WO2011069009A1 - Polyubiquitin levels as a prognostic indicator in leukemia - Google Patents

Abstract

Provided herein are methods for the diagnosis, prognosis, or management of proliferative hematological disorders and other diseases by measuring polyubiquitin in acellular body fluids or cell-containing samples. Further provided are methods of predicting response to therapy in certain populations of leukemia patients.

Description

POLYUBIQUITIN LEVELS AS A PROGNOSTIC

INDICATOR IN LEUKEMIA

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application 61/266,665 filed December 4, 2009, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to the diagnosis, prognosis, and management of hematological disorders, including leukemia.

BACKGROUND OF THE INVENTION

[0003] The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.

[0004] The ubiquitin-proteasome system (UPS) is responsible for the degradation of approximately 80-90% of normal and abnormal intracellular proteins and therefore plays a central role in a large number of physiological processes. For example, the regulated proteolysis of cell cycle proteins, including cyclins, cyclin-dependent kinase inhibitors, and tumor suppressor proteins, is required for controlled cell cycle progression and proteolysis of these proteins occurs via the ubiquitin-proteasome pathway (Deshaies, Trends in Cell Biol., 5:428-434 (1995) and Hoyt, Cell, 91 : 149-151 (1997)). In another example, the activation of the transcription factor NF-κΒ, which itself plays a central role in the regulation of genes involved in the immune and inflammatory responses, is dependent upon the proteasome- mediated degradation of an inhibitory protein, Ικα Β-α (Palombella et al., WO 95/25533). In yet another example, the ubiquitin-proteasome pathway plays an essential role in antigen presentation through the continual turnover of cellular proteins.

[0005] While serving a central role in normal cellular homeostasis, the UPS also mediates the inappropriate or accelerated protein degradation occurring as a result or cause of pathological conditions including cancer, inflammatory diseases, and autoimmune diseases, characterized by deregulation of normal cellular processes. In addition, the cachexia or muscle wasting associated with conditions such as cancer, chronic infectious diseases, fever, muscle atrophy, nerve injury, renal failure, and hepatic failure results from an increase in proteolytic degradation by the UPS. Furthermore, the cytoskeletal reorganization that occurs during maturation of protozoan parasites is proteasome-dependent (Gonzales et ah, J. Exp. Med., 184: 1909 (1996)).

[0006] Central to this system is the 26S proteasome, a multi-subunit proteolytic complex, consisting of one 20S proteasome core and two flanking 19S complexes. The 20S proteasome consists of four rings: two outer a-rings and two inner β-rings surrounding a barrel-shaped cavity. The two inner β-rings form a central chamber that harbors the catalytic site for the chymotryptic, tryptic, and caspase-like activities (von Mikecz, J Cell Sci, 119(10): 1977-84, 2006).

[0007] Proteins targeted for degradation by the proteasome contain a recognition signal. This signal consists of a polyubiquitin chain that is selectively attached to the protein target by the sequential addition of ubiquitin monomers. The polyubiquitin signal is recognized by the 19S complex, which mediates the entry of the target protein into the proteolytic chamber.

SUMMARY OF THE INVENTION

[0008] The present invention is based on the discovery that polyubiquitin may be detected in patient samples and that such levels can have clinical value in the diagnosis and prognosis of certain disease states.

[0009] In one aspect, the present invention provides a method for diagnosing a proliferative hematological disorder in a subject, the method comprising: determining the level of polyubiquitin in an acellular body fluid sample from the subject, and diagnosing the subject as having a proliferative hematological disorder when a difference of the level of

polyubiquitin compared to a reference level indicates a proliferative hematological disorder in the subject. In one embodiment, a level of circulating polyubiquitin in the subject sample that is higher than a reference level measured in healthy control subjects indicates a diagnosis of CLL for the subject. In one embodiment, the reference level is the level of polyubiquitin from a sample from a control subject. In another embodiment, the reference level is the average level of polyubiquitin from samples taken from a population of healthy subjects. The reference sample is preferably an acellular body fluid sample is more preferably is the same acellular body fluid sample source as taken from the subject. [0010] In another aspect, the invention provides a method of determining a prognosis of a subject having a proliferative hematological disorder, wherein the method comprises determining the level of circulating polyubiquitin in a sample from the subject, and providing a prognosis for the subject based on a difference of the level of circulating polyubiquitin compared to a reference level.

[0011] In one embodiment, the proliferative hematological disorder is selected from the group consisting of chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), myelodysplasia syndrome (MDS), and acute lymphoblastic leukemia (ALL). In one embodiment, the proliferative hematological disorder is CLL.

[0012] In one embodiment, the reference level is a cutoff value determined from the level of polyubiquitin present in a comparable sample from a subject or population of subjects with a favorable prognosis, and wherein an increase or decrease in the subject level of

polyubiquitin relative to the cutoff value is used to determine a prognosis for the subject. In one embodiment, the prognosis is selected from the group consisting of survival rate, 5 -year survival rate, and complete remission duration (CRD). In one embodiment, a level of circulating polyubiquitin greater than about 192 ng/mL indicates a better survival rate for the subject compared to subjects having a level of circulating ubiquitin or polyubiquitin less than about 192 ng/mL. In some embodiments, the cutoff value is between about 185-195 ng/mL, about 180-200 ng/mL, or about 175-205 ng/mL.

[0013] In one embodiment, the prognostic methods further comprise determining the level of beta-2 microglobulin in a sample from the subject. In one embodiment, a level of beta-2 microglobulin less than about 3.2 mg/L indicates a better survival rate for the subject compared to subjects having a level of circulating beta-2 microglobulin greater than about 3.2 mg/L.

[0014] In one embodiment, the prognostic test sample is an acellular body fluid sample. In an illustrative embodiment, the acellular body fluid is selected from the group consisting of serum and plasma. BRIEF DESCRIPTION OF THE FIGURES

[0015] FIG. 1 is a chart showing Kaplan-Meier estimates of patient survival grouped by polyubiquitin levels. E, number of patients with event (death); N, total number of patients in the group.

[0016] FIG. 2 is a chart showing Kaplan-Meier estimates of patient survival grouped by β2 microglobulin (B2M) and polyubiquitin levels in patients with chronic lymphocytic leukemia. Patients with ubiquitin levels higher than 192 ng/ml have significantly better survival.

DETAILED DESCRIPTION

[0017] The present invention relates generally to methods of assessing the ubiquitin- proteasome system (UPS) for the diagnosis of disease. As demonstrated herein, increasing or decreasing amounts of the polyubiquitin in acellular body fluids correlates with the presence of disease or the prognosis of a patient suffering from a disease. In particular, methods for diagnosing proliferative hematological disorders, determining the likelihood of survival, and methods for predicting likelihood for responsiveness to therapy are provided.

[0018] The present technology is described herein using several definitions, as set forth throughout the specification. As used herein, unless otherwise stated, the singular forms "a," "an," and "the" include plural reference. Thus, for example, a reference to "a proteasome" is a reference to one or more proteasomes.

[0019] The term "about" as used herein in reference to quantitative measurements or values, refers to the enumerated value plus or minus 10%, unless otherwise indicated.

[0020] The term "antibody" as used herein encompasses both monoclonal and polyclonal antibodies that fall within any antibody classes, e.g., IgG, IgM, IgA, IgE, or derivatives thereof. The term "antibody" also includes antibody fragments including, but not limited to, Fab, F(ab')₂, and conjugates of such fragments, and single-chain antibodies comprising an antigen recognition epitope. In addition, the term "antibody" also means humanized antibodies, including partially or fully humanized antibodies. An antibody may be obtained from an animal, or from a hybridoma cell line producing a monoclonal antibody, or obtained from cells or libraries recombinantly expressing a gene encoding a particular antibody. [0021] The terms "assessing" and "evaluating" are used interchangeably to refer to any form of measurement, and include determining if a characteristic, trait, or feature is present or not. The terms "determining," "measuring," "assessing," and "assaying" are used

interchangeably and include both quantitative and qualitative determinations. Assessing may be relative or absolute. "Assessing the presence of includes determining the amount of something present, as well as determining whether it is present or absent.

