositions described herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.Other exemplary cancers include basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatoma; hepatoma; intraepithelial neoplasia; kidney or renal cancer; laryngeal cancer; leukemia; hepatoma; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); Hodgkin's lymphoma. and lymphomas, including non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and throat); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; and other cancers and sarcomas; and B-cell lymphomas (low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) These include, but are not limited to, NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small noncleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nevus syndrome, edema (such as that associated with brain tumors), and Meigs syndrome.

[0101] In some embodiments, the cancer or sarcoma includes, but is not limited to, cancers and sarcomas found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovary, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, tailbone, testes, thyroid, and uterus. Types of cancer include, but are not limited to, papilloma / carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma / adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, hemangioma, osteoma, chondroma, glioma, lymphoma / leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, basal cell carcinoma, and sinonasal undifferentiated carcinoma. Types of sarcomas include, but are not limited to, soft tissue sarcomas such as alveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma.

[0102] In one embodiment of the methods and compositions described herein, the subject with tumor, cancer or malignant condition is undergoing or has undergone treatment with conventional cancer therapy.In some embodiments, the cancer therapy is chemotherapy, radiation therapy, immunotherapy, targeted therapy / biological therapy (e.g., CAR-T cell), or a combination thereof.

[0103] Prostate cancer: In various examples, the subject has or is at risk of developing a disease or condition associated with precancerous prostate lesions and / or prostate cancer in various associated tissues of the prostate, such as prostate tissue and / or associated lymph nodes.In some embodiments, the method of differentially diagnosing the presence and stage of prostate cancer in a subject, or preventing unnecessary intervention or unnecessary treatment of a disease or condition characterized by precancerous lesions or prostate cancer, comprises isolating, investigating, or characterizing platelet-sequestered proteins.In some embodiments, the disease or condition is characterized by prostate, splenic, gastric, bladder, colon, colorectal, neuroendocrine, or prostatic hyperplasia, such as adenomatous polyps, benign prostatic hyperplasia (BPH), and / or inflammation. In some embodiments, the diagnosis and staging of hyperplasia and tumor lesions are determined by the AJCC (American Joint Committee on Cancer) TNM system, including but not limited to clinical or pathological tumor categories, such as (cT1-4, a-c or pT1-4, a-c), N categories of lymph node spread, such as (N0 or N1), M categories of metastasis, such as (M0 or M1), and any combination of the above categories. In some embodiments, the subject has or is at risk of developing a morbidity due to treatment or lack of treatment of the tumor lesion, such as by PSA screening, digital rectal examination, etc. In some embodiments, the subject has or is at risk of developing an irreversible morbidity of prostate cancer, such as erectile dysfunction or incontinence. In some embodiments, the disease or condition is, results from, is a precursor to, or is a cancerous lesion of, a precancerous lesion, a sarcoma, or a cancer of the prostate, spleen, stomach, bladder, colon, colorectum, neuroendocrine tissue, and / or inflammation in a disease or condition.

[0104] platelet Platelets (thrombocytes) are small irregularly shaped clear cell structures (meaning "cells" without nuclei) 2-3 μm in diameter that are created from pseudopodial extensions of precursor bone marrow cells called "megakaryocytes". In healthy individuals, platelet counts range from 150,000 to 450,000 platelets / microliter of blood, so an adult with 5 liters of blood will have a total of 750 million to 225 million platelets. Thrombocytosis is greater than 450,000 platelets / uL of blood, while thrombocytopenia is less than 150,000 platelets / uL. Although the primary function of platelets is to maintain hemostasis, it also stores and delivers many proteins necessary for tissue repair and regeneration. The classical role of platelets is in the formation of thrombi upon injury to the endothelium, such as a laceration that leads to vascular injury and release of blood. Conversely, inappropriate thrombus formation leads to pathological conditions, such as those seen in acute coronary thrombosis. The ability of platelets to store and transport proteins is exploited in cancer for the storage and transport of cancer-promoting growth factors. As previously shown, angiogenic regulators are actively sequestered in platelets against a concentration gradient (Cervi D et al. 2008, Klement GL et al. 2009). Moreover, the sequestration of angiogenic regulators is selective (Klement GL et al. 2009).

[0105] These findings indicate that platelets are "first responders" to insults, sequestering proteins in a dynamic manner that allows for the diagnosis of disease: platelets carry a highly responsive and continuously changing "cargo" of, for example, protein stimulators and inhibitors of cancer or neoplasia. For example, platelets can carry angiogenic stimulators and inhibitors (e.g., VEGF, bFGF, and PDGF) that play a role in neovascularization and tissue remodeling. As tissue remodeling progresses, the amount and type of stimulators and inhibitors in the platelet cargo change in a predictable manner. The constantly changing platelet protein profile (or protein signature) allows for 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 may allow monitoring of the entire cancer-associated protein profile, including known polypeptides. Such platelet-sequestered proteins can be used to classify platelet-associated proteins by their function and effect (e.g., angiogenesis-regulating, inflammatory, growth-promoting, etc.) and may indicate changes in platelet protein profile that correlate with disease stage and progression. The methods and compositions described herein allow for real-time assessment of efficacy and patient response to therapy to guide patient treatment and clinical trial enrollment and design.

[0106] Previous studies have shown that cancer-associated growth factors are actively sequestered in platelets, which occurs against a concentration gradient in plasma and is more robust and specific than protein concentrations found in plasma or serum. The data described herein in the Examples demonstrate that sequestration of cancer-associated proteins occurs early in malignant progression, but clearly reflects cancer stage and response to treatment.

[0107] Methods for platelet isolation are known to those skilled in the art and are described in "Current Protocols in Immunology by FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, K. Struhl and VB Chanda (Editors), John Wiley & Sons, 2004.," which is incorporated herein by reference. For example, whole blood is drawn from a donor and placed into a vacutainer containing sodium citrate. The whole blood is then centrifuged at low g-force to separate the platelet-rich plasma from the other components in a first step. In a second step of the procedure, the platelet-rich plasma is separated into a new tube and a platelet concentrate is obtained by centrifuging the platelets at a higher speed. The platelet concentrate is then resuspended in a standard lysis buffer and associated proteins are isolated.

[0108] In some embodiments, platelet isolation is by routine phlebotomy procedures (e.g., venipuncture) utilizing collection tubes containing anticoagulants, such as, but not limited to, 3.2% sodium citrate, sodium or lithium heparin without gel, potassium EDTA, sodium fluoride, and sodium or potassium oxalate, or combinations of the above compounds at various concentrations. Note that when serum is created, all clotting factors (including platelets) will clot (e.g., serum tubes are sometimes referred to as "clotting" tubes). Therefore, it cannot be used for platelet isolation. Thus, platelet isolation must always be performed in the absence of activator gel. In some embodiments, platelets are isolated from whole blood, platelet-rich plasma, or washed platelets. In some embodiments, systemic administration or isolation of platelets includes intravenous injection or infusion of platelets to be subsequently isolated for characterization or analysis of platelet-sequestered proteins. In some embodiments, normalization of platelet-associated proteins is by platelet count, such as, for example, manual count or count number obtained by flow cytometry. In some embodiments, normalization of platelet-associated proteins is by volume, e.g., platelet volume, mean platelet volume (MPV), etc. In some embodiments, normalization of platelet-associated proteins is by endogenous proteins (e.g., tubulin or actin, which are platelet scaffolding proteins), exogenous proteins (e.g., labeled heavy peptide controls for quantification of specific proteins), or compounds sequestered by platelets, e.g., derivatives or modifications of exogenously introduced platelet-sequestered proteins. In some embodiments, normalization of platelet-associated proteins is by one or a combination of selectively sequestered platelet proteins, e.g., the sequestered proteins in Tables 1-5. In some embodiments, the subject is a human.

[0109] Polypeptides that exhibit functions in cancer development 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, and physiologically dynamic system that monitors disease onset and biological events that involve genetic changes not only in cancer cells, but also in the physiology of a subject. That is, such proteins can bind to and be released by platelets in a dynamic manner that reflects their expression levels and the disease status of the subject. For example, as cancer progresses, proteins sequestered by platelets indicate a cancerous state, including an increased amount of cancer stimulatory factors and / or a decreased amount of cancer inhibitory factors. Polypeptides sequestered by platelets can represent 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, and metastatic malignant cell transformation, invasion, angiogenesis, and / or metastasis.

[0110] Table 1. Platelet proteins that change between normal and stage T2 prostate cancer TIFF2025500074000002.tif52162TIFF2025500074000003.tif245162TIFF2025500074000004.tif163162

[0111] Table 2. Platelet proteins that change between normal and stage T3 prostate cancer TIFF2025500074000005.tif75162TIFF2025500074000006.tif228162TIFF2025500074000007.tif137162

[0112] Table 3. Platelet proteins that change between stage T2 and stage T3 prostate cancer. TIFF2025500074000008.tif191165

[0113] Table 4. Most frequently occurring protein domains in platelet-sequestered proteins TIFF2025500074000009.tif60165

[0114] Table 5. Exemplary panel of polypeptides for cancer diagnosis and staging TIFF2025500074000010.tif113165

[0115] In one embodiment, polypeptides exhibiting a function in malignant cell transformation are detected in platelets and include one or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2. In one embodiment, such polypeptides exhibiting a function in malignant cell transformation can be used to detect and / or stage prostate cancer.

[0116] In another aspect, the method can be used to diagnose cancer (e.g., prostate cancer), and the polypeptides exhibiting a function in cancer cell invasion (e.g., localized tissue cancer invasion) include one or more of MYCBP2, CD47, FKBP1A, and tribbles homolog 2.

[0117] In another embodiment, the method can be used to diagnose cancer (e.g., prostate cancer), and the polypeptides exhibiting a function in angiogenesis include one or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3.

[0118] In another embodiment, the method can be used to diagnose cancer (e.g., prostate cancer), and the polypeptides exhibiting a function in metastasis include one or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

[0119] In one aspect, the method includes detecting at least one polypeptide from each functional group consisting of polypeptides that exhibit functions in malignant cell transformation, cancer cell invasion (e.g., local tissue cancer invasion), angiogenesis, and metastasis.

[0120] In other embodiments, the method includes detecting at least one polypeptide (e.g., at least two, at least three, at least four, at least five, or all six polypeptides) derived from polypeptides exhibiting a function in malignant cell transformation in combination with at least one polypeptide (e.g., at least two, three, or four) derived from at least one functional group consisting of polypeptides exhibiting a function in cancer cell invasion (e.g., local tissue cancer invasion), angiogenesis, and / or metastasis.

[0121] In other aspects, the method includes detecting at least one polypeptide (e.g., at least two, at least three, or all four polypeptides) derived from polypeptides exhibiting a function in cancer cell invasion (e.g., local tissue cancer invasion) in combination with at least one polypeptide (e.g., at least two, three, four, five, or six) derived from at least one functional group consisting of polypeptides exhibiting a function in malignant cell transformation, angiogenesis, and / or metastasis.

[0122] In other aspects, the method includes detecting at least one polypeptide (e.g., at least two or all three polypeptides) from a set of polypeptides exhibiting a function in angiogenesis in combination with at least one polypeptide (e.g., at least two, three, four, five, or six) from at least one functional group consisting of polypeptides exhibiting a function in malignant cell transformation, cancer cell invasion (e.g., local tissue cancer invasion), and / or metastasis.

[0123] In other embodiments, the method includes detecting at least one polypeptide (e.g., at least two or all three polypeptides) derived from polypeptides exhibiting a function in metastasis in combination with at least one polypeptide (e.g., at least two, three, four, five, or six) derived from at least one functional group consisting of polypeptides exhibiting a function in malignant cell transformation, cancer cell invasion (e.g., local tissue cancer invasion), and / or angiogenesis.

[0124] In one embodiment, the method comprises detecting at least three proteins selected from the group consisting of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, heat shock protein HSP 90-alpha isoform 2, MYCBP2, CD47, FKBP1A, tribbles homolog 2, PF4, TSP-1, basic FGF, VEGF, PDGF beta, integrin beta-3, CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6. In other embodiments, the method comprises detecting at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or all sixteen proteins selected from the group consisting of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, heat shock protein HSP 90-alpha isoform 2, MYCBP2, CD47, FKBP1A, tribbles homolog 2, PF4, TSP-1, basic FGF, VEGF, PDGF beta, integrin beta-3, CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

[0125] In other embodiments, the method comprises detecting at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or all sixteen proteins consisting essentially of polypeptides selected from the group consisting of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, heat shock protein HSP90-alpha isoform 2, MYCBP2, CD47, FKBP1A, tribbles homolog 2, PF4, TSP-1, basic FGF, VEGF, PDGF beta, integrin beta-3, CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

[0126] In certain embodiments, the polypeptides detected in platelets consist of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, heat shock protein HSP 90-alpha isoform 2, MYCBP2, CD47, FKBP1A, tribbles homolog 2, PF4, TSP-1, basic FGF, VEGF, PDGF beta, integrin beta-3, CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

[0127] Balance between stimulatory and inhibitory polypeptides In certain embodiments, the polypeptides detected in the platelets include at least one cancer stimulating polypeptide and at least one cancer inhibiting polypeptide shown in Figure 3 or 5. In one embodiment, at least two, three, four, five, six, or all seven of the cancer stimulating polypeptides in Figure 3 or 5 are detected in combination with at least one cancer inhibiting polypeptide shown in Figure 3 or 5. In an alternative embodiment, at least two, three, four, five, six, or all seven of the cancer inhibiting polypeptides in Figure 3 or 5 are detected in combination with at least one cancer stimulating polypeptide shown in Figure 3 or 5.

[0128] In one embodiment, at least two polypeptides (e.g., at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or all fourteen) selected from the group consisting of CAMSAP2, MYCBP2, HERC1, CD47, AKT2, SGRN, RAP1A, PF-4 isoform 1, PF-4 isoform 4, Rab-14, HSP70 protein 1A / 1B, HSP70 protein 6, HSP70 protein 1A / 1B, HSP90 isoform 2, integrin beta-3, and FKBBP1A are detected in platelets using the methods described herein.

