Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/32—Cardiovascular disorders
  • G01N2800/323—Arteriosclerosis, Stenosis

Definitions

• the present invention relates to the identification and use of diagnostic and prognostic markers for vascular diseases, particularly subclinical atherosclerosis.
• ACS acute coronary syndromes
• myocardial injury or myocardial damage that is commonly secondary to atherosclerosis or hypertension, and is the leading cause of death in the United States.
• ACS is commonly caused by occlusion associated with coronary artery disease cause by atherosclerotic plaque formation and progression to either further occlusion or fissure.
• ACS can be manifested as stable angina, unstable angina, or myocardial infarction.
• ACS is believed to result largely from thrombus deposition and growth within one or more coronary arteries, resulting in a partial or complete occlusion of the artery, and frequently involves rupture of the plaque, resulting in an ischemic injury.
• ACS may also be precipitated by a coronary vasospasm or increased myocardial demand. For review, see, e.g., Davies, Clin. Cardiol. 20 (Supp. I): 12-17 (1997).
• Stable angina is characterized by constricting chest pain that occurs upon exertion or stress, and is relieved by rest or sublingual nitroglycerin.
• Unstable angina is characterized by constricting chest pain at rest that is relieved by sublingual nitroglycerin.
• Anginal chest pain is usually relieved by sublingual nitroglycerin, and the pain usually subsides within 30 minutes.
• Myocardial infarction is characterized by constricting chest pain lasting longer than 30 minutes that can be accompanied by diagnostic electrocardiography (ECG) Q waves.
• ECG electrocardiography
• Unstable angina is thought to represent the clinical state between stable angina and myocardial infarction, and is commonly associated with atherosclerotic plaque rupture and thrombus formation. In this regard, atherosclerotic plaque rupture is the most common cause of myocardial infarction.
• ECG electrocardiography
• AMI acute myocardial infarction
• Patients with ACS frequently have constricting chest pain that often radiates to the neck, jaw, shoulders, or down the inside of the left or both arms and can have accompanying symptoms of dyspnea, diaphoresis, palpitations, light-headedness, and nausea.
• Myocardial ischemia can produce diagnostic ECG changes including Q waves and ST segment changes. Elevations of the plasma concentration of cardiac enzymes may reflect the degree of cardiac tissue necrosis associated with severe unstable angina and myocardial infarction.
• CAC coronary artery calcium
• TAC thoracic aortic calcium
• EBT electron beam tomography
• MSCT multislice computed tomography
• the protein MCP-I was described as a circulating marker useful for diagnosing subclinical atherosclerosis in a subject, or assigning a prognostic risk of one or more future clinical outcomes to a subject suffering from subclinical atherosclerosis. See, e.g., U.S. Patent Application Publication 2004/0203083 entitled "Use of thrombus precursor protein and monocyte chemoattractant protein as diagnostic and prognostic indicators in vascular diseases," which is hereby incorporated by reference in its entirety.
• the present invention relates to the identification and use of diagnostic and/or prognostic markers for one or more vascular diseases.
• Natriuretic peptides, their precursors, and fragments thereof can be used in methods and devices for diagnosing subclinical atherosclerosis in a subject, or assigning a prognostic risk of one or more future clinical outcomes to a subject suffering from subclinical atherosclerosis.
• the time horizon over which such risk stratification may be applied may be from 1 day to 5 years, more preferably from 1 week to 2 years, and most preferably from 1 month to 1 year.
• methods for diagnosing subclinical atherosclerosis comprise performing one or more assays on a test sample obtained from the subject, such assay(s) being configured to detect the presence or amount of one or more natriuretic peptides or markers related thereto, and using results of the assays performed to assign the presence or absence of subclinical atherosclerosis to the subject.
• the assay(s) detecting one or more natriuretic peptides or markers related thereto may be used together with other biochemical markers and/or physical characteristics of the subject in a diagnostic "panel.”
• Such panels may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more or individual assays, at least one of which being an assay configured to detect the presence or amount of one or more natriuretic peptides or markers related thereto.
• Such panels may, then, include values determined from characteristics such as ACS risk factors, CAC scores, and/or one or more additional assays configured to detect one or more additional biochemical markers independently selected from the group consisting of specific markers of cardiac injury, specific markers of neural tissue injury, markers related to blood pressure regulation, markers related to inflammation, markers related to coagulation and hemostasis, and markers related to apoptosis.
• characteristics such as ACS risk factors, CAC scores, and/or one or more additional assays configured to detect one or more additional biochemical markers independently selected from the group consisting of specific markers of cardiac injury, specific markers of neural tissue injury, markers related to blood pressure regulation, markers related to inflammation, markers related to coagulation and hemostasis, and markers related to apoptosis.
• the invention features methods of predicting a risk of one or more clinical outcomes for a subject suffering from subclinical atherosclerosis by performing one or more assays on a test sample obtained from the subject, such assay(s) being configured to detect the presence or amount of one or more natriuretic peptides or markers related thereto, and using results of the assay(s) performed to associate a risk of one or more clinical outcomes to the subject.
• assay(s) being configured to detect the presence or amount of one or more natriuretic peptides or markers related thereto, and using results of the assay(s) performed to associate a risk of one or more clinical outcomes to the subject.
• the analysis of natriuretic peptides or markers related thereto may be used together with other biochemical markers and/or physical characteristics of the subject in a prognostic "panel.”
• preferred assays detect one or more natriuretic peptides selected from the group consisting of atrial natriuretic peptide ("ANP"), pro- ANP, NT-proANP, B-type natriuretic peptide ("BNP”), NT-pro BNP, pro-BNP, and C- type natriuretic peptide.
• ANP atrial natriuretic peptide
• BNP B-type natriuretic peptide
• NT-pro BNP pro-BNP
• C- type natriuretic peptide C- type natriuretic peptide.
• Particularly preferred assays detect one or more natriuretic peptides selected from the group consisting of BNP, NT-pro BNP, and pro-BNP.
• an assay is "configured to detect" a particular marker of interest if that assay generates a detectable signal indicative of the presence or amount of a physiologically relevant concentration of that marker.
• Such an assay may, but need not, specifically detect a particular natriuretic peptide (e.g., detect BNP but not proBNP).
• a particular natriuretic peptide e.g., detect BNP but not proBNP.
• an immunoassay will detect other polypeptides (e.g., related markers) so long as the other polypeptides contain the epitope(s) necessary to bind to the antibody used in the assay.
• an assay that detects BNP may also detect proBNP, together with one or more fragments of BNP or proBNP that may exist in the sample, to the extent that such molecules contain the necessary epitopes to be detected in the assay.
• preferred marker(s) related to blood pressure regulation for use in the methods described herein comprise, for example, one or more marker(s) selected from the group consisting of urotensin II, arginine vasopressin, aldosterone, angiotensin I, angiotensin II, angiotensin III, bradykinin, calcitonin, procalcitonin, calcitonin gene related peptide, adrenomedullin, calcyphosine, endothelin-2, endothelin-3, renin, and urodilatin, or markers related thereto.
• marker(s) selected from the group consisting of urotensin II, arginine vasopressin, aldosterone, angiotensin I, angiotensin II, angiotensin III, bradykinin, calcitonin, procalcitonin, calcitonin gene related peptide, adrenomedullin,
• Preferred marker(s) markers related to inflammation for use in the methods described herein comprise, for example, one or more marker(s) selected from the group consisting of acute phase reactants, cell adhesion molecules such as vascular cell adhesion molecule ("VCAM”), intercellular adhesion molecule- 1 (“ICAM-I”), intercellular adhesion molecule-2 (“ICAM-2”), and intercellular adhesion molecule-3 (“ICAM-3”), C-reactive protein, interleukins such as IL-l ⁇ , IL-6, and IL-8, interleukin-1 receptor agonist, caspase-3, lipocalin-type prostaglandin D synthase, mast cell tryptase, eosinophil cationic protein, KL-6, haptoglobin, tumor necrosis factor ⁇ , tumor necrosis factor ⁇ , Fas ligand, soluble Fas (Apo-1), TRAIL, TWEAK, fibronectin, macrophage migration inhibitory factor (MIF), and
• acute phase reactants refers to proteins whose concentrations are elevated in response to stressful or inflammatory states that occur during various insults that include infection, injury, surgery, trauma, tissue necrosis, and the like. Acute phase reactant expression and serum concentration elevations are not specific for the type of insult, but rather as a part of the homeostatic response to the insult.