[0022] The term "body fluid" or "bodily fluid" as used herein refers to any fluid from the body of an animal. Examples of body fluids include, but are not limited to, plasma, serum, blood, lymphatic fluid, cerebrospinal fluid, synovial fluid, urine, saliva, mucous, phlegm and sputum. A body fluid sample may be collected by any suitable method. The body fluid sample may be used immediately or may be stored for later use. Any suitable storage method known in the art may be used to store the body fluid sample; for example, the sample may be frozen at about -20°C to about -70°C. Suitable body fluids are acellular fluids. "Acellular" fluids include body fluid samples in which cells are absent or are present in such low amounts that the polyubiquitin level determined reflects its level in the liquid portion of the sample, rather than in the cellular portion. Typically, an acellular body fluid contains no intact cells. Examples of acellular fluids include plasma or serum, or body fluids from which cells have been removed.

[0023] The term "clinical factors" as used herein, refers to any data that a medical practitioner may consider in determining a diagnosis or prognosis of disease. Such factors include, but are not limited to, the patient's medical history, a physical examination of the patient, complete blood count, analysis of the activity of enzymes (e.g., liver enzymes), examination of blood cells or bone marrow cells, cytogenetics, and immunophenotyping of blood cells. Levels of circulating polyubiquitin are a clinical factor.

[0024] The term "comparable" or "corresponding" in the context of comparing two or more samples, means that the same type of sample (e.g., plasma) is used in the comparison. For example, a level of polyubiquitin in a sample of plasma can be compared to level of polyubiquitin in another plasma sample. In some embodiments, comparable samples may be obtained from the same individual at different times. In other embodiments, comparable samples may be obtained from different individuals (e.g., a patient and a healthy individual). In general, comparable samples are normalized by a common factor. For example, acellular body fluid samples are typically normalized by volume body fluid and cell-containing samples are normalized by protein content or cell count.

[0025] As used herein, the term "diagnosis" means detecting a disease or disorder or determining the stage or degree of a disease or disorder. Usually, a diagnosis of a disease or disorder is based on the evaluation of one or more factors and/or symptoms that are indicative of the disease. That is, a diagnosis can be made based on the presence, absence or amount of a factor which is indicative of presence or absence of the disease or condition. Each factor or symptom that is considered to be indicative for the diagnosis of a particular disease does not need be exclusively related to the particular disease; i.e. there may be differential diagnoses that can be inferred from a diagnostic factor or symptom. Likewise, there may be instances where a factor or symptom that is indicative of a particular disease is present in an individual that does not have the particular disease. The term "diagnosis" also encompasses

determining the therapeutic effect of a drug therapy, or predicting the pattern of response to a drug therapy. The diagnostic methods may be used independently, or in combination with other diagnosing and/or staging methods known in the medical art for a particular disease or disorder, e.g., a proliferative hematological disorder.

[0026] As used herein, the phrase "difference of the level" refers to differences in the quantity of a particular markers, such as a polyubiquitin, in a sample as compared to a control or reference level. For example, the quantity of polyubiquitin may be present at an elevated amount or at a decreased amount in samples of patients with a proliferative hematological disorder compared to a reference level. In one embodiment, a "difference of a level" may be a difference between the polyubiquitin present in a sample as compared to a control of at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80% or more. In one embodiment, a "difference of a level" may be a statistically significant difference between the polyubiquitin present in a sample as compared to a control. For example, a difference may be statistically significant if the measured level of the

polyubiquitin falls outside of about 1.0 standard deviations, about 1.5 standard deviations, about 2.0 standard deviations, or about 2.5 stand deviations of the mean of any control or reference group. [0027] The term "enzyme linked immunosorbent assay" (ELISA) as used herein refers to an antibody-based assay in which detection of the antigen of interest is accomplished via an enzymatic reaction producing a detectable signal. ELISA can be run as a competitive or noncompetitive format. ELISA also includes a two-site or "sandwich" assay in which two antibodies to the antigen are used, one antibody to capture the antigen and one labeled with an enzyme or other detectable label to detect captured antibody-antigen complex. In a typical two-site ELISA, the antigen has at least one epitope to which unlabeled antibody and an enzyme-linked antibody can bind with high affinity. An antigen can thus be affinity captured and detected using an enzyme-linked antibody. Typical enzymes of choice include alkaline phosphatase or horseradish peroxidase, both of which generated a detectable product upon digestion of appropriate substrates.

[0028] The term "label" as used herein, refers to any physical molecule directly or indirectly associated with a specific binding agent or antigen which provides a means for detection for that antibody or antigen. A "detectable label" as used herein refers any moiety used to signal the amount of complex formation between a target and a binding agent. These labels are detectable by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, electrochemiluminescence or any other appropriate means. Suitable detectable labels include fluorescent dye molecules or fluorophores.

[0029] As used herein, the term "polyubiquitin" refers to a chain of ubiquitin moieties (i.e., two or more) which are transferred or attached to another polypeptide. The polypeptide to which the ubiquitin moieties may be attached is not particularly limited and may include any protein targeted for degradation by the ubiquitin proteasome system. The ubiquitin moiety can comprise a ubiquitin from any species of organism, typically a eukaryotic species. In suitable embodiments the ubiquitin moiety is a mammalian ubiquitin, such as a human ubiquitin. In one embodiment, the ubiquitin moiety comprises a 76 amino acid human ubiquitin.

[0030] The term "proliferative hematological disorder" as used herein means a disorder of a bone marrow or lymph node-derived cell type, such as a white blood cell. A proliferative hematological disorder is generally manifest by abnormal cell division resulting in an abnormal level of a particular hematological cell population. The abnormal cell division underlying a proliferative hematological disorder is typically inherent in the cells and not a normal physiological response to infection or inflammation. A leukemia is a type of proliferative hematological disorder. Exemplary proliferative hematological disorders include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myelodysplasia syndrome, chronic myeloid leukemia, hairy cell leukemia, leukemic manifestations of lymphomas, and multiple myeloma. Lymphoma is a type of proliferative disease that mainly involves lymphoid organs, such as lymph nodes, liver, and spleen. Exemplary proliferative lymphoid disorders include lymphocytic lymphoma (also called chronic lymphocytic leukemia), follicular lymphoma, large cell lymphoma, Burkitt's lymphoma, marginal zone lymphoma, lymphoblastic lymphoma (also called acute lymphoblastic lymphoma).

[0031] The term "prognosis" as used herein refers to a prediction of the probable course and outcome of a clinical condition or disease. A prognosis is usually made by evaluating factors or symptoms of a disease that are indicative of a favorable or unfavorable course or outcome of the disease. The phrase "determining the prognosis" as used herein refers to the process by which the skilled artisan can predict the course or outcome of a condition in a patient. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy. Instead, the skilled artisan will understand that the term "prognosis" refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition.