[0129] In some embodiments, the "balance between stimulatory and inhibitory polypeptides" can be used to predict or indicate the presence of cancer or a given stage of cancer. As an example, an increase in the amount or concentration of one or more cancer stimulatory polypeptides (e.g., above a given threshold) and / or a decrease in the amount or concentration of one or more cancer inhibitory polypeptides (e.g., below a given threshold) tilts the balance toward cancer stimulation, thereby indicating that cancer exists or progresses in the subject. Conversely, a decrease in the amount or concentration of one or more cancer stimulatory polypeptides and / or an increase in the amount or concentration of one or more cancer inhibitory polypeptides tilts the balance toward cancer inhibition, thereby indicating that cancer does not exist, that cancer does not progress in the subject, or that the treatment used to treat the subject for cancer is effective.

[0130] Methods for the detection of polypeptides associated with cancer function - Patents.com Essentially any method for detecting multiple (e.g., at least two) polypeptides that are sequestered by platelets and associated with one or more cancer functions described herein can be used with the methods described herein. Detection of platelet-sequestered proteins can be carried out using any method known in the art. Liquid-based biopsy, microfluidic device, lateral flow assay, or test strip is preferred for detecting and contrasting the amount of cancer stimulators and inhibitors, but the isolation and detection of such proteins by other means is also expressly contemplated herein.

[0131] Capillary action lateral flow test strip etc.: The polypeptides representing one or more cancer functions can be detected using the test device or kit described herein.In one embodiment, the test device comprises a sample receiving zone, to which platelet or blood sample from subject is added, and (ii) a conjugate zone, which comprises a plurality of labeled binding reagents, each of which specifically binds to one of the polypeptides selected from RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, heat shock protein HSP 90-alpha isoform 2, MYCBP2, CD47, FKBP1A, tribbles homolog 2, PF4, TSP-1, basic FGF, VEGF, PDGF beta, integrin beta-3, CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6. In another embodiment, the testing device may include a conjugate zone that includes a plurality of labeled binding reagents that each specifically bind to one of the polypeptides listed in Figure 3 or 5. The testing device or kit may also include a solid support that defines a liquid flow path for the sample and includes test lines corresponding to each of a plurality (e.g., at least two) of polypeptides, each test line including (a) an immobilized additional binding reagent that also specifically binds to one of the plurality of polypeptides, thereby immobilizing the polypeptide at the test line and generating a signal via the labeled binding reagent also specifically bound to the polypeptide; or (b) an immobilized version of one of the plurality of polypeptides or analogs thereof that can compete with the polypeptide in the sample for specific binding to the labeled binding reagent. In some embodiments, the test lines for each polypeptide are spatially separated such that the level of each polypeptide can be measured and distinguished from the levels of the other polypeptide(s).

[0132] In certain embodiments, the boundaries of the sample receiving zone may be marked for the convenience of the user using one or more symbols, such as, for example, 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 in sufficient contact with the sample to be tested, but downstream components (e.g., the test line) are not.

[0133] The testing device or kit may further comprise at least one labeled control binding reagent that binds to a binding partner immobilized on a control line downstream of the test line(s) for at least one or more polypeptides, thereby confirming successful completion of the test. The control line may be spatially separated from the test line for each polypeptide. By way of example, in certain embodiments, the binding partner immobilized on the control line comprises BSA-biotin, and the labeled control binding reagent that binds to the immobilized binding partner comprises an anti-biotin antibody complexed to a gold particle.

[0134] The testing device may further comprise an absorbent material downstream of the test (and control, if present) line(s) for absorbing excess sample. In certain embodiments, the solid support comprises a chromatographic medium or a capillary flow device. The device may be provided in a test strip format in some embodiments.

[0135] In certain embodiments, downstream of the test line (and control line and / or absorbent material, if present), a user-held area is provided. Thus, the user can easily manipulate the testing device without disturbing the sample and its subsequent testing. The area may be called a "handle area" and may be made of any suitable material, such as plastic. The area may be visibly marked as a "handle area" or simply a "handle" for the convenience of the user.

[0136] In some embodiments, the testing device includes a dipstick or test strip with multiple testing zones, each capable of detecting a given polypeptide. When a dipstick or test strip is used, detecting a color change in the dipstick or test strip indicates the measurement of a particular analyte or polypeptide in each testing zone of the panel. Each testing zone may change the amount of colored light reflected from one of the dipstick's components or may change color based on a chemical reaction. For a negative result (i.e., the presence of a given polypeptide is not detected), the strip may remain its original color or may change to a particular color. For a positive result (i.e., the presence of a given polypeptide is detected), the strip may change to a color distinct from a negative result. An example is a strip that turns blue for a negative result and pink for a positive result. In some embodiments, the results are non-qualitative (color vs. no color), but differ by a degree corresponding to the level of the polypeptide present. For example, a dark color may indicate the presence of a high level of a particular polypeptide, and a light color may indicate the presence of a low level of the polypeptide.

[0137] In some embodiments, the dipstick or other dry chemistry device can be inserted into an instrument that quantifies the reflected light per test pad (preferably handheld), and the quantitative value can be recorded. In this manner, the amount of each polypeptide present can be determined to obtain further information regarding the subject's health. In other words, a polypeptide can be associated with, for example, a stage of cancer, not just its presence, but a decrease or increase in its level. Alternatively, the quantification device is included in the panel itself and is not a separate device.

[0138] The quantification device may include or be connected to a computer containing software that allows the data obtained to be used to perform an analysis in an analysis mechanism. The analysis mechanism may calculate the values ​​of each of the polypeptides in the test, perform normalization, and even calculate the relationship of the test results to each other, so that the presence and / or stage of cancer may be determined. The analysis mechanism may also search a database for facts that correlate high or low levels of the polypeptide with cancer, and the appropriate treatment thereof.

[0139] The presence or amount of platelet-sequestered protein can then be indicated to the user. The quantification device further includes an output mechanism for displaying the results in a manner meaningful to an individual, a clinical laboratory technician, or a medical professional. The display can be on a screen included on the panel and can include a printing mechanism for printing the results. Alternatively, the output mechanism can transmit the results via wireless signal or line to a PDA, a smart phone, or a remote computer for printing or display. The results can be incorporated into reports for individual wellness or cancer risk assessment, including but not limited to comparisons with values ​​and ratios calculated to be normal ranges previously established for the results of the test, normal healthy men and women of different ages, ethnic groups (if relevant), and / or other relevant parameters. Such reports also incorporate test history data for individual subjects obtained using the same method(s), which can be used to monitor the effectiveness of treatment.

[0140] A preferred use of a given panel of polypeptides is point-of-testing for cancer risk assessment or cancer staging assessment, which can be performed in a clinic or other medical practitioner. The panel can also be used by individuals to monitor their health in their own homes.

[0141] A desirable panel of platelet-sequestered polypeptides to test for can include the use of a single, easy-to-use, disposable test strip that contains multiple test zones and eliminates the need for individual assays for each of the various polypeptides discussed herein.

[0142] Furthermore, the integration of all of these tests onto a single platform allows for the collection of data from all results and for it to be compiled in a complementary manner such that data from individual test zones enhances the interpretation of other test zones on the strip. For example, the presence or amount of a single platelet-sequestered polypeptide described herein, when viewed alone, may or may not be indicative of the presence or stage of cancer, but when combined with information detailing the amount or presence of multiple other platelet-sequestered proteins, then it is possible to identify a subject as having cancer or a particular stage of cancer.

[0143] If the testing device is a lateral flow device, the testing device may further include visual aids such as printed documents (e.g., printed cards) that display different line intensity patterns that may allow a user (e.g., clinician) to interpret the results of the completed assay(s). As an example to illustrate the concept, when a polypeptide is detected via the sandwich assay format described herein, the lines may be graded (grade lines 1-10), with grade line 1 being the lightest colored line, followed by grade line 2 of increasing color intensity, and so on to grade line 10, which is the darkest (i.e., most strongly colored) line; grade lines 1 and 2 are calibrated to or below a predetermined threshold level, indicating that a particular polypeptide is present but within normal parameters, and therefore, cancer or its function is not detected, while grade lines 3-10 are calibrated above a predetermined threshold level, indicating that a particular polypeptide is present at an abnormally high level, and therefore, cancer may be detected and / or staged. Increasing the intensity of grade lines 3-10 allows a user to understand whether polypeptide levels are continually increasing abnormally, and therefore whether the cancer is progressing and / or the current treatment is ineffective, particularly when multiple samples obtained over time are analyzed using the monitoring methods described herein. A null grade line (grade line 0), in which no colored line is displayed on the visual aid, may also be provided to indicate the absence (or presence of negligible levels) of polypeptide derived from the platelet or blood sample.

[0144] In some embodiments, the detection of a given polypeptide or a group thereof is by enzyme activity, which can be measured directly in platelets or platelet protein samples or using lateral flow assays or liquid bioassays. The enzyme activity can be measured, for example, by detecting the processing of a substrate in the sample, which can be labeled. For example, the assay can be a fluorogenic substrate assay. Examples of suitable assay formats include the assays shown in International Patent Applications WO2009 / 024805, WO2009 / 063208, WO2007 / 128980, WO2007 / 096642, WO2007 / 096637, WO2013 / 156794, WO2015 / 059487, and WO2013 / 156795, the contents of each of which are incorporated herein by reference.

[0145] In some embodiments, the determined levels of the polypeptides are compared to corresponding threshold levels. This allows to identify increases (or decreases) relative to the threshold. The threshold level of a given polypeptide may be defined from a population test or may be specific to an individual (i.e., individualized or derived from a previous cancer risk or staging assessment). Although individualized levels may be more suitable for monitoring applications, monitoring is preferably also achieved by comparison with a predefined threshold level. The threshold levels may be set in relation to a training data set that includes samples defined in relation to a particular cancer status or stage. Thus, it is not necessary to measure the threshold levels for each assay according to the method performed. They may be preprogrammed into the reader device or provided for comparison purposes when performing such a method. Since the threshold levels may vary depending on the measurement technique applied, they are not described as fixed values, but may be determined by the skilled person once a particular measurement technique has been selected. If specific to an individual, the levels may reflect those in samples obtained from the subject at an earlier time point. Hence, the method may rely on an individualized baseline level of the relevant polypeptide or polypeptides from which the threshold is calculated. The calculation may be continuous, performed simultaneously with the test. Thus, the threshold value can be a continuously fluctuating threshold value obtained from a continuously fluctuating baseline.In this situation, it is clear that the level of polypeptide(s) does not need to be measured absolutely, but can be measured absolutely or relatively.The marker simply needs to be measured in a manner that allows comparison to be made with the polypeptide level in samples obtained at different times.

[0146] Alternatively, the threshold level for each marker can be set based on population analysis. The threshold level can be set to maximize the sensitivity and / or specificity of detection, as would be readily understood by one of skill in the art.

[0147] In some embodiments, the threshold level of a polypeptide is set by determining the level of the polypeptide in a sample obtained from a subject at an earlier time point.In its simplest form, the method may rely on a simple comparison between the level of the polypeptide in a test sample and a sample obtained earlier (i.e., a single earlier time point).On the other hand, an earlier time point may include at least two, and possibly three, four, five, six, seven, eight, nine, ten, etc., earlier measurements that are immediately prior to the determination of the level of the polypeptide in the current sample.

[0148] Isolation and detection of proteins sequestered by platelets: If desired, proteins can be isolated from platelets as described in Current Protocols in Immunology by FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, K. Struhl and VB Chanda (Editors), John Wiley & Sons, 2004., which is incorporated herein by reference. In one example described in WO02 / 077176, which is also incorporated herein by reference, the procedure generally involves the extraction of proteins in one solubilization step. The result of this procedure is intact proteins that are substantially free of cross-contamination. The isolated proteins maintain activity, allowing for analysis via any number of assays.

[0149] Buffers for protein isolation steps may include one or more of the following: buffer components, salt(s), detergents, protease inhibitors, and phosphatase inhibitors. Specifically, one buffer effective for extracting proteins to be analyzed by immunohistochemistry includes the buffers Tris-HCI, NaCI, detergents Nonidet (g) P-40, EDTA, and sodium pyrophosphate, protease inhibitors aprotinin and leupeptin, and phosphatase inhibitors sodium deoxycholate, sodium orthovanadate, and 4-2 aminoethylbenzenesulfonyl fluoride (AEBSF). Another salt that may be used is LiCl, while glycerol is a suitable emulsifier that may be added to the fractionation buffer. Additional optional protease inhibitors include soybean trypsin inhibitor and pepstatin. Other suitable phosphatase inhibitors include phenylmethylsulfonyl fluoride, sodium molybdate, sodium fluoride, and beta-glycerol phosphate.

[0150] While simple dissolution in I% SDS solution is effective for 2-D gel analysis, radical analysis using the SELDI® process requires the detergents Triton-X-100 (Sigma, St. Louis, Mo.), MEGA109 (ICN, Aurora, Ohio), and octyl B-glucopyranoside (ESA, Chelmsford, Mass.) in a standard PBS base. Alternative buffers used prior to 2-D gel analysis were 7 M urea, 2 M thiourea, CHAPS, MEGA10, octyl B-glucopyranoside, Tris, DTT, tributylphosphine, and Pharmalytes.

[0151] Once the protein is solubilized, the isolated protein can be characterized using several different immunological or biochemical analyses.Methods for analysis by ELISA and Western blot are known to those skilled in the art and are further described in "Current Protocols in Molecular Biology by FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, K. Struhl and VB Chanda (Editors), John Wiley & Sons, 2004", which is incorporated herein by reference.Methods for performing mass spectrometry are known to those skilled in the art and are further described in Methods of Enzymology, Vol. 193: "Mass Spectrometry" (JA McCloskey, editor), 1990, Academic Press, New York.