• one or more markers related to inflammation may also be selected from the group of acute phase reactants consisting of hepcidin, HSP-60, HSP-65, HSP-70, asymmetric dimethylarginine (an endogenous inhibitor of nitric oxide synthase), matrix metalloproteins 11, 3, and 9, defensin HBD 1, defensin HBD 2, serum amyloid A, oxidized LDL, insulin like growth factor, transforming growth factor ⁇ , e-selectin, glutathione-S-transferase, hypoxia-inducible factor- l ⁇ , inducible nitric oxide synthase ("I-NOS”), intracellular adhesion molecule, lactate dehydrogenase, monocyte chemoattractant peptide-1 ("MCP-I”), n-acetyl aspartate, prostaglandin E2, receptor activator of nuclear factor (“RANK”)
• hepcidin HSP-60, HSP-65, HSP-70,
• Preferred marker(s) related to coagulation and hemostasis for use in the methods described herein comprise, for example, one or more marker(s) selected from the group consisting of plasmin, fibrinogen, D-dimer, ⁇ -thromboglobulin, platelet factor 4, fibrinopeptide A, platelet-derived growth factor, prothrombin fragment 1+2, plasmin- ⁇ 2-antiplasmin complex, thrombin-antithrombin III complex, P-selectin, thrombin, and von Willebrand factor, tissue factor, or markers related thereto.
• one or more marker(s) selected from the group consisting of plasmin, fibrinogen, D-dimer, ⁇ -thromboglobulin, platelet factor 4, fibrinopeptide A, platelet-derived growth factor, prothrombin fragment 1+2, plasmin- ⁇ 2-antiplasmin complex, thrombin-antithrombin III complex, P-selectin, thrombin, and von Willebrand factor, tissue factor
• Preferred marker(s) related to apoptosis for use in the methods described herein comprise, for example, one or more marker(s) selected from the group consisting of spectrin, cathepsin D, caspase 3, s-acetyl glutathione, and ubiquitin fusion degradation protein 1 homolog.
• each assay result obtained may be compared to a "normal" value, or a value indicating a particular disease or outcome.
• a particular diagnosis/prognosis may depend upon the comparison of each assay result to such a value, which may be referred to as a diagnostic or prognostic "threshold.”
• assays for one or more diagnostic or prognostic indicators are correlated to a condition or disease by merely the presence or absence of the indicator(s) in the assay.
• an assay can be designed so that a positive signal only occurs above a particular threshold concentration of interest, and below which concentration the assay provides no signal above background.
• ROC Receiver Operating Characteristic curves
• a threshold is selected, above which (or below which, depending on how a marker changes with the disease) the test is considered to be abnormal and below which the test is considered to be normal.
• the area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition.
• a threshold is selected to provide a ROC curve area of greater than about 0.5, more preferably greater than about 0.7, still more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9.
• the term "about” in this context refers to +/- 5% of a given measurement.
• particular thresholds for one or more markers in a panel are not relied upon to determine if a profile of marker levels obtained from a subject are indicative of a particular diagnosis/prognosis. Rather, the present invention may utilize an evaluation of a marker panel "profile" as a unitary whole. A particular "fingerprint" pattern of changes in such a panel of markers may, in effect, act as a specific diagnostic or prognostic indicator. As discussed herein, that pattern of changes may be obtained from a single sample, or from temporal changes in one or more members of the panel (or a panel response value).
• a panel response value is preferably determined by plotting ROC curves for the sensitivity of a particular panel of markers versus 1- (specificity) for the panel at various cutoffs.
• a profile of marker measurements from a subject is considered together to provide a global probability (expressed either as a numeric score or as a percentage risk) of a diagnosis or prognosis.
• an increase in a certain subset of markers may be sufficient to indicate a particular diagnosis/prognosis in one patient, while an increase in a different subset of markers may be sufficient to indicate the same or a different diagnosis/prognosis in another patient.
• Weighting factors may also be applied to one or more markers in a panel, for example, when a marker is of particularly high utility in identifying a particular diagnosis/prognosis, it may be weighted so that at a given level it alone is sufficient to signal a positive result. Likewise, a weighting factor may provide that no given level of a particular marker is sufficient to signal a positive result, but only signals a result when another marker also contributes to the analysis.
• markers and/or marker panels are selected to exhibit at least about 70% sensitivity, more preferably at least about 80% sensitivity, even more preferably at least about 85% sensitivity, still more preferably at least about 90% sensitivity, and most preferably at least about 95% sensitivity, combined with at least about 70% specificity, more preferably at least about 80% specificity, even more preferably at least about 85% specificity, still more preferably at least about 90% specificity, and most preferably at least about 95% specificity.
• both the sensitivity and specificity are at least about 75%, more preferably at least about 80%, even more preferably at least about 85%, still more preferably at least about 90%, and most preferably at least about 95%.
• the term "about” in this context refers to +/- 5% of a given measurement.
• a positive likelihood ratio, negative likelihood ratio, odds ratio, or hazard ratio is used as a measure of a test's ability to predict risk or diagnose a disease.
• a value of 1 indicates that a positive result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the diseased group; and a value less than 1 indicates that a positive result is more likely in the control group.
• markers and/or marker panels are preferably selected to exhibit a positive or negative likelihood ratio of at least about 1.5 or more or about 0.67 or less, more preferably at least about 2 or more or about 0.5 or less, still more preferably at least about 5 or more or about 0.2 or less, even more preferably at least about 10 or more or about 0.1 or less, and most preferably at least about 20 or more or about 0.05 or less.
• the term "about” in this context refers to +/- 5% of a given measurement.
• markers and/or marker panels are preferably selected to exhibit an odds ratio of at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less.
• the term "about” in this context refers to +/- 5% of a given measurement.
• a value of 1 indicates that the relative risk of an endpoint (e.g., death) is equal in both the "diseased" and “control” groups; a value greater than 1 indicates that the risk is greater in the diseased group; and a value less than 1 indicates that the risk is greater in the control group.
• markers and/or marker panels are preferably selected to exhibit a hazard ratio of at least about 1.1 or more or about 0.91 or less, more preferably at least about 1.25 or more or about 0.8 or less, still more preferably at least about 1.5 or more or about 0.67 or less, even more preferably at least about 2 or more or about 0.5 or less, and most preferably at least about 2.5 or more or about 0.4 or less.
• the term "about” in this context refers to +/- 5% of a given measurement.
• associating a diagnostic or prognostic indicator, with a diagnosis or with a prognostic risk of a future clinical outcome is a statistical analysis.
• a marker level of greater than X may signal that a patient is more likely to suffer from an adverse outcome than patients with a level less than or equal to X, as determined by a level of statistical significance.
• a change in marker concentration from baseline levels may be reflective of patient prognosis, and the degree of change in marker level 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.
• Preferred confidence intervals of the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.
• multiple determinations of diagnostic or prognostic markers can be made, and a temporal change in the marker can be used to determine a diagnosis or prognosis.
• a marker concentration in a subject sample may be determined at an initial time, and again at a second time from a second subject sample.
• an increase in the marker from the initial time to the second time may be indicative of a particular diagnosis, or a particular prognosis.
• a decrease in the marker from the initial time to the second time may be indicative of a particular diagnosis, or a particular prognosis.
