Compositions and methods for identifying and modulating thrombotic conditions in cancer patients

Abstract

The present invention relates to compositions and methods for identifying at risk patients and modulating thrombotic conditions in cancer patients. The examples provided herein include a method of determining the risk of a thrombotic event in a tumor patient, the method comprising: detecting in a sample of a cancer patient PPIA, PDIA3, and an elevated level of at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62 / 945,482, filed December 9, 2019. The contents of these applications are incorporated herein by reference in their entirety for all purposes. TECHNICAL FIELD

[0003] The present disclosure relates to methods for identifying and modulating thrombotic conditions in cancer patients.

[0004] GOVERNMENT SUPPORT

[0005] The present invention was made with government support under Grant Numbers HL112302 and HL143365 awarded by the NIH. The government has certain rights in the invention. BACKGROUND

[0006] Thrombosis is an important cause of death in cancer patients. Bick, N Engl J Med 349:109-111 (2003). For example, serious life-threatening thrombotic events occur in about 6% of cancer patients. Alguire et al., J Clin Oncol 2004 Vol 22 (July 15th Supplement) No. 14S:8082. Cancer patients often exhibit a hypercoagulable state in which the coagulation system has an increased propensity to clot. Rickles and Edwards, Blood 62:14-31 (1983). For at least some cancers, markers of hypercoagulability are associated with poor patient outcomes. Bick, Semin Thromb Hemostat 18:353-372 (1992); Buccheri et al., Cancer 97:3044-3052 (2003); Wojtukiewicz, Blood Coagul Fibrinolysis 3:429-437 (1992). Causes of hypercoagulability include the cancer itself and cancer therapy (e.g., chemotherapy). Hypercoagulability leads to an increased risk of thrombotic events, which can be further exacerbated when patients are bedridden. Anticoagulant therapy confers a survival benefit in some cancers when not contraindicated. Lebeau et al., Cancer 74:38-45 (1994); Chahinian et al., J Clin Oncol 7:993-1002 (1989). However, treatment options are often limited because many cancer patients are at high risk for major bleeding, which precludes the administration of anticoagulants that could otherwise be used prophylactically to reduce the risk of thrombosis. Thus, currently available methods of diagnosing and preventing thrombosis in cancer patients are unsatisfactory, and there is a need for new diagnostics and therapies. Such diagnostics and therapies would improve the survivability of cancer patients and promote better quality of life. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 Heat maps and UPR panels are shown that assess UPR markers in plasma of advanced cancer patients. SUMMARY

[0009] Embodiments provided herein include a method of determining the risk of a thrombotic event in a cancer patient, the method comprising:

[0010] detecting in a sample of a cancer patient an increase in the level of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 compared to a baseline, reference, or control level of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70; and diagnosing the patient as being at risk for a thrombotic event when PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 is elevated above the baseline, control, or reference level.

[0011] Other embodiments provided herein include a method of diagnosing and treating a thrombotic condition in a cancer patient, the method comprising the steps of: a. detecting in a sample of a cancer patient an increase in the level of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 compared to a baseline, reference, or control level of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70; b. diagnosing the patient as being at risk for a thrombotic condition when PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 is elevated above the baseline, control, or reference level; and c. treating the at-risk patient with an effective amount of isoquercitrin and optionally an anti-thrombotic agent.

[0012] Other embodiments provided herein include a method for monitoring a cancer patient undergoing treatment for a risk of a thrombotic condition, the method comprising the steps of: a. detecting in a sample of the cancer patient the level of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 compared to a baseline, reference, or control level of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70; b. diagnosing the patient as having a risk of a thrombotic condition when PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 is elevated above the baseline, control, or reference level; and c. treating the at-risk patient with an effective amount of isoquercetin and optionally an anti-thrombotic agent; wherein the monitoring is repeated at the indicated times per week, every two weeks, every month, or throughout the course of treatment.

[0013] In certain embodiments, the patient does not exhibit a serious adverse event (Grade 3 or 4 toxicity) during treatment.

[0014] In certain embodiments, the patient does not exhibit a primary venous thromboembolism (VTE) during treatment.

[0015] In certain embodiments, the patient does not exhibit a major bleeding during treatment.

[0016] Other embodiments provided herein include a kit comprising a biomarker panel comprising PPIA, PDIA3, and at least one of EIF5A, EIF4a3, EIF4H, UBE2N, UBE2L3, UBE2I, and HSP70 for diagnosing a thrombotic condition in a patient in need thereof.

[0017] Other embodiments provided herein include a kit comprising: (a) a solid support coated with polyclonal or monoclonal antibodies, wherein the antibodies comprise antibodies specific for PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70; (b) a polyclonal or monoclonal antibody-substrate conjugate, wherein the substrate comprises a chromogenic or fluorescent reagent, and wherein the conjugate is reactive with the antibodies of (a); and (c) at least one of PPIA, PDIA3, and EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 as an antigen standard.

[0018] In certain embodiments, the antibody of (a) further comprises an antibody specific for soluble P-selectin.

[0019] In certain embodiments, the solid support is a microtiter plate or a membrane. In certain embodiments, the solid support is a bead or particle. In certain embodiments, the kit is an ELISA kit. In certain embodiments, the solid support is a microbead array.

[0020] Further embodiments provided herein include a method of determining PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 in a serum or plasma sample, the method comprising contacting the sample with a solid support and a conjugate of a kit described herein; wherein the solid support comprises a microtiter plate, wherein the conjugate comprises alkaline phosphatase, wherein the chromogenic reagent comprises p-nitrophenylphosphate; and determining the reaction of the conjugate with the sample.

[0021] Other embodiments provided herein include a method of determining a combination of markers in a biological fluid sample obtained from a human subject, the method comprising performing an immunoassay by contacting the sample with a solid support of a kit described herein.

[0022] In certain embodiments, the immunoassay is an ELISA. In certain embodiments, the solid support is a microbead array. In certain embodiments, the sample is plasma or serum.

[0023] In certain embodiments, the method further comprises contacting the sample with a conjugate of the kit and determining the reaction of the conjugate with the sample.

[0024] In certain embodiments, the method further comprises contacting an antigen standard with the solid support and conjugate and determining the relative level of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 in the sample relative to the antigen standard.

[0025] Detailed Description

[0026] Before the compositions and methods of the present application are described, it is to be understood that this application is not limited to the particular process, dosage form, compositions, or methodologies described, as these can vary. It is also to be understood that the terminology used in the description is for the purpose of describing particular versions or embodiments only and is not intended to limit the scope of the embodiments herein, which will be limited only by the appended claims. Unless defined otherwise, 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 application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present application, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that embodiments herein are not entitled to antedate such disclosure by virtue of prior application.

[0027] It must also be noted that, as used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0028] The term "about" as used herein means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in a range of 45-55%.

[0029] As used herein, the term "subject" includes, but is not limited to, humans (also commonly referred to as "patients") and non-human vertebrates such as wild, domestic and farm animals. In certain embodiments, the subject described herein is an animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep or goat. In certain embodiments, the subject is a companion animal, such as a dog or cat. In certain embodiments, the subject is a livestock animal, such as a cow, pig, horse, sheep or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal, such as a rodent, dog or non-human primate. In certain embodiments, the subject is a non-human transgenic animal, such as a transgenic mouse or a transgenic pig.

[0030] The terms "treatment," "treated," or "treating" or "medical" as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to inhibit, arrest or slow down or reduce the full effects of any unwanted physiological condition, disorder or disease, or to improve, inhibit or otherwise achieve a beneficial or desired clinical outcome. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, improvement or remission of symptoms, diminishment of extent of condition, disorder or disease, stabilization (i.e., not worsening) of the state of condition, disorder or disease, delay or slowing of condition, disorder or disease progression, amelioration of the state of condition, disorder or disease, and remission, whether partial or total, whether detectable or undetectable. Treatment includes eliciting a clinically significant response without an unacceptably high level of side effects. Treatment also includes increasing the length of survival when compared to expected survival without treatment.

[0031] The terms "screening" and "screening" as used herein mean testing a subject or patient to determine whether they have a particular condition or disease or a particular manifestation of a condition or disease. The terms also mean testing an agent to determine whether it has a particular effect or efficacy.

[0032] The terms "identify," "distinguish," "recognize," and the like as used herein mean identifying a disease state or clinical manifestation or severity of a disease state in a subject or patient. The terms are also used in connection with testing agents and their ability to have a particular effect or efficacy.

[0033] The terms "predict," "forecast," "foretell," and the like as used herein mean foretelling on the basis of expert knowledge.

[0034] The terms "reference value" or "control value" as used herein mean the amount or quantity of a particular protein or nucleic acid in a sample from a healthy control or healthy donor or, in some cases, from a late stage cancer patient who has not exhibited a VTE or other thrombotic condition over a period of time.

[0035] The term "healthy control" is a human subject who does not have cancer or any other cancer-related condition.

[0036] The term "isolated," and the like, as used herein, means that the material to which reference is made is free of components, which normally accompany the material as it exists in its native environment. Specifically, an isolated biological material is free of cellular components. In the case of nucleic acid molecules, isolated nucleic acids include PCR products, isolated mRNA, cDNA, isolated genomic DNA, or restriction fragments. In another embodiment, an isolated nucleic acid is preferably excised from the chromosome on which it can exist. An isolated nucleic acid molecule can be inserted into a plasmid, cosmid, artificial chromosome, or the like. Thus, in a specific embodiment, a recombinant nucleic acid is an isolated nucleic acid. An isolated protein can be associated with other proteins or nucleic acids with which it is associated in a cell, or with the cell membrane if it is a membrane-bound protein. An isolated material can, but need not be, purified.

[0037] The term "purified," and the like, as used herein, refers to material that has been isolated under conditions that reduce or eliminate unrelated material, i.e., contaminants. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found in a cell. The term "substantially free of," as used herein, is operative in the context of analytical testing of materials. Preferably, a purified material that is substantially free of contaminants has a purity of at least 50%, more preferably a purity of at least 90%, more preferably a purity of at least 99%. Purity can be assessed by chromatography, gel electrophoresis, immunoassay, compositional analysis, biological assay, and other methods known in the art.

[0038] The term "expression profile" or "gene expression profile" refers to any description or measurement of one or more genes expressed by a cell, tissue, or organism under particular conditions or in response to particular conditions. An expression profile can identify genes that are up-regulated, down-regulated, or unaffected under particular conditions. Gene expression can be detected at the nucleic acid level or the protein level. An expression profile at the nucleic acid level can be achieved using any available technique that measures the level of gene transcripts. For example, the method can utilize in situ hybridization, Northern hybridization, or hybridization to nucleic acid microarrays such as oligonucleotide microarrays or cDNA microarrays. Alternatively, the method can utilize reverse transcriptase-polymerase chain reaction (RT-PCR), for example, quantitative real-time PCR (qPCR) based on fluorescent dyes. PCR). In the Example section provided below, nucleic acid expression profiles were obtained using Affymetrix oligonucleotide microarrays. An expression profile at the protein level can be achieved using any available technique that measures protein levels, for example, using an array of peptide-specific capture reagents.

