WO2011139773A1

WO2011139773A1 - Plasma biomarkers of cognitive impairment in parkinson's disease

Info

Publication number: WO2011139773A1
Application number: PCT/US2011/034143
Authority: WO
Inventors: John Q. Trojanowski; Leslie M. Shaw; Alice Chen-Plotkin; Virginia M.Y. Lee
Original assignee: The Trustees Of The University Of Pennsylvania
Priority date: 2010-04-27
Filing date: 2011-04-27
Publication date: 2011-11-10

Abstract

The present invention includes methods for assessing risk of development of cognitive impairment in a Parkinson's Disease patient comprising measuring the level of a biomarker in a biological fluid such as plasma obtained from the patient. Methods of assessing risk over time are provided, as well methods of assessing the effect of a pharmaceutical agent on cognitive impairment.

Description

TITLE OF THE INVENTION

Plasma Biomarkers of Cognitive Impairment in Parkinson's Disease

BACKGROUND OF THE INVENTION

Parkinson's Disease (PD) is a common neurodegenerative disease selectively affecting dopaminergic neurons of the substantia nigra, resulting in symptoms of

bradykinesia, rigidity, and tremor. Over time, however, the disease process spreads throughout many brain regions (Braak et al, 2003, Neurobiol Aging 24, 197-211), and cognitive symptoms may develop. Most people with Parkinson's Disease (PD) develop cognitive impairment (CI) over time (Williams-Gray et al., 2007, Brain 130, 1787-1798), with up to 44% of PD patients exhibiting frank dementia and falling into the category of PD with Dementia (PDD) (Mayeux et al., 1992, Arch Neurol 49, 492-497), While genotype at the apolipoprotein E gene (ApoE) (Huang et al, 2006, Arch Neurol 63, 189-193) or microtubule associated protein tau gene (MAPI) (Goris et al., 2007, Ami Neurol 62, 145- 153) may affect the risk of developing CI in PD, and PDD may demonstrate reduced cerebro-, spinal fluid (CSF) levels of Αβ 1 -42 (Mollenhauer et al., 2006, Dement Geriatr Cogn Disord 22, 200-208; Siderowf et al, 2010, in press), many questions about the nature of CI and dementia in PD remain unanswered.

The development of CI and dementia in PD is a significant transition for PD patients and their families (Aarsland et al., 1999, Int J Geriatr Psychiatry 14, 866-874), as well as a substantial added cost burden for the healthcare system (Pressley et al., 2003, Neurology 60, 87-93), yet this turning point cannot presently be predicted (Leverenz et al, 2009, Curr Top Med Chem 9, 903-912). Accordingly, there is an unmet need in the art for a method of assessing risk of developing cognitive impairment in a Parkinson's Disease patient. The present invention meets this need,

BRIEF SUMMARY OF THE INVENTION

The present invention encompasses a method of assessing risk of developing cognitive impairment in a patient diagnosed with Parkinson's Disease. The method comprises the steps of obtaining a sample of plasma from the patient's blood, and assessing the level of epidermal growth factor (EGF) in said plasma sample, If the level of EGF is in the first quartile of a reference population of Parkinson's Disease patients without cognitive impairment, the patient is at an increased risk of developing cognitive impairment. In some embodiments, the method further comprises assessing the level of a second protein in the plasma sample, wherein the second protein is selected from the group consisting of: brain- derived neurotrophic factor (BDNF); CD40 ligand; epithelial neutrophil-activating protein 78 (ENA78); Fas antigen (FAS); growth regulated oncogene-alpha (GROalpha); heparin- binding epidermal growth factor (HBEGF); plasminogen activator inhibitor- Ϊ (PAI1);

platelet-derived growth factor (PDGF); RANTES; stem cell factor; thrombospondinl, and combinations thereof. In some embodiments, an immunoassay is used for the assessment of a biomarker level.

In another embodiment, a method of assessing increased risk of developing cognitive impairment in a patient diagnosed with Parkinson's Disease comprises the steps of obtaining a first sample of plasma from said patient's blood at a first time; assessing the level of epidermal growth factor (EGF) in said first plasma sample to obtain a baseline level; obtaining a second sample of plasma from said patient's blood at a second time to obtain a second level; and assessing the level of epidermal growth factor (EGF) in said second plasma sample to obtain a second level. If the second level is less than the baseline level, the patient is at an increased risk of developing cognitive impairment. In one embodiment, the second level is also compared to a reference population of PD patient's without cognitive impairment; if the second level is in the lowest quartile, the patient is at an increased risk of developing cognitive impairment. In some embodiments, the increased risk is about an 8- fold increase in risk.

Other methods and kits useful in practicing the methods are provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in the drawing certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

Figure 1 is a list of the 102 plasma analytes measured by multiplex immunoassay. Figure 2 depicts a dendogram representing hierarchical clustering of expression of the eleven (1 1) proteins whose levels correlate with baseline cognition (age- adjusted DRS scores).

Figures 3A and 3B depict graphs relating to the robustness of epidermal growth factor (EGF) as a plasma biomarker for cognitive impairment in PD, Figure 3A is a graph of ranked EGF levels as assessed by multiplex immunoassay (EGF-RBM) versus ranked EGF levels as assessed by traditional ELISA (EGF-ELISA). Figure 3B is a gi'aph of survival curves of time to PDD-range cognitive performance for 50 patients having baseline cognition in the non-PDD-range. The data are divided into quartiles with respect to plasma EGF expression, where quartile 1 corresponds to lowest EGF expression.

Figure 4 depicts a graph of survival curves of time to PDD-range (i.e., DRS<5) for quartile 1 and quartiles 2-4 of EGF expression for 50 patients having non-PDD- range baseline cognition, based on analysis of data using Cox proportional hazards models.

