WO2020234269A1 - Method for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis - Google Patents

Method for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis Download PDF

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Publication number
WO2020234269A1
WO2020234269A1 PCT/EP2020/063907 EP2020063907W WO2020234269A1 WO 2020234269 A1 WO2020234269 A1 WO 2020234269A1 EP 2020063907 W EP2020063907 W EP 2020063907W WO 2020234269 A1 WO2020234269 A1 WO 2020234269A1
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Prior art keywords
expression
haptoglobin
level
immunoglobulin heavy
fibrinogen
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PCT/EP2020/063907
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French (fr)
Inventor
Joaquín SERENA LEAL
Maria Carme GUBERN MÉRIDA
Yolanda SILVA BLAS
Mikel TERCEÑO IZAGA
Saima BASHIR VITURRO
José CASTILLO SÁNCHEZ
Tomás SOBRINO MOREIRAS
Original Assignee
Fundació Institut D'investigació Biomèdica De Girona Dr. Josep Trueta
Fundación Instituto De Investigación Sanitaria De Santiago De Compostela (Fidis)
Servizo Galego De Saúde
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Publication of WO2020234269A1 publication Critical patent/WO2020234269A1/en

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    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • 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

  • Present invention relates to the field of medicine.
  • Atheromatous disease is the first cause of death and dependency in developed countries.
  • Carotid stenosis is one of the most prevalent and significant causes of stroke.
  • Surgical treatment mainly including carotid endarterectomy (CEA) and stent implantation
  • Carotid artery stenosis is a narrowing or constriction of any part of the carotid arteries, usually caused by atherosclerosis.
  • Thecommon carotid artery is the large artery whose pulse can be felt on both sides of the neck under the jaw. On the right side it starts from the brachiocephalic artery (a branch of the aorta), and on the left side the artery comes directly off the aortic arch. At the throat it forks into the internal carotid artery and the external carotid artery.
  • the internal carotid artery supplies the brain, and the external carotid artery supplies the face.
  • This fork is a common site for atherosclerosis, an inflammatory build-up of atheromatous plaque (abbreviated as plaque) inside the common carotid artery, or the internal carotid arteries that causes them to narrow.
  • the plaque can be stable and asymptomatic, or it can be a source of symptomatic embolization. Emboli break off from the plaque and travel through the circulation to blood vessels in the brain. As the vessels get smaller, an embolus can lodge in the vessel wall and restrict the blood flow to parts of the brain.
  • This ischemia can either be temporary, yielding a transient ischemic attack, or permanent resulting in a thromboembolic stroke.
  • Symptomatic carotid stenosis is commonly defined as stenosis in the internal carotid artery, intracranial or extracranial, leading to symptoms of amaurosis fugax, transient ischemic attacks (TIA), or ischemic stroke ipsilateral to the lesion.
  • TIA transient ischemic attacks
  • Asymptomatic carotid stenosis curses without symptoms but this silent form leads also to stroke.
  • Asymptomatic patients are those that have never had any experience of neurological deficit, either a TIA or a stroke related to an affected vessel.
  • Asymptomatic carotid stenosis is currently classified as asymptomatic progressive carotid stenosis and asymptomatic stable carotid stenosis, the first one involving the progressive growth and break of the vulnerable plaque, increasing risk of stroke.
  • Carotid artery stenosis is usually diagnosed by color flow duplex ultrasound scan of the carotid arteries in the neck. This involves no radiation, no needles and no contrast agents that may cause allergic reactions. This test has good sensitivity and specificity. Other different imaging modalities, such as a computed tomography angiogram (CTA) or magnetic resonance angiogram (MRA) may also be useful. However, this diagnose using images requires of particular instrumentation and it is not applicable at ambulatory or emergency level (ambulances).
  • CTA computed tomography angiogram
  • MRA magnetic resonance angiogram
  • serum biomarkers have been proposed, not only for the diagnosis of carotid stenosis, but also to discriminate between the several forms of the disease, i.e. symptomatic carotid stenosis and asymptomatic carotid stenosis.
  • C-reactive protein measured in serum is proposed as marker for progression of carotid artery disease and useful in the management of the disease and global carotid risk assessment, providing the option for evaluation and follow-up of patients with subclinical carotid stenosis (Arthus et al.,“A prospective evaluation of C-reactive protein in the progression of carotid artery stenosis”, J.Vasc. Surg 2008; vol. no. 47, pp. : 744-751).
  • HDL high-density lipoproteins
  • IL-6 and C-reactive protein certain cytokines
  • Inventors propose a panel of biomarkers that individually and in combination allow correct classification among subjects suffering from symptomatic carotid stenosis and
  • a first aspect of the invention is an in vitro method for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis, comprising the steps of:
  • a second aspect of the invention is an in vitro method for selecting a subject suffering from carotid stenosis as candidate for surgical therapy, comprising the following steps:
  • the levels of expression of CD5 antigen-like match with corresponding reference values of symptomatic carotid stenosis; or alternatively, the level of expression of CD5 antigen-like, is higher than a reference value.
  • This aspect can also be formulated as a method for selecting and/or recommending a therapy regimen for a subject suffering from carotid stenosis, wherein the method comprises:
  • the levels of expression of CD5 antigen-like match with corresponding reference values of symptomatic carotid stenosis; or alternatively, the level of expression of CD5 antigen-like is higher than a reference value.
  • present invention provides also as another aspect the use of CD5 antigen-like as biomarker of differential diagnosis of symptomatic versus asymptomatic carotid stenosis or as biomarkers for selecting a subject suffering from carotid stenosis as candidate for surgical therapy.
  • kits of parts comprising reagent means for detecting the level of expression of CD5 antigen-like.
  • the invention also proposes using means for detecting the level of expression of CD5 antigen-like in an isolated biofluid sample of a subject in the in vitro method of any one of the first and second aspects.
  • Invention also encompasses a method of detecting, in an isolated biofluid sample of a subject suffering from carotid stenosis, the expression of CD5 antigen-like, the method comprising:
  • Another aspect of the invention is an in vitro method for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis, and for selecting a treatment for carotid stenosis, comprising the steps of:
  • CD5 antigen-likeis higher than a reference value or match with corresponding reference values of symptomatic carotid stenosis
  • CD5 antigen-like is lower than a reference value or match with corresponding reference values of asymptomatic carotid stenosis
  • kits of parts comprising reagent means for detecting the level of expression of CD5 antigen-like.
  • indefinite articles“a” and“an” are synonymous with“at least one” or “one or more.” Unless indicated otherwise, definite articles used herein, such as“the” also include the plural of the noun.
  • diagnosis is understood as becoming aware of a particular medical condition complication or risk in a subject; the determination of the nature of the disease or condition; or the distinguishing of one disease or condition from another. It refers both to the process of attempting to determine or identify the possible disease or disorder, and to the opinion reached by this process.
  • a diagnosis in the sense of diagnostic procedure, can be regarded as an attempt at classification of an individual's condition into separate and distinct categories that allow medical decisions about treatment and prognosis to be made. Subsequently, a diagnostic opinion is often described in terms of a disease or other condition. However, a diagnosis can take many forms. It might be a matter of detecting the presence and naming the disease, lesion, dysfunction or disability.
  • the“differential diagnosis” also relates to the distinction among different diseases. In the present case, it relates to distinction among two or more subtypes of carotid stenosis, but is also encompasses distinction among different diseases with common symptoms, such as pancreatitis and pancreas cancer.
  • intensive medical therapy refers to the administration of suitable drugs with the corresponding schedules and doses that assure the control of progression of any disease.
  • the term also includes the follow-on carried out by medical professionals in order to detect any critical change in the state of the subject.
  • the concept intensive medical therapy implies a chronic treatment of a particular condition or disease (i.e. , insulin administration in diabetes type 1 subjects, or acenocumarol in subjects with a high risk of thrombosis).
  • selecting a patient for a therapy relates to the identification of a patient for a therapy designed to cure a disease or palliate the symptoms associated with one or more diseases or conditions.
  • the term“reference value”,“reference level” or“reference control level”, used interchangeably and referred to in the methods of the any of the aspects of the invention is to be understood as a predefined value or range of values (reference interval) of a given molecular marker or a combination of the given molecular markers, in the present case any of the proteins listed in the first or second aspects as well as in particular embodiments, which is derived from the levels of said molecular marker or markers in a sample or group of samples.
  • the "reference expression level” is a predefined value of protein quantity
  • the "reference expression level” is a predefined value of mRNA quantity.
  • the samples are taken from a subject or group of subjects wherein the presence, absence, stage, histological subtype or grade, or course of the disease has been properly performed previously. This reference value is also useful for determining whether the subject has to initiate a medical regimen and how effective the regimen is.
  • the subject or subjects from whom the“reference value” is derived may include subject/s wherein the condition is absent, subject/s wherein the condition is present, or both.