[0039] The terms "gene signature" and "signature gene" are used interchangeably herein and mean a specific transcript that has been found to be differentially expressed in certain cancer patients.

[0040] UPR biomarkers

[0041] The nine UPR biomarkers (humans) that have been found to be elevated in plasma samples of patients with advanced cancer are PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3. These biomarkers have been found to be elevated in plasma samples from patients with advanced cancer who later exhibited VTE, as compared to the levels of the same UPR biomarker proteins that were not elevated in plasma samples from patients with advanced cancer who did not exhibit VTE (e.g., were monitored for VTE for 2 months, but in certain embodiments are desirably tested every two weeks or monthly throughout the treatment period), thus the latter serve as baseline reference samples. The elevated UPR protein levels are referred to herein as the UPR biomarker panel, which is predictive of the occurrence of a thrombotic condition such as VTE. In certain embodiments, the UPR biomarker panel comprises any combination of PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3. In certain embodiments, the UPR biomarker panel comprises at least one of PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3. In certain embodiments, the UPR biomarker panel comprises at least two of PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3. In certain embodiments, the UPR biomarker panel comprises at least three of PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3. In certain embodiments, the UPR biomarker panel comprises at least four of PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3. In certain embodiments, the UPR biomarker panel comprises at least five of PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3. In certain embodiments, the UPR biomarker panel comprises at least six of PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3. In certain embodiments, the UPR biomarker panel comprises at least seven of PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3.In certain embodiments, the UPR biomarker panel comprises at least eight of PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3. In certain embodiments, the UPR biomarker panel comprises PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3. In certain embodiments, the UPR biomarker panel comprises PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70. In certain embodiments, the UPR biomarker panel comprises PPIA, EIF4H, PDIA3, and at least one of EIF5A, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70. It is further noted that in alternative embodiments, the respective nucleic acid levels can be detected instead of the protein levels, and that the respective nucleic acid levels will also serve as predictive biomarkers for patients at risk of thrombotic events.

[0042] The amino acid sequence of PPIA can be found at P62937 and the Gene ID is 5478. The amino acid sequence of EIF5A can be found at P63241 and the Gene ID is 1984. The amino acid sequence of EIF4H can be found at Q15056 and the Gene ID is 7458. The amino acid sequence of EIF4a3 can be found at P38919 and the Gene ID is 9775. The amino acid sequence of UBE2N can be found at P61088 and the Gene ID is 7334. The amino acid sequence of UBE2L3 can be found at P68036 and the Gene ID is 7332. The amino acid sequence of UBE2I can be found at P63279 and the Gene ID is 7329. The amino acid sequence of HSP70 can be found at P0DMV8 / 9 and the Gene ID is 3303. The amino acid sequence of PDIA3 can be found at P30101 and the Gene ID is 2923.

[0043] The terms "gene," "gene transcript," and "transcript" are used somewhat interchangeably in this application. The term "gene," also referred to as "structural gene," means a DNA sequence that encodes or corresponds to a particular amino acid sequence comprising all or a portion of one or more proteins or enzymes, and can or can not include regulatory DNA sequences (e.g., promoter sequences) that determine, for example, under what conditions the gene is expressed. Some genes that are not structural genes can be transcribed from DNA into RNA but not translated into an amino acid sequence. Other genes can function as regulators of structural genes or regulators of DNA transcription. A "transcript" or "gene transcript" is an RNA sequence produced by transcription of a particular gene. Expression of a gene can thus be measured by a transcript.

[0044] The term "antisense DNA" is the non-coding strand that is complementary to the coding strand of double-stranded DNA.

[0045] The term "genomic DNA" as used herein means all DNA from a subject, including coding and non-coding DNA and DNA contained within introns and exons.

[0046] The term "nucleic acid hybridization" refers to the antiparallel hydrogen bonding between two single-stranded nucleic acids, in which A pairs with T (or U in the case of RNA nucleic acids) and C pairs with G. Nucleic acid molecules are "hybridizable" to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with a complementary base of another nucleic acid molecule under defined stringent conditions. Stringency of hybridization is determined, for example, by (i) the temperature at which the hybridization and / or washing is performed, and (ii) the ionic strength and (iii) the concentration of denaturing agent, such as formamide, and other parameters. Hybridization requires that the two strands contain substantially complementary sequences. However, depending on the stringency of hybridization, some degree of mismatch can be tolerated. Under "low stringency" conditions, a greater percentage of mismatches can be tolerated (i.e., without preventing formation of the antiparallel hydrogen bonders).

[0047] The term "inhibit" includes administration of a compound according to the embodiments described herein to prevent the onset of symptoms, to alleviate symptoms, or to eliminate a disease, condition, or disorder.

[0048] "Pharmaceutically acceptable" means the carrier, diluent or excipient must be compatible with the other ingredients of the formulation of the local dosage form and not deleterious to the recipient thereof.

[0049] The term "blood thinning drug" refers to an anti-platelet aggregation drug, such as clopidogrel bisulfate, heparin, warfarin, enoxaparin, abciximab, eptifibatide, tirofiban, prasugrel, ticlopidine, beraprost, prostacyclin, iloprost, treprostinil, aspirin, allopurinol, carbazochrome calcium, indobufen, triflusal, dipyridamole, picotamide, traxopromine, cilostazol, cloricromene, ditazol; or an anti-coagulant, such as acenocoumarol, brodifacoum, dicumarol, ethyl biscoumacetate, phenprocoumon, clorindione, anisindione, phenindione, tioclomarol, bemiparin, certoparin, dalteparin, nartograstim, parnaparin, reviparin, tinzaparin, fondaparinux, idraparinux, danaparoid, sulodexide, ocril, apixaban, betrixaban, edoxaban, otamixaban, rivaroxaban, bivalirudin, lepirudin, desirudin, argatroban, dabigatran, melagatran, hemopar, ximelagatran, defibrotide, ramatroban, antithrombin III, factor V inhibitor, factor IXa inhibitor, factor X inhibitor, factor XI inhibitor, factor XIII inhibitor, or ecarin.

[0050] The term "thrombotic disorder" refers to a number of different conditions that cause or increase the risk of a venous or arterial thrombotic event, including but not limited to atrial fibrillation, thrombosis caused by mechanical heart valves, myocardial infarction, unstable angina, deep vein thrombosis, acute ischemic stroke, pulmonary embolism, atherosclerosis, Factor V Leiden, antithrombin III deficiency, protein C deficiency, protein S deficiency, prothrombin gene mutation (G20210A), hyperhomocysteinemia, anti-phospholipid antibody syndrome, anti-cardiolipin antibodies, thrombotic syndrome, lupus anticoagulant syndrome, malignancy, major surgery, immobilization, use of oral contraceptives, use of thalidomide (especially in combination with dexamethasone), heparin-induced thrombocytopenia, pregnancy, myeloproliferative disorders, inflammatory bowel disease, nephrotic syndrome, paroxysmal nocturnal hemoglobinuria, hypercoagulability syndrome, Waldenstrom's macroglobulinemia, and trauma. The term "thrombotic disorder" also refers to thrombosis caused by cancer, such as multiple myeloma and other hematological cancers, adenocarcinoma, pancreatic cancer, gastric cancer, ovarian cancer, prostate cancer, colon cancer, lung cancer, brain cancer, breast cancer, kidney cancer, skin cancer, cervical cancer, and ear-nose-throat cancer.

[0051] "Vitamin B3" as referred to herein includes various different forms of Vitamin B3, including nicotinamide, nicotinic acid, niacinamide, inositol hexanicotinate.

[0052] As used herein, "vitamin C" includes vitamin C (i.e., L-ascorbic acid, D-ascorbic acid, or both) and salts thereof (e.g., sodium ascorbate).

[0053] As used herein, "folate" includes vitamin B9, folate salts, pteroylglutamic acid, 5-L-5-methyltetrahydrofolic acid, and L-methyl folate.

[0054] The term "improve" is used to indicate that the compounds or methods of the embodiments herein alter the appearance, form, characteristic, and / or physical property of the condition or tissue to which it is provided, administered, or dosed.

[0055] The terms "improve," "treat," and "alleviate" refer to the administration of an effective amount of the isoquercitrin, quercetin, or rutin composition of the present application to a subject in need of improvement in one or more of the above-mentioned conditions or having one or more of the just-mentioned disorders or one or more of the disorders and conditions, with the intent to improve one or more of these conditions, or to prevent, cure, alleviate, relieve, remedy, or ameliorate the symptoms or causes of one or more of these disorders or one or more of these disorders and conditions. The term "administration" encompasses oral or parenteral delivery of the quercetin, isoquercitrin, or rutin composition of the present application (or any suitable derivative thereof) to a subject in any suitable form (e.g., in the form of a food, beverage, tablet, capsule, suspension, and sterile injectable solution). The term "parenteral" refers to subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection, as well as various different infusion techniques. An "effective amount" refers to a dosage of the isoquercitrin, quercetin, or rutin composition sufficient to provide a therapeutic benefit (e.g., to reduce the level of PDI activity and / or soluble P-selectin in the serum in a patient in need, such as a cancer patient having a high level of soluble P-selectin). In certain embodiments, an effective amount of isoquercitrin is about 1000 mg. In certain embodiments, an effective amount of isoquercitrin can be in the range of about 1,000 mg - 2,000 mg. In other embodiments, an effective amount of isoquercitrin is in the range of about 2,000 mg - 2,500 mg. A particularly preferred effective amount of isoquercitrin is 1000 mg.

[0056] Methods of treatment

[0057] Thrombosis is a common complication of advanced cancer, including advanced solid tumor cancers as well as advanced hematological cancers. However, little is known about the underlying mechanisms linking tumor progression to clot formation.

[0058] The present invention relates to compositions and methods for identifying and / or monitoring at-risk patients and modulating thrombotic conditions in cancer patients, particularly in advanced cancer patients. Certain embodiments of the present invention relate to compositions and methods for identifying and / or monitoring at-risk patients and modulating viral-induced thrombotic conditions, genetically-induced thrombotic conditions, or anemia-induced thrombotic conditions in patients.

[0059] Thrombosis involves several sequential steps, which generally begin after a tear in the skin or a blood vessel injury. Platelets in the circulation first encounter the site of damaged endothelial cells, and then a series of events occurs that allows these platelets to become activated. The activated platelets then summon additional platelets to the site of injury, where they aggregate to form a plug until a stable clot forms. Inactive coagulation factors, which are always present in and circulating in the bloodstream, are sequentially activated in a process known as the coagulation cascade. The coagulation cascade ultimately produces a stable fibrin-containing clot.

[0060] Thrombotic disorders are a group of genetic and acquired disorders that cause abnormal activation of the hemostatic system, leading to an increased risk of venous and arterial thrombosis. Cancer is one of the acquired disorders that greatly increases the risk of thrombosis. Tumor cells cause a hypercoagulable state by expressing high levels of tissue factor on their surface. Tissue factor is required to initiate the coagulation cascade described above.

[0061] Factors involved in thrombosis include protein disulfide isomerase (PDI). PDI leaks from activated endothelial cells and platelets, after which it plays a critical role in thrombosis. PDI can activate tissue factor, leading to activation of the coagulation cascade, ultimately leading to fibrin deposition and thrombosis.