Figure 5 depicts a table of data demonstrating that the relationship between cognitive performance and EGF plasma values is not affected by medications of ApoE genotype. EGF levels were compared in groups with/without medication by two-tailed T- tests. EGF levels did not differ significantly between patients with/without each medication, as indicated by the non-significant p-valiies in the last column. EGF levels were compared between different ApoE genotypes (number of ApoE4 alleles) by chi-square testing. AChE inhibitor = acetylcholinesterase inhibitor

DETAILED DESCRIPTION OF THE INVENTION

The invention described lierein arises from the discovery that the levels of eleven specific proteins in the plasma of Parkinson's Disease (PD) patients correlate with cognitive performance, In particular, the level of epidermal growth factor (EGF) in PD plasma has a robust association with cognitive performance. Moreover, as shown herein, low levels of EGF are not only correlated with poor cognitive test scores at baseline, but are also predictive of an increased risk of conversion to dementia-range scores in PD patients whose baseline cognition was not impaired, Accordingly, methods of assessing risk of developing cognitive impairment based on the plasma level of EGF are presented. Methods of monitoring and/or evaluating likelihood of treatment efficacy regarding cognitive impairment are also provided.

Definitions

As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "about" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used,

The term "antibody," as used herein, refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab and F(ab)₂, as well as single chain antibodies (scFv), heavy chain antibodies, such as camelid antibodies, and humanized antibodies (Harlow et al,, 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory

Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426),

By the term "synthetic antibody" as used herein, is meant an antibody which is generated using recombinant DNA teclinology, such as, for example, an antibody expressed by a bacteriophage as described herein, The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art. As used herein, the term "heavy chain antibody" or "heavy chain antibodies" comprises immunoglobulin molecules derived from canielid species, either by immunization with a peptide and subsequent isolation of sera, or by the cloning and expression of nucleic acid sequences encoding such antibodies. The term "heavy chain antibody" or "heavy chain antibodies" further encompasses immunoglobulin molecules isolated from an animal with heavy chain disease, or prepared by the cloning and expression of VH (variable heavy chain immunoglobulin) genes from an animal,

As used herein, an "immunoassay" refers to any binding assay that uses an antibody capable of binding specifically to a target molecule to detect and quantify the target molecule,

By the term "specifically binds," as used herein with respect to an antibody, is meant an antibody which recognizes an specific antigen, but does not substantially recognize or bind other molecules in a sample. For instance, in a sample containing epidermal growth factor (EGF), an antibody that specifically binds to EGF recognizes and binds to EGF but does not substantially recognize or bind to other molecules in the sample.

As used herein, "cognitive impairment" refers to a diminution in one or more of a person's ability to think, concentrate, formulate ideas, reason and remember. It can be assessed by any clinically- validated assessment tool for cognitive impairment in Parkinson's Disease. Non-limiting examples of such tools include the Mattis Dementia Rating Scale-2 (DRS), the Folstein Mini-Mental State Examination (MMSE), the Abbreviated Mental Test (AMT) and the General Practitioner Assessment of Cognition (GPCOG). Any art-validated method of assessing cognitive impairment can be used in connection with the practice of the method described. In a preferred embodiment, cognitive function is assessed using the DRS. In this assessment, cognitive impairment refers to an age-adjusted score cut-off of <5 (see, e.g., Lucas et al„ 1998, J Clin Exp Neuropsychol 20, 536-547 and Llebaria et al., 2008, Mov Disord 23, 1546-1550). The Dementia Rating Scale-2 is a 36-task and 32-stimulus card individually administered instrument designed to assess level of cognitive functioning for individuals with brain dysfunction, The DRS assesses cognitive functioning on five subscaies: Attention (ATT, 8 items); Initiation-Perseveration (I-P, 1 1 items); Construction (CONST, 6 items); Conceptualization (CONCEPT, 6 items); and Memory (MEM, 5 items). The reliability and validity properties of the DRS are excellent. The DRS is very useful in the assessment and progression of dementia, including of Parkinson's disease.

"Increased risk of developing cognitive impairment" is used herein to refer to an increase in the likelihood or possibility of developing cognitive impairment. This risk can be assessed relative to a patient's own risk, or with respect to a reference population that does not have clinical evidence of cognitive impairment. The reference population may be representative of the patient witli regard to approximate age, age group and/or gender.

"Delaying development of cognitive impairment" as used herein refers to a prolonging of the time to the development of cognitive impairment and/or delay in the rate of increased extent of cognitive impairment.

"Alleviating cognitive impairment" as used herein refers to a decrease in the severity of cognitive impairment (i.e., an increase in cognitive function).

As used herein, a "detector molecule" is a molecule that may be used to detect a compound of interest. Non-limiting examples of a detector molecule are molecules that bind specifically to a compound of interest, such as, but not limited to, an antibody, a cognate receptor, and a small molecule.

It is understood that any and all whole or partial integers between any ranges set forth herein are included herein. Description

The present invention provides methods of assessing risk of developing cognitive impairment in a patient diagnosed with Parkinson's Disease and not having cognitive impairment. The methods rely on assessing the level of a biomarker, whose level correlates in a statistically significant manner with the cognitive impairment, in a sample of a biological fluid obtained from the patient, The biological fluid sample can be selected from blood, blood serum ("serum"), blood plasma ("plasma"), and cerebro-spinal fluid (CSF). In preferred embodiments, the biological fluid is plasma. The biological fluid is obtained from the subject using conventional methods in the art. For instance, one skilled in the art knows how to draw blood and how to process it in order to obtain serum and/or plasma for use in the method. Generally speaking, the method preferably maintains the integrity of the biomarkers of the invention such that it can be accurately quantified in the biological fluid. The btomarkers useful in the practice of the methods described herein are plasma proteins selected from the group consisting of: epidermal growth factor (EGF); brain- derived neurotrophic factor (BDNF); CD40 ligand; epithelial neutrophil-activatmg protein 78 (ENA78); Fas antigen (FAS); growth regulated oncogene-alpha (GROalpha); heparin- binding epidermal growth factor (HBEGF); plasminogen activator inhibitor- 1 (PAl-1);

platelet-derived growth factor (PDGF); RANTES; stem cell factor; and thrombospondinl ,

In each of the methods of the invention described herein, the subject can be any mammal that is susceptible to Parkinson's Disease or can be induced to experience Parkinson's Disease symptoms, including cognitive impairment. The subject is preferably a human patient having a clinician's diagnosis of Parkinson's Disease.