  • a first aspect of the invention is an in vitro method for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis, comprising the steps of: (a) determining in an isolated biofluid sample of a subject the level of expression of CD5 antigen-like; and
  • the in vitro method for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis further comprises determining the level of expression of one or more proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein,
  • Immunoglobulin heavy constant gamma 3 Immunoglobulin heavy constant gamma 3, Prothrombin, and Serum amyloid P- component.
  • the method comprises the steps of:
  • transthyretin are determined and the level of expression of CD5 antigen-like is higher than a reference value and the levels of expression of one of apolipoprotein A-IV and transthyretin are lower than a reference value;
  • the method comprises determining the level of expression of C-reactive protein, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of C-reactive protein is higher than a reference value, or match with a value of a symptomatic carotid stenosis subject.
  • the in vitro method comprises determining the level of expression of apolipoprotein A-IV, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of apolipoprotein A-IV is lower than a reference value, or alternatively if the level of expression of apolipoprotein A-IV match with the value of a symptomatic carotid stenosis reference.
  • the in vitro method comprises determining the level of expression of Immunoglobulin heavy constant gamma 3, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of Immunoglobulin heavy constant gamma 3 is higher than a reference value, or alternatively if the level of expression of Immunoglobulin heavy constant gamma 3 match with the value of a symptomatic carotid stenosis reference.
  • the in vitro method comprises determining the level of expression of Prothrombin, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of Prothrombin is higher than a reference value, or alternatively if the level of expression of Prothrombin match with the value of a symptomatic carotid stenosis reference.
  • the in vitro method comprises determining the level of expression of Serum amyloid P-component, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of Serum amyloid P-component is higher than a reference value, or alternatively if the level of expression of Serum amyloid P-component match with the value of a symptomatic carotid stenosis reference.
  • CD5 antigen-like is a secretion protein that regulates lipid synthesis and is expressed mainly by macrophages, it appears in inflamed tissues such as atherosclerosis and its target cells are adipocytes in which it inhibits the synthesis of fatty acids and leads lipolysis.
  • This lipolysis induced by CD5 antigen-like occurs in the progression of obesity and participates in the inflammation associated with obesity. It is key in the metabolic activation of Th17 T-helper cells. It inhibits apoptosis in macrophages. It is involved in the early response to infections as a receptor of recognition pattern and promoting autophagy.
  • CD5 antigen-like is increased its expression in control patients with respect to patients with type 2 diabetes, both groups with an endarterectomy coronary.
  • Human protein is identified in UniProt KB database with accession number 043866, version 1 of June 1 , 1998 of the sequence with 347 amino acids and version 146 of the entry in database.
  • the C-reactive protein has several functions associated with the defense mechanism: it promotes cell agglutination, capsular inflammation of bacteria, phagocytosis and complement fixation.
  • Human protein is identified in UniProt KB database with accession number P02741 , version 1 of January 1 , 1988 of the sequence with 224 amino acids and version 219 of the entry in database. It is a biomarker of inflammatory reaction and an important mediator of atherosclerosis contributing to the development of the plaque and its instability. Since 2010, theplasma level of C-reactive protein has been used as a prognosis in patients with intermediate risk of cardiovascular diseases.
  • Fibrinogen alpha chain lysed by thrombin protease forms monomers that polymerize with beta fibrinogen and gamma fibrinogen to form an insoluble fibrin matrix.
  • Human protein is identified in UniProt KB database with accession number P02671 , version 2 of October 1 , 1996 of the sequence with 866 amino acids and version 236 of the entry in database. It has some role in wound repair stabilizing the lesion and guiding cell migration in epithelialization. Mutations of this protein cause hereditary renal amyloidosis and a high percentage of patients with atherosclerosis have been found in carriers of these mutations.
  • Haptoglobin binds to free hemoglobin in the blood allowing its recycling in the spleen and liver and preventing kidney damage.
  • Human protein is identified in UniProt KB database with accession number P00738, version 1 of July 21 , 1986 of the sequence with 406 amino acids and version 208 of the entry in database. It also acts as an antioxidant, has antibacterial activity and intervenes in the acute response.
  • the hemoglobin / haptoglobin complex is eliminated by the CD163 receptor of Kupffer cells.
  • Variant 2 of haptoglobin is associated with more unstable atheroma plaques, with more iron, with thinner fibrous layers and with a greater probability of bleeding, since a higher incidence of
  • Apolipoprotein A-IV (Uniprot database or other unequivocal identification) is the major component of HDL and chylomicrons. It could be involved in the secretion and catabolism of the latter. It is an atheroprotective and in animal models it has been observed that its administration reduces hemorrhagic disruptions, the fibrous layer and the lipid nucleus in atheromatous plaques, as well as inflammation and associated apoptosis. Human protein is identified in UniProt KB database with accession number P06727, version 3 of March 7, 2006 of the sequence with 396 amino acids and version 206 of the entry in database.
  • Transthyretin is a thyroid hormone-binding protein and probably transports thyroxine from the bloodstream to the brain.
  • Human protein is identified in UniProt KB database with accession number P02766, version 1 of March 20, 1987 of the sequence with 147 amino acids and version 236 of the entry in database. The dissociation of the tetramer leads to the formation of amyloid aggregates and fibrils. It has been seen that the atheroma plaques of women contain a greater amount of Transthyretin, as well as of Apolipoprotein A-IV.
  • Heparin cofactor 2 activated by glycosaminoglycans, is an inhibitor of thrombin, heparin and dermatan sulfate; and, without the presence of glycosaminoglycans, inhibits chymotrypsin. Its N terminal possesses chemotactic activity in monocytes and neutrophils. The plasma levels of this protein are negatively correlated with the instability of the atheroma plaques.
  • Human protein is identified in UniProt KB database with accession number P05546, version 3 of November 1 , 1991 of the sequence with 499 amino acids and version 202 of the entry in database.
  • Immunoglobulin heavy constant gamma 2 corresponds to human protein identified in UniProt KB database with accession number P01859, version 2 of December 16, 2008 of the sequence with 326 amino acids and version 169 of the entry in database.
  • Immunoglobulin heavy constant gamma 4 corresponds to human protein identified in UniProt KB database with accession number P01861 , version 1 of July 21 , 1986 of the sequence with 327 amino acids and version 159 of the entry in database.
  • Immunoglobulin heavy constant mu corresponds to human protein identified in UniProt KB database with accession number P01871 , version 4 of March 15, 2017 of the sequence with 453 amino acids and version 188 of the entry in database.
  • Immunoglobulin heavy constant gamma 3 corresponds to human protein identified in UniProt KB database with accession number P01860, version 2 of July 1 , 2008 of the sequence with 377 amino acids, and version 179 of the entry in database.
  • Prothrombin corresponds to human protein identified in UniProt KB database with accession number P00734, version 2 of January 1 , 1990 of the sequence with 622 amino acids, and version 260 of the entry in database.
  • Serum amyloid P-component corresponds to human protein identified in UniProt KB database with accession number P02743, version 2 of February 1 , 1995 of the sequence with 223 amino acids, and version 218 of the entry in database.
  • Gelsolin corresponds to human protein identified in UniProt KB database with accession number P06396, version 1 of January 1 , 1988 of the sequence with 782 amino acids for the canonical isoform, and version 238 of the entry in database.
  • Immunoglobulin kappa variable 4-1 corresponds to human protein identified in UniProt KB database with accession number P06312, version 1 of January 1 , 1988 of the sequence with 121 amino acids, and version 151 of the entry in database.
  • Caveolin-1 corresponds to human protein identified in UniProt KB database with accession number Q03135, version 4 of January 23, 2007 of the sequence with 178 amino acids for the canonical isoform, and version 225 of the entry in database.
  • isolated biofluid sample as use herein relates to any liquid sample, optionally containing cells, which can be obtained from the patient.
  • biofluid samples are selected from plasma, serum, whole blood, saliva, semen, sputum, cerebral spinal fluid (CSF), tears, mucus, sweat and milk. More particular biofluid samples are selected from plasma, serum, and whole blood.
  • the in vitro method comprises determining the level of expression of CD5 antigen-like and of one or more of of the above-listed proteins; Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Immunoglobulin heavy constant gamma 3, Prothrombin, and Serum amyloid P- component.
  • the method comprises determining at least two proteins being one of them the C-reactive protein and the other CD5 antigen-like.
  • CD5 antigen-like and C-reactive protein More in particular three proteins, including CD5 antigen-like and C-reactive protein; four proteins, including CD5 antigen-like and C-reactive protein; five proteins, including CD5 antigen-like and C-reactive protein; six proteins, including CD5 antigen-like and C-reactive protein; seven proteins, including CD5 antigen-like and C-reactive protein; eight or nine proteins being one of them the levels of expression of C-reactive protein and another one CD5 antigen-like. More in particular the level of expression of all of CD5 antigen-like, C-reactive protein, Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4,
  • Immunoglobulin heavy constant mu and Heparin cofactor 2 are determined. If in addition, the level of expression of one or more of Immunoglobulin heavy constant gamma 3, Prothrombin, and Serum amyloid P-component are determined, combinations of two, three, four, five, six seven, eight, nine, ten, eleven, and twelve, always including CD5 antigen-like are determined according to the method of the invention. In a particular embodiment, the thirteen proteins are determined. Determination of more than one of the expression levels of these listed proteins allows improving sensitivity and/or specificity of the methods of the invention. This is also translated to the manufacture of particular kits for carrying out the method including different combinations depending on the accuracy that is desired.