• the invention in another aspect, relates to methods for selecting a treatment regimen for use in a subject.
• the methods preferably comprise performing a diagnostic or prognostic method as described herein, and selecting one or more treatment regimens that improve the patient's prognosis by reducing the increased disposition for an adverse outcome associated with the diagnosis.
• Such methods may also be used to screen pharmacological compounds for agents capable of improving the patient's prognosis as above.
• the invention relates to methods for monitoring a treatment regimen for use in a subject.
• a diagnostic or prognostic method as described herein can be performed at desired intervals, and a change in the resut, in the form of a change in diagnosis and/or prognosis, can be used to monitor the efficacy of appropriate therapies.
• a change in the resut in the form of a change in diagnosis and/or prognosis
• a change in the resut in the form of a change in diagnosis and/or prognosis
• kits and devices for determining the diagnosis of subclinical atherosclerosis and/or assigning a prognosis to a subject suffering from subclinical atherosclerosis.
• Kits preferably comprise devices and reagents for performing the assays described herein, and instructions for performing the assays.
• the kits may contain one or more means for converting marker level(s) to a diagnosis or prognosis, such as a suggested threshold measurement to be used to indicate a particular diagnosis and/or prognosis.
• Such kits preferably contain sufficient reagents to perform one or more such determinations, and/or Food and Drug Administration (FDA)-approved labeling.
• FDA Food and Drug Administration
• Natriuretic peptides are a group of naturally occurring substances that act in the body to oppose the activity of the renin-angiotensin system. There are three major natriuretic peptides: atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP).
• ANP atrial natriuretic peptide
• BNP B-type natriuretic peptide
• CNP C-type natriuretic peptide
• Mature human A-type natriuretic peptide (also referred to as atrial natriuretic peptide) is a 28 amino acid peptide that is synthesized, stored, and released by atrial myocytes in response to atrial distension, angiotensin II stimulation, endothelin, and sympathetic stimulation (beta-adrenoceptor mediated).
• Mature ANP is synthesized as a precursor molecule (pro-ANP) that is converted to an active form by proteolytic cleavage.
• pro-ANP precursor molecule
• NT-proANP and linear peptide fragments from the N-terminal prohormone segment have also been reported to have biological activity.
• BNP B-type natriuretic peptide
• BNP brain-type natriuretic peptide
• the precursor to BNP is synthesized as a 108-amino acid molecule, referred to as "pro-BNP” that is proteolytically processed into a 76-amino acid N-terminal peptide (amino acids 1-76), referred to as “NT-proBNP” and the 32-amino acid mature hormone, referred to as BNP or BNP 32 (amino acids 77-108). It has been suggested that each of these species - NT pro-BNP, BNP-32, and the pre-pro-BNP - together with various fragments thereof, can circulate in human plasma. See, e.g., Tateyama et al., Biochem. Biophys. Res. Commun.
• BNP 1-1OS is synthesized as a larger precursor pre-pro-BNP having the following sequence (with the "pre” sequence shown in bold):
• CNP C-type natriuretic peptide
• CNP is structurally related to A-type natriuretic peptide (ANP) and B-type natriuretic peptide (BNP); however, while ANP and BNP are synthesized predominantly in the myocardium, CNP is synthesized in the vascular endothelium as a precursor (pro-CNP) (Prickett et al. , Biochem. Biophys. Res. Commun. 286:513-7, 2001). CNP is thought to possess vasodilator effects on both arteries and veins and has been reported to act mainly on the vein by increasing the intracellular cGMP concentration in vascular smooth muscle cells .
• ANP and BNP are released in response to atrial and ventricular stretch, respectively, and will cause vasorelaxation, inhibition of aldosterone secretion in the adrenal cortex, and inhibition of renin secretion in the kidney. Both ANP and BNP will cause natriuresis and a reduction in intravascular volume, effects amplified by the antagonism of antidiuretic hormone (ADH).
• ADH antidiuretic hormone
• the physiologic effects of CNP differ from those of ANP and BNP; CNP has a hypotensive effect, but no significant diuretic or natriuretic actions.
• natriuretic peptides have been found in certain disease states, suggesting a role in the pathophysiology of those diseases, including stroke, congestive heart failure (CHF), cardiac ischemia, systemic hypertension, and acute myocardial infarction.
• CHF congestive heart failure
• cardiac ischemia cardiac ischemia
• systemic hypertension e.g., systemic hypertension
• acute myocardial infarction e.g., WO 02/089657; WO 02/083913; and WO 03/016910, each of which is hereby incorporated in its entirety, including all tables, figures, and claims.
• the prepro-BNP, proBNP (BNP 1-108 )and NT-proBNP (BNP 1 _ 76 )molecules represent BNP -related markers that may be measured either as surrogates for mature BNP or as markers in and of themselves.
• one or more fragments of these molecules including BNP -related polypeptides selected from the group consisting of BNP 77-106 , BNP 79-106 , BNP 76-107 , BNP 69-108 , BNP 79-108 , BNP 80-108 , BNP 81-108 , BNP 83-108 , BNP 39-86 , BNP 53-85 , BNP 66-98 , BNP 30-103 , BNP 11-107 , BNP 9-106 , and BNP 3-108 may also be present in circulation.
• Natriuretic peptide fragments, including BNP fragments may also comprise one or more oxidizable methionines, the oxidation of which to methionine sulfoxide or methionine sulfone produces additional BNP -related markers. See, e.g., U.S. Patent No. 10/419,059, filed April 17, 2003, which is hereby incorporated by reference in its entirety including all tables
• Myocardial ischemia is caused by an imbalance of myocardial oxygen supply and demand. Specifically, demand exceeds supply due to inadequate blood supply. The heart accounts for a small percentage of total body weight, but is responsible for 7% of body oxygen consumption. Cardiac tissue metabolism is highly aerobic and has very little reserve to compensate for inadequate blood supply. When the blood supply is reduced to levels that are inadequate for myocardial demand, the tissue rapidly becomes hypoxic and toxic cellular metabolites can not be removed. Myocardial cells rapidly use oxygen supplies remaining in the local microvasculature, and the length of time that aerobic metabolism continues is indirectly proportional to the degree of arterial occlusion.
• oxidative phosphorylation can not continue because oxygen is no longer available as an electron acceptor, pyruvate can not be converted to acetyl coenzyme A and enter the citric acid cycle.
• Myocardial metabolism switches to anaerobic metabolism using glycogen and glucose stores, and pyruvate is fermented to lactate. Lactate accumulation is the primary cause of chest pain in individuals with ACS. As ischemia continues, cardiac tissue becomes more acidic as lactate and other acidic intermediates accumulate, ATP levels decrease, and available energy sources are depleted. Cardiac tissue can recover if it is reperfused 15-20 minutes after an ischemic event.
• the cell After the cellular glycogen stores have been depleted, the cell gradually displays features of necrosis, including mitochondrial swelling and loss of cell membrane integrity. Upon reperfusion, these damaged cells die, possibly as a result of the cell's inability to maintain ionic equilibrium. A loss of membrane integrity causes the cell's cytosolic contents to be released into the circulation.
• Stable angina, unstable angina, and myocardial infarction all share one common feature that is indicative of clinical ACS: constricting chest pain associated with myocardial ischemia.
• Angina is classified as stable or unstable through a physician's interpretation of clinical symptoms, with or without diagnostic ECG changes.
• the classification of angina as “stable” or “unstable” does not refer to the stability of the plaque itself, but rather, the degree of exertion that is required to elicit chest pain.
• the classification of chest pain as stable or unstable angina (or even mild myocardial infarction) in cases other than definitive myocardial infarction is completely subjective.
• the diagnosis, and in this case the distinction is made not by angiography, which may quantify the degree of arterial occlusion, but rather by a physician's interpretation of clinical symptoms.