In some embodiments of the invention, risk assessment is determined by comparison of the level of a marker to a database of levels for that biomarker in PD patients without clinically-diagnosed cognitive impairment. The database is divided into quartiles, wherein the first quartile represents the lowest expression of the biomarker selected from the group consisting of EGF, CD40 ligand; PAI-1 ; thrombospondinl ; PDGF; RANTES;

GROalpha; stem cell factor; and ENA-78 or wherein the first quartile represents the highest expression of the biomarker FAS in the biological fluid. As demonstrated herein, if the test level falls within the first quartile, the conclusion is that the patient has an increased risk of developing cognitive impairment. In some embodiments, the increased risk is about an 8- fold increase in risk compared to test level that is falls within any of the other three quartiles. In some embodiments, the increased risk is about an 8-fold increase in risk compared to a test level that is falls within any of the other three quartiles and with a median onset between about 7 to 21 months, between about 10 to 18 months or about 14 months.

The database is generated by measuring the same marker under the same conditions in a representative population, Typically the representative population is a population of patients diagnosed with Parkinson's Disease who do not have cognitive impairment, preferably as measured by Mattis Dementia Rating Scale-2 (DRS). The database of ratios is, optionally, sub-divided by age and/or gender. A quartile is the value of the boundary at the 25th, 50th, or 75th percentiles of a frequency distribution divided into four parts, each containing a quarter of the population, For instance, in a database comprising these values: 2.0, 2.2, 2.04, 1.99, 1.8, 1.8, 1.78, 1.75, 1.65, 1.6, 1 .58, 1.45, 1.26, 1.1 , 1 ,05 and 1 , the first quartile is 1.4, the second qiiartile is 1.7 and the third quartile is 1.85.

A ratio of 1 .56 is therefore within the second quartile. As used here, "first quartile" refers to a value within the quartile demarcated by the 25^Ul percentile,

Cognitive impairment can be assessed by any method considered valid by clinicians for evaluating cognitive function in Parkinson's disease patients. In a preferred embodiment, cognitive impairment is assessed using Mattis Dementia Rating Scale-2 (DRS).

As shown herein, EGF is a robust biomarker for assessing risk of developing cognitive impairment in a Parkinson's Disease patient. Specifically, an EGF level in the biological sample of a PD patient who does not cognitive impairment that is within the first quartile of a reference population of patients with Parkinson's Disease without cognitive impairment, the PD patient is at an increased risk of developing cognitive impairment.

Accordingly, in one embodiment, the method comprises assessing the level of EGF in a biological sample such as plasma in order to assess risk. Advantageously, the robust correlation discovered for plasma EGF is independent of both ApoE genotype and treatment with dopaminergic or dementia medications. Thus, the practice of the methods are not limited based on any of these factors, and they can be useful in any Parkinson's Disease patient regardless of ApoE genotype and treatment with dopaminergic or dementia medications.

The methods described herein optionally further comprise assessing the level of a second biomarker shown herein to correlate with cognitive impairment. The second biomarker is selected from the group consisting of: BDNF; CD40 ligand; ENA78; FAS; GROalpha; HBEGF; PAI- 1 ; PDGF; RANTES; stem cell factor; thrombospondinl ; and combinations thereof. As further shown herein, levels of CD40 ligand, PAI- 1 ,

thrombospondinl , and PDGF are particularly correlated with levels of EGF with regard to cognitive impairment, Accordingly, in a preferred embodiment, the method further comprises measuring the level of a second biomarker selected from the group consisting of CD40 ligand, PAI- 1 , thrombospondinl , PDGF, and combinations thereof, Another cluster with particularly correlated expression consists of RANTES, GROalpha, stem cell factor and ENA-78, Accordingly, in another preferred embodiment, the method further comprises measuring the level of a second biomarker selected from the group consisting of RANTES, GROalpha, stem cell factor, ENA-78, and combinations thereof, The invention also provides a method of assessing increased risk of developing cognitive impairment in a PD patient as a function of time. The method comprises assessing the level of a biomarker in a biological fluid at a first point in time to establish a baseline level of the biomarker. The method further comprises assessing the level of the same biomarker at a second point in time in order to identify whether the level of the marker is changing. For a biomarker selected from the group consisting of EGF, CD40 ligand; PAI- 1 ; thrombospondinl ; PDGF; RANTES; GROalpha; stem cell factor; and ENA- 78, if the second level is less than the baseline level, the patient is at an increased risk of developing cognitive impairment. For FAS as the biomarker, if the second level is greater than the baseline level, the patient is at an increased risk of developing cognitive impairment. In an embodiment, if the second level is within the first quartile of a reference population of patients with Parkinson's Disease without cognitive impairment, the PD patient is at an increased risk of developing cognitive impairment. The second assessing step is generally performed at least one day after the baseline assessment. It can also be performed multiples days, weeks, months or years after the baseline assessment. Moreover, the second assessing step can be performed itetatively over time to acquire additional data and thereby monitor the risk. Rate of change in expression levels can be calculated to identify if there is an increasing trend to reduced expression for a biomarker selected from the group consisting of EGF, CD40 ligand; PAI-1 ; thrombospondinl; PDGF; RANTES; GROalpha; stem cell factor; and ENA-78, or a increasing trend to increased expression for the biomarker FAS. In preferred embodiments, the biomarker assessed is EGF,

