Cellular fibronectin as a diagnostic marker in stroke and methods of use thereof

• US 20070005261A1
• Filed: 02/01/2006
• Published: 01/04/2007
• Est. Priority Date: 09/23/2003
• Status: Active Grant

• Current Assignee: Antoni Davalos, Cornelius Diamond, Joaquin Serena, Jose Castillo, Mar Castellanos
• Original Assignee: Antoni Davalos, Cornelius Diamond, Joaquin Serena, Jose Castillo, Mar Castellanos
• Inventors: Castillo, Jose, Serena, Joaquin, Davalos, Antoni, Castellanos, Mar, Diamond, Cornelius
• Granted Patent: US 7,392,140 B2
• Time in Patent Office: Days
• US Class Current: 702/19
• CPC Class Codes: G01N 2800/2871 Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event
• CPC Class Codes: G01N 2800/324 Coronary artery diseases, e.g. angina pectoris, myocardial infarction
• CPC Class Codes: G16H 50/70 for mining of medical data, e.g. analysing previous cases of other patients
• CPC Class Codes: G16Z 99/00 Subject matter not provided for in other main groups of this subclass
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

Methods for the diagnosis and evaluation of stroke and stroke sub-type employ a variety of bio-markers including cellular fibronectin (c-Fn) assembled as a panel for stoke diagnosis and evaluation. Methods are disclosed for selecting markers and correlating their combined levels with a clinical outcome of interest. In various aspects the methods permit early detection and differentiation of stroke subtypes, determination of the prognosis of a patient presenting stroke symptoms, and identification of a patient at risk for early hematoma growth and/or malignant massive cerebral artery infarction. The disclosed methods provide rapid, sensitive and specific assays to greatly increase the number of patients that can receive beneficial stroke treatment and therapy, and to reduce the human and economic costs associated with incorrect stroke diagnosis.

Claims

1. A method of determining presence or risk of cerebral injury in a human subject, the method comprising:

obtaining a test sample from a human subject;

analyzing the obtained test sample for presence or amount of (1) cellular fibronectin and (2) one or more additional markers both proteomic and non-proteomic for, or mass spectrometry peak levels of, any of apoptosis, cellular adhesion, cellular injury, coagulation, glial activation, inflammatory mediation, myelin breakdown, thrombosis, vascular damage, and specific and non-specific markers of cerebral injury; and

then

correlating (1) the presence or amount of said cellular fibronectin and said more additional markers or peak levels, with (2) clinical patient information, other than the cellular fibronectin and additional makers or peak levels for cerebral injury, in order to deduce a probability of present or future risk of a cerebral injury for the subject.

2. The method according to claim 1 wherein the correlating is in order to deduce a probability of present or future risk of cerebral injury of a type drawn from the group consisting of secondary brain edema and early growth of intracerebral hemorrhage (ICH).

3. The method according to claim 2 further comprising:

determining from the deduced probability whether the subject could benefit from aggressive therapies including decompressive hemicraniectomy and hypothermia.

4. The method according to claim 1 wherein the correlating comprises:

determining the expression levels or mass spectrometry peak levels of one or more proteomic marker(s) or mass-to-charge ratio(s) and the numerical quantity of one or more non-proteomic marker(s) or mass-to-charge ratio(s) from humans suspected or known to have some form of cerebral injury;

comparing said determined levels and numerical values to humans known to have said matched type of cerebral injury; and

training an algorithm to identify patterns of differences in said humans which correlate with the prescience or absence of said matched type of cerebral injury, respectively.