  • CD5 for CD5 antigen-like
  • Creactive for C-reactive protein
  • Fibrinogen for Fibrinogen alpha chain
  • gamma2 for Immunoglobulin heavy constant gamma 2
  • the method comprises determining the level of expression of two of the proteins, said two-protein sets (two- membered groups) selected from the group consisting of:1.-CD5, Creactive; 2.-CD5, Fibrinogen; 3.-CD5, Haptoglobin; 4.-CD5, gamma2; 5.-CD5, apo4; 6.-CD5, Transth; 7.- CD5, gamma4; 8.-CD5, mu; 9.-CD5, heparin; 10.- Creactive, Fibrinogen; 11.- Creactive, Haptoglobin; 12.- Creactive, gamma2; 13.- Creactive, apo4; 14.- Creactive, Transth; 15.- Creactive, gamma4; 16.- Creactive, mu; 17.- Creactive, heparin; 18.- Fibrinogen,
  • Haptoglobin 19.- Fibrinogen, gamma2; 20.- Fibrinogen, apo4; 21.- Fibrinogen, Transth; 22.- Fibrinogen, gamma4; 23.- Fibrinogen, mu; 24.- Fibrinogen, heparin; 25.- Haptoglobin, gamma2; 26.- Haptoglobin, apo4; 27.- Haptoglobin, Transth
  • the method comprises determining the level of expression of three of the proteins, said three-protein sets (three-membered groups) selected from the group consisting of: 1.-CD5, Creactive, Fibrinogen; 2.-CD5, Creactive, Haptoglobin; 3.-CD5, Creactive, gamma2; 4.-CD5, Creactive, apo4; 5.-CD5, Creactive, Transth; 6.-CD5, Creactive, gamma4; 7.-CD5, Creactive, mu; 8.-CD5,
  • Fibrinogen Haptoglobin, gamma2; 66.- Fibrinogen, Haptoglobin, apo4; 67.- Fibrinogen, Haptoglobin, Transth; 68.- Fibrinogen, Haptoglobin, gamma4; 69.- Fibrinogen,
  • the method comprises determining the level of expression of four of the proteins, said four-protein sets (four-membered groups) selected from the group consisting of: 1.-CD5, Creactive, Fibrinogen, Haptoglobin; 2.- CD5, Creactive, Fibrinogen, gamma2; 3.-CD5, Creactive, Fibrinogen, apo4; 4.-CD5, Creactive, Fibrinogen, Transth; 5.-CD5, Creactive, Fibrinogen, gamma4; 6.-CD5,
  • CD5 Fibrinogen, Haptoglobin, Transth; 32.-CD5, Fibrinogen, Haptoglobin, gamma4; 33.- CD5, Fibrinogen, Haptoglobin, mu; 34.-CD5, Fibrinogen, Haptoglobin, heparin; 35.-CD5, Fibrinogen, gamma2, apo4; 36.-CD5, Fibrinogen, gamma2, Transth; 37.-CD5, Fibrinogen, gamma2, gamma4; 38.-CD5, Fibrinogen, gamma2, mu; 39.-CD5, Fibrinogen, gamma2, heparin; 40.-CD5, Fibrinogen, apo4, Transth; 41.-CD5, Fibrinogen, apo4, gamma4; 42.- CD5, Fibrinogen, apo4, mu; 43.-CD5, Fibrinogen, apo4, heparin; 44.-CD5, Fibrinogen, Transth,
  • the method comprises determining the level of expression of five of the proteins, said five-protein sets (five-membered groups) selected from the group consisting of: 1.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2; 2.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4; 3.-CD5, Creactive,
  • Fibrinogen, Haptoglobin, Transth 4.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma4; 5.-CD5, Creactive, Fibrinogen, Haptoglobin, mu; 6.-CD5, Creactive, Fibrinogen,
  • the method comprises determining the level of expression of six of the proteins, said six-protein sets (six-membered groups) selected from the group consisting of: 1.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4; 2.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, Transth; 3.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, gamma4; 4.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, mu; 5.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, heparin; 6.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, Transth; 7.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, gamma4; 8.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, mu; 9.
  • Fibrinogen Haptoglobin, gamma2, gamma4, mu; 135.- Creactive, Fibrinogen,
  • the method comprises determining the level of expression of seven of the proteins, said seven-protein sets (seven-membered groups) selected from the group consisting of:1.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, Transth; 2.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, gamma4; 3.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, mu; 4.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, heparin; 5.-CD5, Creactive, Creactive,
  • the method comprises determining the level of expression of eight of the proteins, said eight-protein sets (eight-membered groups) selected from the group consisting of: 1.-CD5, Creactive, Fibrinogen,
  • the method comprises determining the level of expression of nine of the proteins, said nine-protein sets (nine-membered groups) selected from the group consisting of: 1.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, gamma4, mu; 2.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, ga a4, heparin; 3.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, u, heparin; 4.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, gamma4, mu, heparin; 5.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, Transth, gamma4, mu, heparin; 6.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, Trans
  • the subject suffering from carotid stenosis can be correctly classified as an asymptomatic if the detected values correspond to asymptomatic reference values or as symptomatic if the detected in the sample values (also called test values) correspond to symptomatic reference values.
  • the reference value is selected from the level of expression of the one or more proteins in a symptomatic subject, the level of expression of the one or more proteins in an asymptomatic subject, and a control non-suffering carotid stenosis. Said reference value can be a discrete value or a range of values in which one of the conditions to be discriminated are included.
  • the method further comprises the step of determining in the isolated sample the level of expression of one or more of the proteins selected from the group consisting of Immunoglobulin lambda variable 2-18, Immunoglobulin heavy constant delta, Insulin-like growth factor-binding protein 3, Haptoglobin, Alpha-2-HS-glycoprotein, Immunoglobulin kappa variable 3-15, Antithrombin-Ill, Immunoglobulin kappa variable 4-1 , Gelsolin, Immunoglobulin heavy constant gamma 3 and Caveolin-1.
  • the proteins selected from the group consisting of Immunoglobulin lambda variable 2-18, Immunoglobulin heavy constant delta, Insulin-like growth factor-binding protein 3, Haptoglobin, Alpha-2-HS-glycoprotein, Immunoglobulin kappa variable 3-15, Antithrombin-Ill, Immunoglobulin kappa variable 4-1 , Gelsolin, Immunoglobulin heavy constant gamma 3 and Caveolin-1.
  • the method further comprises the step of determining in the isolated sample the level of expression of one or more of the proteins selected from the group consisting of Immunoglobulin kappa variable 4-1 , Gelsolin, Immunoglobulin heavy constant gamma 3 and Caveolin-1.AII these additional proteins are reliable individual markers distinguishing among progressive asymptomatic and stable asymptomatic subjects. Differential diagnosis among asymptomatic subjects allows accommodating a proper therapy regimen recommendation. For example, in some progressive asymptomatic subjects CEA or stenting can be recommended if other additional physical parameters (imaging) or clinical parameters (age, gender, etc.) are in favor of these therapies.
  • the proteins selected from the group consisting of Immunoglobulin kappa variable 4-1 , Gelsolin, Immunoglobulin heavy constant gamma 3 and Caveolin-1.
  • Immunoglobulin kappa variable 4-1 Gelsolin, Immunoglobulin heavy constant gamma 3 are of particular interest because their levels in the isolated biofluid samples correlate with the levels in atheromatous plaque.
  • the method further comprises the step of determining in the isolated sample the level of expression of one or more of the proteins selected from the group consisting of Immunoglobulin lambda variable 2-18, Immunoglobulin heavy constant delta, Insulin-like growth factor-binding protein 3, Haptoglobin, Alpha-2-HS-glycoprotein, Immunoglobulin kappa variable 3-15, Antithrombin-Ill, and Immunoglobulin kappa variable 4-1.
  • the method further comprises the step of determining in the isolated sample the level of expression of one or more of Immunoglobulin kappa variable 4-1 and Gelsolin. Levels of both proteins determined in combination allow a very good classification.
  • these proteins are, in a particular embodiment, of the second aspect, used in combination with the levels of CD5 antigen-like to select a patient as candidate for surgical therapy.
  • the method further comprises the step of determining in the isolated sample the level of expression of Immunoglobulin heavy constant gamma 3.
  • this protein is, in a particular embodiment of the second aspect, used in combination with the levels of CD5 antigen-like to select a patient as candidate for surgical therapy.