Risk assessment over time can also be performed in the presence of pharmaceutical agent to monitor the likelihood that the pharmaceutical agent is delaying development of cognitive impairment in a Parkinson's patient, In this method, a baseline level of the biomarker in a biological fluid is assessed in the absence of the pharmaceutical agent. After administration of the pharmaceutical agent, the level of the biomarker is again assessed at at least one time point to get a treated level. If the treated level is the same or greater than the baseline level for a biomarker selected from the group consisting of EGF, CD40 ligand; PAI-1 ; thrombospondinl ; PDGF; RANTES; GROalpha; stem cell factor; and ENA-78, the likelihood increases that development of cognitive impairment is delayed by the pharmaceutical agent. For FAS as the biomarker, if the treated level is the same or less than the baseline level for FAS, the likelihood increases that development of cognitive impairment is delayed by the pharmaceutical agent, The treated level can alternatively or additionally be compared to a database of such measurements in a population not receiving the

pharmaceutical agent to assess whether cognitive impairment is delayed. If the treated level is greater than an average measurement or range of measurements of the treated level in the untreated population for a biomarker selected from the group consisting of EGF, CD40 ligand; PAH ; thrombospondinl ; PDGF; RANTES; GROalpha; stem cell factor; and ENA- 78, that is also indicative that of an increased likelihood that cognitive impairment development is delayed by the pharmaceutical agent. For FAS as the biomarker, if the treated level is less than an average measurement or range of measurements of the treated level in the untreated population, that is also indicative that of an increased likelihood that cognitive impairment development is delayed by the pharmaceutical agent.

The invention also provides a method assessing the likelihood that a pharmaceutical agent is alleviating cognitive impairment in a Parkinson's patient diagnosed with cognitive impairment. In this method, a baseline level of the biomarker is assess in the absence of the pharmaceutical agent. After administration of the pharmaceutical agent, the level of the biomarker is again assessed at least one time point to obtain a treated level. For a biomarker selected from the group consisting of EGF, CD40 ligand; PAH ;

thrombospondinl ; PDGF; RANTES; GROalpha; stem cell factor; and ENA-78, if the treated level is the same or greater than the baseline level, there is an increased likelihood that the pharmaceutical agent is alleviating cognitive impairment in the Parkinson's patient diagnosed with cognitive impairment. For FAS as the biomarker, if the treated level is the same or less than the baseline level for FAS, there is an increased likelihood that the pharmaceutical agent is alleviating cognitive impairment in the Parkinson's patient diagnosed with cognitive impairment. The treated level can alternatively or additionally be compared to a database of such measurements in a population not receiving the pharmaceutical agent to assess whether cognitive impairment is alleviated. If the treated level is greater than an average

measurement or range of measurements of the treated level in the untreated population for a biomarker selected from the group consisting of EGF, CD40 ligand; PAH ;

thrombospondinl ; PDGF; RANTES; GROalpha; stem cell factor; and ENA-78, that is also indicative of an increased likelihood that the pharmaceutical agent is alleviating cognitive impairment. For FAS as the biomarker, if the treated level is less than an average measurement or range of measurements of the treated level in the untreated population, that is also indicative of an increased likelihood that the pharmaceutical agent is alleviating cognitive impairment.

The methods of the invention include quantitatively measuring the level of a protein biomarker, Methods of quantitatively assessing the level of a protein in a biological fluid such as plasma are well known in the art, In some embodiments, assessing the level of a protein involves the use of a detector molecule for the biomarker. Detector molecules can be obtained from commercial vendors or can be prepared using conventional methods in the art. Exemplary detector molecules include, but are not limited to, an antibody that binds specifically to the biomarker, a naturally-occurring cognate receptor, or functional domain thereof, for the biomarker, and a small molecule that binds specifically to the biomarker,

In a preferred embodiment, the level of a biomarker is assessed using an antibody. Thus, exemplary methods for assessing the level of a biomarker in a biological fluid sample include various immunoassays, for example, immunohistochemistry assays, immuno cytochemistry assays, ELISA, capture ELISA, sandwich assays, enzyme immunoassay, radioimmunoassay, fluorescent immunoassay, and the like, all of which are known to those of skill in the art. See e.g. Harlow el aL, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Harlow e! aL, 1999, Using Antibodies: A

Laboratory Manual, Cold Spring Harbor Laboratory Press, NY.

The generation of polyclonal antibodies is accomplished by inoculating the desired animal with an antigen and isolating antibodies which specifically bind the antigen therefrom.

Monoclonal antibodies directed against one of the eleven biomarkers identified herein may be prepared using any well known monoclonal antibody preparation procedures, such as those described, for example, in Harlow et al, (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY) and in Tuszynski et al. (1988, Blood, 72: 109- 115). Human monoclonal antibodies may be prepared by the method described in U.S. patent publication 2003/0224490. Monoclonal antibodies directed against a biomarker such as EGF are generated from mice immunized with the biomarker using standard procedures as referenced herein. For use in preparing an antibody, a biomarker may be purified from a biological source that endogenously comprises the biomarker, or from a biological source recombinant ly-engineered to produce or over-produce the biomarker, using conventional methods known in the art. Exemplary nucleic acid and protein sequences for the eleven biomarkers described herein are readily available in public sequence databases, such as National Library of Medicine's genetic sequence database GenBank® (Benson et al., 2008, Nucleic Acids Research, 36(Database issue):D25-30). Preferably, antibodies are generated against the human homo logs of each of the eleven biomarkers for practicing the methods using a biological fluid sample from a human patient diagnosed with Parkinson's Disease.

Nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using teclinoiogy which is available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. Immunol. 12(3 ,4): 125- 168) and the references cited therein. Further, the antibody useful in the practice of the invention may be "humanized" using the technology described in Wright et al., (supra) and in the references cited therein, and in Gu et al. (1997, Thrombosis and Hematocyst

77(4):755-759).

To generate a phage antibody library, a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which express the desired protein to be expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes, The procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Sambrook et al. (2001 , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

Bacteriophage which encode the desired antibody may be engineered such that the protein is displayed on the surface thereof in such a manner that it is available for binding to the antigen against which the antibody is directed. Thus, when bacteriophage which express a specific antibody are incubated in the presence of the antigen, for instance, antigen immobilized on a resin or surface, the bacteriophage will bind to the antigen. Bacteriophage which do not express the antibody will not bind to the antigen. Such panning techniques are well blown in the art and are described for example, in Wright et al, (supra).