[0062] Protein disulfide isomerase is a thiol isomerase that is primarily localized to the endoplasmic reticulum, where it plays an essential role in protein folding. However, PDI can also be released from cells under disease states or after tissue injury and contribute to pathological processes. PDI has been implicated in cancer, neurodegenerative diseases, infectious diseases, and thromboembolism. In the case of thromboembolic diseases, PDI is released from activated platelets and endothelial cells and can modulate a number of extracellular coagulation substrates, such as factor XI, tissue factor, factor V, vitronectin, αΙΙbβ3, and ανβ3, through oxidation, reduction, or isomerization. Targeting PDI activity with blocking antibodies or small molecules has been shown to prevent platelet aggregation and fibrin generation at the site of vascular injury in several different animal models of thrombosis.

[0063] There is a need for additional methods and compositions for identifying at-risk patients and preventing and reducing venous or arterial thrombotic events, particularly in patients with advanced cancer, including solid tumor cancers and hematological cancers.

[0064] Thrombosis is a common complication of advanced cancer, including advanced solid tumor cancers as well as advanced blood cancers. However, little is known about the underlying mechanisms linking tumor progression to clot formation.

[0065] The present invention relates to compositions and methods for identifying at risk patients and modulating thrombotic conditions in cancer patients, particularly in advanced cancer patients. Although various different embodiments herein relate to cancer patients, the patients can also be patients not suffering from cancer. In certain embodiments, the patients have a virus-induced thrombotic condition, a genetically-induced thrombotic condition, or an anemia-induced thrombotic condition.

[0066] Certain embodiments of the present invention describe identifying a cancer patient as being at risk for a thrombotic event when a plasma sample from the cancer patient exhibits elevated levels of the UPR biomarkers PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3, or any combination or subset thereof, which are elevated in plasma samples from advanced cancer patients who later exhibit VTE, as compared to the levels of the same UPR biomarker proteins in plasma samples from advanced cancer patients who do not exhibit VTE and thus serve as baseline reference samples, wherein the method further comprises reducing or preventing the formation of a thrombus in the at risk cancer patient by administering to the at risk cancer patient an effective amount of isoquercitrin or a derivative compound according to any of the embodiments described herein or quercetin or a quercetin derivative compound or rutin or a rutin derivative compound. In certain embodiments, the cancer patient is a patient actively undergoing cancer treatment, including receiving chemotherapy and / or radiation and / or immunotherapy and / or cellular therapy.

[0067] Certain embodiments of the present application describe identifying a cancer patient as being at risk for a thrombotic event when a plasma sample from the cancer patient exhibits an elevated level of the UPR biomarkers PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70, which are elevated in plasma samples from advanced cancer patients who later exhibit VTE, as compared to the level of the same UPR biomarker proteins in plasma samples from advanced cancer patients who do not exhibit VTE and thus serve as baseline reference samples, wherein the method further comprises reducing or preventing the formation of a thrombus in the at risk cancer patient by administering to the at risk cancer patient an effective amount of isoquercitrin or a derivative compound according to any of the embodiments described herein or quercetin or a quercetin derivative compound or rutin or a rutin derivative compound. In certain embodiments, the cancer patient is a patient actively undergoing cancer treatment, including receiving chemotherapy and / or radiation and / or immunotherapy and / or cellular therapy.

[0068] In certain embodiments, a sample of biological tissue or bodily fluid is obtained from a subject having cancer. In other embodiments, a protein sample can be obtained from any biological tissue. Examples of biological tissues include, but are not limited to, epidermis, whole blood, and plasma. A protein sample can also be obtained from any bodily fluid. Examples of bodily fluids include, but are not limited to, plasma, saliva, and urine.

[0069] In certain embodiments, according to any of the methods described herein, the patient does not exhibit a serious adverse event (grade 3 or 4 toxicity) during treatment.

[0070] In certain embodiments, according to any of the methods described herein, the patient does not exhibit a primary venous thromboembolism (VTE) during treatment.

[0071] In certain embodiments, according to any of the methods described herein, the patient does not exhibit a VTE at least 30-60 days after treatment.

[0072] In certain embodiments, according to any of the methods described herein, the patient does not exhibit a major bleeding during treatment.

[0073] Rutin, Quercetin, Isoquercetin, and Related Derivatives

[0074] The terms "isoquercitrin," "quercetin," and "rutin" refer to certain active compounds used for administration described herein.

[0075] Isoquercitrin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)oxan-2-yl]oxychrom-4-one) is a 3-O-glucoside of quercetin having the following structure:

[0076]

[0077] Rutin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[a-L-rhamnopyranosyl-(1→6)-β-D- glucopyranosyloxy]-4H-chromen-4-one) is another common glycoside having the following structure with the disaccharide rutinose (a-L-rhamnopyranosyl-(1→6-β-D-glucopyranose) attached to the 3O position of quercetin:

[0078]

[0079] Quercetin is characterized as having the following structure:

[0080]

[0081] In embodiments described herein, the active compound can include quercetin or a quercetin derivative, such as: quercetin-5-O-glucoside, quercetin-7-O-glucoside, quercetin-9-O-glucoside, quercetin-3-O-[a-rhamnopyranosyl-(1→2)-a-rhamnopyranosyl-(1→6)]-β- glucoside, quercetin-3-O-galactoside, quercetin-7-O-galactoside, quercetin-3-O-rhamnoside, isoquercitrin, rutin, and quercetin-7-O-galactoside. Upon digestion, quercetin derivatives are converted in vivo to quercetin glycoside aglycone and / or other active derivatives, including methylated, sulfated, and glucuronated forms that are absorbed in vivo.

[0082] In certain embodiments described herein, the compound used in the methods of the application is isoquercitrin or quercetin. In certain embodiments, the compound is isoquercitrin. In certain embodiments, the compound is rutin. Suitable conjugates or derivatives include methylates, sulfates, and glucuronides.

[0083] In any of the embodiments described herein, the quercetin or quercetin derivative can be added to the composition in pure form or as a component in a mixture, such as a plant extract. Examples of commercially available quercetin include QU995 (containing 99.5% quercetin) and QU985 containing 98.5% quercetin) from Quercegen Pharmaceuticals LLC (Boston, Mass.). Examples of commercially available isoquercitrin compounds include ISQ 995 AN (99.5% pure full natural isoquercitrin) and ISQ 995 CIT (99.5% pure isoquercitrin) available from Quercegen Pharmaceuticals LLC. Other methods and isoquercitrin compositions can be found in U.S. Patent Nos. 7,745,486 and 7,745,487, which are incorporated herein by reference.

[0084] According to any of the embodiments described herein, the isoquercitrin, quercetin or rutin composition or any derivative thereof can be administered by oral or parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implantation) dosage forms, and can be formulated, either individually or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and vehicles appropriate for each route of administration. The compounds and compositions described herein can also be formulated into controlled release dosage forms.

[0085] According to any of the embodiments described herein, the isoquercitrin, quercetin or rutin composition or any derivative thereof can be administered in a wide variety of dosage forms, including, for example, solid dosage forms and liquid dosage forms. Solid dosage forms can include powders, tablets, pills, capsules, suppositories, or dispersible granules. The solid carrier can be one or more substances, which also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the carrier generally includes fine powdered material, such as powdered glucose, maltodextrin, or PVP. Examples of suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, hardeners, gelatin, tragacanth, methylcellulose, and sodium carboxymethylcellulose.

[0086] Liquid dosage forms include, for example, solutions, suspensions, and emulsions. Also included are compositions in solid form intended to be converted to liquid form shortly before ingestion. These forms can contain, in addition to the active ingredient, artificial coloring, flavoring agents, stabilizers, buffers, natural or artificial sweeteners, dispersants, thickeners, solubilizers, and the like.

[0087] The solution or mixture can be administered directly to the nasal cavity using conventional means, such as drops or spray. The compositions can be produced in single or multiple dose forms. Multiple dose forms will include droppers, pipettes, or nebulizers that deliver a predetermined volume of the composition.

[0088] According to any of the embodiments described herein, the isoquercitrin, quercetin or rutin composition or any derivative thereof can be provided in a single dosage unit containing an appropriate amount of the active ingredient.

[0089] The single dose can be provided in a package or as a kit that includes a measuring device, such as a device for measuring an oral or injectable dose (i.e., a graduated cup, a needle, or a syringe). The kit can also include other materials, such as buffers, diluents, filters, and package inserts with instructions for use. The kit can have a label indicating that the composition is used for a particular therapy, and can also indicate instructions for use.

[0090] If desired, the compositions of the present application can also include one or more additional active agents. In suitable cases, any active agent can be administered in the form of the compound itself and / or in the form of a salt, polymorph, ester, amide, prodrug, derivative, and the like, so long as the salt, polymorph, ester, amide, prodrug, or derivative is pharmacologically suitable. In suitable cases, salts, esters, amides, prodrugs, and other derivatives of the active agent can be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry, and described in, for example, J. March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4thedition (New York: Wiley-Interscience, 1992). For any active agent that can exist in enantiomeric form, the active agent can be incorporated into the compositions of the present application as a racemate or in enantiomerically enriched form.

[0091] In certain embodiments, the dose of isoquercitrin, quercetin or rutin composition or any derivative thereof to be administered will depend on the condition to be treated, the particular compound, and other clinical factors such as the age, sex, weight, and health of the subject to be treated, the route of administration of the compound, and the type of composition to be administered (tablet, gel cap, capsule, solution, suspension, inhalant, aerosol, elixir, lozenge, injection, patch, ointment, cream, etc.), according to any of the embodiments described. It should be understood that the present disclosure is applicable to human and animal use. The amount of quercetin or quercetin derivative required for treatment is ultimately determined by the attending physician or clinician, as appropriate, according to any of the embodiments described.

[0092] In certain embodiments, the isoquercitrin, quercetin or rutin composition or any suitable derivative thereof can be in a soft chewable composition comprising isoquercitrin, quercetin or rutin or suitable derivatives thereof, niacinamide, ascorbic acid, sodium ascorbate, folic acid, sugar, corn syrup, sucralose, soy lecithin, sunflower lecithin, corn starch, glycerin, palm oil, xylitol, carrageenan, FD&C Yellow #6, FD&C Yellow #5, or natural or artificial flavoring agents. Optionally, any of the quercetin, quercetin derivative, isoquercitrin, isoquercitrin derivative or rutin or rutin derivative compositions described herein can also comprise a group of vitamins such as vitamin B3, vitamin C and / or folic acid. An exemplary soft chewable composition (5.15 g) comprises 250 mg of isoquercitrin, 12.9 mg of vitamin B3 (i.e., niacinamide) and 382.8 mg of vitamin C (i.e., L-ascorbic acid and sodium ascorbate). In other exemplary embodiments, the components of the exemplary soft chewable are the same, except that the active pharmaceutical agent is replaced with 500 mg or 1000 mg of isoquercitrin. For example, a subject can take 1 to 8 (e.g., 4) of these soft chewable compositions per day. The amount taken can vary depending, for example, on the disorder or condition to be treated and the physical state of the subject. Another exemplary composition of such a soft chewable comprises 5.25 wt% of quercetin, 0.25 wt% of vitamin B3 and 7.81 wt% of vitamin C (i.e., L-ascorbic acid and sodium ascorbate) per chewable, and 200 μg of folic acid.