[0152] The platelet-sequestered proteins described herein can be used to predict, indicate, diagnose, or monitor the 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 assays (ELISAs), fluorescence polarization immunoassays (FPIAs) and homogeneous immunoassays, point-of-care testing using conventional lateral flow immunochromatography (LFA), quantitative point-of-care testing using chemiluminescence, fluorescence, and magnetic particle determinations, as well as latex agglutination, biosensors, gel electrophoresis, mass spectrometry (MS), gas chromatography-mass spectrometry (GC-MS), nanotechnology-based methods, proximity extension assays (e.g., using DNA oligonucleotides linked to antibodies against target molecules that can be quantified with real-time polymerase chain reaction), slow-offrate-modified-aptamer (SOMAmer) assays, nanoscale needles, and the like. Such techniques may include immunofluorescence assays, enzyme immunoassays, radioimmunoassays, chemiluminescence assays, sandwich format assays, approaches using microfluidic or MEMS technology, re-engineering techniques (e.g., devices utilizing sensors for polypeptides used for telemedicine purposes), epitope-based techniques, other fluorescence techniques, microarrays, lab-on-a-chip, and rapid point-of-care screening techniques.

[0153] Exemplary proximity extension assays are commercially available from Olink (Uppsalla, Sweden), while exemplary slow dissociation modified aptamer reagent (SOMAmer) assays include the SOMAscan™ assay commercially available from SOMAlogic (Boulder, Colo.). Exemplary assays that use nanoscale needles that act as label-free biosensors functionalized with capture antibodies that change color upon binding to their targets (which are then quantified) are commercially available from companies such as NanoMosaic (Woburn, Mass.).

[0154] Other detection paradigms that can be used for this purpose include optical methods, electrochemical methods (voltammetric and amperometric techniques), atomic force microscopy, and radio frequency methods such as multipolar resonance spectroscopy. Examples of optical methods are both confocal and non-confocal microscopy, as well as fluorescence, luminescence, chemiluminescence, absorption, reflection, transmission, and birefringence or refraction detection (e.g., surface plasmon resonance, ellipsometry, resonant mirror methods, grating coupler waveguide methods, or interferometry).

[0155] In one embodiment, the sample can be analyzed using a biochip. A biochip generally comprises a solid substrate and has a generally flat surface to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Often, the surface of the biochip comprises a plurality of accessible locations, each of which has a capture reagent bound thereto.

[0156] "Protein biochip" refers to a biochip suitable for capturing polypeptides. Many protein biochips have been reported in the art. These include, for example, protein biochips produced by Ciphergen Biosystems, Inc. (Fremont, Calif.), Packard BioScience Company (Meriden Conn.), Zyomyx (Hayward, Calif.), Phylos (Lexington, Mass.), and Biacore (Uppsala, Sweden). Examples of such protein biochips are described in the following patents or published patent applications: U.S. Patent No. 6,225,047; PCT International Publication No. WO 99 / 51773; U.S. Patent No. 6,329,209, PCT International Publication No. WO 00 / 56934, and U.S. Patent No. 5,242,828.

[0157] In another embodiment, the polypeptides described herein are detected by mass spectrometry, a method that uses a mass spectrometer to detect gas phase ions. Examples of mass spectrometers are time-of-flight, magnetic, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer, and hybrids thereof. In one embodiment, the mass spectrometer is a laser desorption / ionization mass spectrometer. In laser desorption / ionization mass spectrometry, the analyte is placed on the surface of a mass spectrometry probe, a device that is suitable for connecting with the probe interface of a mass spectrometer and presenting the analyte to ionizing energy for ionization and introduction into the mass spectrometer. Laser desorption mass spectrometers use laser energy, typically from an ultraviolet laser, but also from an infrared laser, to desorb the analyte from the surface, volatilizing and ionizing it, making it available to the ion optics of the mass spectrometer.

[0158] In another embodiment, the mass spectrometry technique for use in detecting the polypeptides described herein is "surface-enhanced laser desorption ionization", or "SELDI". This refers to a method of desorption / ionization gas phase ion spectrometry (e.g., mass spectrometry) in which polypeptides are captured on the surface of a SELDI mass spectrometry probe, respectively. There are several versions of SELDI that are known to those skilled in the art and contemplated for use herein, including "affinity capture mass spectrometry", i.e., surface-enhanced affinity capture (SEAC), surface-enhanced neat desorption (SEND), and surface-enhanced photodissociative attachment and release (SEPAR).

[0159] In another embodiment, mass spectrometry can be used to capture the polypeptide on a suitable chromatographic resin. For example, the polypeptide can be captured on a cation exchange resin, such as CM Ceramic HyperD F resin, the resin is washed, the polypeptide is eluted, and detected by MALDI. Alternatively, the method can be preceded by fractionating the sample with an anion exchange resin before application to the cation exchange resin. In another alternative, the sample can be fractionated with an anion exchange resin and directly detected by MALDI. In yet another method, the polypeptide can be captured on an immunochromatographic resin that contains an antibody that binds to the polypeptide, the resin is washed to remove unbound material, the polypeptide is eluted from the resin, and the eluted polypeptide can be detected by MALDI or by SELDI. These methods are known to those skilled in the art and will not be described in detail herein.

[0160] In another embodiment, the polypeptides described herein can be detected and / or measured by immunoassays in which a specific capture reagent, such as an antibody or a binding fragment thereof, is bound to each of the polypeptides in the panel.Antibodies can be produced by methods well known in the art, for example, by immunizing an animal separately 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.

[0161] Also expressly contemplated herein are traditional immunoassays for detecting or measuring the level of polypeptide in a panel, including but not limited to sandwich immunoassays and other enzyme immunoassays, including ELISA or fluorescence-based immunoassays.In SELDI-based immunoassays, antibodies or other binding reagents against polypeptides are added onto the surface of MS probes, such as pre-activated protein chip arrays.Polypeptides can then be specifically captured on biochips and detected by mass spectrometry.

[0162] Detection of a given polypeptide may require the use of a label or detectable moiety. Such detectable moieties may be isotopic labels; magnetic, electrical, or thermal labels; colored or luminescent dyes; and enzymes, all of which allow for detection of the polypeptide(s). In various embodiments, detectable secondary labels are used. Secondary labels are indirectly detected, including, but not limited to, one of the following: binding partner pairs; chemically modifiable moieties; nuclease inhibitors; enzymes such as horseradish peroxidase, alkaline phosphatase, luciferase, and the like. In certain sandwich formats, enzymes may act as secondary labels attached to soluble capture ligands. In various embodiments, the system relies on detecting the precipitation of reaction products or a change in a property of the label, for example, color, for detection. Detection systems for colorimetry may include spectrophotometers, colorimeters, or other devices that measure the absorption or transmission of light at one or more wavelengths.

[0163] As described herein, the assessment of results can be qualitative or quantitative depending on the particular detection method used.

[0164] Although the methods described herein are exemplified using analysis at the protein level, it is expressly contemplated herein that processes in which expression of each member in the panel can be detected and determined at the mRNA level can also be used with the methods described herein.

[0165] Reference Level The results of a polypeptide or panel of polypeptides 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 method, or to a urine test strip or visual reference card for lateral flow style assays. Comparing the expression level or amount of each member of the panel of polypeptides in a biological sample from a subject suspected of having cancer to the expression level or amount of each member of the panel of polypeptides in a control biological sample (e.g., from a subject or population of subjects without detectable cancer) can allow for the diagnosis or staging of cancer in the subject. A control biological sample can also be a reference sample obtained from a subject at an earlier time point (e.g., during the initial diagnosis of a given cancer, or as a baseline before the start of cancer treatment) to allow monitoring of disease progression in the subject. For example, a control biological sample can be a sample obtained from a subject one month, two months, three months, six months, or one year before the sample to be tested. In other embodiments, a control biological sample or reference sample is obtained from a subject during or after administration of a given anti-cancer treatment. In other aspects, the reference sample can be a sample from a patient or a population of patients who do not have detectable cancer (i.e., a negative control). Alternatively, the reference sample can be a sample from a patient or a population of patients who have a known stage of cancer (e.g., prostate cancer), such as, for example, low-grade, moderate-grade, or advanced cancer (e.g., prostate cancer) or a particular stage such as T2a or T3a prostate cancer. It should be understood that there can be more than one reference in a given assay. Such a control can serve as a positive control to ensure that the assay works and / or to allow the "staging" of a given cancer. Thus, in other embodiments, the reference is from a subject known to have cancer. In one embodiment, the cancer is the same cancer that is being monitored using the methods described herein. In another embodiment, the reference is a different cancer than that being tested using the methods described herein.In some embodiments, the reference is derived from a subject with a second cancer or a subject with multiple different cancers.In other embodiments, one or more controls can include a known concentration (or range of concentrations) of each of the polypeptides in the panel to quantitatively detect the level of each polypeptide in the subject being tested.In certain embodiments, the positive control sample or negative control sample is a sample obtained or derived from the tissue or biological fluid or tumor corresponding to the sample to be analyzed according to the method described herein.This sample is typically derived from the same patient at the same time point or different time point.

[0166] In certain embodiments, the levels of one or more of the polypeptides described herein can be compared to a reference value or the level of the polypeptide in a control or reference sample to assess the "risk" of a subject for developing cancer. In other embodiments, the levels of the polypeptides in the panel described herein can be compared to a reference value to determine the prognosis (i.e., chance of survival) of a subject.

[0167] In another embodiment, the reference level for one or more polypeptides is established based on the polypeptide level in a sample obtained from an individual at an early time point, for example, before the start of treatment with a therapeutic agent. Such a method allows the skilled person to monitor the effectiveness of a given therapeutic agent. If the relative amount of the polypeptide in the biological sample changes favorably from the polypeptide level in the biological sample obtained from the same individual at an early first time point; that is, has a trend toward normal polypeptide level, the individual is determined to be responding to treatment for cancer. Similarly, if the polypeptide level in the biological sample changes with respect to the level in the individual obtained at an early time point or in relation to the control level, the disease state of the individual may progress. In some embodiments, if it is determined that the subject is worsening despite treatment with a given anti-cancer agent, the method provided herein allows the skilled person to change the subject to more aggressive chemotherapy treatment.

[0168] Algorithms and Normalization For embodiments that rely on measuring the levels of multiple polypeptides, the final detection or staging of cancer may require that the measured levels are integrated to ideally obtain a simple binary result that is easily interpreted. Using a suitable algorithm, data from the levels of multiple polypeptides (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 polypeptides in Table 5 or Figure 3 or 5) can be interpreted and applied to detect or stage cancer (e.g., prostate cancer). In some embodiments, the polypeptide levels may be interdependent, and thus the algorithm is based on this predicted relationship. In certain embodiments, the determined levels of multiple polypeptides are analyzed in a predetermined order to monitor the subject. This may provide a decision tree for detecting or staging cancer. The level of the first of the multiple polypeptides may affect the subsequent threshold required for other polypeptides in detecting or staging cancer, which will be easily understood by those skilled in the art. The output of the method may also guide future sampling and treatment of the subject.

[0169] In some embodiments, the determination level of a plurality of polypeptides (e.g., at least one to all up to 16 (including 16) polypeptides) is weighted. Weighting is a well-known method of applying a degree of relative importance to each polypeptide in a plurality of polypeptides. The algorithm can be a threshold-based algorithm as discussed herein.

[0170] The measured polypeptide levels can be combined using logistic regression, decision tree analysis, neural network, and / or machine learning. Logistic regression analysis involves building a statistical model that adds different markers together in a weighted manner. Similar to linear regression, which can be solved using the equation y=mx+c, logistic regression allows the addition of multiple polypeptides and only allows binary outcomes, so that y is replaced by logit (defined as In (odds) of being in the positive outcome group). Mathematically, the logistic regression equation is logit=(βnXn)+c. It uses the quantitative data of all polypeptides in a weighted manner in the calculation.

[0171] In decision tree analysis, an individual is evaluated for one polypeptide at a time until the individual reaches a terminal node that classifies the patient into a positive outcome group or a negative outcome group. This uses cutoff values ​​for each individual polypeptide and does not necessarily use all polypeptides in the algorithm, depending on the point at which the individual is categorized. Therefore, this type of analysis is suitable for the embodiment of the present invention in which the level of more than one polypeptide is determined.

[0172] The use of neural networks shows some similarity to logistic regression in that each node is the sum of the inputs (markers) multiplied by weights (beta coefficients). However, the summation is performed several times; there are several nodes, and the inputs to these nodes can be the nodes themselves rather than the measured levels. The nodes are initially input randomly with random weights, and then the difference between the predicted and observed outputs is calculated. If it is not zero (as is likely), the weights are changed in the preceding layer, and then in the layers before that, until the input variables are reached. The outputs are recalculated, the difference is calculated again, and the model weights are re-adjusted. This can continue indefinitely until the difference between the predicted and observed outputs is minimized.

[0173] Anti-cancer treatment Any anti-cancer therapy that is useful, has been used, is currently used, or can be used for the prevention, treatment, and / or management of cancer can be used to prevent, treat, and / or manage cancer according to the methods and assays described herein.Exemplary anti-cancer agents include, but are not limited to, peptides, polypeptides, fusion proteins, nucleic acid molecules, small molecules, mimetics, synthetic drugs, inorganic molecules, and organic molecules.Non-limiting examples of cancer therapy include chemotherapy, radiation therapy, hormonal therapy, biological therapy including anti-angiogenic therapy, targeted therapy, and / or immunotherapy, and surgery.In certain embodiments, a therapeutically effective regimen includes the administration of at least two therapies or a combination of at least two agents.