[0032] The terms "favorable prognosis" and "positive prognosis," or "unfavorable prognosis" and "negative prognosis" as used herein are relative terms for the prediction of the probable course and/or likely outcome of a condition or a disease. A favorable or positive prognosis predicts a better outcome for a condition than an unfavorable or negative prognosis. In a general sense, a "favorable prognosis" is an outcome that is relatively better than many other possible prognoses that could be associated with a particular condition, whereas an unfavorable prognosis predicts an outcome that is relatively worse than many other possible prognoses that could be associated with a particular condition. Typical examples of a favorable or positive prognosis include a better than average cure rate, a lower propensity for metastasis, a longer than expected life expectancy, differentiation of a benign process from a cancerous process, and the like. For example, a positive prognosis is one where a patient has a 50% probability of being cured of a particular cancer after treatment, while the average patient with the same cancer has only a 25% probability of being cured.

[0033] As used herein, "plasma" refers to acellular fluid found in blood. Plasma may be obtained from blood by removing whole cellular material from blood by methods known in the art (e.g., centrifugation, filtration, and the like). As used herein, "peripheral blood plasma" refers to plasma obtained from peripheral blood samples.

[0034] As used herein, "serum" includes the fraction of plasma obtained after plasma or blood is permitted to clot and the clotted fraction is removed.

[0035] The terms "polypeptide," "protein," and "peptide" are used herein interchangeably to refer to amino acid chains in which the amino acid residues are linked by peptide bonds or modified peptide bonds. The amino acid chains can be of any length of greater than two amino acids. Unless otherwise specified, the terms "polypeptide," "protein," and "peptide" also encompass various modified forms thereof. Such modified forms may be naturally occurring modified forms or chemically modified forms. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, ubiquitinated forms, etc. Modifications also include intra-molecular crosslinking and covalent attachment to various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, etc. In addition, modifications may also include cyclization, branching and cross-linking. Further, amino acids other than the conventional twenty amino acids encoded by genes may also be included in a polypeptide.

[0036] As used herein, the term "proteasome" refers to certain large protein complexes within cells or body fluid that degrade proteins that have been tagged for elimination, particularly those tagged by ubiquitination. Proteasomes degrade denatured, misfolded, damaged, or improperly translated proteins. Proteasomal degradation of certain proteins, such as cyclins and transcription factors, serves to regulate the levels of such proteins.

Exemplary proteasomes include the 26S proteasome, 20S proteasome, and the

immunoproteasome .

[0037] The "26S proteasome" consists of 3 subcomplexes. The 26S proteasome consists of a 20S proteasome at the core which is capped at each end by a 19S regulatory particle (RP or PA700). The 19S RP mediates the recognition of the ubiquitinated target proteins, the ATP- dependent unfolding and the opening of the channel in the 20S proteasome, allowing entry of the target protein into the proteolytic chamber.

[0038] The "20S proteasome," which forms the core protease (CP) of the 26S proteasome, is a barrel-shaped complex consisting of four stacked rings, each ring having 7 distinct subunits. The four rings are stacked one on top of the other and are responsible for the proteolytic activity of the proteasome. There are two identical outer a rings, having no known function, and two inner β rings, containing multiple catalytic sites. In eukaryotes, two of these sites on the β rings have chymotrypsin-like activity (Ch-L), two of these sites have trypsin-like activity (Tr-L), and two have caspase-like activity (Cas-L).

[0039] The "immunoproteasome," which is characterized by an ability to generate major histocompatibility complex class I-binding peptides, consists of a 20S proteasome core capped on one end by 19S RP and on the other end by PA28, an activator of the 20S proteasome and an alternative RP. PA28 consists of two homologous subunits (termed a and β) and a separate but related protein termed ΡΑ28γ (also known as the Ki antigen).

[0040] The term "proteasomal peptidase activity" refers to any proteolytic enzymatic activity associated with a proteasome, such as the 26S or 20S proteasomes. The peptidase activities of proteasomes include, for example, chymotrypsin-like activity (Ch-L), trypsin- like activity (Tr-L), and caspase-like activity (Cas-L). In some embodiments, proteasomal peptidase activity is determined by measuring the rate of cleavage of a substrate per unit volume of body fluid assayed. Thus, the activity may be expressed as (moles of product formed)/time/(volume body fluid). For example, the activity may be expressed as

pmol/sec/mL.

[0041] As used herein, the term "reference level" refers to a level of a substance which may be of interest for comparative purposes. In one embodiment, a reference level may be the level of polyubiquitin expressed as an average of the polyubiquitin level from similar samples taken from a control population of healthy (disease-free) subjects. In another embodiment, the reference level may be the level in the same subject at a different time, e.g., before the present assay such as the level determined prior to the subject developing the disease or prior to initiating therapy. In general, samples are normalized by a common factor. For example, acellular body fluid samples are normalized by volume body fluid and cell-containing samples are normalized by protein content or cell count. [0042] As used herein, the term "sample" may include, but is not limited to, bodily tissue or a bodily fluid such as blood (or a fraction of blood such as plasma or serum), lymph, mucus, tears, saliva, sputum, urine, semen, stool, CSF, ascites fluid, or whole blood, and including biopsy samples of body tissue. A sample may be obtained from any subject, e.g., a subject/patient having or suspected to have a proliferative hematological disorder.

[0043] As used herein, the term "subject" refers to a mammal, such as a human, but can also be another animal such as a domestic animal {e.g., a dog, cat, or the like), a farm animal {e.g., a cow, a sheep, a pig, a horse, or the like) or a laboratory animal {e.g., a monkey, a rat, a mouse, a rabbit, a guinea pig, or the like). The term "patient" refers to a "subject" who is, or is suspected to be, afflicted with proliferative hematological disorder.

[0044] As used herein, the term "specific activity" of one or more proteasomal peptidases refers to the proteasomal peptidase activity in the sample that is normalized relative to the proteasomal protein content in the sample. Specific activity of the chymotrypsin-like, trypsin-like, and caspase-like proteasomal peptidases may be designated Ch-L/p, Tr-L/p, or Cas-L/p, respectively. The skilled artisan understands that normalization of the proteasomal peptidase activity to the proteasomal protein content in the sample involves measuring and expressing the amount of proteasomal protein per unit volume of body fluid assayed, in the same type of sample (preferably a split sample) as used to measure enzymatic activity. For example, proteasomal protein may be expressed as picograms (pg) of protein per mL which, when used to normalize a proteasomal peptidase activity expressed in pmol/sec/mL, results in a specific activity expressed in pmol/sec/pg proteasomal protein.

[0045] The phrase "substantially the same as" in reference to a comparison of one value to another value for the purposes of clinical management of a disease or disorder means that the values are statistically not different. Differences between the values can vary, for example, one value may be within 20%, within 10%, or within 5% of the other value.

Overview

[0046] Disclosed herein are methods for detecting the presence or absence of proliferative hematological disorders in subjects based, at least in part, on results of testing methods of the present technology on a sample. Further disclosed herein are methods for monitoring the status of subjects diagnosed with proliferative hematological disorders based at least partially on results of tests on a sample. The test samples disclosed herein are represented by, but not limited in anyway to, sputum, blood (or a fraction of blood such as plasma, serum, or particular cell fractions), lymph, mucus, tears, saliva, urine, semen, ascites fluid, whole blood, and biopsy samples of body tissue. This disclosure is drawn, inter alia, to methods of diagnosing and monitoring proliferative hematological disorders using profiles of the ubiquitin-proteasome system (UPS).

[0047] The ubiquitin-proteasome system (UPS) plays a major role in the most important processes that control cell homeostasis in normal and neoplastic states. The present inventors have discovered that analyzing various components of the UPS can provide a profile that may be used for classifying and stratifying cancer patients for diagnosis, therapy, and prediction of clinical behavior.