[0093] In certain embodiments, the isoquercitrin, quercetin or rutin is administered in a composition comprising vitamin B3, and the composition optionally further comprises vitamin C, optionally further comprises folic acid.

[0094] In certain embodiments, the isoquercitrin, quercetin or rutin is administered in a composition comprising about 20 micrograms to about 3 grams of vitamin B3, and the composition optionally further comprises about 200 micrograms to about 3 grams of vitamin C, optionally further comprises 1000 micrograms to about 3000 micrograms of folic acid (e.g., folate).

[0095] When the above compositions take the form of a powder, they can be conveniently used for preparing beverages, pastes, jellies, capsules or tablets. Lactose and corn starch are commonly used as diluents in capsules and as carriers in tablets. Lubricants such as magnesium stearate, are commonly used in tablets.

[0096] The oral bioavailability of isoquercitrin, quercetin or rutin in the above capsule or tablet formulations can be improved by using certain additives. For example, the capsule or tablet can comprise acid-treated gelatin, citrate, potassium hydroxide and / or cyclodextrin. The preferred amount of these additives per mg of isoquercitrin, quercetin or rutin is 0.01-0.5 mg of potassium hydroxide, 0.01-0.7 mg of acid-treated gelatin, 0.1-1 mg of citrate and 0.01-1 mg of cyclodextrin. In the presence of the additives, isoquercitrin, quercetin or rutin can have a solubility of 2-5% in aqueous solution. In addition, the pH of the isoquercitrin, quercetin or rutin-containing formulation with improved oral bioavailability can be between pH 7 and pH 12.

[0097] The isoquercitrin, quercetin or rutin composition administered in the methods of the present application can be a dietary supplement or a pharmaceutical formulation. As a dietary supplement, additional nutrients such as minerals or amino acids can be included. The pharmaceutical formulation can be a sterile injectable or infusible solution containing the isoquercitrin, quercetin or rutin composition and pharmaceutically acceptable excipients. The isoquercitrin, quercetin or rutin composition can also be a food. As used herein, the term "food" refers broadly to any kind of liquid and solid / semi-solid material used for nourishing humans and animals, maintaining normal or accelerated growth, or maintaining endurance or alertness. Examples of human foods include, but are not limited to, tea-based beverages, fruit juices, coffee, milk, jellies, cookies, cereals, chocolates, snack bars, herbal extracts, dairy products (e.g., ice cream and yogurt), soy products (e.g., tofu), and rice products.

[0098] The dosage of the compound as an active ingredient in the composition of the present application can be varied to accommodate desired dosage regimes. The active ingredient can be administered to a patient (animal and human) in need of such treatment in dosages that provide optimal therapeutic response. The selected dosage depends on the desired therapeutic effect, on the route of administration, and on the duration of the treatment. The dosage will vary from patient to patient depending on the nature and severity of disease, on the patient's body weight, on special diets followed by the patient, on concurrent medication, and on other factors that those skilled in the art will recognize.

[0099] In certain embodiments, the therapeutically effective amount will be about 500 mg up to 5 grams per day. In certain other embodiments, the therapeutically effective amount will be about 4 grams or 3 grams or even 2 grams. In certain embodiments, the therapeutically effective amount will be about 500 mg to about 2000 mg per day.

[0100] In some embodiments, the therapeutically effective dose is between the following lower and upper limits: the lower limit is about 500 mg / day, about 525 mg / day, about 550 mg / day, about 575 mg / day, about 600 mg / day, about 625 mg / day, about 650 mg / day, about 675 mg / day, about 700 mg / day, about 725 mg / day, about 750 mg / day, about 775 mg / day, about 800 mg / day, about 825 mg / day, about 850 mg / day, about 875 mg / day, about 900 mg / day, about 925 mg / day, about 950 mg / day, about 975 mg / day, about 1000 mg / day, about 1025 mg / day, about 1050 mg / day, about 1075 mg / day. g / day, approximately 1100mg / day, 1125mg / day, approximately 1150mg / day, approximately 1175mg / day, approximately 1200mg / day, 1225mg / day, approximately 1250mg / day, approximately 1275mg / day, approximately 1300mg / day, 1325mg / day, approximately 1350mg / day, approximately 1375mg / day, approximately 1400mg / day, 1425mg / day, approximately 1450mg / day, approximately 1475mg / day, approximately 1500mg / day, approximately 1525mg / day, approximately 1550mg / day, approximately 1575mg / day, approximately 1600mg / day, approximately 1625mg / day, approximately 1650mg / day, approximately 1675mg / day, approximately 1700mg / day Approximately 1725 mg / day, approximately 1750 mg / day, approximately 1775 mg / day, approximately 1800 mg / day, approximately 1825 mg / day, approximately 1850 mg / day, approximately 1875 mg / day, approximately 1900 mg / day, approximately 1925 mg / day, approximately 1950 mg / day, approximately 1975 mg / day, approximately 2000 mg / day, approximately 2025 mg / day, approximately 2050 mg / day, approximately 2075 mg / day, approximately 2100 mg / day, 2125 mg / day, approximately 2150 mg / day, approximately 2175 mg / day, approximately 2200 mg / day, 2225 mg / day, approximately 2250 mg / day, approximately 2275 mg / day, approximately 2300 mg / day, 2325 mg / day. Approximately 2350 mg / day, approximately 2375 mg / day, approximately 2400 mg / day, 2425 mg / day, approximately 2450 mg / day, approximately 2475 mg / day, approximately 2500 mg / day, approximately 2525 mg / day, approximately 2550 mg / day, approximately 2575 mg / day, approximately 2600 mg / day, approximately 2625 mg / day, approximately 2650 mg / day, approximately 2675 mg / day, approximately 2700 mg / day, approximately 2725 mg / day, approximately 2750 mg / day, approximately 2775 mg / day, approximately 2800 mg / day, approximately 2825 mg / day, approximately 2850 mg / day, approximately 2875 mg / day, approximately 2900 mg / day, approximately 2925 mg / day, approximately 2950 mg / day.about 2975 mg / day, about 3000 mg / day, about 3025 mg / day, about 3050 mg / day, about 3075 mg / day, about 3100 mg / day, 3125 mg / day, about 3150 mg / day, about 3175 mg / day, about 3200 mg / day, 3225 mg / day, about 3250 mg / day, about 3275 mg / day, about 3300 mg / day, 3325 mg / day, about 3350 mg / day, about 3375 mg / day, about 3400 mg / day, 3425 mg / day, about 3450 mg / day, about 3475 mg / day, about 3500 mg / day, about 3525 mg / day, about 3550 mg / day, about 3575 mg / day, about 3600 mg / day, about 3625 mg / day, about 3650 mg / day, about 3675 mg / day, about 3700 mg / day, about 3725 mg / day, about 3750 mg / day, about 3775 mg / day, about 3800 mg / day, about 3825 mg / day, about 3850 mg / day, about 3875 mg / day, about 3900 mg / day, about 3925 mg / day, about 3950 mg / day, about 3975 mg / day, about 4000 mg / day, about 4025 mg / day, about 4050 mg / day, about 4075 mg / day, about 4100 mg / day, 4125 mg / day, about 4150 mg / day, about 4175 mg / day, about 4200 mg / day, 4225 mg / day, about 4250 mg / day, about 4275 mg / day, about 4300 mg / day, 4325 mg / day, about 4350 mg / day, about 4375 mg / day, about 4400 mg / day, 4425 mg / day, about 4450 mg / day, about 4475 mg / day, about 4500 mg / day, about 4525 mg / day, about 4550 mg / day, about 4575 mg / day, about 4600 mg / day, about 4625 mg / day, about 4650 mg / day, about 4675 mg / day, about 4700 mg / day, about 4725 mg / day, about 4750 mg / day, about 4775 mg / day, about 4800 mg / day, about 4825 mg / day, about 4850 mg / day, about 4875 mg / day, about 4900 mg / day, about 4925 mg / day, about 4950 mg / day, about 4975 mg / day, and about 5000 mg / day; the upper limit is about 5000 mg / day, about 4975 mg / day, about 4950 mg / day, about 4925 mg / day, about 4900 mg / day, about 4875 mg / day, about 4850 mg / day, about 4825 mg / day, about 4800 mg / day, about 4775 mg / day, about 4750 mg / day, about 4725 mg / day, about 4700 mg / day, about 4675 mg / day, about 4650 mg / day, about 4625 mg / day, about 4600 mg / day, about 4575 mg / day,about 3675 mg / day, about 3650 mg / day, about 3625 mg / day, about 3600 mg / day, about 3575 mg / day, about 3550 mg / day, about 3525 mg / day, about 3500 mg / day, 3475 mg / day, about 3450 mg / day, about 3425 mg / day, about 3400 mg / day, about 3375 mg / day, about 3350 mg / day, about 3325 mg / day, about 3300 mg / day, about 3275 mg / day, about 3250 mg / day, about 3225 mg / day, about 3200 mg / day, about 3175 mg / day, about 3150 mg / day, about 3125 mg / day, about 3100 mg / day, about 3075 mg / day, about 3050 mg / day, about 3025 mg / day, about 3000 mg / day, 2975 mg / day, about 2950 mg / day, about 2925 mg / day, about 2900 mg / day, about 2875 mg / day, about 2850 mg / day, about 2825 mg / day, about 2800 mg / day, about 2775 mg / day, about 2750 mg / day, about 2725 mg / day, about 2700 mg / day, about 2675 mg / day, about 2650 mg / day, about 2625 mg / day, about 2600 mg / day, about 2575 mg / day, about 2550 mg / day, about 2525 mg / day, about 2500 mg / day, 2475 mg / day, about 2450 mg / day, about 2425 mg / day, about 2400 mg / day, about 2375 mg / day, about 2350 mg / day, about 2325 mg / day, about 2300 mg / day, about 2275 mg / day, about 2250 mg / day, about 2225 mg / day, about 2200 mg / day, about 2175 mg / day, about 2150 mg / day, about 2125 mg / day, about 2100 mg / day, about 2075 mg / day, about 2050 mg / day, about 2025 mg / day, about 2000 mg / day, 1975 mg / day, about 1950 mg / day, about 1925 mg / day, about 1900 mg / day, about 1875 mg / day, about 1850 mg / day, about 1825 mg / day, about 1800 mg / day, about 1775 mg / day, about 1750 mg / day, about 1725 mg / day, about 1700 mg / day, about 1675 mg / day, about 1650 mg / day, about 1625 mg / day, about 1600 mg / day, about 1575 mg / day, about 1550 mg / day, about 1525 mg / day, about 1500 mg / day, 1475 mg / day, about 1450 mg / day, about 1425 mg / day, about 1400 mg / day, about 1375 mg / day, about 1350 mg / day, about 1325 mg / day, about 1300 mg / day, about 1275 mg / day, about 1250 mg / day, about 1225 mg / day, about 1200 mg / day, about 1175 mg / day, about 1150 mg / day, about 1125 mg / day, about 1100 mg / day, about 1075 mg / day, about 1050 mg / day, about 1025 mg / day, about 1000 mg / day, 975 mg / day, about 950 mg / day, about 925 mg / day, about 900 mg / day, about 875 mg / day, about 850 mg / day, about 825 mg / day, about 800 mg / day, about 775 mg / day, about 750 mg / day, about 725 mg / day, about 700 mg / day, about 675 mg / day, about 650 mg / day, about 625 mg / day, about 600 mg / day, about 575 mg / day, about 550 mg / day, about 525 mg / day, about 500 mg / day, 475 mg / day, about 450 mg / day, about 425 mg / day, about 400 mg / day, about 375 mg / day, about 350 mg / day, about 325 mg / day, about 300 mg / day, about 275 mg / day, about 250 mg / day, about 225 mg / day, about 200 mg / day, about 175 mg / day, about 150 mg / day, about 125 mg / day, about 100 mg / day, about 75 mg / day, about 50 mg / day, about 25 mg / day, about 10 mg / day, about 5 mg / day, about 2.5 mg / day, about 1 mg / day, about 0.5 mg / day, about 0.1 mg / day, about 0.05 mg / day, about 0.01 mg / day, about 0.005 mg / day, and about 0.001 mg / day.about 625 mg / day, about 600 mg / day, about 575 mg / day, about 550 mg / day, about 525 mg / day, and about 500 mg / day.