  • the method further comprises the step of determining in the isolated sample the level of expression of Caveolin-1.
  • Caveolin-1 differentially distinguished asymptomatic stable patients from asymptomatic progressive and symptomatic patients with an accuracy (AUC, 95% Cl) of 0.745 (0.621-0.869), using a cutoff of 0.484 ng/ml.
  • Correspondent sensitivity was of 0.61
  • the specificity was of 0.81 , being the positive predictive value (PPV) of 0.97 and the negative predictive value (NVP) of 0.19.
  • a method for the differential diagnosis of asymptomatic stable carotid stenosis patients from asymptomatic progressive and symptomatics that comprises determining in isolated serum the levels of caveolin-1.
  • an in vitro method for selecting a subject suffering from carotid stenosis as candidate for surgical therapy comprising determining in an isolated biofluid sample of a subject the level of expression of Caveolin-1 , wherein the level is optionally compared with a reference value or interval of values, and the subject is selected as candidate if the levels in the sample match with those of symptomatic or of asymptomaic progressive patients.
  • the method further comprises the step of determining in the isolated sample the level of expression of two or more of the proteins Immunoglobulin lambda variable 2-18, Immunoglobulin heavy constant delta, Insulin-like growth factor-binding protein 3, Haptoglobin, Alpha-2-HS-glycoprotein, Immunoglobulin kappa variable 3-15,
  • the method comprise further determining the level of expression of one or more proteins selected from the group consisting of
  • Immunoglobulin lambda variable 7-43 Apolipoprotein(a), Immunoglobulin lambda variable 9-49, Immunoglobulin lambda-like polypeptide 5, Apolipoprotein A-ll, Immunoglobulin kappa variable 3-11 , Immunoglobulin heavy constant alpha 1 , Galectin-3-binding protein, Immunoglobulin lambda variable 3-25, Alpha-1 -antichymotrypsin, Vitamin K-dependent protein S, Immunoglobulin heavy variable 3-43D, Carboxypeptidase N catalytic chain, Immunoglobulin heavy variable 3-30-5, Immunoglobulin lambda constant 2,
  • Immunoglobulin lambda variable 3-19 Triple functional domain protein, Immunoglobulin lambda variable 6-57, Immunoglobulin heavy variable 1-46, Immunoglobulin kappa variable 1-27, Complement component C8 gamma chain, Complement C1 r
  • Hemoglobin subunit beta Hemoglobin subunit beta, Biotinidase, Immunoglobulin kappa variable 1 D-12, Apolipoprotein F, Complement C4-B, Hemoglobin subunit alpha, Immunoglobulin kappa variable 2D-28, Uncharacterized protein ZSWIM9, Coagulation factor XIII B chain, , Keratin type I cytoskeletal 10, Complement C1q subcomponent subunit C , Alpha-2- macroglobulin, Immunoglobulin heavy variable 6-1 , Complement factor H, Transcription factor E2F8, Apolipoprotein A-l , Ficolin-3, Immunoglobulin lambda variable 8-61 , Fibronectin, Immunoglobulin heavy variable 3-7, Lumican, Thyroxine-binding globulin, Inter-alpha-trypsin inhibitor heavy chain H4, Vitronectin, Retinol-binding protein 4, , and Phosphatidylcho
  • the subject is a mammal.
  • the mammal is a domestic animal mammal.
  • the mammal is a human.
  • a second aspect of the invention is an in vitro method for selecting a subject suffering from carotid stenosis as candidate for surgical therapy comprising determining in an isolated biofluid sample of a subject the level of expression of CD5 antigen-like and comparing the level of expression with a corresponding reference value.
  • this second aspect is an in vitro method for selecting a subject suffering from carotid stenosis as candidate for surgical therapy, comprising the following steps:
  • the levels of expression of CD5 antigen-like match with corresponding reference values of symptomatic carotid stenosis; or alternatively, the level of expression of CD5 antigen-like, is higher than a reference value.
  • the in vitro method further comprises determining the level of expression of one or more proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein,
  • the in vitro method for selecting a subject suffering from carotid stenosis as candidate for surgical therapy comprises the following steps:
  • Immunoglobulin heavy constant gamma 3 Prothrombin, Serum amyloid P-component, Immunoglobulin kappa variable 4-1 , Gelsolin, and Caveolin-1 , and
  • Prothrombin, Serum amyloid P-component, Immunoglobulin kappa variable 4-1 , Gelsolin, and Caveolin-1 match with corresponding reference values of symptomatic carotid stenosis; or alternatively,
  • the subject is selected as candidate for additional determination of the level of expression of one or more proteins in the isolated sample and/or as candidate for intensive medical treatment if:
  • Prothrombin, Serum amyloid P-component, Immunoglobulin kappa variable 4-1 , Gelsolin, and Caveolin-1 match with corresponding reference values of asymptomatic carotid stenosis; or alternatively,
  • Fibrinogen alpha chain Haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Prothrombin, Serum amyloid P-component, Immunoglobulin kappa variable 4-1 , and Gelsolin, are determined, and the levels of one of them are lower than a reference value, and/or the level of expression of CD5 antigen-like and of one or more of apolipoprotein A-IV, transthyretin, Immunoglobulin heavy constant gamma 3 and Caveolin-1 are determined, and the level of expression of CD5 antigen-like is lower than a reference value and the levels of expression of the one of apolipoprotein A-IV, transthyretin
  • Immunoglobulin heavy constant gamma 3 and Caveolin-1 are higher than a reference value.
  • This previous embodiment can also be worded as a method for selecting and/or recommending a therapy regimen for a subject suffering from carotid stenosis, wherein the method comprises determining the level of expression of CD5 antige-like, and of the one or more of all above listed proteins.
  • a particlar embodiment is a method for selecting and/or recommending a therapy regimen for a subject suffering from carotid stenosis, wherein the method comprises:
  • the subject is diagnosed as symptomatic carotid stenosis and an effective treatment comprising surgical therapy is selected or recommended if: the levels of expression of CD5 antigen-like and of one or more of fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2 apolipoprotein A-IV and transthyretin match with corresponding reference values of symptomatic carotid stenosis; or alternatively,
  • the level of expression of CD5 antigen-like and of one or more of fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2 are determined, and the levels of CD5 antigen like and of one of the one or more proteins are higher than a reference value, and/or if the level of expression of CD5 antigen-like and of one or more of apolipoprotein A-IV and transthyretin are determined and the levels of expression of CD5 antigen-like and of one of apolipoprotein A-IV and transthyretinare lower than a reference value; and
  • the subject is diagnosed as asymptomatic carotid stenosis and determination of additional level of expression of one or more proteins in the isolated sample and/or an intensive medical treatment is selected or recommended if: the level of expression of CD5 antigen-like and of one or more of fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, apolipoprotein A-IV and transthyretin match with corresponding reference values of asymptomatic carotid stenosis; or alternatively,
  • the level of expression of CD5 antigen-like and of one or more of fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2 are determined, and the levels of CD5 antigen like and of one of the one or more proteins are lower than a reference value, and/or the level of expression of CD5 antigen-like and of one or more of apolipoprotein A-IV and transthyretin are determined and the levels of expression of CD5 antigen-like and of one of apolipoprotein A-IV and transthyretin are higher than a reference value.
  • the second aspect comprises determining the level of expression of C-reactive protein in the isolated biofluid sample, and wherein the subject is selected as a candidate for surgical therapy if the level of expression of the protein is higher than a reference value or match with a value of a symptomatic carotid stenosis subject.
  • the in vitro method of this second aspect comprises determining the level of expression of Apolipoprotein A- IV in the isolated biofluid sample, and wherein the subject is selected as a candidate for surgical therapy if the level of expression of the Apolipoprotein A- IV is lower than a reference value, or alternatively, if the level of expression of Apolipoprotein A- IV match with the value of a symptomatic carotid stenosis reference.
  • the in vitro method comprises determining the level of expression of Immunoglobulin heavy constant gamma 3, and the subject is is selected as a candidate for surgical therapy if the level of expression of Immunoglobulin heavy constant gamma 3 is higher than a reference value, or alternatively if the level of expression of Immunoglobulin heavy constant gamma 3 match with the value of a symptomatic carotid stenosis reference.
  • the in vitro method comprises determining the level of expression of Prothrombin, and the subject is selected as a candidate for surgical therapy if the level of expression of Prothrombin is higher than a reference value, or alternatively if the level of expression of Prothrombin match with the value of a symptomatic carotid stenosis reference.
  • the in vitro method comprises determining the level of expression of Serum amyloid P-component, and the subject is selected as a candidate for surgical therapy if the level of expression of Serum amyloid P-component is higher than a reference value, or alternatively if the level of expression of Serum amyloid P-component match with the value of a symptomatic carotid stenosis reference.