Processes, such as those described above, have been developed for the production of human antibodies using Ml 3 bacteriophage display (Burton et al., 1994, Adv. Immunol. 57: 191-280). Essentially, a cDNA library is generated from mRNA obtained from a population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin genes and thus, the cDNA encodes the same. Amplified cDNA is cloned into M l 3 expression vectors creating a library of phage which express human Fab fragments on their surface. Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human Fab immunoglobulin. Thus, in contrast to conventional monoclonal antibody synthesis, this procedure immortalizes DNA encoding human immunoglobulin rather than cells which express human immunoglobulin.

The procedures just presented describe the generation of phage which encode the Fab portion of an antibody molecule. However, phage which encode single chain antibodies (scFv/phage antibody libraries) are also useful in preparing Fab molecules useful in the invention. Fab molecules comprise the entire Ig light chain, that is, they comprise both the variable and constant region of the light chain, but include only the variable region and first constant region domain (CHI) of the heavy chain. Single chain antibody molecules comprise a single chain of protein comprising the Ig Fv fragment. An Ig Fv fragment includes only the variable regions of the heavy and light chains of the antibody, having no constant region contained therein. Phage libraries comprising scFv DNA may be generated following the procedures described in Marks et al., 1991, J. Mol. Biol. 222:581 -597.

Panning of phage so generated for the isolation of a desired antibody is conducted in a manner similar to that described for phage libraries comprising Fab DNA. Synthetic phage display libraries in which the heavy and light chain variable regions may be synthesized such that they include nearly all possible specificities (Barbas, 1995, Nature Medicine 1 :837-839; de Kruif et al,, 1995, J. Mol. Biol, 248:97-105) may also be used to prepare an antibody useful in the practice of the invention.

Other methods for assessing the level of a protein include chromatography (e.g., HPLC, gas chromatography, liquid chromatography) and mass spectrometry (e.g., MS, MS-MS). For instance, a chromatography medium comprising a cognate receptor for the biomarker or a small molecule that binds to the biomarker can be used to substantially isolate the biomarker from the sample of biological fluid, Small molecules that bind specifically to a biomarker can be identified using conventional methods in the art, for instance, screening of compounds using combinatorial library methods known in the art, including biological libraries, spatially-addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection,

The level of substantially isolated protein can be quantitated directly or indirectly using a conventional technique in the art such as spectrometry, Bradford protein assay, Lowry protein assay, biuret protein assay, or bicinchoninic acid protein assay, as well as immunodetection methods.

Kits

A kit is envisaged for every method disclosed, The following description of a kit useful for assessing risk of developing cognitive impairment in a mammal by measuring the level of a biomarker in a biological fluid therefore is not intended to be limiting and should not be construed that way.

The kit comprises a negative control solution containing a biomarker at a concentration of about the concentration of the biomarker which is present in a biological fluid sample of a PD patient who does not have cognitive impairment, and does not increased risk for cognitive impairment. Preferably, the biomarker is EGF.

The kit also includes a positive control solution containing the biomarker at a concentration of about the concentration of the biomarker which is present in a biological fluid sample of a PD patient without cognitive impairment who has an increased risk of cognitive impairment,

Additionally, the kit includes an antibody directed against the biomarker for cognitive performance. Preferably, the biomarker is EGF. Methods for the preparation and purification of antibodies are known in the ait, and are described, for example, in Harlow et al,, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y. The antibody can be any type of antibody known in the art. The kit optionally can include at least one sample container for containing a biological fluid sample obtained from the mammal. The kit can, optionally include a secondary antibody directed against the antibody specific for the biomarker.

Furthermore, the kit includes an instructional material for use in the assessment of risk of developing cognitive impairment in a mammal. The instructional material can be a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the method of the invention in the kit for assessment of cardiovascular risk in a mammal. The instructional material of the kit of the invention may, for example, be affixed to a container which contains other contents of the kit, or be shipped together with a container which contains the kit. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the contents of the kit be used cooperatively by the recipient.

EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

The materials and methods used in Experimental Examples 1 -5 below are now described.

Plasma biomarker quantitation: Whole blood samples were obtained under IRB approval, placed immediately on ice, spun down for plasma aliquotting (0,5mL aliquots), and frozen at -80°C within the same day. Plasma aliquots were then stored at - 80°C until analysis. Simultaneous screening of 15 1 proteins by multiplex immunoassay on the Human DiscoveryMAP® (RBM, Autrn, TX) panel using a Luminex®100™ (Luminex, Austin, TX) platform was then performed for all samples, in one batch, by Rules-Based Medicine, Inc,

In brief, the Luminex® 100™ platform technology multiplexes immunoassays on the surface of polystyrene microsphere beads. The microsphere beads are loaded with a ratio of two spectrally distinct fluorochromes yielding up to 100 uniquely color-coded beads. The beads are coated with capture antibodies specific for the assay and run in either standard sandwich or competitive immunoassay format. Capture-antibody microspheres are incubated with blocking solution and diluted plasma sample or calibration controls for one hour. Beads are rinsed and biotinylated detection reagent added, Streptavidm-phycoerthyrin is then added to each well and incubated for 60 minutes. Following wash steps, beads are resuspended in reading solution and read on the luminex instrument. Details of each assay procedure are provided by RBM in validation reports compliant with CLIA standards, Validation details of all the assays are available from RBM (www(dot)rulesbasedmedicine(dot)com).

Of the 151 proteins in the RBM Human DiscoveryMAP® panel, 302 proteins passed the quality control measures (<20% of samples below lowest reliable value as given by RBM, <40% variation across technical duplicates) and were used in this study. For confirmation of EGF values, separate aliquots of samples previously run on the multiplex immunoassay were quantitated on an EGF enzyme-linked immunosorbent assay (ELISA, R&D Systems, Minneapolis, MN).

Cognitive tests: The Mattis Dementia Rating Scale-2 (DRS) was used to evaluate cognition in study patients. A raw score was obtained and adjusted for age as previously described (Lucas et al., 1998, J Clin Exp Neuropsychol 20, 536-547). An age- adjusted score cut-off of <5 was used for cognitive performance in the PDD range, following recommended criteria in the DRS manual. All subjects had baseline DRS testing within 6 months of plasma draw; 61/70 subjects had DRS testing on the same day as the plasma draw - the 9 individuals who were tested on a different day fell into all 4 quartiles of EGF values.