[0048] In the context of cancer diagnosis, it is frequently difficult to have access to the diseased cells. This is true even in hematologic diseases because of the problem of dilution effects by normal cells. In various embodiments, the present methods overcome problems of cancer diagnosis by determining the levels of polyubiquitin in the serum or plasma of patients with proliferating hematological disorders. By studying the levels of ubiquitin and/or proteasome enzymatic activities in the plasma, a UPS profile of the leukemic blasts can be determined. Assays of polyubiqutin level for diagnosing proliferative hematological disorders is described in further detail below and in the Examples.

[0049] In some embodiments, the methods may be used to assay polyubiquitin which can predict survival of a subject having a proliferative hematological disorder. In one aspect, the methods generally provide for the detection, measuring, and comparison of polyubiquitin in a patient sample. Accordingly, the various aspects relate to the collection, preparation, separation, identification, characterization, and comparison of the abundance of polyubiquitin and other UPS proteins and/or activities in a test sample. The technology further relates to detecting and/or monitoring a sample containing polyubiquitin and one or more UPS proteins or activities, which are useful, alone or in combination, to determine the presence or absence of a proliferative hematological disorder or any progressive state thereof.

Sample Preparation

[0050] Test samples of acellular body fluid or cell-containing samples may be obtained from an individual or patient. Methods of obtaining test samples are well-known to those of skill in the art and include, but are not limited to, aspirations or drawing of blood or other fluids. Samples may include, but are not limited to, whole blood, serum, plasma, saliva, cerebrospinal fluid (CSF), pericardial fluid, pleural fluid, urine, and eye fluid.

[0051] In embodiments in which the proteasome activity will be determined using an acellular body fluid, the test sample may be a cell-containing liquid or an acellular body fluid (e.g., plasma or serum). In some embodiments in which the test sample contains cells, the cells may be removed from the liquid portion of the sample by methods known in the art (e.g. , centrifugation) to yield acellular body fluid for the proteasome activity measurement. In suitable embodiments, serum or plasma are used as the acellular body fluid sample.

Plasma and serum can be prepared from whole blood using suitable methods well-known in the art. In these embodiments, data may be normalized by volume of acellular body fluid.

[0052] The cell-containing sample includes, but is not limited to, blood, urine, organ, and tissue samples. In suitable embodiments, the cell-containing sample is a blood sample, such as a blood cell lysate. Cell lysis may be accomplished by standard procedures. In certain embodiments, the cell-containing sample is a whole blood cell lysate. In other embodiments, the cell-containing sample is a white blood cell lysate. Methods for obtaining white blood cells from blood are known in the art (Rickwood et ah, Anal. Biochem., 123:23-31 (1982); Fotino et ah, Ann. Clin. Lab. Sci., 1 : 131 (1971)). Commercial products useful for cell separation include without limitation Ficoll-Paque (Pharmacia Biotech) and NycoPrep (Nycomed). In some situations, white blood cell lysates provide better reproducibility of data than do whole blood cell lysates.

[0053] Variability in sample preparation of cell-containing samples can be corrected by normalizing the data by, for example, protein content or cell number. In certain

embodiments, polyubiquitin in the sample may be normalized relative to the total protein content or proteasomal protein content in the sample. Total protein content in the sample can be determined using standard procedures, including, without limitation, Bradford assay and the Lowry method. In other embodiments, polyubiquitin in the sample may be normalized relative to cell number.

Measuring Polyubiquitin Level

[0054] In one embodiment, the quantity or concentration polyubiquitin may be measured by determining the amount of one or more ubiquitin proteins in a sample. The polypeptides can be detected by an antibody which is detectably labeled, or which can be subsequently labeled. A variety of formats can be employed to determine whether a sample contains a proteasomal protein or proteins that bind to a given antibody. Immunoassay methods useful in the detection of ubiquitin proteins include, but are not limited to, e.g., dot blotting, western blotting, protein chips, immunoprecipitation (IP), competitive and non-competitive protein binding assays, enzyme-linked immunosorbent assays (ELISA), and others commonly used and widely-described in scientific and patent literature, and many employed commercially.

[0055] Proteins from samples can be isolated using techniques that are well-known to those of skill in the art. The protein isolation methods employed can, e.g., be including, but not limited to, e.g., those described in Harlow & Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988). In some embodiments, proteasomal protein is extracted from the acellular body fluid sample. Plasma purification methods are known in the art such. See e.g., Cohn, E.J., et ah, Am. Chem. Soc,

62:3396-3400.(1940); Cohn, E.J., et al., J. Am. Chem. Soc, 72:465-474 (1950); Pennell, R.B., Fractionation and isolation of purified components by precipitation methods, pp. 9-50. In The Plasma Proteins, Vol. 1, F.W. Putman (ed.). Academic Press, New York (1960); and US Patent No. 5,817,765.

[0056] Antibodies can be used in methods, including, but not limited to, e.g. , western blots or ELISA, to detect the expressed protein complexes. In such uses, it is possible to immobilize either the antibody or proteins on a solid support. Supports or carriers include any support capable of binding an antigen or an antibody. Well-known supports or carriers include, but are not limited to, e.g., glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.

[0057] Methods of generating antibodies are well known in the art, see, e.g., Sambrook, et ah, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, NY. Antibodies may be detectably labeled by methods known in the art. Labels include, but are not limited to, radioisotopes such as ³H, ¹⁴C, ³⁵S, ³²P, ¹²³I, ¹²⁵I, ¹³¹I), enzymes {e.g., peroxidase, alkaline phosphatase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase and glucose oxidase), enzyme substrates, luminescent substances {e.g., luminol), fluorescent substances {e.g., FITC, rhodamine, lanthanide phosphors), biotinyl groups (which can be detected by marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods), predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags) and colored substances. In binding these labeling agents to the antibody, the maleimide method (Kitagawa, T., et al., J. Biochem., 79:233-236 (1976)), the activated biotin method (Hofmann, K., et ah, J. Am. Chem. Soc, 100:3585 (1978)) or the hydrophobic bond method, for instance, can be used.

[0058] In some embodiments, labels are attached via spacer arms of various lengths to reduce potential steric hindrance. Antibodies may also be coupled to electron dense substances, such as ferritin or colloidal gold, which are readily visualized by electron microscopy.

[0059] Where a radioactive label is used as a detectable substance, proteins may be localized by autoradiography. The results of autoradiography may be quantitated by determining the density of particles in the autoradiographs by various optical methods, or by counting the grains.

[0060] The antibody or sample may be immobilized on a carrier or solid support which is capable of immobilizing cells, antibodies, etc. For example, the carrier or support may be nitrocellulose, or glass, polyacrylamides, gabbros, and magnetite. The support material may have any possible configuration including spherical (e.g. bead), cylindrical (e.g. inside surface of a test tube or well, or the external surface of a rod), or flat (e.g. sheet, test strip). Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against one or more proteins that comprise a proteasome. Antibodies to proteasomal proteins are available commercially through multiple sources. For example, polyclonal antibodies directed to proteasome core subunit are available from Biomol International, Cat. No. PW8155-0100 (Plymouth, PA). Monoclonal antibodies directed to proteasome a subnit are available from Biomol International, Cat. No. PW8100 (Plymouth, PA).

[0061] Immunoassays, or assays to detect an antigen using an antibody, are well known in the art and can take many forms, e.g., radioimmunoassay, immunoprecipitation, Western blotting, enzyme-linked immunosorbent assay (ELISA), and 2-site or sandwich

immunoassay.

[0062] In one embodiment, a sandwich ELISA is used. In this assay, two antibodies to different segments, or epitopes, of the antigen are used. The first antibody (capture antibody) is coupled to a solid support. When a sample of bodily fluid is contacted with the capture antibody on the solid support, the antigen contained in the bodily fluid is captured on the solid support through a specific interaction between antigen and antibody, resulting in the formation of a complex. Washing of the solid support removes unbound or non-specifically bound antigen. Subsequent exposure of the solid support to a detectably-labeled second antibody (detection antibody) to the antigen (generally to a different epitope than the capture antibody) enables the detection of bound or captured antigen.