[0174] Examples of anti-cancer treatments include: acivicin; aclarubicin; acodazole hydrochloride; acronine; adzelesin; aldesleukin; altretamine; ambomycin; amethanthrone acetate; aminoglutethimide; amsacrine; anastrozole; anthracyclines; anthramycin; asparaginase; asperlin; azacitidine (Vidaza); azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide mesylate; bisphosphonates (e.g., pamidronate (Aredria), sodium clondronate). (Bonefos), zoledronic acid (Zometa), alendronate (Fosamax), etidronate, ibandronate, cimadronate, risedromate, and tiludromate); biceresin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calsterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; ciloremycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine (Ara-C); dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine (Dacogen); Demethylating agents, dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; EphA2 inhibitors; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; elbrozole; esorubicin hydrochloride; estramustine; estramustine sodium phosphate; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride;Histone deacetylase inhibitors (HDAC-I) hydroxyurea; idarubicin hydrochloride; ifosfamide; irmofosine; imatinib mesylate (Gleevec, Glivec); interleukin II (including recombinant interleukin II, i.e., rIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha-n1; interferon alpha-n3; interferon beta-Ia; interferon gamma-Ib; iproplatin; irinotecan hydrochloride; lanreotide acetate; lenalidomide (Revlimid); letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; anti-CD2 antibodies (e.g., siplizumab (MedImmune Inc.; International Publication No. WO 02 / 098370, which is incorporated herein by reference in its entirety); megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocalcin; mitochromine; mitogillin; mitomarcine; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxaliplatin; oxisuran; paclitaxel; pegaspargase; periomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxant ron hydrochloride;plicamycin;promestane;porfimer sodium;porfiromycin;prednimustine;procarbazine hydrochloride;puromycin;puromycin hydrochloride;pirazofurin;ribopurin;logretimide;safingol;safingol hydrochloride;semustine;simtrazene;sparphosate sodium;sparsomycin;spirogermanium hydrochloride;spiromustine;spiroplatin;streptonigrin;streptozocin;sulofenur;tallysomycin;tecogalan sodium;tegafur;teroxantrone hydrochloride;temoporfin;teniposide;teroxylon;testolactone;thiamiprine;thioguanine;thiotepa;tiazofurin;These include, but are not limited to, tirapazamine; toremifene citrate; trestron acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tuburozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrocidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride;

[0175] Additional exemplary anti-cancer agents include 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adzelesin; aldesleukin; ALL-TIC antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1 (anti ... protein-1);antiandrogens, prostate cancer;antiestrogens;antineoplastons;antisense oligonucleotides;aphidicolin glycinate;apoptotic gene modulators;apoptotic regulators;apurinic acid;ara-CDP-DL-PTBA;arginine deaminase;asulaculin;atamestane;atrimustine;axinastatin 1;axinastatin 2;axinastatin 3;azasetron;azatoxin;azatyrosine;baccatin Ill derivatives;balanol;batimastat;BCR / ABL antagonists beta-lactam derivatives;beta-arretin;betaclamycin B;betulinic acid;bFGF inhibitors;bicalutamide;bisantrene;bisaziridinylspermine;bisnafide;bisstraten A;bisceresin;breflate;bropirimine;budotitanium;buthionine sulfoximine;calcipotriol;calphostin C;camptothecin derivatives;canarypox IL-2;capecitabine;carboxamide-amino-triazole;carboxyamidotriazole;CaRest M3;CARN 700;cartilage-derived inhibitor;carzelesin;casein kinase inhibitor (ICOS);castanospermine;cecropin B;cetrorelix;chlorin;chloroquinoxaline sulfonamide;cicaprost;cis-porphyrin;cladribine;clomiphene analogues;clotrimazole;collismycin A;collismycin B;combretastatin A4;combretastatin analogues;conagenin;crambesidin 816;crisnatol;cryptophycin 8;cryptophycin A derivatives;curacin A;Cyclopentanthraquinone; cycloplatam; cypemycin; cytarabine ocphosphate; cytolytic factor, cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diazicon; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, dioxamycin; diphenylspiromustine; docetaxel; Docosanol; Dolasetron; Doxifluridine; Droloxifene; Dronabinol; Duocarmycin SA; Ebselen; Ecomustine; Edelfosine; Edrecolomab; Eflornithine; Elemene; Emiteflu; Epirubicin; Epristeride; Estramustine analogs; Estrogen agonists; Estrogen antagonists; Etanidazole; Etoposide phosphate; Exemestane; Fadrozole; Fazarabine; Fenretinide; Filgrastim; Finasteride; Flavopiridol; Frezelastine; Fluasterone; Fludarabine; Fluorodaunorunicin hydrochloride hydrochloride);forfenimex;formestane;fostriecin;fotemustine;gadolinium texaphyrin;gallium nitrate;galocitabine;ganirelix;gelatinase inhibitors;gemcitabine;glutathione inhibitors;HMG CoA reductase inhibitors (e.g., atorvastatin, cerivastatin, fluvastatin, lescol, lupitor, lovastatin, rosuvastatin, and simvastatin); hepsulfame; heregulin; hexamethylene bisacetamide; hypericin; ibandronate; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridone; imiquimod; immunostimulating peptides; insulin-like growth factor-1 receptor inhibitors; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-iropract; irsogladine;isobengazole; isohomohalichondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanrcotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibitory factor; leukocyte alpha interferon; leuprolide + estrogen + progesterone; leuprorelin; levamisole; LFA-3TIP (Biogen, Cambridge, Mass.; International Publication No. WO 93 / 0686 and U.S. Patent No. 6,162,432);liarozole;linear polyamine analogs;lipophilic disaccharide peptides;lipophilic platinum compounds;lissoclinamide 7;lobaplatin;lombricin;lometrexol;lonidamine;losoxantrone;lovastatin;loxoribine;lurtotecan;lutetium texaphyrin;lisofylline;lytic peptides;maytansine;mannostatin A;marimastat;masoprocol;maspin;matrilysin inhibitors;matrix metalloproteinase inhibitors;menogaril ;merbarone;meterelin;methioninase;metoclopramide;MIF inhibitor,mifepristone;miltefosine;mirimostim;mismatched double-stranded RNA;mitoguazone;mitolactol;mitomycin analogue;mitonafide;mitotoxin fibroblast growth factor-saporin;mitoxantrone;mofalotene;molgramostim;monoclonal antibody,human chorionic gonadotropin;monophosphoryl lipid A+myobacterium cell wall sk;mopidamol;multidrug resistance gene inhibitor,multiple tumor suppressor 1(multiple tumor suppressor 1) based therapy;mustard anticancer drugs;mycaperoxide B;mycobacterial cell wall extract;myriaporone;N-acetyldinaline;N-substituted benzamides;nafarelin;nagrestip;naloxone + pentazocine;napavin;naphterpin;nartograstim;nedaplatin;nemorubicin;neridronic acid;neutral endopeptidase;nilutamide;nisamycin;nitric oxide modulators;nitroxide antioxidants;nitrullyn;O6-benzylguanine;Octreotide;Oxenone;Oligonucleotides;Onapristone;Oracin;Oral cytokine inducers;Ormaplatin;Osateron;Oxaliplatin;Oxaunomycin;Paclitaxel;Paclitaxel analogs;Paclitaxel derivatives;Palauamine;Palmitoyl rhizoxin;Pamidronic acid;Panaxytriol;Panomyphen;Parabactin;Pazeliptin;Pegaspargase;Perdecin;Pentosan polysulfate sodium;Pentostatin;Pentrozole;Perflubron;Perphosphamide;Perillyl alcohol;Phenazinomycin;Phenylacetic acid;Phosphatase inhibitors;Picibanil;Pilocarpine hydrochloride;Pirarubicin;Piritrexim;Placetin A (placetin A);Placetin B (placetin B);Plasminogen activator inhibitors;Platinum complexes;Platinum compounds;Platinum-triamine complexes;Porfimer sodium;Porfiromycin;Prednisone;Propyl bis-acridone;Prostaglandin J2;Proteasome inhibitors;Protein A-based immunomodulators;Protein kinase C inhibitors;Microalgae protein kinase C inhibitors;Protein tyrosine phosphatase inhibitors;Purine nucleoside phosphorylase inhibitors;Purpurin;Pyrazoloacridines;Pyridoxylated hemoglobin polyoxyethylene;raf antagonists;Raltitrexed;Ramosetron;Ras farnesyl protein transferase inhibitors;Ras inhibitors;Ras-GAP inhibitors;Demethylated reterliptin;Rhenium Re 186 etidronate; rhizoxin; ribozyme; RII retinamide; logretimide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintpin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetic; semustine; senescence derived inhibitor 1; sense oligonucleotide; signal transduction inhibitor; signal transduction modulator; gamma secretase inhibitor, single-chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; sorberol; somatomedin binding protein; sonermin;Sparfosic acid;Spicamycin D;Spiromustine;Splenopentin;Spongistatin 1;Squalamine;Stem cell inhibitors;Stem cell division inhibitors;Stipiamide;Stromelysin inhibitors;Sulfinosine;Superactive vasoactive intestinal peptide antagonists;Suradista;Suramin;Swainsonine;Synthetic glycosaminoglycans;Talimustine;S-fluorouracil;Leucovorin;Tamoxifen methiodide;Tauromustine;Tazarotene ;Tecogalan sodium;Tegafur;Terlapyrylium;Telomerase inhibitors;Temoporfin;Temozolomide;Teniposide;Tetrachlorodecaoxide;Tetrazomine;Taliblastine;Thiocoraline;Thrombopoietin;Thrombopoietin mimetics;Thymalfasin;Thymopoietin receptor agonists;Thymotrinan;Thyroid stimulating hormone;Ethyl etiopurinse;Tirapazamine;Titanocene bichloride bichloride); topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrophostin; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitor; urokinase receptor antagonists; vapreotide; variolin B; vector systems, erythrocyte gene therapy; thalidomide; veraresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; anti-integrin antibodies (e.g., anti-integrin αvβ3 antibodies); vorozole; zanoteron; zeniplatin; zilascorub; and zinostatin stimalamer.

[0176] In some embodiments, the anti-cancer agent is administered in combination with a steroid or other therapeutic agent.

[0177] Pharmaceutical Composition, Administration, and Efficacy A pharmaceutical or therapeutic composition comprising a therapeutic agent for the treatment of cancer may comprise a physiologically tolerable carrier, in which the therapeutic agent is dissolved or dispersed as an active ingredient(s). In a preferred embodiment, the pharmaceutical composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes. As used herein, the terms "pharmaceutical acceptable", "physiologically tolerable", and grammatical variations thereof, when referring to compositions, carriers, diluents, and reagents, are used interchangeably to denote that the material can be administered to or on a mammal without producing undesirable physiological effects, such as nausea, dizziness, stomach upset, and the like. A pharmaceutical acceptable carrier does not promote an enhanced immune response to the agent with which it is mixed, unless this is desired. The preparation of pharmacological or pharmaceutical compositions comprising active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on the method of manufacture. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, although solid forms suitable for dissolving or suspending in liquid prior to use may also be prepared. Preparations may also be emulsified or given as liposomal compositions. The active ingredient may be mixed with an excipient that is pharma- ceutically acceptable and compatible with the active ingredient, in an amount suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the composition may contain small amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient. Therapeutic compositions containing therapeutic agents for the treatment of cancer may contain pharma- ceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids, such as, for example, hydrochloric or phosphoric acid, or organic acids, such as acetic acid, tartaric acid, mandelic acid, and the like.Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0178] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no other material than active ingredient and water, or buffers such as phosphate buffered saline, including sodium phosphate, physiological saline, or both at physiological pH values. Furthermore, aqueous carriers can include more than one buffer salt, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions can also include liquid phases in addition to water or instead of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active agent used in the methods described herein that will be effective in treating cancer or its symptoms depends on the nature of the disorder or condition, and can be determined by standard clinical procedures.

[0179] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion.The preparation for injection can be provided in unit dosage form, for example, in ampoules or multi-dose containers, optionally containing added preservatives.The composition can be a suspension, solution or emulsion in oily or aqueous medium, and can contain compounding agents such as suspending agents, stabilizing agents and / or dispersing agents.

[0180] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions.

[0181] Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

[0182] Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, for example, a sterile pyrogen-free water-based solution, before use.

[0183] In some embodiments, the therapeutic agent can be delivered in an immediate release form. In other embodiments, the therapeutic agent can be delivered in a controlled or sustained release system. Controlled or sustained release pharmaceutical compositions can have a common goal of improving drug therapy over the results achieved by their non-controlled or non-sustained release counterparts. The advantages of controlled or sustained release compositions include extended activity of the therapeutic agent, reduced dosage frequency, and improved compliance. In addition, controlled or sustained release compositions can favorably affect other characteristics such as the time of onset of action or blood levels of the therapeutic agent, thus reducing the incidence of adverse side effects. Controlled or sustained release of active ingredients can be stimulated by various conditions, including but not limited to pH change, temperature change, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0184] In one embodiment, a pump may 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)). In another embodiment, polymeric materials may 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. Chem. 23:61 (1983); Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); and Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled or sustained release system may be placed near the target of infection, for example, the bone marrow, and thus require only a small fraction of the systemic dose.

[0185] When in tablet or pill form, the pharmaceutical compositions described herein can be coated (e.g., enteric coated) to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over an extended period of time. A selectively permeable membrane covering an osmotically active driving compound is also suitable for orally administered compositions. In these latter platforms, fluid from the capsule's surrounding environment is absorbed by the driving compound, which swells and displaces the agent or agent composition through an opening. These delivery platforms can provide an essentially zero-order delivery profile, as opposed to the spiked profile of immediate release formulations. Time-delay materials 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 aspect, the excipients are of pharmaceutical grade.

[0186] The pharmaceutical compositions described herein may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

[0187] The dosage range suitable for a given therapeutic agent depends on its efficacy, and includes a large enough amount to obtain the desired effect, such as the reduction of at least one symptom of cancer.The dosage of therapeutic agent should not be so large as to cause unacceptable or life-threatening harmful side effects, and should also be used under the strict supervision of medical professionals.In general, dosage will vary with the type of anticancer agent, as well as the age, condition and sex of the patient.Dosage can be determined by those skilled in the art, and can also be adjusted by individual physicians in the event of any complications.