ApoE genotyping: ApoE genotyping was done for study participants using EDTA blood samples and TaqMan® (Applied Biosystems, Foster City, CA) quantitative PCR assays to genotype ApoE nucleotides 334 T/C and 472 CT with an ABI 7900 real-time thermocycler on DNA freshly prepared from EDTA whole blood.

Statistical analyses: Linear regression analyses evaluating the association of levels of each protein to age-adjusted DRS scores were performed in R, The model used for screening designated age and gender as covariates and evaluated the association of each protein individually to DRS scores. For the top 11 proteins, the association between each protein and DRS score was further evaluated in models incorporating age, gender, and either UPDRS-motor scores or disease duration as covariates,

To evaluate whether the association between EGF levels and DRS scores was affected by medications, presence/absence of each medication or class of medication at the time of plasma draw (levodopa, dopamine agonists, anti-psychotics medications, antidepressant medications, statins, acetylcholinesterase inhibitors, memantine, and

benzodiazepines) was established by chart review, and each was entered as an additional categorical yes/no factor in a linear model with age, gender, and EGF levels as continuous variables predicting DRS performance. In addition, EGF levels were compared between groups (with/without medication) by two-tailed T-tests after establishing that distributions met assumptions of normalcy. ApoE genotype was similarly evaluated, with categories based on the number of ApoE4 alleles, and chi-square testing used to compare EGF levels among groups. An alternative model of ApoE genotype effect (in which ApoE2 alleles are protective, ApoE3 alleles are neutral, and ApoE4 alleles are deleterious) performed similarly,

The Partek® Genomics Suite™ was used to perform hierarchical cluster analysis (Euclidean distance) of co-expression among the top 1 1 proteins, and to generate graphics (Partek GS, copyright 2010, St, Louis, MO), Survival analysis curves were compared with log-rank tests, treating each EGF quartile as a separate group. Based on the observation that only the lowest quartile of EGF values differed in outcomes, further analyses were performed comparing the lowest EGF quartile to all other quartiles combined. To evaluate the effects of baseline DRS performance, age, and gender on the relationship between EGF quartile and conversion to PDD-range DRS, Cox proportional hazards models were used,

The experimental results are now presented.

Experimental Example 1 : Potential plasma biomarkers of cognitive impairment in

Parkinson's Disease

To identify plasma proteins that might function as a biomarker of cognitive impairment in Parkinson's Disease, 70 archived plasma samples from patients with concurrent cognitive testing on the Mattis Dementia Rating Scale-2 (DRS) (Lucas et al., 1998, J Clin Exp Neuropsychol 20, 536-547; Liebaria et al., 2008, Mov Disord 23, 1546- 1550) were obtained. Of these 70 subjects, 16 (23%) had cognitive scores within the PDD- range (age-adjusted DRS<5), and 54 did not (age-adjusted DRS>5). Demographic characteristics (age at plasma draw, age at disease onset, disease duration, UPDRS motor score, gender, and medication regimens) and ApoE genotypes were similar between these two groups (Table 1).

Using these 70 samples, plasma levels of 102 proteins (Figure 1) were quantitated on a multiplex bead-based immunoassay as previously described (Hu et al., 2010, Acta Neuropathol., 2010 Mar 16, Epub ahead of print). Levels of each protein were evaluated individually for correlation to age-adjusted DRS, using linear regression in a model accounting for age and gender.

Eleven out of 102 proteins screened showed correlations between plasma level and age-adjusted DRS scores (nominal p<0.05) in linear regression models with age and gender as covariates (Table 2, Model 1). The 1 1 proteins are: brain-derived neurotrophic factor (BDNF); CD40 ligand; epidermal growth factor (EGF); epithelial neutrophil-activatmg protein 78 (ENA78); Fas antigen (FAS); growth regulated oncogene-alpha (GROalpha); heparin-binding epidermal growth factor (HBEGF); plasminogen activator inhibitor- 1 (PAI1); platelet-derived growth factor (PDGF); RANTES; stem ceil factor; and

thrombospondinl .

Epidermal growth factor (EGF, p<0.001) and CD40 ligand (p=0.006) exhibited the most significant associations. Of the 1 1 , levels of 3 proteins (CD40 ligand, EGF, and HBEGF, bold bordered rows) also showed significant correlations with age- adjusted DRS scores in two additional models including either disease duration (Table 2, Model 2) or UPDRS motor scores (Table 2, Model 3) as additional covariates, Notably, a preponderance of these proteins associated with cognitive performance are growth factors.

The data for the eleven (l l ) proteins showing correlations between plasma level and age-adjusted DRS scores can be plotted as a heat map. The map can further include expression data for CD 40 and EGF-R, which are receptors for CD40 ligand and EGF, respectively. The heat map can be organized as 70 columns, representing each of the 70 samples and 1 1 rows representing expression level of a given protein in each of the 70 samples. The rows can be arranged to show expression data for, top to bottom, EGF, CD40 ligand, PAI-1 , thrombospondinl , PDGF, RANTES, GROalpha, stem cell factor, ENA-78, FAS, CD40, HB-EGF, and EGF-R, where one color represents low expression, a second color represents high expression and shades of gray denote intermediate expression. The columns can be arranged so that individuals with age-adjusted DRS>5 (i.e., non-PDD -range) are grouped together, and individuals with age-adjusted DRS<5 (i.e., PDD-range). The nine individuals who were DRS>5 at baseline and who subsequently converted to DRS<5 (i.e., PDD-range) during follow-up can be indicated.

When the data are plotted as described, it is evident that individuals with baseline PDD-range cognition almost uniformly had low expression for EGF and co- expressed proteins (CD40 ligand, PAI-1 , thrombospondinl and PDGF). It is also evident that the nine individuals who were DRS>5 at baseline and who subsequently converted to DRS<5 had lower expression levels for EGF, CD40 ligand, PAI- 1 , thrombospondinl , PDGF, RANTES, GROalpha, stem cell factor, and ENA-78 and a higher expression level for FAS.