• Stable angina is characterized by constricting chest pain that occurs upon exertion or stress, and is relieved by rest or sublingual nitroglycerin. Coronary angiography of patients with stable angina usually reveals 50-70% obstruction of at least one coronary artery. Stable angina is usually diagnosed by the evaluation of clinical symptoms and ECG changes. Patients with stable angina may have transient ST segment abnormalities, but the sensitivity and specificity of these changes associated with stable angina are low.
• Unstable angina is characterized by constricting chest pain at rest that is relieved by sublingual nitroglycerin. Anginal chest pain is usually relieved by sublingual nitroglycerin, and the pain usually subsides within 30 minutes.
• class I characterized as new onset, severe, or accelerated angina
• class II subacute angina at rest characterized by increasing severity, duration, or requirement for nitroglycerin
• class III characterized as acute angina at rest.
• Unstable angina represents the clinical state between stable angina and AMI and is thought to be primarily due to the progression in the severity and extent of atherosclerosis, coronary artery spasm, or hemorrhage into non-occluding plaques with subsequent thrombotic occlusion.
• Coronary angiography of patients with unstable angina usually reveals 90% or greater obstruction of at least one coronary artery, resulting in an inability of oxygen supply to meet even baseline myocardial oxygen demand.
• Slow growth of stable atherosclerotic plaques or rupture of unstable atherosclerotic plaques with subsequent thrombus formation can cause unstable angina. Both of these causes result in critical narrowing of the coronary artery.
• Unstable angina is usually associated with atherosclerotic plaque rupture, platelet activation, and thrombus formation. Unstable angina is usually diagnosed by clinical symptoms, ECG changes, and changes in cardiac markers (if any). Treatments for patients with unstable angina include nitrates, aspirin, GPIIb/IIIa inhibitors, heparin, and beta-blockers. Thrombolytic therapy has not been demonstrated to be beneficial for unstable angina patients, and calcium channel blockers may have no effect. Patients may also receive angioplasty and stents. Finally, patients with unstable angina are at risk for developing AMI.
• Myocardial infarction is characterized by constricting chest pain lasting longer than 30 minutes that can be accompanied by diagnostic ECG Q waves. Most patients with AMI have coronary artery disease, and as many as 25% of AMI cases are "silent" or asymptomatic infarctions, and individuals with diabetes tend to be more susceptible to silent infarctions. Population studies suggest that 20-60% of nonfatal myocardial infarctions are silent infarctions that are not recognized by the patient.
• Atypical clinical presentations of AMI can include congestive heart failure, angina pectoris without a severe or prolonged attack, atypical location of pain, central nervous system manifestations resembling stroke, apprehension and nervousness, sudden mania or psychosis, syncope, weakness, acute indigestion, and peripheral embolization.
• AMI is usually diagnosed by clinical symptoms, ECG changes, and elevations of cardiac proteins, most notably cardiac troponin, creatine kinase-MB and myoglobin. Treatments of AMI have improved over the past decade, resulting in improved patient outcome and a 30% decrease in the death rate associated with AMI.
• Treatment of AMI patients is accomplished by administering agents that limit infarct size and improve outcome by removing occlusive material, increasing the oxygen supply to cardiac tissue, or decreasing the oxygen demand of cardiac tissue.
• Treatments can include the following: supplemental oxygen, aspirin, GPIIb/IIIa inhibitors, heparin, thrombolytics (tPA), nitrates (nitroglycerin), magnesium, calcium channel antagonists, ⁇ -adrenergic receptor blockers, angiotensin-converting enzyme inhibitors, angioplasty (PTCA), and intraluminal coronary artery stents.
• Stable angina and unstable angina are characterized angiographically as 50-70% and 90% or greater arterial occlusion, respectively, and myocardial infarction is characterized by complete or nearly complete occlusion.
• a common misconception is that stable angina and unstable angina refer to plaque stability, or that they, along with myocardial infarction, are separate diseases. Because stable angina often progresses to unstable angina, and unstable angina often progresses to myocardial infarction, stable angina, unstable angina, and myocardial infarction can all be characterized as coronary artery disease of varying severity.
• the progression of coronary artery disease from mild unstable angina to severe unstable angina and myocardial infarction is related to plaque instability and the degree of arterial occlusion.
• Myocardial ischemia is the major determinant in the pathogenesis of stable angina, unstable angina, and myocardial infarction, and they should not be thought of as individual diseases. Rather, they reflect the increasing severity of myocardial damage from ischemia.
• Inflammatory mechanisms play a pivotal role in the atherosclerotic process. At the base of atherogenesis there are complex interactions between macrophages, T lymphocytes and smooth muscle cells. A growing body of experimental evidence suggests that inflammation is involved in the pathogenesis of ACS and influences its clinical evolution. In patients with ACS, coronary atherosclerotic plaques are characterized by an abundant inflammatory infiltrate. Moreover, in these patients systemic signs of inflammatory reaction can be observed: activated circulating inflammatory cells (neutrophil, monocytes and lymphocytes) and increased concentrations of pro-inflammatory cytokines, such as interleukin (IL)-I and 6, and of acute phase reactants, in particular C-reactive protein (CRP).
• IL interleukin
• CRP C-reactive protein
• the first mechanism involves the activation of platelets to facilitate adherence to the site of vessel injury.
• the activated platelets then aggregate to form a platelet plug that reduces or temporarily stops blood loss.
• the processes of platelet aggregation, plug formation and tissue repair are all accelerated and enhanced by numerous factors secreted by activated platelets.
• Platelet aggregation and plug formation is mediated by the formation of a fibrinogen bridge between activated platelets.
• Concurrent activation of the second mechanism, the coagulation cascade results in the generation of fibrin from fibrinogen and the formation of an insoluble fibrin clot that strengthens the platelet plug.
• the coagulation cascade is an enzymatic pathway that involves numerous serine proteinases normally present in an inactive, or zymogen, form.
• the presence of a foreign surface in the vasculature or vascular injury results in the activation of the intrinsic and extrinsic coagulation pathways, respectively.
• a final common pathway is then followed, which results in the generation of fibrin by the serine proteinase thrombin and, ultimately, a crosslinked fibrin clot.
• one active enzyme is formed initially, which can activate other enzymes that activate others, and this process, if left unregulated, can continue until all coagulation enzymes are activated. Fortunately, there are mechanisms in place, including fibrinolysis and the action of endogenous proteinase inhibitors that can regulate the activity of the coagulation pathway and clot formation.
• Fibrinolysis is the process of proteolytic clot dissolution. In a manner analogous to coagulation, fibrinolysis is mediated by serine proteinases that are activated from their zymogen form. The serine proteinase plasmin is responsible for the degradation of fibrin into smaller degradation products that are liberated from the clot, resulting in clot dissolution. Fibrinolysis is activated soon after coagulation in order to regulate clot formation. Endogenous serine proteinase inhibitors also function as regulators of fibrinolysis.
• Platelets are round or oval disks with an average diameter of 2-4 ⁇ m that are normally found in blood at a concentration of 200, 000-300, 000/ ⁇ l. They play an essential role in maintaining hemostasis by maintaining vascular integrity, initially stopping bleeding by forming a platelet plug at the site of vascular injury, and by contributing to the process of fibrin formation to stabilize the platelet plug.
• vascular injury occurs, platelets adhere to the site of injury and each other and are stimulated to aggregate by various agents released from adherent platelets and injured endothelial cells. This is followed by the release reaction, in which platelets secrete the contents of their intracellular granules, and formation of the platelet plug.
• thrombin in the coagulation cascade allows for consolidation of the plug, followed by clot retraction and stabilization of the plug by crosslinked fibrin.
• Active thrombin generated in the concurrent coagulation cascade, also has the ability to induce platelet activation and aggregation.
• the first step of the common pathway of the coagulation cascade involves the proteolytic cleavage of prothrombin by the factor Xa/factor Va prothrombinase complex to yield active thrombin.