[0188] Typically, dosages of a given therapeutic agent may range from 0.001 mg / kg to 5 g / kg of body weight. In some embodiments, dosage ranges are 0.001 mg / kg to 1 g / kg of body weight, 0.001 mg / kg to 0.5 g / kg of body weight, 0.001 mg / kg to 0.1 g / kg of body weight, 0.001 mg / kg to 50 mg / kg of body weight, 0.001 mg / kg to 25 mg / kg of body weight, 0.001 mg / kg to 10 mg / kg of body weight, 0.001 mg / kg to 5 mg / kg of body weight, 0.001 mg / kg to 1 mg / kg of body weight, 0.001 mg / kg to 0.1 mg / kg of body weight, 0.001 mg / kg to 0.005 mg / kg of body weight. Alternatively, in some embodiments, the dosage range is 0.1 g / kg body weight to 5 g / kg body weight, 0.5 g / kg body weight to 5 g / kg body weight, 1 g / kg body weight to 5 g / kg body weight, 1.5 g / kg body weight to 5 g / kg body weight, 2 g / kg body weight to 5 g / kg body weight, 2.5 g / kg body weight to 5 g / kg body weight, 3 g / kg body weight to 5 g / kg body weight, 3.5 g / kg body weight to 5 g / kg body weight, 4 g / kg body weight to 5 g / kg body weight, 4.5 g / kg body weight to 5 g / kg body weight, 4.8 g / kg body weight to 5 g / kg body weight. In one embodiment, the dosage range is 5 μg / kg body weight to 30 μg / kg body weight. Alternatively, the dosage range is set to maintain a serum level of 5 μg / mL to 30 μg / mL.

[0189] Currently available treatments, including experimental treatments, for cancer or symptoms thereof, and their dosages, routes of administration, and recommended usage are known in the art and / or described in references such as the Physician's Desk Reference (60th ed., 2017). For experimental treatments, appropriate dosages can be estimated based on dose-response modeling in animal models or in silico modeling of drug effects.

[0190] The administration of the above doses or doses used by a skilled clinician can be repeated for a finite and defined period of time. In some embodiments, the doses are given once a day or multiple times a day, for example, but not limited to, three times a day. Typically, the dosage regimen is determined based on the half-life of the agent and the minimum therapeutic concentration of the agent in blood, serum, or localized in a given biological tissue. In a preferred embodiment, the above doses are administered daily for several weeks or months. The duration of treatment depends on the subject's clinical progression and continued responsiveness to treatment. An initial higher therapeutic dose is followed by a continuous lower maintenance dose is contemplated.

[0191] A therapeutically effective amount is an amount of an agent sufficient to obtain a statistically significant measurable change in a given symptom of cancer (see "Efficacy Measurements" below). Such an effective amount can be determined in clinical trials and animal studies for a given agent. For example, a reduction in a given symptom of cancer can indicate adequate therapeutic effectiveness of the agent(s).

[0192] Agents useful in the methods and compositions described herein can be administered topically, intravenously (by bolus or continuous infusion), orally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavity, delivered by peristaltic means if desired, or by other means known to those skilled in the art. Agents can be administered systemically if desired.

[0193] Therapeutic compositions containing at least one therapeutic agent can be conventionally administered in unit doses. The term "unit dose", when used in relation to therapeutic compositions, refers to a physically discrete unit suitable as a single dosage for a subject, each unit containing a predetermined amount of therapeutic agent calculated to obtain a desired therapeutic effect in association with a necessary physiologically acceptable diluent, i.e., carrier or vehicle.

[0194] The composition is administered in a therapeutically effective amount in a manner compatible with the dosage formulation.The amount and timing of administration depend on the subject to be treated, the ability of the subject's system to utilize active ingredient, and the degree of desired therapeutic effect.The agent can be targeted by using targeting moiety such as antibody or targeted liposome technology.

[0195] The exact amount of active ingredient required to be administered depends on the judgment of the practitioner and is specific to each individual.Meanwhile, the dosage range suitable for systemic application is disclosed herein and depends on the route of administration.The regimen suitable for administration can also vary, but typically includes an initial administration, followed by one or more repeated doses at intervals by subsequent injections or other administrations.Alternatively, continuous intravenous infusion sufficient to maintain blood concentration within the range specified for in vivo treatment is contemplated.

[0196] In some embodiments, a combination of anti-cancer therapeutics is used in the treatment of cancer in a subject diagnosed as described herein.

[0197] In some embodiments, therapeutically effective agents are administered to a subject simultaneously with a combination therapy.As used herein, the term "simultaneously" is not limited to the administration of two or more agents at exactly the same time, but rather, intended to be administered to a subject in an order and time interval that allows them to act together (e.g., synergistically, so as to provide more benefit than if they were administered in other ways).For example, a combination of therapeutic agents can be administered at the same time, or sequentially in any order at different times in time; while if not administered at the same time, they should be administered close enough in time that desired therapeutic effect is obtained, preferably in a synergistic manner.The agents can be administered separately in any suitable form and by any suitable route.It will be understood that if each of the therapeutic agents in the combination is not administered in the same pharmaceutical composition, they can be administered to a subject in need thereof in any order. For example, a first therapeutic agent may 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 prior to), concomitantly therewith, 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) administration of a second therapeutic agent to a subject in need thereof, or vice versa. In other embodiments, delivery of either therapeutic agent ends before delivery of the other agent / treatment begins. In some aspects of either case, treatment is more effective because of the combined administration. For example, the therapeutic agents used in combination are more effective than would be seen with either agent alone. In some aspects, delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than would be seen with either therapeutic agent alone. The effect of such combinations can be partially additive, fully additive, or greater than additive.The agents and / or other therapeutic agents, procedures, or modalities may be administered during periods of active disease or may be administered during periods of persistent or reduced disease activity.

[0198] When administered in combination, one or more of the therapeutic agents may be administered in an amount or dosage that is greater than, less than, or the same as the amount or dosage of a given agent used individually, for example, as monotherapy.In certain embodiments, the dosage or dosage of a first therapeutic agent, when administered in combination with a second therapeutic agent, is less than the amount or dosage of the first agent when used individually (e.g., at least 20%, at least 30%, at least 40%, or at least 50%).In other embodiments, the amount or dosage of a first therapeutic agent, when administered in combination with a second therapeutic agent, produces a desired effect (e.g., improved cognitive function), which is less than the amount or dosage of the first (or second) agent required to achieve the same therapeutic effect when administered alone (e.g., at least 20%, at least 30%, at least 40%, or at least 50%).

[0199] The effectiveness of a given treatment for cancer can be determined by a skilled clinician. However, a treatment is considered to be "effective treatment", as the term is used herein, if any one or all of the signs or symptoms of cancer are changed in a beneficial manner, or other clinically recognized symptoms or markers of disease are improved or alleviated, for example, by at least 10%, after treatment with a therapeutic agent for cancer. Efficacy can also be measured by the absence of individual deterioration (i.e., the progression of disease is stopped or at least slowed down), as assessed by disease stabilization or the need for medical intervention. Methods for measuring such indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of disease in individuals or animals (some non-limiting examples include humans or mammals), including (1) suppressing disease, for example, stopping or slowing the progression of cancer, or (2) relieving disease, for example, causing regression of symptoms, and (3) preventing or reducing the likelihood of disease onset, or preventing secondary diseases / disorders associated with infection (e.g., anemia).

[0200] An effective amount for the treatment of a disease, as that term is defined herein, means that the amount is sufficient to provide effective treatment for the disease when administered to a mammal in need thereof. The effectiveness of an agent can be determined, for example, by assessing physical indicators of the disease, such as anemia, white blood cell level or identity, pain, fatigue, fever, and the like.

[0201] Treatment according to the method provided herein can reduce or eliminate one or more symptoms associated with cancer, such as fatigue, pain, tumor size, tumor growth, etc. In one embodiment, the cancer is prostate cancer, and one or more symptoms associated with prostate cancer include urination trouble, increased frequency of urination, pelvic pain or discomfort, decreased force of urination, difficulty in starting or stopping urine flow, blood in semen, and bone pain.

[0202] Devices and Kits For example, kits are provided herein that include quantification of individual platelet-associated polypeptides or a subset of platelet-sequestered proteins using whole blood-derived antibody tests targeting the proteins described herein that are sequestered by platelets. The kits may optionally include agents or devices for quantification of platelet proteins. For example, the kits may optionally include agents or devices for whole blood collection, lysis of platelets, such as (0.01% TritonX), and quantification, such as (by flow cytometry or chemical assay). In other examples, the kits may include one or more sterile applicators, such as syringes or needles. In addition, the kits may optionally include other agents, such as, for example, anesthetics or antibiotics. The kits may also include a package insert that instructs the user of the kit, such as, for example, (physician, laboratory technician), to carry out the methods disclosed herein.

[0203] In some embodiments, the testing device comprises a disposable, single-use device to which the sample is applied. Typically, such testing devices may comprise a sample receiving zone to which the sample is added. Such devices typically also incorporate a solid support that defines a liquid / capillary flow path for the sample when applied to the sample receiving zone. The sample receiving zone may be an integral part of the solid support. The solid support may comprise a chromatographic medium, such as a membrane material (e.g., nitrocellulose), in some embodiments. The sample applied to the sample application zone typically rehydrates the reagents required to detect the marker. The reagents include a binding reagent that specifically interacts with the polypeptide or a substrate for an effector molecule if activity is measured. An additional reagent that is further immobilized along the flow path binds to the complex of the polypeptide and the binding reagent. The binding reagent is labeled to generate a signal at the site of immobilization (through binding to the additional reagent) of the complex of the polypeptide and the binding reagent. Suitable labels include fluorescent labels, magnetic labels, latex, or gold, as will be readily understood by those skilled in the art.

[0204] When enzyme activity is assayed, the binding reagent and / or the further binding reagent can only bind to the substrate after it is modified by enzyme activity, or only when the substrate is not modified by enzyme activity. Examples of enzyme activity assays include those shown in International Patent Applications WO2009 / 024805, WO2009 / 063208, WO2007 / 128980, WO2007 / 096642, WO2007 / 096637, WO2013 / 156794, WO2015 / 059487, and WO2013 / 156795 (the contents of each of which are incorporated herein by reference).

[0205] The binding reagent and the additional reagent are typically antibodies.Therefore, in certain embodiments, one or more testing devices, testing kits, or testing substance compositions can include lateral flow test strips.In some embodiments, a single lateral flow test strip can be used to detect all the polypeptides to be determined in the test sample.In other embodiments, a separate lateral flow test strip is provided for each polypeptide to be determined.

[0206] The device, kit, or composition of matter may also include a control zone to confirm whether the sample has passed through the device successfully. If not, the system or the reader of the test kit or testing device may indicate to the user, for example via a display, that the result is invalid. The device, kit, or composition of matter may act as a competitive assay or a sandwich assay. ELISA (enzyme-linked immunosorbent assay) is an example of a suitable assay format that may be incorporated into the testing device used with the methods described herein. Also, typically, all the reagents for detecting the levels of multiple polypeptides are pre-loaded onto the testing device, kit, or composition of matter so that they can interact with the sample once they are added to the device (e.g., via the sample receiving zone). This minimizes intervention and therefore minimizes subject-induced errors. Thus, effectively, the device may require the user to only provide a sample and then observe the output of the assay.

[0207] The system, test kit, testing device, and testing substance composition may incorporate a reader (together with a processor and a storage medium) suitable for providing a quantitative output; this output may be an absolute or relative output. A suitable reader may incorporate an illuminator for exposing the device to light of a particular wavelength(s) and a detector suitable for reflecting or emitting light. The system, test kit, testing device, and testing substance composition may also incorporate a processor and computer application suitable for outputting a result based on the detection signal. Thus, the processor running the computer application is operatively connected to the reader. By "operable connection" is intended a functional connection that allows for the exchange of signals or information between elements.

[0208] One or more of the testing devices, kits, or compositions of matter may include enzyme detection devices. These devices may be particularly useful in determining enzyme activity.

[0209] A system or test kit may incorporate a suitable number of test devices to allow the determination of each polypeptide. This is particularly true when the polypeptides are detected using different platforms. Thus, in some embodiments, one or more test devices include one or more lateral flow activity assays, ELISAs, fluorogenic substrate assays, etc. In some embodiments, one or more test devices include one or more lateral flow activity assays, ELISAs, or competitive assays. In some embodiments, one or more test devices include one or more lateral flow assays and ELISAs.

[0210] Computer Systems Testing devices and kits are provided herein for carrying out the methods described herein.Computer applications for use with the systems and test kits are also provided herein.Computer applications can also be used in the testing devices or test kits described herein, for example by incorporating a reader.Thus, in certain aspects, computer-implemented methods, systems, and computer program products can be embodied in computer applications, such as in the context of a computer, for example by running a processor and executing thereon.

[0211] As used herein, a processor may be included within any computer, server, embedded system, or computing system. A computer may include various internal or auxiliary components, such as, for example, a system bus, system memory, storage media, input / output interfaces, and a network interface for communicating with a network.

[0212] The computer may be implemented as, for example, a conventional computer system, an embedded controller, a laptop, a server, a custom machine, any other hardware platform such as a laboratory computer or device, or any combination thereof. The computer may be, for example, a distributed system configured to function using multiple computers interconnected via a data network or bus system.

[0213] The processor may be configured to execute code or instructions to perform the operations and functionality described herein, manage request flow and address translation, perform calculations, and generate commands. The processor may be configured to manage and control the operation of components in a computing device. The processor may 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, gate logic, a discrete hardware component, any other processing unit, or any combination or multiplicity thereof. The processor may be a single processing unit, multiple processing units, a single processing core, multiple processing cores, a special purpose processing core, a coprocessor, or any combination thereof. According to certain example embodiments, the processor may be a virtualized computing device running in one or more other computing devices along with other components of the computing device.