[0101] The compound can be administered in a regimen of 1 to 4 times per day, for example, once, twice, three times, or four times per day.

[0102] The efficacy of administration of quercetin to reduce hypercoagulability in cancer patients was evaluated (see Zwicker et al., JCI Insight. 2019; 4(4):e125851, and Clinicaltrials.gov NCT02195232). Venous thromboembolism (VTE) is common in cancer patients and is a leading cause of death in this population. In high-risk cancer patients, especially with driving surveillance for radiologic protocols for deep vein thrombosis, the incidence of VTE often exceeds 20% within the first few months of chemotherapy. The bleeding risk is also elevated in cancer patients, which limits the adoption of conventional primary thromboprophylaxis in cancer outpatients receiving chemotherapy. Developing diagnostic and therapeutic approaches to reduce the incidence of VTE without increasing the risk of major bleeding would have a broad impact on the care of patients with advanced malignancies and any cancer patient.

[0103] As used herein, the type of cancer can be selected from the group consisting of estrogen receptor-dependent breast cancer, estrogen receptor-independent breast cancer, hormone receptor-dependent prostate cancer, hormone receptor-independent prostate cancer, brain cancer, kidney cancer, glioblastoma, colon cancer, familial adenomatous polyposis (FAP), colorectal cancer, pancreatic cancer, bladder cancer, esophageal cancer, gastric cancer, urogenital cancer, gastrointestinal cancer, uterine cancer, ovarian cancer, astrocytoma, glioma, skin cancer, squamous cell carcinoma, keratoacanthoma, Bowen's disease, cutaneous T-cell lymphoma, melanoma, basal cell carcinoma, actinic keratosis, ichthyosis, acne, acne vulgaris, sarcoma, Kaposi's sarcoma, osteosarcoma, head and neck cancer, small cell lung cancer, non-small cell lung cancer, leukemia, lymphoma, and / or other blood cell cancer.

[0104] Other cancers that would benefit from the methods described herein include cancers associated with certain viruses (and include improving precancerous conditions during viral infection). Such disorders include disorders associated with human T-cell leukemia virus type, also known as human T-lymphotropic virus (HTLV-1), which is associated with adult T-cell leukemia / lymphoma. Another such cancer includes cancers associated with human papillomavirus (HPV), which has at least 12 strains that can cause cancer in both men and women, including anal, cervical, penile, throat, vaginal, and vulvar cancers. Other disorders include disorders associated with human herpesvirus 8 (HHV-8), which is associated with Kaposi sarcoma in people with weakened immune systems, such as HIV patients. Likewise, there are a number of cancers associated with HIV, which is believed to impair the immune system and reduce defenses against other tumor viruses. Cancers associated with HIV include Kaposi sarcoma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma, cervical cancer, as well as anal, liver, oral, throat, and lung cancers. In addition, hepatitis C is a major cause of liver cancer and can cause non-Hodgkin's lymphoma, and thus can benefit from the methods described herein. Similarly, hepatitis B is a major cause of liver cancer, and these disorders can benefit from the methods described herein. Finally, Epstein-Barr virus (EBV) infection increases the risk of Burkitt's lymphoma, certain types of Hodgkin's and non-Hodgkin's lymphoma, and stomach cancer, which can also benefit from the methods described herein.

[0105] In certain embodiments, the cancer is a metastatic cancer. A "metastatic cancer" is a cancer that can form or often forms metastases. A metastatic cancer that has spread from the body site where it started, the primary site, to other sites of the body is also called a metastatic cancer. When cancer cells break away from a tumor, they can move through the blood or lymphatic system to other areas of the body. These cancer cells can then form new tumors in other areas of the body.

[0106] In certain embodiments, the cancer is a metastatic cancer selected from the group consisting of Hodgkin's lymphoma, colorectal cancer, cervical cancer, lung cancer, skin cancer such as squamous cell carcinoma or basal cell carcinoma, head and neck cancer, gastric cancer, pancreatic cancer, head and neck squamous cell carcinoma, and breast cancer in metastatic form.

[0107] In certain embodiments, the metastatic cancer is colorectal cancer, pancreatic cancer, or non-small cell lung cancer.

[0108] In certain embodiments, the cancer is classifiable as Stage III or Stage IV according to the TNM anatomical / prognostic group system of the cancer staging system of the American Joint Committee on Cancer. In additional embodiments, the cancer is classifiable as Stage IV according to the TNM anatomical / prognostic group system of the cancer staging system of the American Joint Committee on Cancer.

[0109] In certain embodiments, the cancer is a metastatic cancer selected from the group consisting of Hodgkin's lymphoma, colorectal cancer, cervical cancer, lung cancer, skin cancer such as squamous cell carcinoma or basal cell carcinoma, head and neck cancer, gastric cancer, pancreatic cancer, and breast cancer in metastatic form, wherein the metastatic cancer is classifiable as Stage IV according to the TNM anatomical / prognostic group system of the cancer staging system of the American Joint Committee on Cancer (7th edition, 2010, Springer).

[0110] In certain embodiments, the isoquercitrin, quercetin or rutin composition is used in combination with detection of the UPR biomarker panel for reducing or preventing thrombotic conditions and in combination with other therapies for treating cancer in cancer patients, including patients with metastases already formed, for example, treating metastatic forms of Hodgkin's lymphoma, colorectal cancer, cervical cancer, head and neck cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer and breast cancer in a mammal, typically a human subject. In other embodiments, the isoquercitrin, quercetin or rutin composition is used in patients without metastatic cancer, but rather exhibiting cancer only at the primary site. Furthermore, it is contemplated that the methods and treatments described herein will be effective in treating any solid or blood cell cancer, as all patients with these cancers, whether or not metastatic, will benefit from a reduction in the level of plasma PDI and / or soluble P-selectin, and in addition will benefit from diagnosis, monitoring, reduction or elimination of venous thromboembolism (VTE) or other thrombotic conditions without increasing the risk of major bleeding. It should be noted that cancer patients often exhibit high levels of soluble P-selectin and are therefore at high risk of developing venous thromboembolism (VTE) and related thrombotic conditions. Thus, in certain embodiments, the combination of the UPR biomarker panel with high levels of soluble P-selectin can be a useful screening tool to identify cancer patients at risk of thrombotic events for whom prophylactic treatment would be beneficial.

[0111] In preferred embodiments of the application, reagents that specifically bind to the UPR biomarker proteins PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70 and PDIA3, which are elevated in plasma samples from advanced cancer patients who later exhibit VTE, as compared to the levels of the same UPR biomarker proteins in plasma samples from advanced cancer patients who do not exhibit VTE and thus serve as baseline reference samples, or any subset or combination thereof, and optionally one or more other biomarkers, are immobilized on a solid support, such as a polystyrene surface. Preferred embodiments of the application provide a protein microarray or protein array device for simultaneous binding and quantification of a panel of markers for assessing the risk of thrombotic conditions. The protein array device is composed of molecules (capture agents) bound to defined sites on a support material. Specific binding reagents, which will preferably be biotinylated, are bound as very small spots on a solid phase coated with streptavidin. The array is then exposed to the sample. Capture agents, such as antibodies, are able to bind the proteins of interest from the biological sample. Binding of the specific analyte proteins to individual spots can then be monitored by quantification of the signal generated by each spot.

[0112] In another preferred embodiment of the application, reagents that specifically bind to the UPR biomarker proteins PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70, which are elevated in plasma samples from advanced cancer patients who later exhibit VTE, as compared to the levels of the same UPR biomarker proteins in plasma samples from advanced cancer patients who do not exhibit VTE and thus serve as baseline reference samples, and optionally one or more other biomarkers, are immobilized on a solid support, such as a polystyrene surface. Preferred embodiments of the application provide a protein microarray or protein array device for simultaneous binding and quantification of panels of markers for assessing the risk of thrombotic conditions. The protein array device is composed of molecules (capture agents) bound to defined sites on a support material. Specific binding reagents, which will preferably be biotinylated, are bound as very small spots on a solid phase coated with streptavidin. The array is then exposed to the sample. Capture agents, e.g. antibodies, are able to bind the proteins of interest from the biological sample. Binding of the specific analyte proteins to individual spots can then be monitored by quantification of the signal generated by each spot.

[0113] In yet another embodiment, the application relates to a protein array device comprising at least suitable specific binding partners for measuring the expression levels of UPR biomarkers and optionally suitable specific binding partners for one or more other markers useful for assessing the risk of thrombotic conditions in cancer patients, in particular advanced cancer patients.

[0114] Suitable immunoassays commonly used in the art for detecting protein expression levels in plasma samples include, for example, but are not limited to, western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS), immunoradiometric assay, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays, imaging mass cytometry, complement fixation assays, and immunoelectrophoresis assays. According to this aspect of the disclosure, the UPR biomarker expression levels measured in a patient sample (from a cancer patient) can be further compared to the UPR biomarker protein expression levels measured in a baseline, reference, or control sample, e.g., levels of any combination or subset of PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3, and optionally P-selectin, from a cancer patient who has not exhibited a VTE or other thrombotic condition for at least 8 weeks; and the patient is diagnosed as being at risk for a thrombotic event when any combination or subset of PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3, and optionally P-selectin, is elevated above the baseline, control, or reference levels.

[0115] Suitable immunoassays commonly used in the art for detecting protein expression levels in plasma samples include, for example, but are not limited to, western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS), immunoradiometric assay, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays, imaging mass cytometry, complement fixation assays, and immunoelectrophoresis assays. According to this aspect of the disclosure, the UPR biomarker expression levels measured in a patient sample (from a cancer patient) can be further compared to the UPR biomarker protein expression levels measured in a baseline, reference, or control sample, e.g., levels of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70, and optionally P-selectin, from a cancer patient who has not exhibited a VTE or other thrombotic condition for at least 8 weeks; and the patient is diagnosed as being at risk for a thrombotic event when PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70, and optionally P-selectin, is elevated above the baseline, control, or reference levels.