5. The method according to claim 4 wherein the training of the algorithm on characteristic protein levels or patterns of differences includes the steps of

obtaining numerous examples of (i) said proteomic and non-proteomic data, and (ii) historical clinical results corresponding to this proteomic and non-proteomic data;

constructing an algorithm suitable to map (i) said protein expression levels or mass spectrometry peak mass-to-charge ratio(s) and said non-proteomic values as inputs to the algorithm, to (ii) the historical clinical results as outputs of the algorithm;

exercising the constructed algorithm to so map (i) the said protein expression levels or mass spectrometry peak mass-to-charge ratio(s) and said non-proteomic values as inputs to (ii) the historical clinical results as outputs; and

conducting an automated procedure to vary the mapping function, inputs to outputs, of the constructed and exercised algorithm in order that, by minimizing an error measure of the mapping function, a more optimal algorithm mapping architecture is realized;

wherein realization of the more optimal algorithm mapping architecture, also known as feature selection, means that any irrelevant inputs are effectively excised, meaning that the more optimally mapping algorithm will substantially ignore said protein expression levels or mass spectrometry peak mass-to-charge ratio(s) and said non-proteomic values that are irrelevant to output clinical results; and

wherein realization of the more optimal algorithm mapping architecture, also known as feature selection, also means that any relevant inputs are effectively identified, making that the more optimally mapping algorithm will serve to identify, and use, those input protein expression levels or mass spectrometry peak mass-to-charge ratio(s) and said non-proteomic values that are relevant, in combination, to output clinical results that would result in a clinical detection of disease, disease diagnosis, disease prognosis, or treatment outcome or a combination of any two, three or four of these actions.

6. The method according to claim 5 wherein the constructed algorithm is drawn from the group consisting essentially of:

linear or nonlinear regression algorithms;

linear or nonlinear classification algorithms;

ANOVA;

neural network algorithms;

genetic algorithms;

support vector machines algorithms;

hierarchical analysis or clustering algorithms;

hierarchical algorithms using decision trees;

kernel based machine algorithms such as kernel partial least squares algorithms, kernel matching pursuit algorithms, kernel fisher discriminate analysis algorithms, or kernel principal components analysis algorithms;

Bayesian probability function algorithms;

Markov Blanket algorithms;

a plurality of algorithms arranged in a committee network; and

forward floating search or backward floating search algorithms.

7. The method according to claim 5 wherein the feature selection process employs an algorithm drawn from the group consisting essentially of:

linear or nonlinear regression algorithms;

linear or nonlinear classification algorithms;

ANOVA;

neural network algorithms;

genetic algorithms;

support vector machines algorithms;

hierarchical analysis or clustering algorithms;

hierarchical algorithms using decision trees;

kernel based machine algorithms such as kernel partial least squares algorithms, kernel matching pursuit algorithms, kernel fisher discriminate analysis algorithms, or kernel principal components analysis algorithms;

Bayesian probability function algorithms;

Markov Blanket algorithms;

recursive feature elimination or entropy-based recursive feature elimination algorithms;

a plurality of algorithms arranged in a committee network; and

forward floating search or backward floating search algorithms.

8. The method according to claim 5 wherein a tree algorithm is trained to reproduce the performance of another machine-learning classifier or regressor by enumerating the input space of said classifier or regressor to form a plurality of training examples sufficient (1) to span the input space of said classifier or regressor and (2) train the tree to emulate the performance of said classifier or regressor.

9. The method according to claim 2

wherein the correlating so as to deduce the risk of cerebral injury is particularly so as to deduce a risk of early hematoma growth following intercerebral hemorrhage;

and wherein the method further comprises;

diagnosing a risk of early hematoma growth following intercerebral hemorrhage by measuring the level of cellular fibronectin (c-Fn) alone;

evaluating the patient'"'"'s risk of hemorrhage following thrombolytic therapy from said measured level of c-Fn; and

administering stroke therapy as appropriate to the evaluated risk of cerebral injury.

10. The method according to claim 9 wherein the one or more additional markers includes, in addition to cellular fibronectin (c-Fn), the proteomic markers MMP-9, IL-6, TNF-α

, TAFI, and PAI-1.

11. The method according to claim 9 wherein the one or more additional markers includes, in addition to cellular fibronectin (c-Fn), a proteomic marker of endothelial injury.