  • the recommended surgical therapy is selected from the group consisting of CEA, stenting and combinations thereof.
  • the method further comprises determining one or more clinical parameters of the subject.
  • Particular clinical parameters include, without being a limitative listgender, age, echography determination of plaque features, and vascular risk profile, among others.
  • the combination of determining the level of expression of above-listed proteins in an isolated biofluid sample, together with additional clinical parameters is, in another particular embodiment, carried out by means of decision algorithms allowing improvement of accuracy of patient classification.
  • values detected for each of the proteins can be discretized and/or applied to them particular weight factors by means of mathematical formula, optionally considering these additional clinical parameters, in order to give a cutoff or value allowing classification.
  • the methods further comprise the step of treating by surgical therapy the selected candidate patients if the level of expression of the one or more proteins match with a value of a symptomatic carotid stenosis subject, or if they are higher or lower than a reference level depending on the protein as above indicated.
  • Particular surgical therapies include CEA, stenting and combinations thereof.
  • methods include the step of determining additional proteins allowing
  • All these particular embodiments including treatment can be formulated as a method of treatment by surgical therapy of a patient suffering carotid stenosis, said method of treatment comprising the steps of carrying out any of the methods as defined in the first and/or second aspects of the invention or any of their particular embodiments.
  • biofluid sample and the number of proteins whose levels are determined indicated for the first aspect do also apply to this second aspect.
  • particular listed combinations for the first aspect also apply to this second and additional aspects of the invention or to reformulations of these aspects.
  • the level of expression of one or more of the proteins is carried out at protein level by means for detecting and quantifying proteins, which are selected from the group consisting of immunoassays, protein migration, chromatography, mass spectrometry, turbidimetry, nephelometry and combination thereof.
  • the level of expression of one or more of the proteins is carried out at nucleic acid level by means of polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the expression levels are determined by means of the quantification of the levels of mRNA encoded by said genes.
  • the latter can be quantified by means of using conventional methods, for example, methods comprising the amplification of mRNA and the quantification of the amplification product of said mRNA, such as electrophoresis and staining, or alternatively, by means of Northern blot and the use of suitable probes, Northern blot and use of specific probes of the mRNA of the genes of interest or of their corresponding cDNA/cRNA, mapping with the SI nuclease, RT-PCR, hybridization, microarrays, etc.
  • the levels of the cDNA/cRNA corresponding to said mRNA encoded by the marker genes can also be quantified by means of using conventional techniques; in this event, the method of the invention includes a step of synthesis of the corresponding cDNA by means of reverse transcription (RT) of the corresponding mRNA followed by the synthesis (RNA polymerase) and amplification of the cRNA complementary to said cDNA.
  • RT reverse transcription
  • RNA polymerase RNA polymerase
  • control RNA relates to RNA whose expression levels do not change or change only in limited amounts.
  • the control RNA is mRNA derived from housekeeping genes and which code for proteins which are constitutively expressed and carry out essential cellular functions.
  • housekeeping genes for use in the present invention include 18-S ribosomal protein, b-2-microglobulin, ubiquitin, cyclophilin, GAPDH, PSMB4, tubulin and b-actin.
  • the expression levels of the marker genes by means of the determination of the expression levels of the proteins encoded by said genes, since if the expression of genes is increased, an increase of the amount of corresponding protein should occur and if the expression of genes is decreased, a decrease of the amount of corresponding protein should occur.
  • the determination of the expression levels of the proteins can be carried out by tests selected from the group consisting of an immunological test, bioluminescence, fluorescence, chemiluminescence, electrochemistry and mass spectrometry.
  • tests selected from the group consisting of an immunological test, bioluminescence, fluorescence, chemiluminescence, electrochemistry and mass spectrometry.
  • POCT point of care test format
  • particular quantitative tests are selected from the group consisting of an immunological test, bioluminescence, fluorescence, chemiluminescence, electrochemistry and mass spectrometry.
  • the level of expression is determined by immunological techniques such as enzyme-linked immunosorbent assay (ELISA), enzyme immunodot assay, agglutination assay, antibody-antigen-antibody sandwich assay, antigen-antibody-antigen sandwich assay, immunocromatography, or other immunoassay formats well-known to the ordinarily skilled artisan, such as radioimmunoassay, as well as protein microarray formats, such as single molecular assay (SIMOA), Western Blot or immunofluorescence.
  • ELISA enzyme-linked immunosorbent assay
  • enzyme immunodot assay enzyme immunodot assay
  • agglutination assay such as enzyme-linked immunosorbent assay (ELISA), enzyme immunodot assay, agglutination assay, antibody-antigen-antibody sandwich assay, antigen-antibody-antigen sandwich assay, immunocromatography, or other immunoassay formats well-known to the ordinarily skilled artisan, such as radioimmuno
  • ELISA electrophoreses under denaturing conditions and immobilized on a membrane, generally nitrocellulose by the incubation with an antibody specific and a developing system (e.g. chemiluminescent).
  • an antibody specific and a developing system e.g. chemiluminescent
  • the analysis by immunofluorescence requires the use of an antibody specific for the target protein for the analysis of the expression.
  • ELISA is based on the use of antigens or antibodies labelled with enzymes so that the conjugates formed between the target antigen and the labelled antibody results in the formation of enzymatically-active complexes.
  • SIMOA single-molecule arrays
  • in vitro methods of the invention that provide a differential diagnostic andinformation for selecting a therapy, they further comprise the steps of (i) collecting the diagnostic information, and (ii) saving the information in a data carrier.
  • a“data carrier” is to be understood as any means that contain meaningful information data for the differential diagnosis and/or prognosis of carotid stenosis, such as paper.
  • the carrier may also be any entity or device capable of carrying the differential diagnosis data or information for selecting a therapy.
  • the carrier may comprise a storage medium, such as a ROM, for example a CD ROM or a semiconductor ROM, or a magnetic recording medium, for example a floppy disc or hard disk.
  • the carrier may be a transmissible carrier such as an electrical or optical signal, which may be conveyed via electrical or optical cable or by radio or other means.
  • the carrier When the diagnosis/prognosis data are embodied in a signal that may be conveyed directly by a cable or other device or means, the carrier may be constituted by such cable or other device or means.
  • Other carriers relate to USB devices and computer archives. Examples of suitable data carrier are paper, CDs, USB, computer archives in PCs, or sound registration with the same information.
  • the invention also proposes using means for detecting the level of expression of CD5 antigen-like, and optionally of one or more proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2,
  • Apolipoprotein A-IV Transthyretin, Immunoglobulin heavy constant gamma 4,
  • Immunoglobulin heavy constant mu Heparin cofactor 2 detecting C-reactive protein, Prothrombin, Serum amyloid P-component, and Immunoglobulin kappa variable 4-1 in an isolated sample of a subject in the in vitro method of any one of the first and second aspects.
  • said means comprise a corresponding antibody or a fragment thereof able to specifically bind to a corresponding protein of the list.
  • said means form part of a kit.
  • kits of parts comprising reagent means for detecting the level of expression of CD5 antigen-like, and optionally reagent means for detecting one or more of the proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV,
  • the kit of parts according to the invention further comprises reagent means for detecting the level of expression of one or more of the proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4,
  • kit of parts comprises reagent means for detecting the level of expression of CD5 antigen-like and reagent means for detecting the level of expression of one two, three, four, five, six, seven, eight nine, ten, eleven, twelve proteins selected from Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin,
  • Immunoglobulin heavy constant gamma 4 Immunoglobulin heavy constant mu 4
  • Heparin cofactor 2 Immunoglobulin heavy constant mu 4
  • Heparin cofactor 2 Immunoglobulin heavy constant mu 4
  • Immunoglobulin kappa variable 4-1 Immunoglobulin kappa variable 4-1.
  • it comprises reagents means for detecting the level of expression of CD5 antigen-like and Apolipoprotein A-IV, and optionally C-reactive protein, Prothrombin, Serum amyloid P-component and Immunoglobulin kappa variable 4- 1.
  • the kit of parts accodting to the invention comprises reagent means for detecting the level of expression of one or more of the proteins selected from the group consisting of Immunoglobulin lambda variable 2-18, Immunoglobulin heavy constant delta, Insulin-like growth factor-binding protein 3, Haptoglobin, Alpha-2-HS-glycoprotein, Immunoglobulin kappa variable 3-15,
  • kit refers to a product containing the different reagents (or reagent means) necessary for carrying out the methods of the invention packed so as to allow their transport and storage.
  • Materials suitable for packing the components of the kit include crystal, plastic (e.g. polyethylene, polypropylene, polycarbonate), bottles, vials, paper, or envelopes.
  • kits of the invention can contain instructions for the simultaneous, sequential or separate use of the different components which are in the kit.
  • Said instructions can be in the form of printed material or in the form of an electronic support capable of storing instructions susceptible of being read or understood, such as, for example, electronic storage media (e.g. magnetic disks, tapes), or optical media (e.g. CD- ROM, DVD), or audio materials.