• Thrombin is a serine proteinase that proteolytically cleaves fibrinogen to form fibrin, which is ultimately integrated into a crosslinked network during clot formation.
• the coagulation cascade can be activated through either the extrinsic or intrinsic pathways. These enzymatic pathways share one final common pathway.
• the first step of the common pathway involves the proteolytic cleavage of prothrombin by the factor Xa/factor Va prothrombinase complex to yield active thrombin.
• Thrombin is a serine proteinase that proteolytically cleaves fibrinogen.
• markers refers to proteins, polypeptides, phospholipids, or small molecules to be used as targets for screening test samples obtained from subjects.
• Proteins or polypeptides used as markers in the present invention are contemplated to include any fragments thereof, in particular, immunologically detectable fragments.
• the term "related marker” as used herein refers to one or more fragments of a particular marker or its biosynthetic parent that may be detected as a surrogate for the marker itself or as independent markers.
• human BNP is derived by proteolysis of a 108 amino acid precursor molecule, referred to hereinafter as BNP 1-1 Os- Mature BNP, or "the BNP natriuretic peptide," or "BNP-32" is a 32 amino acid molecule representing amino acids 77-108 of this precursor, which may be referred to as BNP 77 . 108 .
• the remaining residues 1-76 are referred to hereinafter as BNP 1-76 .
• markers described herein are synthesized as larger precursor molecules, which are then processed to provide mature marker; and/or are present in circulation in the form of fragments of the marker.
• related markers to each of the markers described herein may be identified and used in an analogous fashion to that described above for BNP. Additionally, related markers may be the result of covalent modification of the parent marker, for example by oxidation of methionine residues, ubiquitination, etc.
• marker fragments are an ongoing process that may be a function of, inter alia, the elapsed time between onset of an event triggering marker release into the tissues and the time the sample is obtained or analyzed; the elapsed time between sample acquisition and the time the sample is analyzed; the type of tissue sample at issue; the storage conditions; the quantity of proteolytic enzymes present; etc., it may be necessary to consider this degradation when both designing an assay for one or more markers, and when performing such an assay, in order to provide an accurate prognostic or diagnostic result.
• individual antibodies that distinguish amongst a plurality of marker fragments may be individually employed to separately detect the presence or amount of different fragments.
• the results of this individual detection may provide a more accurate prognostic or diagnostic result than detecting the plurality of fragments in a single assay. For example, different weighting factors may be applied to the various fragment measurements to provide a more accurate estimate of the amount of natriuretic peptide originally present in the sample.
• the BNP concentration may be used to determine if therapy is effective (e.g., by monitoring BNP to see if an elevated level is returing to normal upon treatment).
• therapy e.g., by monitoring BNP to see if an elevated level is returing to normal upon treatment.
• the same "false positive" BNP result discussed above may lead the physician to continue, increase, or modify treatment because of the false impression that current therapy is ineffective.
• troponin exists in muscle mainly as a "ternary complex" comprising three troponin polypeptides (T, I and C). But troponin I and troponin T circulate in the blood in forms other than the I/T/C ternery complex. Rather, each of (i) free cardiac-specific troponin I, (ii) binary complexes (e.g., troponin I/C complex), and (iii) ternary complexes all circulate in the blood.
• the "complex state" of troponin I and T may change over time in a patient, e.g., due to binding of free troponin polypeptides to other circulating troponin polypeptides. Immunoassays that fail to consider the "complex state" of troponin may not detect all of the cardiac-specific isoform of interest.
• the methods described hereinafter utilize one or more markers that are derived from the subject.
• subject-derived marker refers to protein, polypeptide, phospholipid, nucleic acid, prion, or small molecule markers that are expressed or produced by one or more cells of the subject.
• the presence, absence, amount, or change in amount of one or more markers may indicate that a particular disease is present, or may indicate that a particular disease is absent.
• Additional markers may be used that are derived not from the subject, but rather that are expressed by pathogenic or infectious organisms that are correlated with a particular disease.
• Such markers are preferably protein, polypeptide, phospholipid, nucleic acid, prion, or small molecule markers that identify the infectious diseases described above.
• ACS risk factors refers to characteristics of a subject that have been associated with an increased predisposition to suffer from ACS in comparison to subjects lacking the risk factor.
• CAC score refers to a measure of coronary artery calcification performed using electron-beam computed tomography or similar technique using the Agatston scoring method.
• a CAC score reflects the volume and density of calcification.
• a value that is approximately between the 40 th and 80 th percentile of subjects lacking clinical atherclerosis may be used to identify subclinical disease.
• altering the threshold for a positive test trades off sensitivity for specificity.
• test sample refers to a sample of bodily fluid obtained for the purpose of diagnosis, prognosis, or evaluation of a subject of interest, such as a patient. In certain embodiments, such a sample may be obtained for the purpose of determining the outcome of an ongoing condition or the effect of a treatment regimen on a condition.
• Preferred test samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusions.
• test samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.
• a "plurality" as used herein refers to at least two.
• a plurality refers to at least 3, more preferably at least 5, even more preferably at least 10, even more preferably at least 15, and most preferably at least 20.
• a plurality is a large number, i.e., at least 100.
• subject refers to a human or non-human organism.
• methods and compositions described herein are applicable to both human and veterinary disease.
• a subject is preferably a living organism, the invention described herein may be used in post-mortem analysis as well.
• Preferred subjects are "patients," i.e., living humans that are receiving medical care. This includes persons with no defined illness who are being investigated for signs of pathology.
• diagnosis refers to methods by which the skilled artisan can estimate and/or determine whether or not a patient is suffering from a given disease or condition.
• the skilled artisan often makes a diagnosis on the basis of one or more diagnostic indicators, i.e., a marker, the presence, absence, amount, or change in amount of which is indicative of the presence, severity, or absence of the condition.
• a "prognosis” refers to assignment of a probability that a given course or outcome will occur. This is often determined by examining one or more "prognostic indicators.” These are markers, the presence or amount of which in a patient (or a sample obtained from the patient) signal a probability that a given course or outcome will occur. For example, when one or more prognostic indicators reach a sufficiently high level in samples obtained from such patients, the level may signal that the patient is at an increased probability for experiencing a future stroke in comparison to a similar patient exhibiting a lower marker level.
• a level or a change in level of a prognostic indicator which in turn is associated with an increased probability of morbidity or death, is referred to as being "associated with an increased predisposition to an adverse outcome" in a patient.
• Preferred prognostic markers can predict the onset of delayed neurologic deficits in a patient after stroke, or the chance of future stroke.
• correlating refers to comparing the presence or amount of the marker(s) in a patient to its presence or amount in persons known to suffer from, or known to be at risk of, a given condition; or in persons known to be free of a given condition.
• a marker level in a patient sample can be compared to a level known to be associated with a specific diagnosis.
• the sample's marker level is said to have been correlated with a diagnosis; that is, the skilled artisan can use the marker level to determine whether the patient suffers from a specific type diagnosis, and respond accordingly.
• the sample's marker level can be compared to a marker level known to be associated with a good outcome (e.g., the absence of disease, etc.).
• a profile of marker levels are correlated to a global probability or a particular outcome.
• determining the diagnosis refers to methods by which the skilled artisan can determine the presence or absence of a particular disease in a patient.
• diagnosis does not refer to the ability to determine the presence or absence of a particular disease with 100% accuracy, or even that a given course or outcome is more likely to occur than not. Instead, the skilled artisan will understand that the term “diagnosis” refers to an increased probability that a certain disease is present in the subject.
• a diagnosis indicates about a 5% increased chance that a disease is present, about a 10% chance, about a 15% chance, about a 20% chance, about a 25% chance, about a 30% chance, about a 40% chance, about a 50% chance, about a 60% chance, about a 75% chance, about a 90% chance, and about a 95% chance.