Moreover, hierarchical clustering indicates that many of the 11 cognition- associated proteins are co-expressed (Figure 2). In particular, CD40 ligand, EGF, PAI- 1 , Thrombospondin-1, and PDGF represent one cluster with particularly correlated expression (bolded lines), Notably, plasma levels of the soluble receptors for EGF and CD40 ligand - EGF receptor (EGF-R) and CD40, respectively - were neither associated with DRS scores, nor correlated with expression of their ligands (Table 2). In addition, the association between cognitive test performance and plasma protein level persisted for EGF, CD40 ligand, and heparin-binding EGF (HBEGF) in models that accounted for either disease duration or UPDRS-motor scores, in addition to age and gender (Table 2). Interestingly, HBEGF expression was not correlated with EGF expression, although they may act on the same receptor. This result implies differential regulation of these two proteins, despite both demonstrating relationships with cognition. Experimental Example 2: Independent confirmation of plasma EGF levels confirms robustness of cognition relationship

The screen performed in Experimental Example 1 used a multiplex immunoassay. This method allows for the simultaneous measurement of many (> 100) proteins, but require independent follow-up confirmation. The robustness of the top analyte (EGF) was therefore evaluated using a second method for measuring plasma protein levels. Duplicate aliquots of 24 plasma samples were qnantitated for EGF levels by multiplex immunoassay and by traditional ELISA. Readings on each assay were ranked from 1 to 24, and ranks by multiplex immunoassay (EGF-RBM, X-axis) were plotted against ranks by ELISA (EGF-ELISA, Y-axis). See Figure 3A. Cross-platform correlation was excellent, as indicated by an r² value of 0.91 for rank correlation. These data indicate that EGF is technically robust as a biomarker for cognitive impairment in Parkinson's Disease.

Experimental Example 3: EGF is not a robust classifying biomarker for PDD-range DRS

In the preceding analyses, DRS scores were treated as a continuous variable. Dichotomizing scores into PDD-range (age-adjusted DRS<5) and non-PDD-range (age- adjusted DRS>5), it was observed that subjects with DRS scores in the PDD-range almost uniformly had low levels of EGF, CD40 ligand, and other co-expressed proteins. Therefore, an evaluation was made to determine whether levels of our top plasma analyte (EGF) could serve as a classifier for PDD-range DRS. The conclusion was that trade-offs between specificity and sensitivity limited the utility of EGF as a classifying biomarker for baseline PDD-range performance, with a maximum classification accuracy of 79%.

Experimental Example 4: EGF levels are predictive of conversion from non-PDD to PDD- range DRS scores

Follow-up DRS testing was available for 66 of the 70 samples, with a median follow-up of 16 months and interquartile range (IQR) of 13-26 months. Therefore, an analysis was performed to ascertain if EGF levels were predictive of conversion from non- PDD-range DRS scores to PDD-range DRS scores (i.e, <5) among subjects with age-adjusted DRS scores>5 at baseline.

Strikingly, survival analyses of time to PDD-range exhibited markedly different outcomes for those subjects with the lowest plasma EGF levels (Figure 3B, quartile 1 data). Specifically, non-PDD-range subjects with EGF levels in the lowest quartile (EGF<150, n=12) were eight times more likely to convert to PDD-range, with a median conversion time of 14 months (p<0.001, hazard ratio 8.34, 95% CI 4.26-122.90). The other three quartiles of EGF levels did not differ significantly from each other (Figure 3B). The association between EGF quartile and risk for conversion persisted in models adjusting for age and baseline DRS score (p=0.033 for EGF quartile; Figure 4); or age, gender, and baseline DRS score (p=0,037 for EGF quartile).

Experimental Example 5: EGF levels are predictive of conversion from non-PDD to PDD- range DRS scores

To further explore the association between low EGF levels and CI in PD, the effects of concurrent medications (levodopa, dopamine agonists, anti-psychotics medications, anti-depressant medications, statins, acetylcholinesterase inhibitors, memantine, and benzodiazepines) and ApoE genotype were analyzed. Each factor was entered as an additional categorical factor in a linear model with age, gender, and EGF levels as continuous variables predicting DRS performance. The results of the analysis are presented in Figure 5.

EGF levels did not differ significantly between patients with/without each medication, and the presence/absence of each medication did not affect the association of EGF levels and DRS performance (Figure 5). Of factors analyzed, only the use of anti- depressants was significantly associated with DRS performance, with poorer DRS performance among patients taking anti-depressants.

The impact of ApoE genotype (genotyped in 69 of the 70 subjects) on the relationship between DRS score and EGF levels was similarly evaluated. Again, no effect and no correlation was detected between ApoE genotype and EGF levels (Figure 5), In contrast, as previously reported, ApoE genotype was correlated with plasma levels of ApoE in the dataset (p=0.012). Thus, the association between low EGF levels and poor cognitive test performance appears to be independent of both medication use and ApoE genotype.

In summary, eleven plasma proteins have been identified that correlate with cognitive performance in Parkinson's Disease patients. Epidermal growth factor exhibits the most robust correlation of these eleven proteins. EGF as a biomarker of cognitive impairment is independent of ApoE genotype and of treatment with dopaminergic or dementia medications. Moreover, low levels of EGF precede the development of cognitive impairment. These data indicate that assessing the level of EGF in a patient diagnosed with Parkinson's Disease, who does have cognitive impairment, can be used to assess the risk of that patient developing cognitive impairment. The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the tine spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

What is claimed is: 1. A method of assessing risk of developing cognitive impairment in a patient diagnosed with Parkinson's Disease, said method comprising:

obtaining a sample of plasma from the patient's blood,

assessing the level of epidermal growth factor (EOF) in said plasma sample,

wherein if the level of BOP is in the first quartile of a reference population of

Parkinson's Disease patients without cognitive impairment, the patient is at an increased risk of developing cognitive impairment.