  • the media can contain internet addresses that provide said instructions.
  • the reagent means (or simply reagents) of the kit include compounds that bind specifically to the marker proteins.
  • said compounds are antibodies, aptamers or fragments thereof.
  • the reagent is an antibody or fragments thereof.
  • the reagent means are one or more antibodies that specifically recognize the proteins of interest.
  • the antibodies of the kit of the invention can be used according to techniques known in art for determining the protein expression levels, such as, for example, flow cytometry, Western blot, ELISA, radioimmune assay (RIA), competitive Enzymatic immuno assay (EIA), double antibody sandwich RLISA (DAS-ELISA), techniques based on the use of biochips, protein microarrays, or assays of colloidal precipitation in reactive strips.
  • the antibodies can be fixed to a solid support such as a membrane, a plastic or a glass, optionally treated to facilitate the fixation of said antibodies to the support.
  • Said solid support comprises, at least, a set of antibodies which specifically recognize the marker (i.e. the protein of interest), and which can be used for detecting the levels of expression of said marker.
  • kits of the invention comprise reagents for detecting a protein encoded by a constitutive gene.
  • additional reagents allows normalizing the measurements performed in different samples (for example, the sample to be analyzed and the control sample) to rule out that the differences in the expression of the biomarkers are due to a different quantity of total protein amount in the sample more than the real differences in the relative levels of expression.
  • the constitutive genes in the present invention are genes that are always active or being transcribed constantly and which encode for proteins that are expressed constitutively and carry out essential cellular functions.
  • Proteins that are expressed constitutively and can be used in the present invention include, without limitation, b-2-microglobulin (B2M), ubiquitin , 18-S ribosomal protein, cyclophilin, GAPDH, PSMB4, tubulin and actin.
  • B2M b-2-microglobulin
  • ubiquitin ubiquitin
  • 18-S ribosomal protein ubiquitin
  • cyclophilin cyclophilin
  • GAPDH GAPDH
  • PSMB4 tubulin and actin.
  • the reagent means for assaying the levels of the different biomarkers comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the total amount of reagents for assaying biomarkers forming the kit.
  • kits comprising reagents for assaying the levels of the thirteen proteins (i.e. CD5 antigen-like, Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2,
  • Apolipoprotein A-IV Transthyretin, Immunoglobulin heavy constant gamma 4,
  • the reagents specific for said biomarkers comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the antibodies present in the kit.
  • kits are, thus, simplified kits including mainly the reagent means for detecting the levels of CD5 antigen like or of the thirteen proteins.
  • the invention relates to the use of said kit of the invention for differentiating symptomatic carotid stenosis from asymptomatic carotid stenosis or for selecting a patient suffering carotid stenosis for surgical therapy (CEA or stenting).
  • the invention relates to the use of the kit of the invention in any of the methods of the invention.
  • the invention also includes as an aspect the use of CD5 antigen-like as biomarker of differential diagnosis of symptomatic versus asymptomatic carotid stenosis or as biomarker for selecting a subject suffering from carotid stenosis as candidate for surgical therapy.
  • this use is in combination with one or more of
  • Fibrinogen alpha chain Haptoglobin, Immunoglobulin heavy constant gamma 2
  • Apolipoprotein A-IV Transthyretin, Immunoglobulin heavy constant gamma 4,
  • Immunoglobulin heavy constant mu Heparin cofactor 2, C-reactive protein, Prothrombin, Serum amyloid P-component, and Immunoglobulin kappa variable 4-1 as biomarkers of differential diagnosis of symptomatic versus asymptomatic carotid stenosis or as biomarkers for selecting a subject suffering from carotid stenosis as candidate for surgical therapy.
  • present invention relates to the capability of distinguishing symptomatic carotid stenosis subjects from asymptomatic carotid stenosis subjects by determining the level of expression of one or more of the proteins selected from CD5 antigen-like, Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, and Heparin cofactor 2, and optionally in combination with the levels of expression of C-reactive protein, Prothrombin, Serum amyloid P-component, and Immunoglobulin kappa variable 4-1.
  • the proteins selected from CD5 antigen-like, Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, and Hepar
  • the capability of distinguishing symptomatic carotid stenosis subjects from asymptomatic carotid stenosis subjects by determining the level of expression of two or more of the proteins. More in particular three, four, five, six, seven, eight or nine proteins, or ten, eleven, telwe or thireen if protein levels of C-reactive protein, Prothrombin, Serum amyloid P-component, and Immunoglobulin kappa variable 4-1 are also determined.
  • Exclusion criteria were: Patients treated >30 days of last symptomatology, treated with carotid angioplasty or with a disease affecting inflammatory molecules. Patients with symptomatic carotid stenosis and 2 potential causes of stroke. Endarterectomy samples were collected and serum samples (2 vials of 4.5 ml for biochemistry analysis of serum and 2 vials of BD Vacutainer PPTTM Plasma for the analysis of markersin plasma) were obtained immediately before carotid endarterectomy. As controls, serum samples were collected from patients with asymptomatic stable carotid stenosis >70% (Nr of patients 5) whose were stable in at least two separate controls.
  • Bold values indicate that the fold change is symptomatic vs asymptomatic and the underwritten and cursive values indicates that the fold change is asymptomatic progressive vs. symptomatic
  • inventors detected that also a differential expression can be observed at protein level between asymptomatic progressive and asymptomatic stable subjects.
  • Proteins with a fold change greater than or equal to 1.5 between asymptomatic progressive and asymptomatic stable Proteins with a fold change greater than or equal to 1.5 between asymptomatic progressive and asymptomatic stable.
  • Bold values indicate fold change for asymptomatic progressive vs. asymptomatic stable and underwritten and cursive values fold change for asymptomatic stable vs. asymptomatic progressive.
  • Next table 5.1 to 5.4 show the fold change between groups.
  • diagnosing the subject of asymptomatic carotid stenosis if the level of expression of one or more of CD5 antigen-like, fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2 are determined, and the levels of one of them are lower than a reference value, and/or one or more of apolipoprotein A-IV and transthyretin are determined and the levels of expression of one of them are higher than a reference value.
  • Clause 2. The in vitro method according to clause 1 , which further comprises determining the level of expression of C-reactive protein, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of C-reactive protein is higher than a reference value, or alternatively if the level of expression of C-reactive protein match with the value of a symptomatic carotid stenosis reference.
  • Clause 3. The in vitro method according to any one of clauses 1-2, wherein the isolated biofluid sample is selected from plasma, serum, whole blood, saliva, semen, sputum, cerebral spinal fluid (CSF), tears, mucus, sweat and milk.
  • Clause 4. The in vitro method according to any one of clauses 1-3, wherein the isolated biofluid sample is selected from plasma, serum and whole blood.
  • Clause 5 The in vitro method according to any one of clauses 1-4, wherein if the subject is diagnosed as suffering from asymptomatic carotid stenosis, the method further comprises the step of determining in the isolated sample the level of expression of one or more of the proteins selected from the group consisting oflmmunoglobulin lambda variable 2-18, Immunoglobulin heavy constant delta, Insulin-like growth factor-binding protein 3, Haptoglobin, Alpha-2-HS-glycoprotein, Immunoglobulin kappa variable 3-15,
  • Clause 6. An in vitro method for selecting a subject suffering from carotid stenosis as candidate for surgical therapy, comprising the following steps: (a) determining in an isolated biofluid sample of a subject the level of expression of one or more proteins selected from the group consisting of CD5 antigen-like, Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2; and
  • Immunoglobulin heavy constant gamma 4 Immunoglobulin heavy constant mu and Heparin cofactor 2 are determined, and the levels of one of them are higher than a reference value, and/or if one or more of apolipoprotein A-IV and transthyretin are determined and the levels of expression of one of them are lower than a reference value.
  • Clause 7. The in vitro method according to clause 6, in which the subject is selected as candidate for additional determination of the level of expression of one or more proteins in the isolated sample and/or as candidate for intensive medical treatment if: the level of expression of one or more of CD5 antigen-like, fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, apolipoprotein A-IV and transthyretin match with corresponding reference values of asymptomatic carotid stenosis; or alternatively,
  • the level of expression of one or more of CD5 antigen-like, fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2 are determined, and the levels of one of them are lower than a reference value, and/or one or more of apolipoprotein A-IV and transthyretin are determined and the levels of expression of one of them are higher than a reference value.
  • Clause 8. The in vitro method according to any one of clauses 6-7, which further comprises determining the level of expression of C-reactive protein in the isolated biofluid sample, and wherein the subject is selected as a candidate for surgical therapy if the level of expression of the protein is higher than a reference value, or alternatively, if the level of expression of C-reactive protein match with the value of a symptomatic carotid stenosis reference.
  • Clause 9. -The method according to any of clauses 1- 8 further comprising determining one or more clinical parameters.