• the term "about” in this context refers to +/- 2%.
• determining the prognosis refers to methods by which the skilled artisan can determine the likelihood of one or more future clinical outcomes for a patient.
• prognosis refers to an increased probability that a certain clinical outcome will occur at a future date in the subject.
• a prognosis indicates about a 5% increased chance of a certain clinical outcome compared to a "control" population, about a 10% chance, about a 15% chance, about a 20% chance, about a 25% chance, about a 30% chance, about a 40% chance, about a 50% chance, about a 60% chance, about a 75% chance, about a 90% chance, and about a 95% chance.
• the term "about” in this context refers to +/- 2%.
• discrete refers to areas of a surface that are noncontiguous. That is, two areas are discrete from one another if a border that is not part of either area completely surrounds each of the two areas.
• the term "independently addressable” as used herein refers to discrete areas of a surface from which a specific signal may be obtained.
• the term "antibody” as used herein refers to a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. See, e.g. Fundamental Immunology, 3 rd Edition, W.E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97.
• antibody includes antigen-binding portions, i.e., "antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
• Antigen binding sites e.g., fragments, subs
• the term "specifically binds" is not intended to indicate that an antibody binds exclusively to its intended target. Rather, an antibody “specifically binds” if its affinity for its intended target is about 5 -fold greater when compared to its affinity for a non-target molecule.
• the affinity of the antibody will be at least about 5 fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for a target molecule than its affinity for a non- target molecule.
• Specific binding between an antibody or other binding agent and an antigen means a binding affinity of at least 10 6 M "1 .
• Preferred antibodies bind with affinities of at least about 1O M " , and preferably between about 10 M “1 to about 10 9 M “1 , about 10 9 M “1 to about 10 10 M “1 , or about 10 10 M “1 to about 10 11 M "
• K equilibrium association constant
• n number of ligand binding sites per receptor molecule
• r/c is plotted on the Y-axis versus r on the X-axis thus producing a Scatchard plot.
• the affinity is the negative slope of the line, koff can be determined by competing bound labeled ligand with unlabeled excess ligand (see, e.g., U.S. Pat No. 6,316,409).
• the affinity of a targeting agent for its target molecule is preferably at least about 1 x 10 ⁇ 6 moles/liter, is more preferably at least about 1 x 10 ⁇ 7 moles/liter, is even more preferably at least about 1 x 10 ⁇ 8 moles/liter, is yet even more preferably at least about 1 x 10 ⁇ 9 moles/liter, and is most preferably at least about 1 x 10 ⁇ 10 moles/liter.
• Antibody affinity measurement by Scatchard analysis is well known in the art. See, e.g., van Erp et ah, J. Immunoassay 12: 425-43, 1991; Nelson and Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.
• data for a number of potential markers may be obtained from a group of subjects by testing for the presence or level of certain markers.
• the group of subjects is divided into two sets.
• the first set includes subjects who have been confirmed as having a disease, outcome, or, more generally, being in a first condition state.
• this first set of patients may be those diagnosed with subclinical atherosclerosis that later died as a result of complications from vascular disease prior to a particular study endpoint.
• subjects in this first set will be referred to as "diseased.”
• the second set of subjects is simply those who do not fall within the first set. Subjects in this second set will hereinafter be referred to as "non-diseased". Preferably, the first set and the second set each have an approximately equal number of subjects. This set may be normal patients, and/or patients suffering from subclinical atherosclerosis and that lived to a particular endpoint of interest.
• the data obtained from subjects in these sets preferably includes levels of a plurality of markers.
• data for the same set of markers is available for each patient.
• This set of markers may include all candidate markers that may be suspected as being relevant to the detection of a particular disease or condition. Actual known relevance is not required.
• Embodiments of the methods and systems described herein may be used to determine which of the candidate markers are most relevant to the diagnosis of the disease or condition.
• the levels of each marker in the two sets of subjects may be distributed across a broad range, e.g., as a Gaussian distribution. However, no distribution fit is required.
• a single marker often is incapable of definitively identifying a subject as falling within a first or second group in a prospective fashion. For example, if a patient is measured as having a marker level that falls within an overlapping region in the distribution of diseased and non-diseased subjects, the results of the test may be useless in diagnosing the patient.
• An artificial cutoff may be used to distinguish between a positive and a negative test result for the detection of the disease or condition. Regardless of where the cutoff is selected, the effectiveness of the single marker as a diagnosis tool is unaffected. Changing the cutoff merely trades off between the number of false positives and the number of false negatives resulting from the use of the single marker. The effectiveness of a test having such an overlap is often expressed using a ROC (Receiver Operating Characteristic) curve. ROC curves are well known to those skilled in the art.
• the horizontal axis of the ROC curve represents (1 -specificity), which increases with the rate of false positives.
• the vertical axis of the curve represents sensitivity, which increases with the rate of true positives.
• the value of (1 -specificity) may be determined, and a corresponding sensitivity may be obtained.
• the area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.
• the measurement of the level of a single marker may have limited usefulness, e.g., it may be non-specifically increased due to inflammation.
• the measurement of additional markers provides additional information, but the difficulty lies in properly combining the levels of two potentially unrelated measurements.
• data relating to levels of various markers for the sets of diseased and non- diseased patients may be used to develop a panel of markers to provide a useful panel response.
• the data may be provided in a database such as Microsoft Access, Oracle, other SQL databases or simply in a data file.
• the database or data file may contain, for example, a patient identifier such as a name or number, the levels of the various markers present, and whether the patient is diseased or non-diseased.
• an artificial cutoff region may be initially selected for each marker.
• the location of the cutoff region may initially be selected at any point, but the selection may affect the optimization process described below. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer.
• the cutoff region is initially centered about the center of the overlap region of the two sets of patients.
• the cutoff region may simply be a cutoff point.
• the cutoff region may have a length of greater than zero.
• the cutoff region may be defined by a center value and a magnitude of length.
• the initial selection of the limits of the cutoff region may be determined according to a pre-selected percentile of each set of subjects. For example, a point above which a pre-selected percentile of diseased patients are measured may be used as the right (upper) end of the cutoff range.
• Each marker value for each patient may then be mapped to an indicator.
• the indicator is assigned one value below the cutoff region and another value above the cutoff region. For example, if a marker generally has a lower value for non-diseased patients and a higher value for diseased patients, a zero indicator will be assigned to a low value for a particular marker, indicating a potentially low likelihood of a positive diagnosis.
• the indicator may be calculated based on a polynomial. The coefficients of the polynomial may be determined based on the distributions of the marker values among the diseased and non-diseased subjects.
• the relative importance of the various markers may be indicated by a weighting factor.
• the weighting factor may initially be assigned as a coefficient for each marker. As with the cutoff region, the initial selection of the weighting factor may be selected at any acceptable value, but the selection may affect the optimization process. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer.
• acceptable weighting coefficients may range between zero and one, and an initial weighting coefficient for each marker may be assigned as 0.5.
• the initial weighting coefficient for each marker may be associated with the effectiveness of that marker by itself. For example, a ROC curve may be generated for the single marker, and the area under the ROC curve may be used as the initial weighting coefficient for that marker.
• a panel response may be calculated for each subject in each of the two sets.
• the panel response is a function of the indicators to which each marker level is mapped and the weighting coefficients for each marker.
• the panel response (R) for each subject (j) is expressed as:
• Rj ⁇ Wilij, where i is the marker index, j is the subject index, W 1 is the weighting coefficient for marker i, I is the indicator value to which the marker level for marker i is mapped for subject j, and ⁇ is the summation over all candidate markers i.
• This panel response value may be referred to as a "panel index.”
• an extraordinarily high or low marker levels do not change the probability of a diagnosis of diseased or non-diseased for that particular marker.