[0116] In another embodiment, the UPR biomarker expression levels are measured using one- and two-dimensional electrophoresis gel analysis, high performance liquid chromatography (HPLC), reverse phase HPLC, fast protein liquid chromatography (FPLC), mass spectrometry (MS), tandem mass spectrometry, liquid chromatography-MS (LC-MS), surface enhanced laser desorption / ionization (SELDI), MALDI, and / or protein sequencing.

[0117] According to certain aspects of the present disclosure, the UPR biomarker expression levels, in particular in plasma samples, can also or alternatively be measured by detecting and quantifying the nucleic acid levels of the respective UPR biomarker panel using nucleic acid detection assays. In one embodiment, RNA, e.g., mRNA, levels are measured. RNA is preferably reverse transcribed to synthesize complementary DNA (cDNA), which is then amplified and detected or directly detected. The detected cDNA is measured and the cDNA levels are used as an indicator of the RNA or mRNA levels present in the sample. Reverse transcription can be performed alone or in combination with an amplification step, e.g., reverse transcription polymerase chain reaction (RT-PCR), which can be further modified to be quantitative, e.g., quantitative RT-PCR, as described in U.S. Patent No. 5,639,606, which is incorporated herein in its entirety by reference.

[0118] It can be beneficial or otherwise desirable to extract RNA from plasma samples prior to analysis or for analysis. RNA molecules can be isolated from the sample and their concentration (i.e., total RNA) quantified using any procedure known in the art, with the particular extraction procedure selected based on the particular biological sample. In certain cases, it can also be possible to analyze nucleic acids without extraction from the sample using certain techniques.

[0119] In one embodiment, mRNA is directly analyzed without using an amplification step. Direct analysis can be performed using different methods, including but not limited to nanostring technology (Geiss et al., Direct Multiplexed Measurement of Gene Expression with Color-Coded Probe Pairs, Nat Biotechnol 26(3):317-25 (2008)). Nanostring technology enables the identification and quantification of individual target molecules in a biological sample by attaching a color-coded fluorescent reporter to each target molecule. This approach is analogous to the concept of measuring inventory by scanning barcodes. The reporters can be manufactured using hundreds or even thousands of different codes, allowing for highly multiplexed analysis. In another embodiment, direct analysis can be performed using immunohistochemistry techniques.

[0120] In another embodiment, it can be beneficial or otherwise desirable to reverse transcribe and amplify the RNA prior to detection / molecule. Methods of nucleic acid amplification, including quantitative amplification, are commonly used and well known in the art. Quantitative amplification allows for quantitative determination of the relative amount of RNA in a cell.

[0121] Nucleic acid amplification methods include, but are not limited to, polymerase chain reaction (PCR) (U.S. Patent No. 5,219,727, incorporated herein by reference in its entirety) and variations thereof, such as in situ polymerase chain reaction (U.S. Patent No. 5,538,871, incorporated herein by reference in its entirety), quantitative polymerase chain reaction (U.S. Patent No. 5,219,727, incorporated herein by reference in its entirety), nested polymerase chain reaction (U.S. Patent No. 5,556,773), self-sustained sequence replication and variations thereof (Guatelli et al., Isothermal, In vitro Amplification of Nucleic Acids by a Multienzyme Reaction Modeled after Retroviral Replication, Proc Natl Acad Sci USA 87(5): 1874-8 (1990), incorporated herein by reference in its entirety), transcription amplification and variations thereof (Kwoh et al., Transcription-based Amplification System and Detection of Amplified Human Immunodeficiency Virus type 1 with a Bead-Based Sandwich Hybridization Format, Proc Natl Acad Sci USA 86(4): 1173-7 (1989), incorporated herein by reference in its entirety), Qb replicase and variations thereof (Miele et al., Autocatalytic Replication of a Recombinant RNA, J Mol Biol 171(3): 281-95 (1983), incorporated herein by reference in its entirety), cold PCR (Li et al., Replacing PCR with COLD-PCR Enriches Variant DNA Sequences and Redefines the Sensitivity of Genetic Testing, Nat Med 14(5): 579-84 (2008), incorporated herein by reference in its entirety), or any other nucleic acid amplification method known in the art.Depending on the amplification technique employed, the amplified molecules are detected during amplification (e.g., real-time PCR) or after amplification using detection techniques known to those skilled in the art. Suitable nucleic acid detection assays include, for example, but are not limited to, northern blotting, microarray, serial analysis of gene expression (SAGE), next generation RNA sequencing (e.g., deep sequencing, whole transcriptome sequencing, exome sequencing), gene expression analysis by massively parallel signature sequencing (MPSS), immuno-derivative colorimetric assays, and mass spectrometry (MS) methods (e.g.,. Systems).

[0122] Certain embodiments provided herein include a method of determining a risk of a thrombotic event in a cancer patient, the method comprising: detecting in a sample of the cancer patient a level of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 compared to a baseline, reference, or control level of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70; and diagnosing the patient as having a risk of a thrombotic event when PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 is elevated above the baseline, control, or reference level.

[0123] Other embodiments provided herein include a method of diagnosing and treating a thrombotic condition in a cancer patient, the method comprising the steps of: a. detecting in a sample of the cancer patient a level of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 compared to a baseline, reference, or control level of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70; b. diagnosing the patient as having a risk of a thrombotic condition when PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 is elevated above the baseline, control, or reference level; and c. treating the at risk patient with an effective amount of isoquercitrin and optionally an anti-thrombotic agent.

[0124] Other embodiments provided herein include a method of monitoring a cancer patient undergoing treatment for a risk of a thrombotic condition, the method comprising the steps of: a. detecting in a sample of the cancer patient the level of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 compared to a baseline, reference, or control level of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70; b. diagnosing the patient as having a risk of a thrombotic condition when PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 is elevated above the baseline, control, or reference level; and c. treating the at-risk patient with an effective amount of isoquercetin and optionally an anti-thrombotic agent; wherein the monitoring is repeated at the indicated times per week, every two weeks, every month, or throughout the course of treatment.

[0125] In certain embodiments, the patient does not exhibit a serious adverse event (Grade 3 or 4 toxicity) during treatment.

[0126] In certain embodiments, the patient does not exhibit a primary venous thromboembolism (VTE) during treatment.

[0127] In certain embodiments, the patient does not exhibit a major bleeding during treatment.

[0128] Other embodiments provided herein include a kit comprising a biomarker panel comprising PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 for use in diagnosing a thrombotic condition in a patient in need thereof.

[0129] Other embodiments provided herein include a kit comprising: (a) a solid support coated with a polyclonal or monoclonal antibody, wherein the antibody comprises an antibody specific for PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70; (b) a polyclonal or monoclonal antibody-substrate conjugate, wherein the substrate comprises a chromogenic or fluorescent reagent, and wherein the conjugate is reactive with the antibody of (a); and (c) at least one of PPIA, PDIA3, and EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 as an antigen standard.

[0130] In certain embodiments, the antibody of (a) further comprises an antibody specific for soluble P-selectin.

[0131] In certain embodiments, the solid support is a microtiter plate or a membrane. In certain embodiments, the solid support is a bead or particle. In certain embodiments, the kit is an ELISA kit. In certain embodiments, the solid support is a microbead array.

[0132] Other embodiments provided herein include a method of determining PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 in a serum or plasma sample, the method comprising contacting the sample with a solid support and a conjugate of a kit described herein; wherein the solid support comprises a microtiter plate, wherein the conjugate comprises alkaline phosphatase, wherein the chromogenic reagent comprises p-nitrophenylphosphate; and determining the reaction of the conjugate with the sample.

[0133] Other embodiments provided herein include a method of determining a combination of markers in a biological fluid sample obtained from a human subject, the method comprising performing an immunoassay by contacting the sample with a solid support of a kit described herein.

[0134] In certain embodiments, the immunoassay is an ELISA. In certain embodiments, the solid support is a microbead array. In certain embodiments, the sample is plasma or serum.

[0135] In certain embodiments, the method further comprises contacting the sample with a conjugate of the kit and determining the reaction of the conjugate with the sample.

[0136] In certain embodiments, the method further comprises contacting the antigen standard with the solid support and a conjugate and determining the relative level of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 in the sample relative to the antigen standard.

[0137] Certain embodiments provided herein include a method of determining a cancer patient's risk of a thrombotic event, the method comprising: detecting in a sample of a cancer patient an increase in the level of PPIA and PDIA3 compared to a baseline, reference, or control level of PPIA and PDIA3; if PPIA and PDIA3 are elevated above the baseline, control, or reference level, detecting in the sample of the cancer patient an increase in the level of at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 compared to a baseline, reference, or control level of at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70; and diagnosing the patient as being at risk of a thrombotic event when PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 are elevated above the baseline, control, or reference level.

[0138] Other embodiments provided herein include a method of diagnosing and treating a thrombotic condition in a cancer patient, the method comprising the steps of: a. detecting in a sample of a cancer patient an increase in the level of PPIA and PDIA3 compared to a baseline, reference, or control level of PPIA and PDIA3; if PPIA and PDIA3 are elevated above the baseline, control, or reference level, detecting in the sample of the cancer patient an increase in the level of at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 compared to a baseline, reference, or control level of at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70; b. diagnosing the patient as being at risk of a thrombotic event when PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 are elevated above the baseline, control, or reference level; and c. treating the at-risk patient with an effective amount of isoquercitrin and optionally an anti-thrombotic agent.

[0139] Other embodiments provided herein include a method of monitoring a cancer patient undergoing treatment for a risk of a thrombotic event, the method comprising the steps of: a. detecting in a sample of the cancer patient an increase in the level of PPIA and PDIA3 compared to a baseline, reference, or control level of PPIA and PDIA3; and detecting in the sample of the cancer patient an increase in the level of at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 compared to a baseline, reference, or control level of at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 if PPIA and PDIA3 are elevated above the baseline, control, or reference level; b. diagnosing the patient as having a risk of a thrombotic event when PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 are elevated above the baseline, control, or reference level; and c. treating the at risk patient with an effective amount of isoquercetin and optionally an anti-thrombotic agent; wherein the monitoring is repeated at the indicated times per week, every two weeks, monthly, or throughout the course of treatment.

[0140] In certain embodiments, the patient does not exhibit a serious adverse event (Grade 3 or 4 toxicity) during treatment.

[0141] In certain embodiments, the patient does not exhibit a primary venous thromboembolism (VTE) during treatment.

[0142] In certain embodiments, the patient does not exhibit a major bleeding during treatment. It is contemplated that all of the assays disclosed herein can be in the form of a kit for use by a health care provider and / or diagnostic laboratory.

[0143] Assays for the detection and quantification of one or more UPR biomarkers can be incorporated into a kit. Such a kit comprises probes for one or more UPR biomarker proteins or genes (i.e., PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70), reagents for the isolation and purification of the proteins or nucleic acids from biological tissue or bodily fluids, reagents for assaying the isolated and purified proteins or nucleic acids, instructions for use, and either reference values for the included proteins or genes or means for obtaining reference values in control samples.