  • Clause 10. -The in vitro method according to any one of clauses 1-9, wherein determining the level of expression of one or more of the proteins is carried out at protein level by means for detecting and quantifying proteins selected from the group consisting of immunoassays, protein migration, chromatography, mass spectrometry, turbidimetry, nephelometry and combination thereof.
  • Immunoglobulin heavy constant gamma 4 Immunoglobulin heavy constant mu and Heparin cofactor 2, optionally in combination with C-reactive protein, as biomarkers of differential diagnosis of symptomatic versus asymptomatic carotid stenosis or as biomarkers for selecting a subject suffering from carotid stenosis as candidate for surgical therapy.
  • kit of parts comprising reagent means for detecting the level of expression of one or more of the proteins selected from the group consisting of CD5 antigen-like, Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2,
  • Apolipoprotein A-IV Transthyretin, Immunoglobulin heavy constant gamma 4,
  • Immunoglobulin heavy constant mu and Heparin cofactor 2 and optionally reagent means for detecting C-reactive protein.

Abstract

Invention relates to in vitro methods for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis, comprising the steps of determining the levels of expression of several proteins and comparing them with corresponding reference levels. Particular markers include CD5 antigen-like, Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2. The invention also relates to an in vitro method for selected a patient suffering from carotid stenosis as candidate to surgery therapy. Particular kits for carrying out the methods are also disclosed.

Description

Method for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis
This application claims the benefit of European Patent Application EP19382404.2, filed on 20th May 2019.
Technical Field
Present invention relates to the field of medicine. In particular, it relates to diagnosis of patients suffering from carotid stenosis and to the correct classification among the several manifestations of this disease.
Background Art Atheromatous disease is the first cause of death and dependency in developed countries. Carotid stenosis is one of the most prevalent and significant causes of stroke. Surgical treatment, mainly including carotid endarterectomy (CEA) and stent implantation
(stenting), is effective in symptomatic carotid stenosis >70% (class I evidence), with a marginal benefit in stenosis of 50-70%, and arguable benefit in patients with
asymptomatic stenosis >70%. Identification of biological predictors of vulnerable plaque would permit the identification of high and low risk patients, allowing a better selection of patients to indicate surgical or intensive medical treatment.
Carotid artery stenosis is a narrowing or constriction of any part of the carotid arteries, usually caused by atherosclerosis. Thecommon carotid artery is the large artery whose pulse can be felt on both sides of the neck under the jaw. On the right side it starts from the brachiocephalic artery (a branch of the aorta), and on the left side the artery comes directly off the aortic arch. At the throat it forks into the internal carotid artery and the external carotid artery. The internal carotid artery supplies the brain, and the external carotid artery supplies the face. This fork is a common site for atherosclerosis, an inflammatory build-up of atheromatous plaque (abbreviated as plaque) inside the common carotid artery, or the internal carotid arteries that causes them to narrow. The plaque can be stable and asymptomatic, or it can be a source of symptomatic embolization. Emboli break off from the plaque and travel through the circulation to blood vessels in the brain. As the vessels get smaller, an embolus can lodge in the vessel wall and restrict the blood flow to parts of the brain. This ischemia can either be temporary, yielding a transient ischemic attack, or permanent resulting in a thromboembolic stroke. Symptomatic carotid stenosis is commonly defined as stenosis in the internal carotid artery, intracranial or extracranial, leading to symptoms of amaurosis fugax, transient ischemic attacks (TIA), or ischemic stroke ipsilateral to the lesion. Asymptomatic carotid stenosis curses without symptoms but this silent form leads also to stroke. Asymptomatic patients are those that have never had any experience of neurological deficit, either a TIA or a stroke related to an affected vessel. Asymptomatic carotid stenosis is currently classified as asymptomatic progressive carotid stenosis and asymptomatic stable carotid stenosis, the first one involving the progressive growth and break of the vulnerable plaque, increasing risk of stroke.
Clinically, risk of stroke from carotid artery stenosis is evaluated by the presence or absence of symptoms and the degree of stenosis on imaging.
Carotid artery stenosis is usually diagnosed by color flow duplex ultrasound scan of the carotid arteries in the neck. This involves no radiation, no needles and no contrast agents that may cause allergic reactions. This test has good sensitivity and specificity. Other different imaging modalities, such as a computed tomography angiogram (CTA) or magnetic resonance angiogram (MRA) may also be useful. However, this diagnose using images requires of particular instrumentation and it is not applicable at ambulatory or emergency level (ambulances).
For this reason, serum biomarkers have been proposed, not only for the diagnosis of carotid stenosis, but also to discriminate between the several forms of the disease, i.e. symptomatic carotid stenosis and asymptomatic carotid stenosis.
For example C-reactive protein measured in serum is proposed as marker for progression of carotid artery disease and useful in the management of the disease and global carotid risk assessment, providing the option for evaluation and follow-up of patients with subclinical carotid stenosis (Arthus et al.,“A prospective evaluation of C-reactive protein in the progression of carotid artery stenosis”, J.Vasc. Surg 2008; vol. no. 47, pp. : 744-751).
As previously enunciated, discrimination among symptomatic and the different forms of asymptomatic carotid stenosis is of capital importance, since clinical therapeutically approaches are different in order to be effective and to avoid serious complications.
Thus, circulating biomarkers allowing discrimination among symptomatic and
asymptomatic patients have been identified, including high-density lipoproteins (HDL) cholesterol and certain cytokines (IL-6 and C-reactive protein). HDL was found as significantly higher (p<0.001) in asymptomatic patients, whereas IL-6 and C-reactive protein were found higher (p=0.04 and p=0.07, respectively) in samples of symptomatic patients than in asymptomatic patients (Musialek et al.,“Multimarker Approach in
Discriminating Patients with Symptomatic and Asymptomatic Atherosclerotic Carotid Artery Stenosis”, J. Clin. Neurol 2013, vol. no. 9, pp.: 165-175). High sensitivity C-reactive protein (hs-CRP) in plasma was also proposed as differential biomarker among symptomatic and asymptomatic patients, being higher in symptomatic patients and associated with the presence of neurological events, stroke or TIA (Rekasem et al.,“C- Reactive Protein is Elevated in Symptomatic Compared with Asymptomatic Patients with Carotid Artery Disease”, Eur. J. Vase Endovasc Surg 2002, vol. no. 23, pp.: 505-509).
Although several efforts have been conducted to find non-invasive biomarkers for carotid stenosis and for discriminating among the several manifestations of the disease, there is still a need of alternative reliable markers, improving correct diagnosis and associated- therapeutic approaches.
Summary of Invention
Inventors propose a panel of biomarkers that individually and in combination allow correct classification among subjects suffering from symptomatic carotid stenosis and
asymptomatic carotid stenosis. The proposed biomarkers, which are expressed proteins detectable in biofluid samples (i.e. plasma, serum, whole blood) are reliable biomarkers, since they have been identified from comparison with also differentiating biomarkers in atheromatous plaque between symptomatic and asymptomatic subjects. Thus, they do represent a real image of the risk of suffering stroke or other neurological events associated or due to the different physical parameters or stages of the plaques narrowing carotid artery. Thus, there are provided herewith new biomarkers that used in combination or even individually suppose a reliable and highly specific tool for the discrimination among subjects suffering from carotid stenosis. Correct classification allows deciding the more appropriate therapeutic regimen. A first aspect of the invention is an in vitro method for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis, comprising the steps of:
(a) determining in an isolated biofluid sample of a subject the level of expression of CD5 antigen-like; and
(b) comparing the level of expression of CD5 antigen-like with a corresponding reference value, and: (b1) diagnosing the subject of symptomatic carotid stenosis or of asymptomatic carotid stenosis if the levels of expression of CD5 antigen-like match with corresponding reference values of symptomatic carotid stenosis or asymptomatic carotid stenosis; or, alternatively,
(b2) diagnosing the subject of symptomatic carotid stenosis if the level of expression of CD5 antigen-like is higher than a reference value; and
diagnosing the subject of asymptomatic carotid stenosis if the level of expression of CD5 antigen-like is lower than a reference value.
A second aspect of the invention is an in vitro method for selecting a subject suffering from carotid stenosis as candidate for surgical therapy, comprising the following steps:
(a) determining in an isolated biofluid sample of a subject the level of expression of CD5 antigen-like; and
(b) comparing the level of expression of CD5 antigen-like with a corresponding reference value, and wherein the subject is selected as a candidate for surgical therapy if:
the levels of expression of CD5 antigen-like match with corresponding reference values of symptomatic carotid stenosis; or alternatively, the level of expression of CD5 antigen-like, is higher than a reference value.
This aspect can also be formulated as a method for selecting and/or recommending a therapy regimen for a subject suffering from carotid stenosis, wherein the method comprises:
(a) determining in an isolated biofluid sample of the subject the level of expression of CD5 antigen-like; and
(b) comparing the level of expression with a corresponding reference value, and wherein the subject is diagnosed as symptomatic carotid stenosis and an effective treatment comprising surgical therapy is selected or recommended if:
the levels of expression of CD5 antigen-like, match with corresponding reference values of symptomatic carotid stenosis; or alternatively, the level of expression of CD5 antigen-like is higher than a reference value.