• a marker value above a certain level generally indicates a certain condition state. Marker values above that level indicate the condition state with the same certainty. Thus, an extraordinarily high marker value may not indicate an extraordinarily high probability of that condition state.
• the use of an indicator which is constant on one side of the cutoff region eliminates this concern.
• the panel response may also be a general function of several parameters including the marker levels and other factors including, for example, race and gender of the patient. Other factors contributing to the panel response may include the slope of the value of a particular marker over time. For example, a patient may be measured when first arriving at the hospital for a particular marker. The same marker may be measured again an hour later, and the level of change may be reflected in the panel response. Further, additional markers may be derived from other markers and may contribute to the value of the panel response. For example, the ratio of values of two markers may be a factor in calculating the panel response.
• An objective function may be defined to facilitate the selection of an effective panel.
• the objective function should generally be indicative of the effectiveness of the panel, as may be expressed by, for example, overlap of the panel responses of the diseased set of subjects and the panel responses of the non-diseased set of subjects. In this manner, the objective function may be optimized to maximize the effectiveness of the panel by, for example, minimizing the overlap.
• the ROC curve representing the panel responses of the two sets of subjects may be used to define the objective function.
• the objective function may reflect the area under the ROC curve.
• the ROC curve may be used to define the objective function.
• the point at which the slope of the ROC curve is equal to one may be a useful feature.
• the point at which the product of sensitivity and specificity is a maximum sometimes referred to as the "knee,” may be used.
• the sensitivity at the knee may be maximized.
• the sensitivity at a predetermined specificity level may be used to define the objective function.
• Other embodiments may use the specificity at a predetermined sensitivity level may be used.
• combinations of two or more of these ROC-curve features may be used.
• one of the markers in the panel is specific to the disease or condition being diagnosed.
• the panel response may be set to return a "positive" test result.
• the threshold is not satisfied, however, the levels of the marker may nevertheless be used as possible contributors to the objective function.
• An optimization algorithm may be used to maximize or minimize the objective function. Optimization algorithms are well-known to those skilled in the art and include several commonly available minimizing or maximizing functions including the Simplex method and other constrained optimization techniques. It is understood by those skilled in the art that some minimization functions are better than others at searching for global minimums, rather than local minimums.
• the location and size of the cutoff region for each marker may be allowed to vary to provide at least two degrees of freedom per marker. Such variable parameters are referred to herein as independent variables.
• the weighting coefficient for each marker is also allowed to vary across iterations of the optimization algorithm. In various embodiments, any permutation of these parameters may be used as independent variables.
• the sense of each marker may also be used as an independent variable. For example, in many cases, it may not be known whether a higher level for a certain marker is generally indicative of a diseased state or a non-diseased state. In such a case, it may be useful to allow the optimization process to search on both sides. In practice, this may be implemented in several ways. For example, in one embodiment, the sense may be a truly separate independent variable which may be flipped between positive and negative by the optimization process. Alternatively, the sense may be implemented by allowing the weighting coefficient to be negative.
• the optimization algorithm may be provided with certain constraints as well.
• the resulting ROC curve may be constrained to provide an area-under- curve of greater than a particular value.
• ROC curves having an area under the curve of 0.5 indicate complete randomness, while an area under the curve of 1.0 reflects perfect separation of the two sets.
• a minimum acceptable value such as 0.75
• Other constraints may include limitations on the weighting coefficients of particular markers. Additional constraints may limit the sum of all the weighting coefficients to a particular value, such as 1.0.
• the iterations of the optimization algorithm generally vary the independent parameters to satisfy the constraints while minimizing or maximizing the objective function.
• the number of iterations may be limited in the optimization process.
• the optimization process may be terminated when the difference in the objective function between two consecutive iterations is below a predetermined threshold, thereby indicating that the optimization algorithm has reached a region of a local minimum or a maximum.
• the optimization process may provide a panel of markers including weighting coefficients for each marker and cutoff regions for the mapping of marker values to indicators. Certain markers may be then be changed or even eliminated from the panel, and the process repeated until a satisfactory result is obtained. The effective contribution of each marker in the panel may be determined to identify the relative importance of the markers. In one embodiment, the weighting coefficients resulting from the optimization process may be used to determine the relative importance of each marker. The markers with the lowest coefficients may be eliminated or replaced.
• the lower weighting coefficients may not be indicative of a low importance.
• a higher weighting coefficient may not be indicative of a high importance.
• the optimization process may result in a high coefficient if the associated marker is irrelevant to the diagnosis. In this instance, there may not be any advantage that will drive the coefficient lower. Varying this coefficient may not affect the value of the objective function.
• a "gold standard” test criterion may be selected which allows selection of subjects into two or more groups for comparison by the foregoing methods.
• this gold standard may be a CAC score or other method of identifying subclinical atherosclerosis.
• Measures of test accuracy may be obtained as described in Fischer et al., Intensive Care Med. 29: 1043-51, 2003, and used to determine the effectiveness of a given marker or panel of markers. These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas. As discussed above, preferred tests and assays exhibit one or more of the following results on these various measures:
• ROC curve area of at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95;
• a positive likelihood ratio (calculated as sensitivity/(l -specificity)) of at least 5, more preferably at least 10, and most preferably at least 20, and a negative likelihood ratio (calculated as (l-sensitivity)/specificity) of less than or equal to 0.3, more preferably less than or equal to 0.2, and most preferably less than or equal to 0.1.
• a panel consisting of the markers referenced herein and/or their related markers may be constructed to provide relevant information related to the diagnosis of interest.
• Such a panel may be constructed using 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more individual markers.
• the analysis of a single marker or subsets of markers comprising a larger panel of markers could be carried out by one skilled in the art to optimize clinical sensitivity or specificity in various clinical settings. These include, but are not limited to ambulatory, urgent care, critical care, intensive care, monitoring unit, inpatient, outpatient, physician office, medical clinic, and health screening settings.
• one skilled in the art can use a single marker or a subset of markers comprising a larger panel of markers in combination with an adjustment of the diagnostic threshold in each of the aforementioned settings to optimize clinical sensitivity and specificity.
• These devices and methods can utilize labeled molecules in various sandwitch, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of an analyte of interest. Additionally, certain methods and devices, such as biosensors and optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labeled molecule. See, e.g., U.S. Patents 5,631,171; and 5,955,377, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims.
• robotic instrumentation including but not limited to Beckman Access, Abbott AxSym, Roche ElecSys, Dade Behring Stratus systems are among the immunoassay analyzers that are capable of performing the immunoassays taught herein.
• the markers are analyzed using an immunoassay, and most preferably sandwich immunoassay, although other methods are well known to those skilled in the art (for example, the measurement of marker RNA levels).
• the presence or amount of a marker is generally determined using antibodies specific for each marker and detecting specific binding.
• Any suitable immunoassay may be utilized, for example, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs), competitive binding assays, and the like.
• ELISA enzyme-linked immunoassays
• RIAs radioimmunoassays
• Specific immunological binding of the antibody to the marker can be detected directly or indirectly.
• Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody.
• Indirect labels include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase and the like.
• immobilized antibodies specific for the markers is also contemplated by the present invention.
• the antibodies could be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay place (such as microtiter wells), pieces of a solid substrate material or membrane (such as plastic, nylon, paper), and the like.
• An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip could then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.
• suitable apparatuses include clinical laboratory analyzers such as the ElecSys (Roche), the AxSym (Abbott), the Access (Beckman), the AD VIA® CENTAUR® (Bayer) immunoassay systems, the NICHOLS ADVANTAGE® (Nichols Institute) immunoassay system, etc.
• Preferred apparatuses perform simultaneous assays of a plurality of markers using a single test device.
• Particularly useful physical formats comprise surfaces having a plurality of discrete, adressable locations for the detection of a plurality of different analytes.
• Such formats include protein microarrays, or "protein chips" (see, e.g., Ng and Hag, J. Cell MoI.