[0063] In an illustrative embodiment, an electro-chemiluminescent sandwich immunoassay is used. In this assay, two antibodies to different segments, or epitopes, of the antigen are used. For instance, antibody is coated on plates to capture the ubiquitin protein. The antibody may be a mouse monoclonal antibody to ubiquitin. A sample is contacted to the plate, and after incubation under appropriate binding conditions, the plate is washed. After the wash, primary detection antibody, which also binds to the ubiquitin, is added to each well and incubated. After another wash, a Sulfo-tag labeled secondary antibody (capable of binding to the primary antibody) is added to each well and incubated for another hour. After a final wash, a MSD read buffer is added and signal is detected by MSD Sector2400 (MSD, Gaithersburg, Maryland).

[0064] Relative or actual amounts of polyubiquitin in body fluids can be determined by methods well known in the art. See, e.g., Drach, J., et ah, Cytometry, 10(6):743-749 (1989). For example, a standard curve can be obtained using known amounts of ubiquitin, i.e., ubiquitin standards. The actual amount of the polyubiquitin in a body fluid may thus be determined using the standard curve. Another approach that does not use a standard curve is to determine the dilution of body fluid that gives a specified amount of signal. The dilution at which 50% of the signal is obtained is often used for this purpose. In this case, the dilution at 50% maximal binding of polyubiquitin in a patient body fluid is compared with the dilution at 50% of maximal binding for polyubiquitin obtained in the same assay using a reference sample (i.e., a sample taken from the corresponding bodily fluid of normal individuals, free of proliferative disorders).

[0065] Monoclonal or polyclonal antibodies may be used as the capture and detection antibodies in sandwich immunoassay systems. Monoclonal antibodies are specific for single epitope of an antigen and allow for detection and quantitation of small differences in antigen. Polyclonal antibodies can be used as the capture antibody to capture large amounts of antigen or can be used as the detection antibody. A monoclonal antibody can be used as the either the capture antibody or the detection antibody in the sandwich assay to provide greater specificity. In some embodiments, polyclonal antibodies are used as the capture antibody and monoclonal antibodies are used as the detection antibody.

[0066] One consideration in designing a sandwich ELIS A is that the capture and detection antibodies should be generated against or recognize "non-overlapping" epitopes. The phrase "non-overlapping" refers to epitopes, which are segments or regions of an antigen that are recognized by an antibody, that are sufficiently separated from each other such that an antibody for each epitope can bind simultaneously. That is, the binding of one antibody (e.g., the capture antibody) to a first epitope of the antigen should not interfere with the binding of a second antibody (e.g., the detection antibody) to a second epitope of the same antigen. Capture and detection antibodies that do not interfere with one another and can bind simultaneously are suitable for use in a sandwich ELISA.

[0067] Methods for immobilizing capture antibodies on a variety of solid surfaces are well- known in the art. The solid surface may be composed of any of a variety of materials, for example, glass, quartz, silica, paper, plastic, nitrocellulose, nylon, polypropylene,

polystyrene, or other polymers. The solid support may be in the form of beads,

microparticles, microspheres, plates which are flat or comprise wells, shallow depressions, or grooves, microwell surfaces, slides, chromatography columns, membranes, filters, or microchips. In one embodiment, the solid support is a microwell plate in which each well comprises a distinct capture antibody to a specific marker so that multiple markers may be assayed on a single plate. In another embodiment, the solid support is in the form of a bead or microp article. These beads may be composed of, for example, polystyrene or latex. Beads may be of a similar size or may be of varying size. Beads may be approximately 0.1 μιη - 10 μιη in diameter or may be as large as 50 μιη - 100 μιη in diameter.

[0068] Methods of identifying the binding of a specific binding agent to polyubiquitin are known in the art and vary dependent on the nature of the label. In suitable embodiments, the detectable label is a fluorescent dye. Fluorescent dyes are detected through exposure of the label to a photon of energy of one wavelength, supplied by an external source such as an incandescent lamp or laser, causing the fluorophore to be transformed into an excited state. The fluorophore then emits the absorbed energy in a longer wavelength than the excitation wavelength which can be measured as fluorescence by standard instruments containing fluorescence detectors. Exemplary fluorescence instruments include spectrofiuorometers and microplate readers, fluorescence microscopes, fluorescence scanners, and flow cytometers.

[0069] In one embodiment, a sandwich assay is constructed in which the capture antibody is coupled to a solid support such as a bead or microparticle. Captured antibody-antigen complexes, subsequently bound to detection antibody, are detected using flow cytometry and is well-known in the art. Flow cytometers hydrodynamically focus a liquid suspension of particles (e.g., cells or synthetic microparticles or beads) into an essentially single-file stream of particles such that each particle can be analyzed individually. Flow cytometers are capable of measuring forward and side light scattering which correlates with the size of the particle. Thus, particles of differing sizes or fluorescent characteristics may be used in invention methods simultaneously to detect distinct markers. Fluorescence at one or more wavelengths can be measured simultaneously. Consequently, particles can be sorted by size and the fluorescence of one or more fluorescent labels can be analyzed for each particle. Exemplary flow cytometers include the Becton-Dickinson Immunocytometry Systems FACSCAN. Equivalent flow cytometers can also be used in the invention methods.

Measuring proteasome activity

[0070] In some embodiments, the level of proteasomes or proteasomal activity is also measured in the patient sample. Proteasome activity in the test sample can be measured by any assay method suitable for determining 20S or 26S proteasome peptidase activity. (See, e.g., Vaddi et al, U.S. Patent No. 6,613,541; McCormack et al, Biochemistry, 37:7792-7800 (1998)); Driscoll and Goldberg, J. Biol. Chem., 265:4789 (1990); Orlowski et al,

Biochemistry, 32: 1563 (1993)). In a suitable embodiment, a substrate having a detectable label is provided to the reaction mixture and proteolytic cleavage of the substrate is monitored by following disappearance of the substrate or appearance of a cleavage product. Detection of the label may be achieved, for example, by fluorometric, colorimetric, or radiometric assay.

[0071] Substrates for use in determining proteasomal peptidase activity may be chosen based on the selectivity of each peptidase activity. For example, the chymotrypsin-like peptidase preferentially cleaves peptides on the carboxyl side of tyrosine, tryptophan, phenylalanine, leucine, and methionine residues. The trypsin- like peptidase preferentially cleaves peptides on the carboxyl side of arginine and lysine residues. The caspase-like peptidase (or peptidylglutamyl-peptide hydrolase) preferentially cleaves peptides at glutamic acid and aspartic acid residues. Based on these selectivities, the skilled artisan can choose a specific substrates for each peptidase.

[0072] Suitable substrates for determining 26S proteasome activity include, without limitation, lysozyme, a-lactalbumin, β-lactoglobulin, insulin b-chain, and ornithine decarboxylase. When 26S proteasome activity is to be measured, the substrate is typically ubiquitinated or the reaction mixture further contains ubiquitin and ubiquitination enzymes.