[0214] The storage medium may be selected from flash memory, other non-volatile memory devices, solid state drives ("SSD"), any magnetic storage device, any optical storage device, any electrical storage device, any semiconductor storage device, any physically based storage device, any other data storage device, or any combination or multiple thereof. The storage medium may store one or more operating systems, application programs, and program modules, such as modules, data, or any other information. The storage medium may be part of the computer or connected to the computer. The storage medium may also be part of one or more other computers in communication with the computer, such as a server, a database server, a cloud storage device, a network-attached storage device, etc.

[0215] Thus, the storage medium may be an example of a machine-readable medium or computer-readable medium on which instructions or code may be stored for execution by a processor. Machine-readable medium or computer-readable medium may generally refer to any medium or media used to provide instructions to a processor. Such machine-readable medium or computer-readable medium associated with a module may include a computer software product.

[0216] An input / output ("I / O") interface may be configured to couple to one or more external devices, receive data from one or more external devices, and transmit data to one or more external devices. Such external devices in conjunction with various internal devices may also be known as peripheral devices. An I / O interface may include both electrical and physical connections for operatively coupling various peripheral devices to a computer or processor. An I / O interface may be configured to convey data, address, and control signals between the peripheral devices, the computer, or the processor. An I / O interface may be configured to implement any standard interface, such as Small Computer System Interface ("SCSI"), Serial Attached SCSI ("SAS"), Fibre 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 may be configured to implement only one interface or bus technology.

[0217] Alternatively, the I / O interface may be configured to implement multiple interface or bus technologies. The I / O interface may be configured to operate as part of, all of, or in conjunction with a system bus. The I / O interface may include one or more buffers for buffering transmissions between one or more external devices, internal devices, computers, or processors.

[0218] The I / O interfaces may connect the computer to a variety of input devices, including a mouse, touch screen, scanner, electronic digitizer, sensor, receiver, touch pad, track ball, camera, microphone, keyboard, any other pointing device, or any combination thereof. The I / O interfaces may connect the computer to a variety of output devices, including video displays, speakers, printers, projectors, haptic feedback devices, automation controls, robotic components, actuators, motors, fans, solenoids, valves, pumps, transmitters, signal emitters, lights, etc.

[0219] A computing device may operate in a networked environment using logical connections through a network interface to one or more other systems or computing devices across the network. A network may include a wide area network (WAN), a local area network (LAN), an intranet, the Internet, a wireless access network, a wired network, a mobile network, a telephone network, an optical network, or a combination thereof. A network may be packet-switched, circuit-switched, of any topology, and may use any communication protocol. Communication links within a network may include various digital or analog communication media, such as fiber optic cables, free space optical communications, wave guides, conductors, wireless links, antennas, radio frequency communications, and the like.

[0220] The processor may be connected to the other elements of the computing device or to various peripherals discussed herein via a system bus. It should be understood that the system bus may be within the processor, external to the processor, or both. According to some aspects, the processor, other elements of the computing device, or various peripherals discussed herein may be integrated into a single device, such as a system-on-chip ("SOC"), system-on-package ("SOP"), or ASIC device.

[0221] The embodiments may include computer programs embodying the functions described and illustrated herein, which are implemented in a computer system including instructions stored in a machine-readable medium and a processor that executes the instructions. However, it will be apparent that there may be many different ways to implement the embodiments in computer programming, and the embodiments should not be construed as being limited to any one set of computer program instructions. Moreover, a skilled programmer could write such a computer program to implement one or more of the disclosed embodiments described herein. Thus, the disclosure of a specific set of program code instructions is not deemed necessary for a proper understanding of how to make and use the embodiments. Furthermore, one skilled in the art will understand that one or more aspects of the embodiments described herein can be implemented by hardware, software, or a combination thereof, which may be embodied in one or more computing systems. Furthermore, any reference to an operation being performed by a computer should not be construed as being performed by a single computer, since more than one computer can perform the operation.

[0222] The embodiment described herein may be used with computer hardware and software to perform the methods and processing functions described herein. The systems, methods, and procedures described herein may be embodied in a programmable computer, computer executable software, or digital circuitry. The software may be stored on a computer-readable medium. For example, the computer-readable medium may include RAM, ROM, hard disk, removable media, flash memory, memory stick, optical media, magneto-optical media, CD-ROM, etc. The digital circuitry may include integrated circuits, gate arrays, building logic blocks, field programmable gate arrays (FPGAs), etc.

[0223] The methods, systems, test kits, testing devices, test kits, and testing substance compositions may incorporate a means for automatic identification and data capture (AIDC), such as a radio frequency identification tag or card (RIF).

[0224] In certain embodiments, the system, test kit, testing device, or testing kit further comprises a display for output from the processor. This is intended to provide a simple visual and / or audible readout of the assay performed on the sample. The display can be operatively connected to the processor running the computer application. The output or readout can be instructions to the subject in some embodiments. The output can be color-coded or quantified to reflect the various possible outcomes of the monitoring discussed herein. In some embodiments, a combination of both quantitative and qualitative information types can be presented. Thus, the display can present both quantitative and qualitative readouts in some embodiments. In some embodiments, the predicted outcome and the probability value for a particular outcome can also be output. The display is typically an integral part of the reader device.

[0225] In some aspects, the present technology may be defined in any of the following numbered paragraphs: 1. A method for diagnosing cancer in a subject, comprising: (a) measuring in a sample of platelets from the subject the level of a set of polypeptides that includes a subset of polypeptides that exhibit functions in cancer development, including each of malignant cell transformation, invasion, angiogenesis, and metastasis; (b) comparing the levels of the set of polypeptides measured in step (a) with the levels of the same set of polypeptides in a reference preparation of platelets, plasma, circulating cells, or with a reference value or range for each polypeptide. Including, a difference in the level of the polypeptide exhibiting one or more of the functions relative to the reference is indicative of a cancer status in the subject. The method. 2. The method of item 1, wherein the set of polypeptides includes a plurality of polypeptides associated with each function. 3. The polypeptide exhibiting a function in cancer development includes a cancer-stimulating and / or cancer-inhibiting polypeptide; When the balance of cancer stimulatory polypeptides versus cancer inhibitory polypeptides is imbalanced such that the cancer stimulatory polypeptides are increased and / or the cancer inhibitory polypeptides are decreased relative to the reference, cancer progression is indicated. Method 1 or 2. 4. The method of any one of items 1 to 3, wherein an increase in one or more cancer stimulatory polypeptides or a decrease in one or more cancer inhibitory polypeptides is indicative of cancer progression. 5. The method of any one of items 1 to 3, wherein a decrease in one or more cancer stimulatory polypeptides or an increase in one or more cancer inhibitory polypeptides indicates cancer regression or therapeutic response. 6. The method of any one of items 1 to 5, wherein the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in malignant cell transformation include one or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2. 7. The method of any one of items 1 to 6, wherein the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in malignant cell transformation include two or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2. 8. The method of any one of items 1 to 7, wherein the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in malignant cell transformation include each of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2. 9. The method of any one of items 1 to 8, wherein the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in cancer cell invasion include one or more of MYCBP2, CD47, FKBP1A, and Tribbles homolog 2. 10. The method of any one of items 1 to 9, wherein the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in cancer cell invasion include two or more of MYCBP2, CD47, FKBP1A, and tribbles homolog 2. 11. The method of any one of items 1 to 10, wherein the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in angiogenesis include one or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3. 12. The method of any one of items 1 to 11, wherein the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in angiogenesis include two or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3. 13. The method of any one of items 1 to 12, wherein the cancer is prostate cancer and the polypeptides exhibiting a function in angiogenesis include each of PF4, TSP-1, and integrin beta-3, and optionally basic FGF, VEGF, and / or PDGF beta. 14. The method of any one of items 1 to 13, wherein the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in metastasis include one or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6. 15. The method of any one of items 1 to 14, wherein the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in metastasis include two or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6. 16. The method of any one of items 1 to 15, wherein the method is for diagnosing prostate cancer and the polypeptides exhibiting a function in metastasis include each of CAMSAP2, SRGN, and synaptobrevin homolog YKT6, and optionally one or more of RANK-L, TGF-beta, or IGF-1. 17. The method of any one of items 1 to 16, wherein the aggressiveness or stage of the cancer is indicated by which of the polypeptides of said functional classes falls within a reference range for a subject having a particular stage of cancer. 18. The method of item 17, wherein the relative malignancy increases when markers from said functional classes are found to vary in the following order: polypeptides related to malignant cell transformation, polypeptides related to invasion, polypeptides related to angiogenesis, and polypeptides related to metastasis. 19. Administering an anti-cancer agent if a difference in the level of a polypeptide exhibiting one or more of said functions relative to said reference is indicative of cancer in said subject. 19. The method of any one of items 1 to 18, further comprising: 20. A device for diagnosing cancer, comprising: Reagents sufficient to detect the presence and / or amount of a set of polypeptides in a sample of platelets or platelet proteins from a subject, the set including a subset of polypeptides that exhibit functions in cancer development, including each of malignant cell transformation, invasion, angiogenesis, and metastasis. The device comprising: 21. The device of item 20, wherein the set of polypeptides includes multiple polypeptides associated with each function. 22. The device of item 20 or 21, wherein the polypeptides exhibiting a function in cancer development include cancer-stimulating polypeptides and cancer-inhibiting polypeptides. 23. The device of any one of items 20 to 22, wherein the polypeptide exhibiting a function in malignant cell transformation comprises one or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2. 24. The device of any one of items 20 to 23, wherein the polypeptides exhibiting a function in malignant cell transformation include two or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2. 25. The device of any one of items 20 to 24, wherein the polypeptides exhibiting a function in malignant cell transformation include each of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2. 26. The device of any one of items 20 to 25, wherein the polypeptide exhibiting a function in cancer cell invasion includes one or more of MYCBP2, CD47, FKBP1A, and tribbles homolog 2. 27. The device of any one of items 20 to 26, wherein the polypeptides exhibiting a function in cancer cell invasion include two or more of MYCBP2, CD47, FKBP1A, and tribbles homolog 2. 28. The device of any one of items 20 to 27, wherein the polypeptide exhibiting a function in angiogenesis includes one or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3. 29. The device of any one of items 20 to 28, wherein the polypeptides exhibiting a function in angiogenesis include two or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3. 30. The device of any one of items 20 to 29, wherein the polypeptides exhibiting a function in angiogenesis include each of PF4, TSP-1, and integrin beta-3, and optionally one or more of basic FGF, VEGF, and PDGF beta. 31. The device of any one of items 20 to 30, wherein the polypeptide exhibiting a function in metastasis comprises one or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6. 32. The device of any one of items 20 to 31, wherein the polypeptides exhibiting a function in metastasis include two or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6. 33. The device of any one of items 20 to 32, wherein the polypeptides exhibiting a function in metastasis include each of CAMSAP2, SRGN, and synaptobrevin homolog YKT6, and optionally one or more of RANK-L, TGF-beta, and / or IGF-1. 34. The device of any one of items 20 to 33, comprising a solid substrate comprising a reagent that allows detection of the presence and / or amount of the set of polypeptides. 35. The device of item 26, wherein the solid substrate comprises a lateral flow test strip, a microfluidic chamber, a urine test strip, a bead, or an enzyme-linked immunosorbent assay (ELISA). 36. The device of item 34 or 35, wherein the solid substrate is coated with a glycosaminoglycan. 37. A kit for detecting the presence and / or amount of a set of polypeptides, including a subset of polypeptides exhibiting functions in cancer development, including each of transformation, invasion, angiogenesis, and metastasis, in a sample of platelets or platelet proteins from a subject, comprising: a reagent sufficient to detect in a sample of platelets or platelet proteins from the subject the presence and / or amount of said set of polypeptides, said set including a subset of polypeptides that exhibit a function in cancer development, including each of transformation, invasion, angiogenesis, and metastasis; and Packaging materials for this purpose The kit comprising: 38. A kit for staging cancer, comprising: Reagents necessary to detect in a platelet sample the presence and / or amount of a plurality of sets of polypeptides in functional cancer development categories including each of malignant cell transformation, invasion, angiogenesis, and metastasis The kit comprising: 39. The kit of item 38, comprising at least one solid support containing sufficient reagents for detecting the presence and / or amount of the set of polypeptides. 40. The kit of item 39, wherein the solid support comprises a lateral flow test strip, a microfluidic chamber, a urine test strip, a bead, or an enzyme-linked immunosorbent assay (ELISA). 41. The kit of item 39 or 40, wherein the solid substrate is coated with a glycosaminoglycan. 42. The kit of any one of items 38 to 41, comprising a reagent for detecting actin. 43. The kit of any one of items 38 to 42, wherein the polypeptide exhibiting a function in malignant cell transformation comprises one or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2. 44. The kit of any one of items 38 to 43, wherein the polypeptide exhibiting a function in malignant cell transformation comprises two or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2. 45. The kit of any one of items 38 to 44, wherein the polypeptides exhibiting a function in malignant cell transformation include each of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2. 46. ​​The kit of any one of items 38 to 45, wherein the polypeptide exhibiting a function in cancer cell invasion comprises one or more of MYCBP2, CD47, FKBP1A, and tribbles homolog 2. 47. The kit according to any one of items 38 to 46, wherein the polypeptide exhibiting a function in cancer cell invasion includes two or more of MYCBP2, CD47, FKBP1A, and tribbles homolog 2. 48. The kit of any one of items 38 to 47, wherein the polypeptide exhibiting a function in angiogenesis comprises one or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3. 49. The kit of any one of items 38 to 48, wherein the polypeptide exhibiting a function in angiogenesis includes two or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3. 50. The kit of any one of items 38 to 49, wherein the polypeptides exhibiting a function in angiogenesis include each of PF4, TSP-1, and integrin beta-3, and optionally one or more of basic FGF, VEGF, and PDGF beta. 51. The kit of any one of items 38 to 50, wherein the polypeptide exhibiting a function in metastasis comprises one or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6. 52. The kit of any one of items 38 to 51, wherein the polypeptide exhibiting a function in metastasis comprises two or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6. 53. The kit of any one of items 38 to 52, wherein the polypeptides exhibiting a function in metastasis include each of CAMSAP2, SRGN, and synaptobrevin homolog YKT6, and optionally one or more of RANK-L, TGF-beta, and / or IGF-1. 54. A solid support comprising reagents sufficient to detect in a sample of platelet proteins from a subject a polypeptide in each of a set of functional cancer development categories including malignant cell transformation, invasion, angiogenesis, and metastasis. 55. The solid support of item 54, wherein reagents sufficient to detect the presence and / or amount of said set of polypeptides are disposed on said support in four regions, one region each for reagents sufficient to detect polypeptides in functional carcinogenesis categories including malignant cell transformation, invasion, angiogenesis, and metastasis, each region containing reagents sufficient to detect the presence and / or amount of one or more polypeptides in one of the respective functional carcinogenesis categories. 56. The solid support of item 55, wherein each region contains sufficient pooled reagents to detect the presence of multiple polypeptides in a respective functional carcinogenesis category. 57. The solid support of item 55 or 56, wherein the amount of detectable signal in each region upon detection of the set of polypeptides in a platelet or platelet protein sample provides an indication of the presence and / or stage of cancer in the subject from which the platelets were obtained. 58. A method for diagnosing or staging cancer in a subject, comprising the step of contacting a solid support of any one of items 54 to 57 with a sample of platelets or platelet proteins obtained from the subject, the contacting allowing detection of the presence and / or amount of platelet proteins in the sample in one or more of the functional cancer development categories. 59. A kit comprising the solid support of any one of items 54 to 57 and packaging materials therefor. 60. A method of monitoring a cancer treatment for effectiveness in a subject, comprising: (a) measuring in a first sample of platelets from the subject a level of a set of polypeptides that includes a subset of polypeptides that exhibit functions in cancer development, including each of malignant cell transformation, invasion, angiogenesis, and metastasis; (b) measuring the level of the set of polypeptides, the set including a subset of polypeptides that exhibit a function in cancer development, the subset including each of malignant cell transformation, invasion, angiogenesis, and metastasis, in a second sample of platelets from the subject taken after administration of a cancer therapy to the subject following step (a); and (c) comparing the levels of said polypeptide measured in steps (a) and (b). Including, a change in the level of a polypeptide in one or more of the subsets of polypeptides measured in steps (a) and (b) provides an indication of the effectiveness of the cancer therapy. The method. 61. The method of item 60, wherein the polypeptides exhibiting a function in cancer development include cancer-stimulating polypeptides and cancer-inhibiting polypeptides. 62. The method of item 61, wherein a decrease in the level of one or more cancer-stimulating polypeptides or an increase in the level of one or more cancer-inhibiting polypeptides measured in step (b) relative to that measured in step (a) indicates that the cancer therapy is effective. 63. The method of item 60, wherein a positive prognostic change in the level of a polypeptide representing malignant cell transformation, invasion, angiogenesis, or metastasis indicates that the therapy is effective, and a negative prognostic change in the level of a polypeptide representing malignant transformation, invasion, angiogenesis, or metastasis indicates that the therapy is not effective. 64. continuing to administer the therapy at its current dosage and frequency, or reducing the dosage and / or frequency of administration of the therapy, if the comparing step indicates that the therapy is effective; or increasing the dosage and / or frequency of the therapy, or administering another therapy alone or in combination with the therapy, if the comparing step indicates that the therapy is ineffective. The method of any one of items 60 to 63, further comprising:

[0226] The present technology is further illustrated by the following examples, which should not be construed as limiting. The contents of all references cited throughout this application, as well as figures and tables, are hereby incorporated by reference. EXAMPLES

[0227] Today, people who are fighting cancer, such as prostate cancer, have limited options for rapid, accurate, and reliable methods to monitor their disease. For example, current clinical methods for determining whether a prostate cancer patient should undergo surgery or respond to chemotherapy include frequent and inaccurate PSA tests, invasive and subjective digital examinations, painful tissue biopsies, or simply hoping that the patient will overcome the tumor, all of which can cause severe stress to patients who are already suffering. Provided herein is a method for efficiently and accurately identifying and monitoring diseases, such as cancer. By evaluating platelets in a small amount of blood (e.g., a teaspoon), the inventors have identified a series of proteins whose presence in platelets strongly indicates certain types of cancer, such as breast, prostate, and colorectal cancer. Furthermore, the inventors have mapped and measured the "in platelets" or "platelet sequestration" concentrations of certain proteins and protein combinations in prostate cancer patients. These protein combinations allow for a high degree of certainty and predictability regarding the stage, rate of evolution, and / or regression of the disease.

[0228] Tumors can be understood as "wounds that never heal" [1], suggesting that understanding wound healing may lead to clues to inhibit tumor growth. A major component of wound healing in tumor biology is cellular fragments called platelets. Platelets have been shown to naturally adhere to tumors and regulate tumor growth. Platelets from healthy human subjects contain primarily tumor suppressors, suggesting that platelets may act as a surveillance system in tumor dormancy while promoting tumor growth as cancer progresses. We have shown for the first time that platelets actively sequester and concentrate a variety of specific tumor angiogenesis-related proteins against a concentration gradient. This sequestration represents a novel and more accurate source of proteins that are highly suitable for the detection of proteins that are differentially expressed between cancer patients and healthy controls ([2-4]). However, previous studies of platelet proteins have not characterized this ability of platelets to be utilized as a source of polypeptides, nor the methodology for diagnosing cancer in a precise stage-specific manner. Utilizing platelets from healthy subjects and platelets from prostate cancer patients via discovery-based mass spectrometry approaches, we identified platelet protein sequestration and novel stage-specific domains of prostate polypeptides. We identified novel domains associated with specific sequestration by platelets, including heparan sulfate binding domain, C-type lectin binding domain, p-selectin binding domain, elastin binding domain, and intracellular plexin-D1 binding domain. We also identified 70 proteins selectively sequestered by platelets to specifically distinguish platelets from healthy subjects and platelets from subjects with stage T2a prostate cancer. We also identified 68 proteins selectively sequestered by platelets that selectively distinguish healthy subjects from stage T3 prostate cancer patients. Finally, we identified 31 proteins selectively sequestered by platelets that selectively distinguish stage T2 prostate cancer patients from stage T3 prostate cancer patients.

[0229] Currently, serum and plasma proteins are being evaluated as sources and methodologies of clinically applicable polypeptides for cancer detection and monitoring due to their ease of use [5]. While reliable for detection and outcome of late-stage disease, large tumors [6]; [7], these proteins do not correlate with early tumor growth and / or tumor invasion [8]. The search for serum or plasma proteins that are expected to be differentially expressed between patients with or without cancer continues. However, plasma and serum analysis is hampered by the vast number of proteins found in any given specimen and the large dynamic range over which they are found [9]. Platelets are anucleated cell fragments produced by bone marrow megakaryocytes and are released directly into the circulation by platelet precursor cell formation. Several platelet-derived proteins, such as platelet factor 4 (PF4) and platelet basic protein (PBP), are synthesized by megakaryocytes and concentrated in platelets during platelet formation [10-12]. Other proteins, such as albumin, IgG, and fibrinogen, are synthesized by other cells and taken up by platelets in the periphery [13-16]. Platelets can be isolated from whole blood by multiple centrifugations in clinical facilities, and contamination of isolated platelets with plasma proteins or other blood cells, such as red or white blood cells, is minimal. Platelets are easily separated from white blood cells prior to storage or processing, which reduces the confounding effects of other cell types. A major challenge in platelet research is ensuring that analysis of platelet protein content is performed on resting, inactivated platelets, since release of proteins from activated platelets can be variable. One example is the differential release of VEGF and endostatin upon agonist-induced platelet activation

[17] . Under normal physiological conditions, the sequestered protein content of platelets is fairly stable, but the methodology of isolation can affect the identity and concentration of sequestered proteins.Although several studies have now classified the various proteins present in resting platelets [18-20], they have not characterized stage-specific platelet proteins in prostate cancer or the use of platelet-sequestered proteins to accurately predict or indicate a specific stage of cancer. Active sequestration and release of many angiogenic regulatory proteins may play a critical role in angiogenesis and tumor survival [2, 21-25].

[0230] The present disclosure provides methods and compositions for use in the diagnosis and staging of precancerous lesions and prostate cancer, or conditions associated with prostate cancer and / or inflammation in a subject (e.g., a human subject) using platelet-selectively sequestered proteins. The methods include isolation of platelet-sequestered proteins from the subject (such as by isolation from whole blood, isolation from platelet-rich plasma, plateletpheresis, or differentiation from patient megakaryocytes, or by identification in whole blood (e.g., whole blood kits that test for platelet proteins)).

[0231] The present disclosure provides methods and compositions for use in the diagnosis and staging of precancerous lesions and prostate cancer, or conditions associated with prostate cancer and / or inflammation in a subject (e.g., a human subject) using platelet selectively sequestered proteins. In some embodiments, platelet sequestered proteins are quantified by mass spectrometry and / or antibody-based methods and / or kits, such as (ELISA, immunoblot, flow cytometry), and utilized to diagnose the presence and stage of prostate cancer. Protein concentrations are determined for a panel of desired polypeptides (e.g., those listed in Tables 1-3) in platelets from healthy individuals and individuals suspected of having a neoplasm associated with the prostate or another tissue described herein. The protein concentrations can then be normalized to platelet counts using a correction factor. The correction factor is derived from the ratio of platelet counts from a standard range of healthy patients. A statistically significant deviation of more than 20% using standard statistical methods, such as (ANOVA, Student's t-test, Tukey), is then utilized to determine the probability of the presence of prostate cancer and the clinical stage of prostate cancer.

[0232] The inventors identified proteins that are statistically significantly increased or decreased by more than 20% in platelets from 5 patients with stage T2 prostate cancer and 5 patients with stage T3 prostate cancer compared to platelets from 5 healthy individuals. The inventors have previously demonstrated the ability of platelets to selectively sequester proteins against a concentration gradient toward the physiological level. Previous studies have established the presence of angiogenesis regulators in platelets obtained from patients with cancer. However, these previous studies and claims have not established a mechanism of sequestration beyond heparan sulfate, have not identified non-angiogenesis-related proteins selectively sequestered by platelets, and have not established the association of platelet-associated proteins and specific stages of disease in humans. As described in Tables 1 and 2, the inventors identified proteins selectively sequestered in platelets that distinguish healthy individuals from individuals with stage T2 prostate cancer or individuals with stage T3 prostate cancer. The inventors have also identified platelet-sequestered proteins that distinguish between stage T2 and stage T3 prostate cancer.

[0233] Normalization: We normalized platelet-associated proteins between healthy subjects and individuals with prostate cancer. Platelet-sequestered proteins were isolated, digested, and quantified using mass spectrometry at two different locations and by two different methodologies. Only proteins identified by both methodologies and locations were analyzed. Proteins were quantified using standard "heavy" reference proteins of known concentration. Protein sequence identity was established from a minimum of three peptides identified as protein-specific by sequence alignment. Once protein identity was established, platelet protein levels were normalized by a normalization factor. The normalization factor was calculated for each individual patient from the number of platelets in one microliter of blood and multiplied by a correction factor so that each sample contained 200,000 platelets / uL of whole blood. Additional normalization methods included, but were not limited to, normalization to total protein, albumin, and normalization to sequestered proteins. Only proteins that were significant in multiple normalization methods were utilized.

[0234] Human subjects and preparation of human platelets and plasma: All samples were collected from healthy human volunteers or patients with stage T2 or T3 prostate cancer. The study was approved by the Institutional Review Boards of Tufts Medical Center, and study subjects provided written informed consent to participate in the study. Whole blood was collected in citrated vacutainer tubes and centrifuged at 150 g for 20 minutes to obtain platelet-rich plasma (PRP). To isolate platelets from platelet-poor plasma (PPP), PRP was centrifuged at 900 g for 10 minutes. Platelet counts were quantified using a hemocytometer and confirmed by flow cytometry from whole blood and PRP. Isolated platelets and PPP were frozen at -80°C until further analysis. Samples were obtained from five control individuals with no history of cancer and five individuals with stage T2a prostate cancer, as well as patients with stage T3 prostate cancer who underwent surgery due to a clinical diagnosis of prostate cancer. Initial diagnosis was determined via tissue biopsy along with tumor diagnosis or confirmed by postoperative pathology review of resected growths. Platelet counts were performed for all patients at the time of blood draw. Neither tumor nor normal patient samples were measured for inflammatory markers. Healthy volunteers had no history of cancer and were not using any anti-inflammatory medications.

[0235] Example 1. Platelet proteins that change between normal and stage T2 prostate cancer Identical isolated platelets and platelet-poor plasma normal and patient samples were analyzed by Waters SCIEX Mass Spectrometer and Thermo Orbitrap LC-MS / MS according to the manufacturer's recommended protocol. Only proteins that were not found in plasma or were greater or less than 100% greater in platelets compared to plasma were included. The Waters data contained 1369 unique protein IDs and the Thermo Orbitrap data contained 1199 unique protein IDs. A total of 442 proteins seen by both Waters and Thermo Orbitrap technologies were utilized in this study. Protein concentrations were normalized as described herein under the heading of normalization. Statistical significance was determined by either two-tailed Student's t-test or two-way ANOVA with Tukey's post-hoc test or repeated measures test (Prism GraphPad Software, La Jolla, CA) to ensure the validity of multiple comparison tests. For homogeneity, significance was assigned based on p<0.05. Only proteins that were present in all five patients and that were 20% more or less abundant in platelets from cancer patients compared to platelets from healthy subjects were utilized. Table 1 lists the proteins that differed between normal and stage T2a prostate cancer.

[0236] Table 2 lists proteins that were different between normal and stage T3 prostate cancer. Table 3 lists proteins that were different between stage T2 and stage T3 prostate cancer.