12. The method of claim 1 wherein the analyzing of one or more additional markers in addition to cellular-fibronectin is of one or more markers selected from the group consisting of two or more of the following:

Glial fibrillary acidic protein, apolipoprotein CI (ApoC-1), apolipoprotein CIII (ApoC-III), serum amyloid A (SAA), Platelet factor 4 (PF4), platelet-derived growth factor, antithrombin-III fragment (AT-III fragment), bradykinin, renin, haptoglobin, Creatine kinase brain band (CK-BB), adenylate kinase, lactate dehydrogenase, troponin 1, troponin T, Brain Derived Neurotrophic Factor, CPK, LDH Isoenzymes, Thrombin-Antithrombin III, calcitonin, procalcitonin, c-tau, Protein C, Protein S, fibrinogen, Factor VIII, activated Protein C resistance, E-selectin, P-selectin, von Willebrand factor (vWF), platelet-derived microvesicles (PDM), plasminogen activator inhibitor-1 (PAI-1), angiotensin I, angiotensin II, angiotensin III, annexin V, arginine vasopressin, B-type natriuretic peptide (BNP), pro-BNP, atrial natriuretic peptide (ANP), N-terminal pro-ANP, pro-ANP, C-type natriuretic peptide, (CNP), c-fos, c-jun, ubiquitin, cytochrome C, beta-enolase, cardiac troponin I, cardiac troponin T, urotensin II, creatine kinase-MB, glycogen phosphorylase-BB, KL-6, endothelin-1, endothelin-2, and endothelin-3, A-, F-, and H-Fatty acid binding protein (A-, F-, H-FABP), phosphoglyceric acid mutase-MB, aldosterone, S-100beta (S100□

), myelin basic protein, NR2A or NR2B NMDA receptor or fragment thereof (a subtype of N-methyl-D-aspartate (NMDA) receptors), Intracellular adhesion molecule (ICAM or CD54), Neuronal cell adhesion molecule, (NCAM or CD56), C-reactive protein, caspase-3, cathepsin D, hemoglobin alpha.sub.2, human lipocalin-type prostaglandin D synthase, interleukin-1 beta, interleukin-1 receptor angonist, interleukin 2, interleukin 2 receptor, interleukin-6, IL-1, IL-8, IL-10, monocyte chemotactic protein-1, soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, MMP-2, MMP-3, MMP-9, MMP-12, MMP-9, tissue factor (TF), NDKA, RAGE, RNA-BP, TRAIL, TWEAK, UFD1, fibrin D-dimer (D-dimer), total sialic acid (TSA), TpP, heat shock protein 60, heat shock protein 70, tumor necrosis factor alpha, tumor necrosis factor receptors 1 and 2, VEGF, Calbindin-D, Proteolipid protein RU Malendialdehyde, neuron-specific enolase gamma gamma isoform (NSE γ

.γ

isoform), thrombus precursor protein, Chimerin, Fibrinopeptide A (FPA), plasmin-α

2AP complex (PAP), plasmin inhibitory complex (PIC), beta-thromboglobulin ({tilde over (β

)}TG), Prothrombin fragment 1+2, PGI2, Creatinine phosphokinase brain band, neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), neurokinin A, neurokinin B, neurotensin, neuropeptide Y, Lactate dehydrogenase (LDH), soluble thrombomodulin (sTM), Insulin-like growth factor-1 (IGF-1), protein kinase C gamma (PKC-γ

, Secretagogin, PGE2,8-epi PGF.sub.2alpha and Transforming growth factor β

eta (TGF-β

) or markers related thereto.

13. The method of claim 11

wherein the correlating is further so as to determine diagnostic or prognostic outcome;

and

wherein the correlating is performed in accordance with an algorithm drawn from the group consisting essentially of;

linear or nonlinear regression algorithms;

linear or nonlinear classification algorithms;

ANOVA;

neural network algorithms;

genetic algorithms;

support vector machines algorithms;

hierarchical analysis or clustering algorithms;

hierarchical algorithms using decision trees;

kernel based machine algorithms such as kernel partial least squares algorithms, kernel matching pursuit algorithms, kernel fisher discriminate analysis algorithms, or kernel principal components analysis algorithms;

Bayesian probability function algorithms;

Markov Blanket algorithms;

recursive feature elimination or entropy-based recursive feature elimination algorithms;

a plurality of algorithms arranged in a committee network; and

forward floating search or backward floating search algorithms.