[0073] In some embodiments, the substrate is a peptide less than 10 amino acids in length. In one embodiment, the peptide substrate contains a cleavable fluorescent label and release of the label is monitored by fiuorometric assay. Non-limiting examples of substrates to measure trypsin-like activity include N-(N-benzoylvalylglycylarginyl)-7-amino-4-methylcoumarin (Bz-Val-Gly-Arg-AMC), N-(N-carbobenzyloxycarbonylleucylleucylarginyl)-7-amino-4- methylcoumarin (Z-Leu-Leu-Arg-AMC), Ac-Arg-Leu-Arg-AMC, and Boc-Leu-Arg-Arg- AMC. Non-limiting examples of substrates to measure caspase-like activity include N-(N- carbobenzyloxycarbonylleucylleucylglutamyl)-2-naphthylamine (Z-Leu-Leu-Glu-2NA), N- (N-carbobenzyloxycarbonylleucylleucylglutamyl)-7-amino-4-methylcoumarin (Z-Leu-Leu- Glu-AMC), and acetyl-L-glycyl-L-prolyl-L-leucyl-L-aspartyl-methylcoumarin (Ac-Gly-Pro- Leu-Asp-AMC). Non- limiting examples of substrates to measure chymotrypsin-like activity include N-(N-succinylleucylleucylvalyltyrosyl)-7-amino-4-methylcoumarin (Suc-Leu-Leu- Val-Tyr-AMC), Z-Gly-Gly-Leu-2NA, Z-Gly-Gly-Leu-AMC, and Suc-Arg-Pro-Phe-His-Leu- Leu-Val-Tyr-AMC.

[0074] Suitable substrates for measuring the chymotrypsin-like, caspase-like, and trypsin- like activities of the proteasome are Suc-Leu-Leu-Val-Tyr-AMC, Z-Leu-Leu-Glu-AMC, and Bz-Val-Gly-Arg-AMC, respectively, and the release of the cleavage product, AMC, can be monitored at 440 nm (λ_εχ = 380 nm). Cleavage due to a particular peptidase may be determined by, for example, using a substrate specific for that peptidase and assaying that activity independent of other peptidases.

[0075] In certain embodiments, the reaction mixture further contains a 20 S proteasome activator. Activators include those taught in Coux et al. {Ann. Rev. Biochem., 65:801-847 (1995)), such as PA28 or sodium dodecyl sulfate (SDS). However, SDS is not compatible with Bz-Val-Gly-Arg-AMC, therefore when Bz-Val-Gly-Arg-AMC is chosen as the substrate, PA28 is used instead of SDS to activate the proteasome.

Diagnosis of Disease States

[0076] In some embodiments, the level of polyubiquitin in a test sample is used to diagnose a disease. The polyubiquitin level may be compared to an appropriate reference value to determine if the levels of polyubiquitin are elevated or reduced relative to the reference value. Typically, the reference value is the polyubiquitin measured in a comparable sample from one or more healthy individuals. An increase or decrease in the polyubiquitin may be used in conjunction with clinical factors other than polyubiquitin level to diagnose a disease.

[0077] Association between a pathological state (e.g., a proliferative hematological disorder) and the aberration of a polyubiquitin level can be readily determined by

comparative analysis in a normal population and an abnormal or affected population. Thus, for example, one can study the polyubiquitin level in both a normal population and a population affected with a particular pathological state. The study results can be compared and analyzed by statistical means. Any detected statistically significant difference in the two populations would indicate an association. For example, if the polyubiquitin is statistically significantly higher in the affected population than in the normal population, then it can be reasonably concluded that higher polyubiquitin is associated with the pathological state.

[0078] Statistical methods can be used to set thresholds for determining when the polyubiquitin level in a subject can be considered to be different than or similar to a reference level. In addition, statistics can be used to determine the validity of the difference or similarity observed between a patient's polyubiquitin level and the reference level. Useful statistical analysis methods are described in L.D. Fisher & G. vanBelle, Biostatistics: A Methodology for the Health Sciences (Wiley-lnterscience, NY, 1993). For instance, confidence ("/?") values can be calculated using an unpaired 2-tailed t test, with a difference between groups deemed significant if the p value is less than or equal to 0.05. As used herein a "confidence interval" or "CI" refers to a measure of the precision of an estimated or calculated value. The interval represents the range of values, consistent with the data that is believed to encompass the "true" value with high probability (usually 95%). The confidence interval is expressed in the same units as the estimate or calculated value. Wider intervals indicate lower precision; narrow intervals indicate greater precision. Preferred confidence intervals of the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%. A "p-value" as used herein refers to a measure of probability that a difference between groups happened by chance. For example, a difference between two groups having a p-value of 0.01 (or p=0.01) means that there is a 1 in 100 chance the result occurred by chance. Preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001. Confidence intervals and p- values can be determined by methods well-known in the art. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. Exemplary statistical tests for associating a prognostic indicator with a predisposition to an adverse outcome are described hereinafter.

[0079] Once an association is established between a polyubiquitin level and a pathological state, then the particular physiological state can be diagnosed or detected by determining whether a patient has the particular aberration, i.e. elevated or reduced polyubiquitin levels. The term "elevated levels" or "higher levels" as used herein refers to levels of polyubiquitin that are higher than what would normally be observed in a comparable sample from control or normal subjects {i.e., a reference value). In some embodiments, "control levels" {i.e., normal levels) refer to a range of polyubiquitin levels that would be normally be expected to be observed in a mammal that does not have a proliferative hematological disorder. A control level may be used as a reference level for comparative purposes. "Elevated levels" refer to polyubiquitin levels that are above the range of control levels. The ranges accepted as "elevated levels" or "control levels" are dependent on a number of factors. For example, one laboratory may routinely determine the polyubiquitin level in a sample that is different than the polyubiquitin level obtained for the same sample by another laboratory. Also, different assay methods may achieve different value ranges. Value ranges may also differ in various sample types, for example, different body fluids or by different treatments of the sample. One of ordinary skill in the art is capable of considering the relevant factors and establishing appropriate reference ranges for "control values" and "elevated values" of the present invention. For example, a series of samples from control subjects and subjects diagnosed with proliferative hematological disorders can be used to establish ranges that are "normal" or "control" levels and ranges that are "elevated" or "higher" than the control range

[0080] Similarly, "reduced levels" or "lower levels" as used herein refer to polyubiquitin levels that are lower than what would normally be observed in a comparable sample from control or normal subjects {i.e., a reference value). In some embodiments, "control levels" (i.e. normal levels) refer to a range of polyubiquitin levels that would be normally be expected to be observed in a mammal that does not have a proliferative hematological disorder and "reduced levels" refer to proteasome activity levels that are below the range of such control levels.

[0081] The polyubiquitin level in a test sample can be used in conjunction with clinical factors other than polyubiquitin level to diagnose a disease. Clinical factors of particular relevance in the diagnosis of proliferative hematological disorders include, but are not limited to, the patient's medical history, a physical examination of the patient, complete blood count, examination of bone marrow cells, cytogenetics, and immunophenotyping of blood cells.

Monitoring Pro ression and/or Treatment

[0082] In one aspect, the polyubiquitin level in a biological sample of a patient is used to monitor the effectiveness of treatment or the prognosis of disease. In some embodiments, the polyubiquitin level in a test sample obtained from a treated patient can be compared to the level from a reference sample obtained from that patient prior to initiation of a treatment. Clinical monitoring of treatment typically entails that each patient serve as his or her own baseline control. In some embodiments, test samples are obtained at multiple time points following administration of the treatment. In these embodiments, measurement of polyubiquitin of in the test samples provides an indication of the extent and duration of in vivo effect of the treatment.

Determining Prognosis

[0083] A prognosis may be expressed as the amount of time a patient can be expected to survive. Alternatively, a prognosis may refer to the likelihood that the disease goes into remission or to the amount of time the disease can be expected to remain in remission.