[0144] A preferred kit for patient classification for thrombosis risk and clinical presentation will comprise probes for any combination or subset of proteins or genes from the UPR biomarker panel (i.e. PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70 and PDIA3), and optionally probes or reagents for further detection of soluble P-selectin.

[0145] A preferred kit for patient classification for thrombosis risk and clinical presentation will comprise probes for at least two proteins or genes from the UPR biomarker panel (i.e. PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I and HSP70), and optionally probes or reagents for further detection of soluble P-selectin.

[0146] In another embodiment, the kit will comprise reagents for testing the UPR biomarker panel (i.e. PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70 and PDIA3) for increased levels.

[0147] In another embodiment, the kit will comprise reagents for testing the UPR biomarker panel (i.e. PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I and HSP70) for increased levels.

[0148] Such a kit can comprise antibodies recognizing the peptides of interest, reagents for isolating and purifying proteins from biological tissues or fluids, reagents for assaying the isolated and purified proteins, instructions for use, and reference values for the amount or level of the peptides in control samples or means for obtaining reference values.

[0149] Another kit for monitoring treatment of disease activity or progression comprises probes for at least one protein or gene from the UPR biomarker panel (i.e. PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70 and PDIA3).

[0150] Another kit for monitoring treatment of disease activity or progression comprises probes for at least two proteins or genes from the UPR biomarker panel (i.e. PPIA, PDIA3, and at least one of EIF5A, EIF4a3, UBE2N, UBE2L3, UBE2I and HSP70).

[0151] Such kits can comprise antibodies that recognize the peptide of interest, reagents for isolating and / or purifying proteins from biological tissues or fluids, reagents for performing assays on the isolated and purified proteins, instructions for use, and reference values for the amount or level of peptide in control samples or means for obtaining reference values.

[0152] One embodiment of these kits will have probes attached to a solid state. Another embodiment will have probes in a microarray format, where nucleic acid probes for one or more genes from one or more gene signatures will be ordered on a surface or substrate.

[0153] For use in the methods described herein, the kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, etc., each of which contains one of the independent elements to be used in the methods described herein. The probes, antibodies, and other reagents of the kits described herein can be provided in any appropriate form including frozen, lyophilized, or in a pharmaceutically acceptable buffer such as TBS or PBS. The kits can also include other reagents to be used in the in vitro or in vivo utilization of the reagents such as buffers (i.e., TBS, PBS), blocking agents (solutions containing skim milk, normal serum, Tween-20 detergent, BSA, or casein), and / or detection reagents (i.e., goat anti-mouse IgG antibody-biotin, streptavidin-HRP conjugate, allophycocyanin, B-phycoerythrin, R-phycoerythrin, peroxidase, fluorescent agents (i.e., DyLight, Cy3, Cy5, FITC, HiLyteFluor 555, HiLyte Fluor 647), and / or staining kits (i.e., ABC Staining Kit, Pierce)) and / or instructions for using the antibodies, probes, and other reagents in the above-described common assays such as liquid or gas chromatography, spectroscopy, electrochemical assays, flow cytometry analysis, ELISA, immunoblotting (i.e., western blot), immunocytochemistry, immunohistochemistry.

[0154] In one embodiment, the kit provides the reagents in purified form. In another embodiment, the reagents are immunoreagents provided in biotinylated form, either alone or with an avidin conjugated detection reagent (i.e., an antibody). In another embodiment, the kit comprises fluorescently labeled immunoreagents that can be used to directly detect antigens. Buffers and the like required for use of any of these systems are well known in the art and can be prepared by the end user or provided as components of the kit. The kit can also include a solid support containing positive and negative control proteins and / or tissue samples. For example, a kit for performing a dot-blot or western-blot type assay can include control cell or tissue lysates for SDS-PAGE or a nylon or other membrane containing pre-immobilized control samples and extra space for experimental samples.

[0155] In certain embodiments, the kit will generally contain the above-described container(s) and one or more other containers containing materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. Additionally, labels can be provided on the containers to indicate that the compositions are used for a specific application, and can also indicate directions for use, such as the directions described above. Instructions and / or other information also can be included on a leaflet which is attached to the kit.

[0156] Another embodiment provides a kit comprising a panel of antibodies that specifically bind to 9 UPR biomarkers, PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3, and optionally one or more additional markers (e.g., antibodies to soluble P-selectin). In one embodiment, the kit further comprises a solid support to which the antibodies are immobilized. Examples of solid supports include, but are not limited to, microtiter plates, beads, membranes, or other supports known to those skilled in the art. In one embodiment, the antibodies are immobilized by binding to antigens immobilized on the solid support. In one embodiment, the antibodies are immobilized by binding to beads or particles, such as luminex. In one embodiment, the kit further comprises a chromogenic substrate.

[0157] Another embodiment provides a kit comprising antibodies that specifically bind to UPR biomarkers PPIA, EIF4H, PDIA3, and at least one of EIF5A, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70, and optionally one or more additional markers (e.g., antibodies to soluble P-selectin). In one embodiment, the kit further comprises a solid support to which the antibodies are immobilized. Examples of solid supports include, but are not limited to, microtiter plates, beads, membranes, or other supports known to those skilled in the art. In one embodiment, the antibodies are immobilized by binding to antigens immobilized on the solid support. In one embodiment, the antibodies are immobilized by binding to beads or particles, e.g., luminex. In one embodiment, the kit further comprises a chromogenic substrate.

[0158] Another illustrative embodiment is an ELISA kit for screening a plasma molecular profile predictive of a thrombotic condition (e.g., VTE) in a cancer patient undergoing treatment by detecting an increase in the levels of PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3 and / or an increase in the level of P-selectin in the plasma or serum, the kit comprising: (a) a microtiter plate coated with polyclonal or monoclonal antibodies specific for PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3 and optionally soluble P-selectin disclosed herein; (b) polyclonal or monoclonal antibody-alkaline phosphatase conjugates reactive with PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3 and optionally soluble P-selectin disclosed herein; (c) p-nitrophenyl phosphate; and (d) PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3 as antigen standards.

[0159] Another illustrative embodiment is an ELISA kit for screening a plasma molecular profile predictive of a thrombotic condition, e.g., VTE, in a cancer patient undergoing treatment by detecting an increase in the level of PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70, and optionally an increase in the level of soluble P-selectin in plasma or serum, the kit comprising: (a) a microtiter plate coated with polyclonal or monoclonal antibodies specific for PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70, and optionally soluble P-selectin; (b) polyclonal or monoclonal antibody-alkaline phosphatase conjugates reactive with PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70, and optionally soluble P-selectin; (c) p-nitrophenyl phosphate; and (d) at least one of PPIA, PDIA3, and EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 as an antigen standard.

[0160] Another illustrative embodiment is an ELISA kit for screening a plasma molecular profile associated with a thrombotic condition, e.g., VTE, in a patient by detecting PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3 in plasma or serum, the kit comprising: (a) a microtiter plate coated with polyclonal or monoclonal antibodies specific for PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3; (b) polyclonal or monoclonal antibody-alkaline phosphatase conjugates reactive with PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3; (c) p-nitrophenyl phosphate; and (d) PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3 as an antigen standard.

[0161] Another illustrative embodiment is an ELISA kit for screening a plasma molecular profile associated with a thrombotic condition, e.g., VTE, in a patient by detecting PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 in plasma or serum, the kit comprising: (a) a microtiter plate coated with polyclonal or monoclonal antibodies specific for PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70; (b) polyclonal or monoclonal antibody-alkaline phosphatase conjugates reactive with PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70; (c) p-nitrophenyl phosphate; and (d) at least one of PPIA, PDIA3, and EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 as an antigen standard.

[0162] Another illustrative embodiment is a Luminex kit for screening a molecular profile associated with a thrombotic condition, e.g., VTE, in a patient in plasma, serum, and / or biological fluids by detecting PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3 and optionally P-selectin, the kit comprising: (a) a microbead array coated with polyclonal or monoclonal antibodies specific for PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3 and optionally P-selectin; (b) polyclonal or monoclonal antibody-fluorescent dye conjugates reactive with PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3 and optionally P-selectin; and (c) PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70, and PDIA3 and optionally P-selectin as an antigen standard.

[0163] Another illustrative embodiment is a Luminex kit for screening a molecular profile associated with a thrombotic condition, such as VTE, in a patient in plasma, serum, and / or biological fluids by detecting PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70, and optionally P-selectin, the kit comprising: (a) a microbead array coated with polyclonal or monoclonal antibodies specific for PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70, and optionally P-selectin; (b) polyclonal or monoclonal antibody-fluorochrome conjugates reactive with PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70, and optionally P-selectin; and (c) PPIA, PDIA3, and at least one of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70, and optionally P-selectin as antigen standards.

[0164] In certain embodiments, the various different embodiments described herein can be combined with other diagnostic tests including, but not limited to, a complete blood count (CBC), a troponin test, a CKP isozyme test, a comprehensive metabolome, or any combination thereof. Examples

[0165] Example 1

[0166] Tissue factor (TF) is the initiator of the coagulation cascade and is essential for hemostasis. Under pathological conditions, TF is released into the circulation on small membrane vesicles called microparticles (MPs). Recent studies have shown that elevated levels of MP-TF can trigger thrombosis.

[0167] The unfolded protein response (UPR) is associated with malignant transformation in pancreatic cancer, but it has not previously been assessed whether activation of the UPR is associated with cancer thrombosis. To determine whether UPR signaling plays a role in the prothrombotic transformation of pancreatic cancer, pancreatic adenocarcinoma cells (HPAF-II cells) were exposed to three UPR inducers that act through independent mechanisms (tunicamycin, triptolide, and thapsigargin). The generation of thrombin in the particulate fraction was increased 3-fold, confirming that induction of the UPR led to the release of thrombogenic material into the supernatant. Knockdown of UPR components including IRE1a (80% ± 3% reduction) or PERK (60% ± 10% reduction) by siRNA inhibited the release of thrombogenic material. Chemical inhibition of the UPR also inhibited the release of thrombogenic material from HPAF-II cells. Exposure to the IRE1a inhibitor MKC-3946 led to a 70% ± 10% reduction in thrombin generation, and incubation with the PERK inhibitor GSK2606414 led to an 80% ± 5% reduction in thrombin generation. Characterization of the thrombogenic activity revealed that it was present on extracellular vesicles (EVs) and was inhibited by anti-tissue factor (anti-TF) antibodies. Flow cytometry indicated a 3-fold increase in the generation of EVs bearing TF after UPR induction. Electron microscopy showed that HPAF II EVs were in the 100-500 μm range and exhibited increased clustering after UPR induction. Three-color immunofluorescence microscopy of HPAF II cells using actin, nucleus, and TF markers showed that induction of the UPR led to TF-rich, actin-deficient membrane vesicles. No apoptosis detected by caspase-3 cleavage was observed under these conditions. Brefeldin A, which inhibits vesicular transport between the endoplasmic reticulum and the Golgi apparatus, inhibited the generation of UPR-induced EVs bearing TF, indicating that UPR-mediated vesicular transport contributes to the formation of EVs bearing TF.