[0237] We compared proteins found in plasma with proteins found in platelets. Only proteins found in platelets and not in plasma were used. We identified each protein domain found in proteins sequestered by platelets. Only protein domains found in 5 or more proteins sequestered by platelets were utilized and are listed in Table 4.

[0238] Quantification of platelet-associated proteins. Due to the protein characteristics of platelets from cancer subjects, normalization to total protein amount can be problematic. In some embodiments, alternative methods are used to normalize platelet count. One exemplary method is to equalize platelet samples using β-actin, which is the protein with the least variability between platelets from normal subjects and platelets from cancer subjects.

[0239] Modifications and variations of the described disclosure that do not depart from the scope and spirit of the disclosure will be apparent to those skilled in the art. Although the disclosure has been described in connection with specific embodiments, it is understood that the claimed disclosure should not be unduly limited to such specific embodiments. Modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the disclosure. Additional embodiments are set forth in the claims.

[0240] References TIFF2025500074000011.tif98165TIFF2025500074000012.tif238165TIFF2025500074000013.tif111165

Claims

1. A method for diagnosing cancer in a subject, (a) A step of measuring the level of a set of polypeptides in a sample of platelets derived from the subject, which includes a subset of polypeptides representing functions in cancer development, including malignant cell transformation, invasion, angiogenesis, and metastasis; (b) A step of comparing the level of polypeptides in the set measured in step (a) with the level of polypeptides in the same set in a reference preparation of platelets, plasma, or circulating cells, or a reference value or range for each polypeptide. Includes, The difference in the level of polypeptides representing one or more of the functions relative to the reference indicates the cancer status in the subject. The aforementioned method.

2. The method according to claim 1, wherein the set of polypeptides comprises a plurality of polypeptides associated with each function.

3. The polypeptide exhibiting the function in cancer development includes cancer-stimulating and / or cancer-inhibiting polypeptides. Cancer progression is indicated when the balance between cancer-stimulating polypeptides and cancer-inhibiting polypeptides is disrupted, such that cancer-stimulating polypeptides increase and / or cancer-inhibiting polypeptides decrease relative to a reference. The method according to claim 1.

4. The method according to claim 3, wherein an increase in one or more cancer-stimulating polypeptides or a decrease in one or more cancer-inhibiting polypeptides indicates cancer progression.

5. The method according to claim 3, wherein a decrease in one or more cancer-stimulating polypeptides or an increase in one or more cancer-inhibiting polypeptides results in cancer regression or a therapeutic response.

6. The method according to claim 1, wherein the method diagnoses prostate cancer, and the polypeptide representing a function in malignant cell transformation comprises one or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

7. The method according to claim 1, wherein the method diagnoses prostate cancer, and the polypeptides representing function in malignant cell transformation include two or more of the following: RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

8. The method according to claim 1, wherein the method diagnoses prostate cancer, and the polypeptides representing a function in malignant cell transformation each include RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

9. The method according to claim 1, wherein the method diagnoses prostate cancer, and the polypeptide representing a function in cancer cell invasion comprises one or more of MYCBP2, CD47, FKBP1A, and Tribbles homolog 2.

10. The method according to claim 1, wherein the method diagnoses prostate cancer, and the polypeptides that represent a function in cancer cell invasion include two or more of MYCBP2, CD47, FKBP1A, and tribbles homolog 2.

11. The method according to claim 1, wherein the method diagnoses prostate cancer, and the polypeptide representing a function in angiogenesis comprises one or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3.

12. The method according to claim 1, wherein the method diagnoses prostate cancer, and the polypeptides representing functions in angiogenesis include two or more of the following: PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3.

13. The method according to claim 1, wherein the cancer is prostate cancer, and the polypeptide representing the function in angiogenesis comprises PF4, TSP-1, and integrin beta-3, respectively, and optionally basic FGF, VEGF, and / or PDGF beta.

14. The method according to claim 1, wherein the method diagnoses prostate cancer, and the polypeptide representing a function in metastasis comprises one or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

15. The method according to claim 1, wherein the method diagnoses prostate cancer, and the polypeptides representing function in metastasis include two or more of the following: CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

16. The method according to claim 1, wherein the method diagnoses prostate cancer, and the polypeptide representing a function in metastasis comprises each of CAMSAP2, SRGN, and synaptobrevin homolog YKT6, and optionally one or more of RANK-L, TGF-beta, or IGF-1.

17. The method according to claim 1, wherein the malignancy or stage of the cancer is indicated by which of the polypeptides of the functional class falls within a reference range for a subject having cancer of a particular stage.

18. The method according to claim 17, wherein relative malignancy increases when markers from the functional classes are found to vary in the order of polypeptides related to malignant cell transformation, polypeptides related to invasion, polypeptides related to angiogenesis, and polypeptides related to metastasis.

19. The step of administering an anticancer drug if the difference in the level of polypeptides representing one or more of the functions compared to the reference indicates cancer in the subject. The method according to any one of claims 1 to 18, further comprising:

20. A device for diagnosing cancer, A sufficient reagent to detect the presence and / or amount of a set of polypeptides, including a subset of polypeptides representing functions in cancer development, such as malignant cell transformation, invasion, angiogenesis, and metastasis, in a sample of platelets or platelet proteins derived from the subject. The device, including the device.

21. The device according to claim 20, wherein the set of polypeptides comprises a plurality of polypeptides associated with each function.

22. The device according to claim 20, wherein the polypeptide representing a function in cancer development includes a cancer-stimulating polypeptide and a cancer-inhibiting polypeptide.

23. The device according to claim 20, wherein the polypeptide representing a function in malignant cell transformation comprises one or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

24. The device according to claim 20, wherein the polypeptide representing a function in malignant cell transformation comprises two or more of the following: RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

25. The device according to claim 20, wherein the polypeptides representing a function in malignant cell transformation each include RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

26. The device according to claim 20, wherein the polypeptide representing a function in cancer cell invasion comprises one or more of MYCBP2, CD47, FKBP1A, and Tribbles homolog 2.

27. The device according to claim 20, wherein the polypeptides representing a function in cancer cell invasion include two or more of MYCBP2, CD47, FKBP1A, and Tribles homolog 2.

28. The device according to claim 20, wherein the polypeptide representing a function in angiogenesis comprises one or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3.

29. The device according to claim 20, wherein the polypeptides representing functions in angiogenesis include two or more of the following: PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3.

30. The device according to claim 20, wherein the polypeptide representing a function in angiogenesis comprises each of PF4, TSP-1, and integrin beta-3, and optionally one or more of basic FGF, VEGF, and PDGF beta.

31. The device according to claim 20, wherein the polypeptide representing the function in translocation comprises one or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

32. The device according to claim 20, wherein the polypeptide representing the function in translocation comprises two or more of the following: CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

33. The device according to claim 20, wherein the polypeptide representing the function in transfer comprises each of CAMSAP2, SRGN, and the synaptobrevin homolog YKT6, and optionally one or more of RANK-L, TGF-beta, and / or IGF-1.

34. The device according to any one of claims 20 to 33, comprising a solid substrate containing a reagent that enables detection of the presence and / or amount of polypeptides of the set.

35. The device according to claim 34, wherein the solid substrate comprises a lateral flow test strip, a microfluidic chamber, a urine test strip, beads, or an enzyme-linked immunosorbent assay (ELISA).

36. The device according to claim 34, wherein the solid substrate is coated with a glucosaminoglycan.

37. A kit for detecting the presence and / or amount of a set of polypeptides in a sample of platelets or platelet proteins of a subject origin, the set comprising a subset of polypeptides representing functions in cancer development, including transformation, invasion, angiogenesis, and metastasis, A reagent sufficient to detect the presence and / or amount of polypeptides in a sample of platelets or platelet proteins derived from the subject, including a subset of polypeptides representing functions in cancer development, such as transformation, invasion, angiogenesis, and metastasis, respectively, as well as Packaging materials for that purpose The kit includes the above.

38. A kit for cancer staging, Reagents required to detect the presence and / or quantity of multiple sets of polypeptides in platelet samples in functional carcinogenesis categories, including malignant cell transformation, invasion, angiogenesis, and metastasis. The kit includes the above.

39. The kit according to claim 38, comprising at least one solid support containing a reagent sufficient to detect the presence and / or amount of polypeptides of the set.

40. The kit according to claim 39, wherein the solid support comprises a lateral flow test strip, a microfluidic chamber, a urine test strip, beads, or an enzyme-linked immunosorbent assay (ELISA).

41. The kit according to claim 39, wherein the solid support is coated with a glycosaminoglycan.

42. The kit according to claim 38, comprising a reagent for detecting actin.

43. A kit according to any one of claims 38 to 42, wherein the polypeptide representing a function in malignant cell transformation comprises one or more of RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

44. The kit according to any one of claims 38 to 42, wherein the polypeptides representing a function in malignant cell transformation include two or more of the following: RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

45. The kit according to any one of claims 38 to 42, wherein the polypeptides representing a function in malignant cell transformation each include RAB1B, RAP1A, heat shock protein 70 kDa protein 1B, heat shock 70 kDa protein 6, heat shock 70 kDa protein 1-like, and heat shock protein HSP 90-alpha isoform 2.

46. The kit according to any one of claims 38 to 42, wherein the polypeptide representing a function in cancer cell invasion comprises one or more of MYCBP2, CD47, FKBP1A, and Tribbles homolog 2.

47. A kit according to any one of claims 38 to 42, wherein the polypeptides that represent a function in cancer cell invasion include two or more of MYCBP2, CD47, FKBP1A, and tribbles homolog 2.

48. The kit according to any one of claims 38 to 42, wherein the polypeptide representing a function in angiogenesis comprises one or more of PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3.

49. The kit according to any one of claims 38 to 42, wherein the polypeptides representing functions in angiogenesis include two or more of the following: PF4, TSP-1, basic FGF, VEGF, PDGF beta, and integrin beta-3.

50. The kit according to any one of claims 38 to 42, wherein the polypeptides representing a function in angiogenesis each include PF4, TSP-1, and integrin beta-3, and optionally one or more of basic FGF, VEGF, and PDGF beta.

51. The kit according to any one of claims 38 to 42, wherein the polypeptide representing a function in translocation comprises one or more of CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

52. A kit according to any one of claims 38 to 42, wherein the polypeptide representing a function in translocation comprises two or more of the following: CAMSAP2, SRGN, RANK-L, TGF-beta, IGF-1, and synaptobrevin homolog YKT6.

53. A kit according to any one of claims 38 to 42, wherein the polypeptide representing a function in transfer comprises each of CAMSAP2, SRGN, and the synaptobrevin homolog YKT6, and optionally one or more of RANK-L, TGF-beta, and / or IGF-1.

54. A solid support containing sufficient reagents to detect polypeptides in each of a set of functional carcinogenesis categories, including malignant cell transformation, invasion, angiogenesis, and metastasis, in a sample of platelet protein derived from a target.

55. The solid support according to claim 54, wherein a reagent sufficient to detect the presence and / or amount of the set of polypeptides is arranged on the support in four regions, each region being for a reagent sufficient to detect polypeptides in functional carcinogenesis categories including malignant cell transformation, invasion, angiogenesis, and metastasis, and each region containing a reagent sufficient to detect the presence and / or amount of one or more polypeptides in one of the respective functional carcinogenesis categories.

56. The solid support according to claim 55, wherein each region contains a pooled reagent sufficient to detect the presence of multiple polypeptides in their respective functional carcinogenesis categories.

57. The solid support according to claim 55, wherein the amount of detectable signal in each region when polypeptides of the set are detected in a sample of platelets or platelet proteins provides an indicator of the presence and / or stage of cancer in the subject from which the platelets were obtained.

58. A method for diagnosing or staging cancer in a subject, comprising the step of contacting a solid support according to any one of claims 54 to 57 with a sample of platelets or platelet proteins obtained from the subject, wherein the contact step enables the detection of the presence and / or amount of platelet proteins in the sample in one or more of the functional carcinogenesis categories.

59. A kit comprising a solid support according to any one of claims 54 to 57 and packaging materials therefor.

60. A method for monitoring the effectiveness of cancer treatment in a target group, (a) A step of measuring the level of a set of polypeptides in a first sample of platelets derived from the subject, which includes a subset of polypeptides representing functions in cancer development, including malignant cell transformation, invasion, angiogenesis, and metastasis, respectively; (b) measuring the levels of polypeptides in a subset of polypeptides representing functions in cancer development, including malignant cell transformation, invasion, angiogenesis, and metastasis, in a second sample of platelets derived from the subject, taken after administration of cancer therapy to the subject following step (a); and (c) A step of comparing the levels of the polypeptide measured in steps (a) and (b). Includes, Changes in polypeptide levels in one or more subsets of the polypeptide measured in steps (a) and (b) provide an indicator of the effectiveness of the cancer therapy. The aforementioned method.

61. The method according to claim 60, wherein the polypeptide representing the function in cancer development includes cancer-stimulating polypeptides and cancer-inhibiting polypeptides.

62. The method according to claim 61, wherein a decrease in the level of one or more cancer-stimulating polypeptides measured in step (b) relative to what was measured in step (a), or an increase in the level of one or more cancer-inhibiting polypeptides, indicates that the cancer therapy is effective.

63. The method according to claim 60, wherein a positive prognostic change in the level of polypeptides representing malignant cell transformation, invasion, angiogenesis, or metastasis indicates that the therapy is effective, and a negative prognostic change in the level of polypeptides representing malignant cell transformation, invasion, angiogenesis, or metastasis indicates that the therapy is ineffective.

64. If the comparison step indicates that the therapy is effective, the step of continuing to administer the therapy at the current dosage and frequency, or reducing the dosage and / or frequency of administration of the therapy; or If the comparison step indicates that the therapy is ineffective, the step of increasing the dosage and / or frequency of the therapy, or administering another therapy alone or in combination with the therapy. The method according to any one of claims 60 to 63, further comprising:

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