Prognosis can be expressed in various ways; for example, prognosis can be expressed as a percent chance that a patient will survive after one year, five years, ten years or the like. Alternatively, prognosis may be expressed as the number of years, on average that a patient can expect to survive as a result of a condition or disease. The prognosis of a patient may be considered as an expression of relativism, with many factors affecting the ultimate outcome. For example, for patients with certain conditions, prognosis can be appropriately expressed as the likelihood that a condition may be treatable or curable, or the likelihood that a disease will go into remission, whereas for patients with more severe conditions prognosis may be more appropriately expressed as likelihood of survival for a specified period of time.

[0084] Additionally, a change in a clinical factor from a baseline level may impact a patient's prognosis, and the degree of change in level of the clinical factor may be related to the severity of adverse events. Statistical significance is often determined by comparing two or more populations, and determining a confidence interval and/or a p value.

[0085] Multiple determinations of polyubiquitm levels can be made, and a temporal change in activity can be used to determine a prognosis. For example, comparative measurements are made of the polyubiquitm level of an acellular body fluid in a patient at multiple time points, and a comparison of a polyubiquitm value at two or more time points may be indicative of a particular prognosis.

[0086] A prognosis is often determined by examining one or more clinical factors and/or symptoms that correlate to patient outcomes. As described herein, the polyubiquitm level is a clinical factor useful in determining prognosis. The skilled artisan will understand that associating a clinical factor with a predisposition to an adverse outcome may involve statistical analysis.

[0087] In certain embodiments, the levels of polyubiquitm are used as indicators of an unfavorable prognosis. According to the method, the determination of prognosis can be performed by comparing the measured polyubiquitin level to levels determined in

comparable samples from healthy individuals or to levels known to corresponding with favorable or unfavorable outcomes. The absolute polyubiquitin levels obtained may depend on an number of factors, including, but not limited to, the laboratory performing the assays, the assay methods used, the type of body fluid sample used and the type of disease a patient is afflicted with. According to the method, values can be collected from a series of patients with a particular disorder to determine appropriate reference ranges of polyubiquitin for that disorder. One of ordinary skill in the art is capable of performing a retrospective study that compares the determined polyubiquitin levels to the observed outcome of the patients and establishing ranges of levels that can be used to designate the prognosis of the patients with a particular disorder. For example, polyubiquitin levels in the lowest range would be indicative of a more favorable prognosis, while polyubiquitin levels in the highest ranges would be indicative of an unfavorable prognosis. Thus, in this aspect the term "elevated levels" refers to levels of polyubiquitin that are above the range of the reference value. In some embodiments patients with "high" or "elevated" polyubiquitin levels have levels that are higher than the median activity in a population of patients with that disease. In certain embodiments, "high" or "elevated" polyubiquitin levels for a patient with a particular disease refers to levels that are above the median values for patients with that disorder and are in the upper 40% of patients with the disorder, or to levels that are in the upper 20%> of patients with the disorder, or to levels that are in the upper 10%> of patients with the disorder, or to levels that are in the upper 5% of patients with the disorder.

[0088] Because the level of polyubiquitin in a test sample from a patient relates to the prognosis of a patient in a continuous fashion, the determination of prognosis can be performed using statistical analyses to relate the determined polyubiquitin levels to the prognosis of the patient. A skilled artisan is capable of designing appropriate statistical methods. For example, the methods may employ the chi-squared test, the Kaplan-Meier method, the log-rank test, multivariate logistic regression analysis, Cox's proportional-hazard model and the like in determining the prognosis. Computers and computer software programs may be used in organizing data and performing statistical analyses.

[0089] In certain embodiments, the prognosis of CLL patients can be correlated to the clinical outcome of the disease using the polyubiquitin level and other clinical factors.

Simple algorithms have been described and are readily adapted to this end. The approach by Giles et. ah, British Journal of Hemotology, 121 :578-585, is exemplary. As in Giles et al., associations between categorical variables (e.g. , proteasome activity levels and clinical characteristics) can be assessed via crosstabulation and Fisher's exact test. Unadjusted survival probabilities can be estimated using the method of Kaplan and Meier. The Cox proportional hazards regression model also can be used to assess the ability of patient characteristics (such as proteasome activity levels) to predict survival, with 'goodness of fit' assessed by the Grambsch-Therneau test, Schoenfeld residual plots, martingale residual plots and likelihood ratio statistics (see Grambsch et al, 1995). In some embodiments, this approach can be adapted as a simple computer program that can be used with personal computers or personal digital assistants (PDA). The prediction of patients' survival time in based on their polyubiquitin levels can be performed via the use of a visual basic for applications (VBA) computer program developed within Microsoft® Excel. The core construction and analysis may be based on the Cox proportional hazard models. The VBA application can be developed by obtaining a base hazard rate and parameter estimates. These statistical analyses can be performed using a statistical program such as the SAS®

proportional hazards regression, PHREG, procedure. Estimates can then be used to obtain probabilities of surviving from one to 24 months given the patient's covariates. The program can make use of estimated probabilities to create a graphical representation of a given patient's predicted survival curve. In certain embodiments, the program also provides 6- month, 1-year and 18-month survival probabilities. A graphical interface can be used to input patient characteristics in a user- friendly manner.

[0090] In some embodiments of the invention, multiple prognostic factors, including polyubiquitin level, are considered when determining the prognosis of a patient. For example, the prognosis of an AML, ALL, CLL, or MDS patient may be determined based on polyubiquitin and one or more prognostic factors selected from the group consisting of cytogenetics, performance status, AHD (antecedent hematological disease), age, and diagnosis (e.g., MDS v. AML). In certain embodiments, other prognostic factors may be combined with the polyubiquitin level in the algorithm to determine prognosis with greater accuracy.

Kits

[0091] A kit may be used for conducting the diagnostic and prognostic methods described herein. Typically, the kit should contain, in a carrier or compartmentalized container, reagents useful in any of the above-described embodiments of the diagnostic method. The carrier can be a container or support, in the form of, e.g., bag, box, tube, rack, and is optionally compartmentalized. The carrier may define an enclosed confinement for safety purposes during shipment and storage. In one embodiment, the kit includes an antibody selectively immunoreactive with a proteasome. The antibodies may be labeled with a detectable marker such as radioactive isotopes, or enzymatic or fluorescence markers.

Alternatively, secondary antibodies such as labeled anti-IgG and the like may be included for detection purposes. In addition, reagents to detect the level of polyubiquitin may be provided. Optionally, the kit can include standard polyubiquitin prepared or purified for comparison purposes. Instructions for using the kit or reagents contained therein are also included in the kit. EXAMPLE

[0092] The present methods and kits, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present methods and kits. The following is a description of the materials and experimental procedures used in the example.

Materials and Methods

[0093] Patients and Samples. All samples from patients and healthy volunteers were collected under an internal review board-approved protocol with written informed consent. Patient samples were collected during the period 2001-2003 without selection prior to initiating therapy at MD Anderson Cancer Center (Houston, TX). All patients were newly diagnosed, but the majority were referred after diagnosis by their local physician within a few days of their diagnosis. Plasma was separated from EDTA peripheral blood tubes by centrifuging at 1500 x g for 10 min at 4°C. Plasma samples obtained from apparently healthy volunteers were used as controls for each study. Plasma samples were stored at -70°C until analysis.