14. The method of claim 13 wherein the correlating so as to further determine diagnostic outcome is, in addition to risk of cerebral injury, expanded to predict risk of malignant massive cerebral artery infarction.

15. The method of claim 1 wherein correlating is of clinical patient information selected from a group consisting of Complete blood count (CBC), Coagulation test, Blood chemistry (glucose, serum electrolytes {Na, Ca, K}), Leukocyte and Neutrophil counts, platelet count, and Blood lipids tests.

16. The method of claim 1 wherein the correlating is of clinical patient information is selected from a group consisting of age, weight, height, body mass index, computed tomography scan information, Magnet Resonance Image scan information, gender, time from onset of stroke-like symptoms, time to recanalization, ethnicity, heart rate, blood pressure, respiration rate, blood oxygenation, previous personal and/or familial history of cardiac events, recent cranial trauma and unequal eye dilation.

17. The method of claim 1

wherein the analyzing is of both proteomic and non-proteomic markers;

and

wherein the correlating is further so as to deduce diagnostic or prognostic outcome.

18. The method of claim 1

wherein the obtaining of the test sample from the subject is of a test sample selected from the group consisting of blood, urine, blood plasma, blood serum, cerebrospinal fluid, saliva, perspiration or brain tissue, or a derivative thereof.

19. The method of claim 1

wherein the obtaining of the test sample from the subject is within a specific time window from onset of symptoms;

and

wherein the correlating is between (1) proteomic and non-marker marker values, and (2) the probability of present or future risk of a cerebral injury for the subject, for said specific time window from onset of symptoms.

20. The method of claim 1

wherein the correlating is in accordance with an algorithm drawn from the group consisting essentially of:

linear or nonlinear regression algorithms;

linear or nonlinear classification algorithms;

ANOVA;

neural network algorithms;

genetic algorithms;

support vector machines algorithms;

hierarchical analysis or clustering algorithms;

hierarchical algorithms using decision trees;

kernel based machine algorithms such as kernel partial least squares algorithms, kernel matching pursuit algorithms, kernel fisher discriminate analysis algorithms, or kernel principal components analysis algorithms;

Bayesian probability function algorithms;

Markov Blanket algorithms;

recursive feature elimination or entropy-based recursive feature elimination algorithms;

a plurality of algorithms arranged in a committee network; and

forward floating search or backward floating search algorithms.

21. A kit for determining presence or risk of cerebral injury in a human subject comprising:

a device having reagents at each of a plurality of discrete locations, each reagent and corresponding location configured and arranged to immobilize for detection one of said plurality of subject-derived markers, supports the analysis of both (1) cellular fibronectin and (2) additional markers; and

a computer algorithm, residing on a computer, calculating in consideration of analyzed cellular fibronectin and additional markers a probability of present or future risk of cerebral injury for the subject.

22. The kit according to claim 21 wherein the device at each of said plurality of discrete locations comprises:

a poly- or monoclonal antibody specific for an individual protein or protein fragment and that binds one of said plurality of subject-derived markers.

23. The kit according to claim 21 wherein the device comprises:

an immunoassay.

24. The kit according to claim 21 wherein the device is capable of determining the levels of cellular fibronectin and said additional markers within 20 minutes or less.

25. A method of determining presence or risk of cerebral injury in a human subject comprising:

determining a presence or amount of cellular fibronectin in a human subject; and

predicting the risk of cerebral injury following thrombolyic therapy in accordance with the determined presence or amount.

26. The method according to claim 25

wherein the predicting is that early hematoma growth is more probable than not when the determined presence is of an amount of cellular fibronectin greater than or equal to 6 μ

g/mL in serum.

27. The method according to claim 25

wherein the predicting is that malignant massive cerebral artery infarction is more probable than not when the determined presence is of an amount of cellular fibronectin greater than or equal to 16.6 μ

g/mL in serum.

28. The method according to claim 25

wherein the predicting is that early hematoma growth is more probable than not when the determined presence is of an amount of IL-6 greater than or equal to 24 pg/mL in serum.