• each discrete surface location may comprise antibodies to immobilize one or more analyte(s) (e.g., a marker) for detection at each location.
• Surfaces may alternatively comprise one or more discrete particles (e.g., microparticles or nanoparticles) immobilized at discrete locations of a surface, where the microparticles comprise antibodies to immobilize one analyte (e.g., a marker) for detection.
• Preferred assay devices of the present invention will comprise, for one or more assays, a first antibody conjugated to a solid phase and a second antibody conjugated to a signal development element. Such assay devices are configured to perform a sandwich immunoassay for one or more analytes. These assay devices will preferably further comprise a sample application zone, and a flow path from the sample application zone to a second device region comprising the first antibody conjugated to a solid phase.
• Flow of a sample along the flow path may be driven passively (e.g., by capillary, hydrostatic, or other forces that do not require further manipulation of the device once sample is applied), actively (e.g., by application of feree generated via mechanical pumps, electroosmotic pumps, centrifugal force, increased air pressure, etc.), or by a combination of active and passive driving forces.
• sample applied to the sample application zone will contact both a first antibody conjugated to a solid phase and a second antibody conjugated to a signal development element along the flow path (sandwich assay format). Additional elements, such as filters to separate plasma or serum from blood, mixing chambers, etc., may be included as required by the artisan.
• a panel consisting of the markers referenced above may be constructed to provide relevant information related to differential diagnosis. Such a panel may be constucted using 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more or individual markers.
• the analysis of a single marker or subsets of markers comprising a larger panel of markers could be carried out by one skilled in the art to optimize clinical sensitivity or specificity in various clinical settings. These include, but are not limited to ambulatory, urgent care, critical care, intensive care, monitoring unit, inpatient, outpatient, physician office, medical clinic, and health screening settings.
• one skilled in the art can use a single marker or a subset of markers comprising a larger panel of markers in combination with an adjustment of the diagnostic threshold in each of the aforementioned settings to optimize clinical sensitivity and specificity.
• the clinical sensitivity of an assay is defined as the percentage of those with the disease that the assay correctly predicts
• the specificity of an assay is defined as the percentage of those without the disease that the assay correctly predicts (Tietz Textbook of Clinical Chemistry, 2 nd edition, Carl Burtis and Edward Ashwood eds., W.B. Saunders and Company, p. 496).
• markers could be carried out in a variety of physical formats as well.
• the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples.
• single sample formats could be developed to facilitate immediate treatment and diagnosis in a timely fashion, for example, in ambulatory transport or emergency room settings.
• the present invention provides a kit for the analysis of markers.
• a kit for the analysis of markers.
• Such a kit preferably comprises devises and reagents for the analysis of at least one test sample and instructions for performing the assay.
• the kits may contain one or more means for using information obtained from immunoassays performed for a marker panel to rule in or out certain diagnoses.
• Other measurement strategies applicable to the methods described herein include chromatography (e.g., HPLC), mass spectrometry, receptor-based assays, and combinations of the foregoing.
• the generation and selection of antibodies may be accomplished several ways. For example, one way is to purify polypeptides of interest or to synthesize the polypeptides of interest using, e.g., solid phase peptide synthesis methods well known in the art. See, e.g., Guide to Protein Purification, Murray P. Deutcher, ed., Meth. Enzymol. VoI 182 (1990); Solid Phase Peptide Synthesis, Greg B. Fields ed., Meth. Enzymol. VoI 289 (1997); Kiso et ah, Chem. Pharm. Bull. (Tokyo) 38: 1192-99, 1990; Mostafavi et ah, Biomed. Pept.
• the selected polypeptides may then be injected, for example, into mice or rabbits, to generate polyclonal or monoclonal antibodies.
• the selected polypeptides may then be injected, for example, into mice or rabbits, to generate polyclonal or monoclonal antibodies.
• One skilled in the art will recognize that many procedures are available for the production of antibodies, for example, as described in Antibodies, A Laboratory Manual, Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988), Cold Spring Harbor, N.Y.
• binding fragments or Fab fragments which mimic antibodies can also be prepared from genetic information by various procedures (Antibody Engineering: A Practical Approach (Borrebaeck, C, ed.), 1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920 (1992)).
• phage display technology to produce and screen libraries of polypeptides for binding to a selected target. See, e.g, Cwirla et al, Proc. Natl. Acad. Sci. USA 87, 6378-82, 1990; Devlin et al, Science 249, 404-6, 1990, Scott and Smith, Science 249, 386-88, 1990; and Ladner et al., U.S. Pat. No. 5,571,698.
• a basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide.
• This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide.
• the establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides.
• Phage displaying a polypeptide with affinity to a target bind to the target and these phage are enriched by affinity screening to the target.
• the identity of polypeptides displayed from these phage can be determined from their respective genomes.
• a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means. See, e.g., U.S. Patent No. 6,057,098, which is hereby incorporated in its entirety, including all tables, figures, and claims.
• the antibodies that are generated by these methods may then be selected by first screening for affinity and specificity with the purified polypeptide of interest and, if required, comparing the results to the affinity and specificity of the antibodies with polypeptides that are desired to be excluded from binding.
• the screening procedure can involve immobilization of the purified polypeptides in separate wells of microtiter plates. The solution containing a potential antibody or groups of antibodies is then placed into the respective microtiter wells and incubated for about 30 min to 2 h.
• the microtiter wells are then washed and a labeled secondary antibody (for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 min and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immobilized polypeptide(s) are present.
• the antibodies so identified may then be further analyzed for affinity and specificity in the assay design selected. In the development of immunoassays for a target protein, the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ; certain antibody pairs (e.g., in sandwich assays) may interfere with one another sterically, etc., assay performance of an antibody may be a more important measure than absolute affinity and specificity of an antibody.
• the panels and markers of the present invention may be used to monitor a course of treatment. For example, inproved or worsened prognostic state may indicate that a particular treatment is or is not efficacious.
• the study population included 1377 adults (mean age 59 years, 53% male) free of coronary heart disease, and who underwent computed tomography (CT) for measurement of coronary artery calcium (CAC) and thoracic aortic calcium (TAC) and who had risk factor measures (blood pressure, lipids, glucose, and medical history) available.
• CT computed tomography
• CAC coronary artery calcium
• TAC thoracic aortic calcium
• risk factor measures blood pressure, lipids, glucose, and medical history
• the imaging protocol involved an experienced licensed radiologic technician acquiring a single scan on each patient, consisting of -30-40 3 mm slices or 2.5 mm slices for electron beam tomography and MSCT, respectively.
• the software calculated lesion- specific scores as the product of the area of each calcified focus and peak computed tomography number (categories: 0 HU, 1-99 HU, 100-399 HU, and >400 HU). These were summed across all lesions identified within the left main, left anterior descending, left circumflex, and right coronary arteries to provide arterial-specific calcium scores, and across arteries to provide the total CAC score.
• BNP levels were significantly higher among those with vs. without any CAC (18.3 vs. 12.5 pg/ml, p ⁇ 0.001), significant CAC (42.6 vs. 15.3 pg/ml, p ⁇ 0.001), any TAC (20.3 vs. 11.6 pg/ml, p ⁇ 0.001), and significant TAC (28.9 vs. 13.7 pg/ml, p ⁇ 0.001).
• BNP levels >75 th percentile vs. ⁇ 75 th percentile, 12.1 vs.
• the study population included Our population included 2, 103 asymptomatic adults (54% male) with an average Framingham risk score of 8.6 ⁇ 7 % who underwent computed (CT) for evaluation of coronary artery calcium (CAC).
• CT computed
• CAC coronary artery calcium
• RR for cardiovascular event-free survival in subjects with BNP >40 pg/ml was 4.5 (1.6-13.2, p ⁇ 0.0001).
• a BNP >40 pg/ml added additional prognostic information to CAC and FRS.

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