[0094] Measurement of Ubiquitin Protein. The level of polyubiquitin in plasma was quantitated by a sandwich immunoassay also using electro-chemiluminescence technology. Briefly, after 2 h blockage of the MSD GAM plate, an anti-ubiquitin monoclonal antibody (clone FK1, Biomol, Cat. No. PW8805) was coated overnight onto the MSD GAM single spot plate at 4°C on a shaker. Ubiquitin positive control (Cat. No. 89899, Pierce, Rockford, IL) was used as a calibrator to create a 7 point standard curve using Hel cell lysate. Plasma samples were diluted 1 :2 using MSD lysis buffer. Calibrator, reference standards, and plasma samples were added to the plate and incubated for 3 hours on a shaker at room temperature. In this incubation, ubiquitin (poly-ubiquitin) was specifically captured with the anti- polyubiquitin. After washing, sulfo-tag labeled anti-ubiquitin was added to the plate and incubated for 1 h. After a final wash, the MSD read buffer was added to the plate and signal was read on the MSD Sector2400. The ubiquitin levels were determined by reading against a standard curve and converting to ng ubiquitin/mL plasma.

[0095] IgVn Mutation Status. IgV_H mutation status was evaluated using PCR amplification and sequencing. Sequences with >2% mutations as compared with the corresponding germ- line IgV_H sequences were considered mutated. Cases with V_H3-21 were classified as unmutated, irrespective of the mutation status.

[0096] Statistical Methods. Clinical and biological characteristics were analyzed for their association with response and survival using log-rank test and multivariate Cox proportional hazards models (Cox, J Royal Stat Soc, 34: 187-220 (1972)). Estimates of survival curves from the time of initiating therapy were calculated according to the Kaplan-Meier product- limit method (Kaplan and Meier, J. American Statistical Association, 53:457-481 (1958)). Univariate and multivariate Cox proportional hazard models were developed; predictive variables with P values of less than 0.10 for the univariate Cox proportional hazards model were included in a multivariate model.

High Levels of Circulating Ubiquitin Occur in Patients with CLL

[0097] The ubiquitin-proteasome pathway is implicated in the pathogenesis of many hematological malignancies. The level of ubiquitin protein in the plasma of CLL patients was measured and correlated levels with clinical behavior. Using the Meso Scale Discovery (MSD) platform, the polyubiquitin levels in plasma samples from 148 patients with CLL was quantified and compared levels with 101 normal control patients. The results were compared with various laboratory parameters and outcomes.

[0098] The CLL patients were representative of patients seen in an academic referral center (Table 1). The control group had roughly equal numbers of males and females, with an age range of 20 to 72 years (median = 38 y).

Table 1. Characteristics of 148 Patients with CLL

Characteristic No. (%) of Patients

Male 61 (91)

Rai stage

0 40 (27)

I 47 (32)

II 24 (16)

III 12 (8)

IV 25 (17)

Previously treated 49 (33)

Splenomegaly 41 (28)

Hepatomegaly 25 (18)

IgV_H mutation status

No 58 (68)

Yes 27 (35)

Missing 63

Median (range)

Age, year 62 (33 - 82)

WBC, x 10⁶/L 25.80 (8 - 417)

HGB, g/dL 13.0 (6.4 - 17.1)

Platelets, x 10⁶/L 169.5 (8 - 417)

B2M, mg/L 3.2 (1.4 - 18.10)

WBC, white blood cell count; HGB, hemoglobin; B2M, β2 -microglobulin

[0099] The data showed that polyubiquitm levels were significantly higher in CLL patients than in normal control subjects (Table 2).

Group Median Minimum Maximum P value

Control 57.35 22.00 118.00

CLL 158.70 35.00 281.20 <0.001

Circulating Ubiquitin Levels Correlate with Survival

[0100] Polyubiquitm levels correlated negatively with survival in Cox proportional hazard models (Table 3; P = 0.04). Patients with poly-Ub levels in the upper quartile (>194 ng/mL) had significantly longer survival than did patients with lower levels (P = 0.004) (FIG. 1). Polyubiquitm remained an independent prognostic factor in multivariate models

incorporating IgV_H mutation status, prior therapy, and B2M. Using both B2M and polyubiquitm levels, patients could be stratified into 3 groups with significant differences in survival (FIG. 2): survival was longest (no deaths) in patients with B2M levels <3.2 mg/L and polyubiquitm levels >193 ng/mL, and shortest in those with B2M levels >3.2 mg/L and polyubiquitm levels <193 ng/mL (median survival = 34 mo). Table 3. Cox Pro ortional Hazards Models for Survival in Patients with CLL

[0101] The findings of this study confirm the importance of the proteasome-ubiquitin system in the pathophysiology of CLL. Plasma provides advantages over tumor tissue as a sample type, owing to ease and convenience of sample collection. Polyubiquitin levels correlated positively with survival, both as a continuous variable and when a cut-point was used. More importantly, the ability of polyubiquitin to predict survival was independent of the IgV_H mutation status, B2M levels, or whether the patient had been previously treated. Combining B2M and poly-Ub in one model allowed stratification into groups 3 groups based on survival. These findings suggest that polyubiquitin can be used in methods for the prognosis of patients with proliferative haematological disorders.

* * * *

[0102] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents. [0103] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0104] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0105] Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

CLAIMS What is claimed is:

1. A method for diagnosing a proliferative hematological disorder in a subject, the

method comprising: determining the level of polyubiquitin in an acellular body fluid sample from the subject, and diagnosing the subject as having a proliferative hematological disorder when an increase in the level of polyubiquitin compared to a reference level indicates a proliferative hematological disorder in the subject.

2. The method of claim 1, wherein the proliferative hematological disorder is selected from the group consisting of chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), myelodysplasia syndrome (MDS), and acute lymphoblastic leukemia (ALL).

3. The method of any one of claims 1-2, wherein the proliferative hematological

disorder is chronic lymphocytic leukemia (CLL).

4. The method of any one of claims 1-3, wherein the acellular body fluid is selected from the group consisting of serum and plasma.

5. The method of any one of claims 1-4, wherein the reference level is derived from the level of polyubiquitin from a sample from a healthy control subject or a population of healthy control subjects.

6. A method of determining a prognosis of a subject having a proliferative hematological disorder, wherein the method comprises:

determining the level of circulating polyubiquitin in a sample from the subject, and providing a prognosis for the subject based on a difference of the level of circulating polyubiquitin compared to a reference level.

7. The method of claim 6, wherein the proliferative hematological disorder is selected from the group consisting of chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), myelodysplasia syndrome (MDS), and acute lymphoblastic leukemia (ALL).

8. The method of any one of claims 6-7, wherein the proliferative hematological disorder is CLL.

9. The method of any one of claims 6-8, wherein the reference level is a cutoff value determined from the level of polyubiquitin present in a comparable sample from a subject or population of subjects with a favorable prognosis, and wherein an increase or decrease in the subject level of polyubiquitin relative to the cutoff value is used to determine a prognosis for the subject.

10. The method of any one of claims 6-9, wherein the reference level of polyubiquitin is about 175-205 ng/mL and a subject having a polyubiquitin level greater than the reference level indicates a better survival rate for the subject compared to subjects having a polyubiquitin level less than the reference level.

11. The method of any one of claims 6-10, wherein the reference level of polyubiquitin is about 192 ng/mL and a subject having a polyubiquitin level greater than the reference level indicates a better survival rate for the subject compared to subjects having a polyubiquitin level less than the reference level.

12. The method of any one of claims 6-11, wherein the prognosis is selected from the group consisting of survival rate, 5-year survival rate, and complete remission duration (CRD).

13. The method of any one of claims 6-12, further comprising determining the level of β2 microglobulin in a sample from the subject.

14. The method of claim 13, wherein a level of β2 microglobulin less than about 3.2 mg/L indicates a better survival rate for the subject compared to subjects having a level of circulating β2 microglobulin greater than about 3.2 mg/L.

15. The method of any one of claims 6-14, wherein the test sample is an acellular body fluid sample.

16. The method of claim 15, wherein the acellular body fluid is selected from the group consisting of serum and plasma.