[0168] To assess the possibility of a link between the UPR and cancer thrombosis in a clinical setting, plasma collected from pancreatic cancer patients who were prospectively monitored for the development of venous thromboembolism (including lower extremity ultrasound at baseline and at 2 months) was analyzed. Proteomic analysis using Somalogic technology was used to assess ~1300 analytes in plasma from 9 pancreatic cancer patients who subsequently developed venous thromboembolism and 10 patients with similar pancreatic cancer characteristics who remained free of venous thromboembolism.

[0169] Plasma samples from patients with advanced pancreatic cancer were analyzed by proteomic analysis using Somascan (from SomaLogic, Inc. Boulder, Co. See also Gold, L., Walker, J.J., Wilcox, S.K. & Williams, S. Advances in human proteomics at high scale with the SOMAscan proteomics platform. N. Biotechnol. 29, 543-9 (2012)). Baseline plasma was drawn (baseline ultrasound showed no signs of DVT) and patients were monitored for VTE development for 8 weeks (see MicroTec trial described in Zwicker et al. Br. J. Haematol. 2013 Feb; 160(4):530-7). The UPR panel of nine proteins based on a commercially available list of UPR genes was evaluated. As shown in Figure 1 Table 1 below, p values for each UPR marker are shown.

[0170] Table 1 - p values for each UPR marker

[0171] Protein p-value PPIA 0.0002 EIF5A 1.0 EIF4H 0.009 EIF4A3 0.10 UBE2N 0.48 UBE2L3 1.0 UBE2I 0.10 HSP70 0.26 PDIA3 0.009

[0172] As shown in Figure 1 Evaluation of the nine UPR markers present in the SOMA-Scan panel demonstrated significant upregulation in the plasma of patients who developed clots compared to patients who did not develop clots (p=0.0001), particularly elevated levels of PPIA, EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, HSP70 and PDIA3. These data support a model in which activation of the UPR leads to increased vesicular trafficking, resulting in the release of EVs bearing TF. These observations suggest a mechanistic link between tumor progression in pancreatic cancer and cancer-associated thrombosis. It is likely that similar results will be found in other cancer patients, including patients without advanced disease. In addition, it would be beneficial to continue monitoring patients for elevations in UPR marker levels every 2 weeks or monthly or as long as they are being treated or at risk for thrombosis. Blood was drawn into 3.2% citrate by peripheral venipuncture. Plasma was separated at 2100g for 20 minutes within one hour of sample collection. A second centrifugation was performed at 2100g for 20 minutes to produce platelet-free plasma and aliquoted for storage at -80°C until analysis.

[0173] The primary VTE endpoint included any symptomatic proximal or distal deep venous thrombosis, symptomatic PE diagnosed by autopsy, or fatal PE, or asymptomatic proximal DVT diagnosed by protocol-specified ultrasound at the end of the study. All suspected VTEs were evaluated by an independent adjudication committee, which included central radiology review of imaging. Criteria for new-onset VTE included any of the following: A) new non-compressibility of a segment of deep veins of the lower extremity found by compression ultrasound (thrombosis of the distal lower extremity was only considered a primary VTE endpoint if symptomatic); B) intraluminal defects on two or more views of a pulmonary angiogram, abrupt cutoff of contrast on one or more vessels greater than 2.5 mm in diameter on pulmonary angiography; high-probability VQ lung scan showing one or more segmental perfusion defects with corresponding normal ventilation (mismatched defects); or abnormal spiral CT showing a thrombus (subsegmental or greater) in a pulmonary vessel. All other venous or arterial events were recorded and analyzed as secondary endpoints. Criteria for major bleeding conformed to the ISTH definition (Schulman S and Kearon C, J Thromb Haemost. 2005; 3(4):692-4). All toxicities were graded according to the NCI Common Terminology Criteria for Adverse Events (CTCAE). Study oversight was performed by an independent data safety monitoring committee of the Dana Farber Harvard Cancer Center.

[0174] General Methods

[0175] Standard methods in molecular biology are described in Sambrook, Fritsch and Maniatis (1982 & 1989 2nd Ed., 2001 3rd Ed.), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001), Molecular Cloning, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993), Recombinant DNA, Vol. 217, Academic Press, San Diego, CA. Standard methods also appear in Ausbel et al., (2001), Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), carbohydrate conjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0176] Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization have been described (Coligan et al., (2000), Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post- translational modification, production of fusion proteins, glycosylation of proteins have been described (see, e.g., Coligan et al., (2000), Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al., (2001), Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co., (2001), Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001), BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies have been described (Coligan et al., (2001), Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane, (1999), Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Coligan et al., (2001), Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0177] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent was specifically and individually indicated to be incorporated by reference. This incorporation by reference of each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent is made by the Applicant pursuant to 37 C.F.R. § 1.57(b)(1) for each of which is identified as complying with 37 C.F.R. § 1.57(b)(2), even though such applications are not immediately adjacent to the specific incorporation by reference. The general incorporation by reference in this specification is not in any way intended to weaken the specific incorporation by reference if any. Citation of the references herein does not constitute an admission that the referenced reference is prior art nor does it constitute any admission as to the correctness or date of publication of the referenced document.

[0178] The scope of the application is not intended to be limited to the particular embodiments described in this document. Indeed, various modifications of the application in addition to those described herein will become apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims.

[0179] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the application. Various modifications of the application in addition to those described herein will become apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. The use of a reagent for detecting at least two of PPIA, PDIA3, and EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 in samples from cancer patients, when the levels of these substances are elevated compared to baseline, reference, or control levels, in the preparation of a kit for determining the risk of thrombotic events in said cancer patients, wherein the kit diagnoses said patients as having a risk of thrombotic events when at least two of PPIA, PDIA3, and EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 are elevated above baseline, control, or reference levels.

2. A reagent, isoquercitrin, and optionally an antithrombotic agent for detecting at least two of PPIA, PDIA3, and EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 in samples from cancer patients, when the levels of these substances are increased compared to baseline, reference, or control levels of at least two of PPIA, PDIA3, and EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70. The use of the agent in the preparation of a kit for diagnosing and treating thrombotic status in cancer patients, wherein the kit diagnoses the patient as being at risk of thrombotic status when at least two of PPIA, PDIA3, and EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 are elevated above baseline, control, or reference levels; and the at-risk patient is treated with an effective amount of isoquercetin and optionally the antithrombotic agent.

3. A reagent, isoquercitrin, and optionally an antithrombotic agent for improving the level of at least two of PPIA, PDIA3, and EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 in samples from said cancer patients compared to baseline, reference, or control levels of at least two of PPIA, PDIA3, and EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 in preparation for monitoring patients undergoing treatment. The kit is used to assess the risk of thrombotic status in cancer patients, wherein the kit diagnoses the patient as having a thrombotic status when at least two of PPIA, PDIA3, and EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 rise above baseline, control, or reference levels; and the patient is treated with an effective amount of isoquercitrin and optionally an antithrombotic agent; wherein the monitoring is repeated weekly, bi-weekly, monthly, or at indicated intervals throughout the treatment course.

4. The use as described in claim 2 or 3, wherein the patient does not exhibit serious adverse events during treatment.

5. The use as claimed in claim 4, wherein the patient does not exhibit grade 3 or 4 toxicity during treatment.

6. The use as claimed in claim 2 or 3, wherein the patient does not exhibit primary venous thromboembolism (VTE) during treatment.

7. The use as claimed in claim 2 or 3, wherein the patient does not exhibit significant bleeding during treatment.

8. Use of a biomarker set in the preparation of a kit for diagnosing thrombotic status in cancer patients, wherein the biomarker set comprises PPIA, PDIA3, and at least two of EIF5A, EIF4a3, EIF4H, UBE2N, UBE2L3, UBE2I, and HSP70.

9. The use of claim 8, wherein the kit comprises: (a) a solid-phase support coated with a polyclonal or monoclonal antibody, wherein the antibody comprises an antibody specifically targeting PPIA, PDIA3, and at least two of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70; (b) a polyclonal or monoclonal antibody-substrate conjugate, wherein the substrate comprises a chromogenic reagent or a fluorescent reagent, and wherein the conjugate is reactive to the antibody of (a); and (c) PPIA, PDIA3, and at least two of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 as antigen standards.

10. The use of claim 9, wherein the antibody of claim (a) further comprises an antibody specifically targeting soluble P-selectin.

11. The use according to claim 9, wherein the solid support is a microtiter plate or membrane.

12. The use of claim 9, wherein the solid support is a bead or a particle.

13. The use according to claim 9, wherein the kit is an ELISA kit.

14. The use of claim 9, wherein the solid support is a microbead array.

15. Use of reagents for determining PPIA, PDIA3, and at least two of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 in serum or plasma samples in the preparation of a kit for diagnosing thrombotic conditions. The kit comprises: (a) a solid-phase support coated with a polyclonal or monoclonal antibody, wherein the antibody comprises an antibody specifically targeting PPIA, PDIA3, and at least two of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70; (b) a polyclonal or monoclonal antibody-substrate conjugate, wherein the substrate comprises a chromogenic or fluorescent reagent, and wherein the conjugate is reactive to the antibody in (a); and (c) PPIA, PDIA3, and at least two of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 as antigen standards. The kit is used in a method comprising contacting the sample with a solid support and a conjugate of the kit; wherein the solid support comprises a microtiter plate, wherein the conjugate comprises alkaline phosphatase, wherein the colorimetric reagent comprises p-nitrophenyl phosphate; and determining the reaction of the conjugate with the sample.

16. Use of reagents for determining combinations of biomarkers in biological fluid samples obtained from cancer patients in the preparation of kits for diagnosing thrombotic conditions. in, The combination of markers includes PPIA, PDIA3, and at least two of EIF5A, EIF4a3, EIF4H, UBE2N, UBE2L3, UBE2I, and HSP70. The kit comprises: (a) a solid-phase support coated with a polyclonal or monoclonal antibody, wherein the antibody comprises an antibody specifically targeting PPIA, PDIA3, and at least two of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70; (b) a polyclonal or monoclonal antibody-substrate conjugate, wherein the substrate comprises a chromogenic or fluorescent reagent, and wherein the conjugate is reactive to the antibody in (a); and (c) PPIA, PDIA3, and at least two of EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I, and HSP70 as antigen standards. The kit is used in a method comprising performing an immunoassay by contacting the sample with a solid support of the kit.

17. The use as claimed in claim 16, wherein the immunoassay is an ELISA.

18. The use of claim 16, wherein the solid support is a microbead array.

19. The use of claim 16, wherein the solid support is a microtiter plate or a membrane.

20. The use of claim 16, wherein the solid support is a bead or a particle.

21. The use of claim 16, wherein the antibody of said (a) further comprises an antibody specifically targeting soluble P-selectin.

22. The use according to claim 16, wherein the sample is plasma or serum.

23. The use of claim 16, wherein the kit is used in a method further comprising contacting the sample with a conjugate of the kit and determining the reaction between the conjugate and the sample.

24. The use of claim 23, wherein the kit is used in a method further comprising contacting the antigen standard with the solid support and the conjugate, and determining the relative levels of at least two of PPIA, PDIA3, and EIF5A, EIF4H, EIF4a3, UBE2N, UBE2L3, UBE2I and HSP70 in the sample relative to the antigen standard.

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