2. The method of claim 1, further comprising wiscsiing the level of a second protein in the plasma sample, wherein the second protein is selected from the group consisting of. brain-derived rieuiotrophlc factor (BDNF); CD40 ligand; epithelial neutrophii- activatittg protein 78 (BNA78); Pas antigen (FAS); growth regulated orKogene-alpha (OROalpha); beparin-binding epidermal growth factor (HBBOF); plasminogen activator inhibitor- 1 (PAI1); platelet-derived growth factor (PDOP); RANTES; stem cell factor, thrombospondinl, and combinations thereof.

3. The method of claim 2, wherein the second protein is selected from the group consisting oft CD40 ligand, PAI-1, thrombospondinl, PDOP and combinations thereof.

4. The method of claim 2, wherein the second protein is selected from the group consisting of RANTES, OROalpha, stem cell factor, ENA-78, and combinations thereof:

5. The method of claim 1 , wherein the step of assessing the level of epidermal growth factor (EOF) in said plasma sample comprises an immunoassay.

6. A method of assessing increased risk of developing cognitive impairment in a patient diagnosed with Parkinson's Disease, said method comprising:

obtaining a first sample of plasma from said patient's blood at a first time,

assessing the level of epidermal growth factor (EGF) in said first plasma sample to obtain a baseline level,

obtaining a second sample of plasma from said patient's blood at a second time to obtain a second level,

assessing the level of epidermal growth factor (EGF) in said second plasma sample to obtain a second level,

wherein if the second level is less than the baseline level, the patient is at an increased risk of developing cognitive impairment.

7. The method of claim 6, wherein the second level is the lowest quartile of a reference population of patients with Parkinson's Disease without cognitive impairment.

8. The method of claim 6, further comprising assessing the level of a second protein in the plasma sample, wherein the second protein is selected from the group consisting of: brain-derived neurotrophic factor (BDNF); CD40 ligand; epithelial neutrophil- activating protein 78 (ENA78); Fas antigen (FAS); growth regulated oncogene-alpha (GROalpha); heparin-binding epidermal growth factor (HBEGF); plasminogen activator inhibitor- 1 (PAI 1); platelet- derived growth factor (PDGF); RANTES; stem cell factor;

thiombospondinl , and combinations thereof.

9. The method of claim 8, wherein the second protein is selected from the group consisting of: CD40 ligand, PAI-1, thrombospondinl , PDGF and combinations thereof.

10. The method of claim 8, wherein the second protein is selected from the group consisting of RANTES, GROalpha, stem cell factor, ENA-78, and combinations thereof,

1 1. The method of claim 6, wherein the steps of assessing the level of epidermal growth factor (EGF) in said plasma sample comprises an immunoassay.

12, A method of assessing the likelihood that a pharmaceutical agent is efficacious in delaying development of cognitive impairment in a Parkinson's Disease patient without cognitive impairment, said method comprising:

obtaining a first sample of plasma from said patient's blood in the absence of said pharmaceutical agent,

administering said pharmaceutical agent to said patient,

obtaining a second sample of plasma from said patient's blood after administration of said pharmaceutical agent,

assessing the level of EGF in said second plasma sample to obtain a treated level,

wherein the likelihood that development of cognitive impairment is delayed by the pharmaceutical agent is increased if the treated level is about the same or greater than the baseline level.

13, The method of claim 12, further comprising obtaining at least a third sample of plasma from said patient's blood after administration of said pharmaceutical agent, and assessing the level of EGF in said second plasma sample to obtain a second treated level,

14, The method of claim 12, further comprising assessing the level of a second protein in the plasma sample, wherein the second protein is selected from the group consisting of: brain-derived neurotrophic factor (BDNF); CD40 ligand; epithelial neutrophil- activating protein 78 (ENA78); Fas antigen (FAS); growth regulated oncogene-alpha (GROalpha); heparin-bmding epidermal growth factor (HBEGF); plasminogen activator inhibitor- 1 (PAI1); plate let- derived growth factor (PDGF); RANTES; stem cell factor; thrombospondinl , and combinations thereof.

15. The method of claim 14, wherein the second protein is selected from the group consisting of: CD40 ligand, PAI-1 , thrombospondinl, PDGF and combinations thereof.

16. The method of claim 14, wherein the second protein is selected from the group consisting of RANTES, GROalpha, stem cell factor, ENA-78, and combinations thereof.

17. The method of claim 12, wherein the steps of assessing the level of epidermal growth factor (EGF) in said plasma sample comprises an immunoassay,

18. A method of assessing the likelihood that a pharmaceutical agent is efficacious in alleviating cognitive impairment in a Parkinson's patient diagnosed with cognitive impairment, said method comprising:

administering said pharmaceutical agent to said patient, obtaining a second sample of plasma from said patient's blood after administration of said pharmaceutical agent,

wherein the likelihood that the pharmaceutical agent alleviates cognitive impairment is increased if the treated level is about the same or greater than the baseline level.

19. The method of claim 18, further comprising obtaining at least a third sample of plasma from said patient's blood after administration of said pharmaceutical agent, and assessing the level of EGF in said second plasma sample to obtain a second treated level,

20. The method of claim 18, further comprising assessing the level of a second protein in the plasma sample, wherein the second protein is selected from the group consisting of: brain-derived neurotrophic factor (BDNF); CD40 ligand; epithelial neutrophil- activating protein 78 (ENA78); Fas antigen (FAS); growth regulated oncogene -alpha (GROalpha); heparin-binding epidermal growth factor (HBEGF); plasminogen activator inhibitor- 1 (PAI1); platelet-derived growth factor (PDGF); RANTES; stem cell factor;

thrombospondinl , and combinations thereof.

21, The method of claim 20, wherein the second protein is selected from the group consisting of: CD40 ligand, PAI-1 , thrombospondinl , PDGF and combinations thereof.

22. The method of claim 20, wherein the second protein is selected from the group consisting of RANTES, GROalpha, stem cell factor, ENA-78, and combinations thereof.

23. The method of claim 18, wherein the steps of assessing the level of epidermal growth factor (EGF) in said plasma sample comprises an immunoassay.