Thus, present invention provides also as another aspect the use of CD5 antigen-like as biomarker of differential diagnosis of symptomatic versus asymptomatic carotid stenosis or as biomarkers for selecting a subject suffering from carotid stenosis as candidate for surgical therapy.
Another aspect of the invention is a kit of parts comprising reagent means for detecting the level of expression of CD5 antigen-like.
The invention also proposes using means for detecting the level of expression of CD5 antigen-like in an isolated biofluid sample of a subject in the in vitro method of any one of the first and second aspects.
Invention also encompasses a method of detecting, in an isolated biofluid sample of a subject suffering from carotid stenosis, the expression of CD5 antigen-like, the method comprising:
(a) obtaining a biofluid sample from the subject; and (b) detecting whether the protein is present in the isolated biofluid sample by: (i) contacting said sample with means capable of binding the corresponding protein and detecting said binding; or (ii) contacting said sample with means capable of binding corresponding RNA going to be translated to the corresponding protein and detecting said binding.
Another aspect of the invention is an in vitro method for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis, and for selecting a treatment for carotid stenosis, comprising the steps of:
(a) detecting in an isolated biofluid sample of a subject suffering from carotid stenosis the level of expression of CD5 antigen-like;
(b) comparing the level of expression of the protein with a corresponding reference value, wherein:
(i) the subject is diagnosed of symptomatic carotid stenosis if the level of
expression of CD5 antigen-likeis higher than a reference value or match with corresponding reference values of symptomatic carotid stenosis; and
(ii) the subject is diagnosed of asymptomatic carotid stenosis if the level of
expression of CD5 antigen-like is lower than a reference value or match with corresponding reference values of asymptomatic carotid stenosis; and
(c) recommending the subject for surgical therapy if is diagnosed of symptomatic carotid stenosis; or recommending the subject for further biomarker analysis and/or for intensive medical therapy if the subject is diagnosed of asymptomatic carotid stenosis.
Finally, another aspect of the invention is a kit of parts comprising reagent means for detecting the level of expression of CD5 antigen-like.
Detailed description of the invention
All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.
As used herein, the indefinite articles“a” and“an” are synonymous with“at least one” or “one or more.” Unless indicated otherwise, definite articles used herein, such as“the” also include the plural of the noun.
As used herein“diagnosis” is understood as becoming aware of a particular medical condition complication or risk in a subject; the determination of the nature of the disease or condition; or the distinguishing of one disease or condition from another. It refers both to the process of attempting to determine or identify the possible disease or disorder, and to the opinion reached by this process. A diagnosis, in the sense of diagnostic procedure, can be regarded as an attempt at classification of an individual's condition into separate and distinct categories that allow medical decisions about treatment and prognosis to be made. Subsequently, a diagnostic opinion is often described in terms of a disease or other condition. However, a diagnosis can take many forms. It might be a matter of detecting the presence and naming the disease, lesion, dysfunction or disability. It might be an exercise to attribute a category for management or for prognosis. It may indicate either degree of abnormality on a continuum or kind of abnormality in a classification. It may also include the distinction between subcategories among a particular diagnosis, such a differential diagnosis. Generically, the“differential diagnosis” also relates to the distinction among different diseases. In the present case, it relates to distinction among two or more subtypes of carotid stenosis, but is also encompasses distinction among different diseases with common symptoms, such as pancreatitis and pancreas cancer.
The term“intensive medical therapy” refers to the administration of suitable drugs with the corresponding schedules and doses that assure the control of progression of any disease. The term also includes the follow-on carried out by medical professionals in order to detect any critical change in the state of the subject. Generally, the concept intensive medical therapy implies a chronic treatment of a particular condition or disease (i.e. , insulin administration in diabetes type 1 subjects, or acenocumarol in subjects with a high risk of thrombosis). The term "selecting a patient for a therapy", as used herein, relates to the identification of a patient for a therapy designed to cure a disease or palliate the symptoms associated with one or more diseases or conditions.
In the present invention, the term“reference value”,“reference level” or“reference control level”, used interchangeably and referred to in the methods of the any of the aspects of the invention is to be understood as a predefined value or range of values (reference interval) of a given molecular marker or a combination of the given molecular markers, in the present case any of the proteins listed in the first or second aspects as well as in particular embodiments, which is derived from the levels of said molecular marker or markers in a sample or group of samples. If the level of expression is determined at the protein level, then the "reference expression level" is a predefined value of protein quantity, whereas if the level of expression is determined at the mRNA level, then the "reference expression level" is a predefined value of mRNA quantity. The samples are taken from a subject or group of subjects wherein the presence, absence, stage, histological subtype or grade, or course of the disease has been properly performed previously. This reference value is also useful for determining whether the subject has to initiate a medical regimen and how effective the regimen is. The subject or subjects from whom the“reference value” is derived may include subject/s wherein the condition is absent, subject/s wherein the condition is present, or both. The skilled person in the art, making use of the general knowledge, is able to choose the subject or group of subjects more adequate for obtaining the reference value for each of the methods of the present invention. Methods for obtaining the reference value from the group of subjects selected are well-known in the state of the art (Burtis C. A. et al. , 2008, Chapter 14, section “Statistical Treatment of Reference Values”). In a particular case“reference value” is a cut-off value defined by means of a conventional ROC analysis (Receiver Operating Characteristic analysis). As the skill person will appreciate, optimal cut-off value will be defined according to the particular applications of the diagnostic or prognostic method: purpose, target population for the diagnosis or prognosis, balance between specificity and sensibility, etc.
As indicated, a first aspect of the invention is an in vitro method for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis, comprising the steps of: (a) determining in an isolated biofluid sample of a subject the level of expression of CD5 antigen-like; and
(b) comparing the level of expression of CD5 antigen-like with a corresponding reference value, and:
(b1) diagnosing the subject of symptomatic carotid stenosis or of asymptomatic carotid stenosis if the levels of expression of CD5 antigen-like match with corresponding reference values of symptomatic carotid stenosis or asymptomatic carotid stenosis; or, alternatively,
(b2) diagnosing the subject of symptomatic carotid stenosis if the level of expression of CD5 antigen-like is higher than a reference value; and
diagnosing the subject of asymptomatic carotid stenosis if the level of expression of CD5 antigen-like is lower than a reference value.
In a particular embodiment of the first aspect, the in vitro method for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis further comprises determining the level of expression of one or more proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein,
Immunoglobulin heavy constant gamma 3, Prothrombin, and Serum amyloid P- component.
In another particular embodiment of the first aspect, optionally in combination with any embodiments above or below, the method comprises the steps of:
(a) determining in an isolated biofluid sample of a subject the level of expression of CD5 antigen-like, and further the level of expression of one or more proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein,
Immunoglobulin heavy constant gamma 3, Prothrombin, and Serum amyloid P- component; and
(b) comparing the level of expression of CD5 antigen-like, and of the one or more proteins with a corresponding reference value, and: (b1) diagnosing the subject of symptomatic carotid stenosis or of asymptomatic carotid stenosis if the levels of expression of CD5 antigen-like, and of the one or more of the other proteins match with corresponding reference values of symptomatic carotid stenosis or asymptomatic carotid stenosis; or, alternatively,
(b2) diagnosing the subject of symptomatic carotid stenosis if the level of expression of CD5 antigen-like, and the level of expression of one or more of fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C- reactive protein, Immunoglobulin heavy constant gamma 3, Prothrombin, and Serum amyloid P-component are determined, and the levels of CD5 antigen-like and of one of the one or more determined are higher than a reference value, and/or the level of expression of CD5 antigen-like and of one or more of apolipoprotein A-IV and
transthyretin are determined and the level of expression of CD5 antigen-like is higher than a reference value and the levels of expression of one of apolipoprotein A-IV and transthyretin are lower than a reference value; and
diagnosing the subject of asymptomatic carotid stenosis if the level of expression of CD5 antigen-like, and the level of expression of one or more of fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Immunoglobulin heavy constant gamma 3, Prothrombin, and Serum amyloid P-component are determined, and the levels of CD5 antigen-like and of one of the one or more determined are lower than a reference value, and/or the level of expression of CD5 antigen-like and the level of expression of one or more of apolipoprotein A-IV and transthyretin are determined and the level of expression of CD5 antigen-like is lower than a reference value and the levels of expression of one of the apolipoprotein A-IV and transthyretin are higher than a reference value.
In another particular embodiment of the first aspect, the method comprises determining the level of expression of C-reactive protein, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of C-reactive protein is higher than a reference value, or match with a value of a symptomatic carotid stenosis subject.
In also another particular embodiment of the method of the first aspect, the in vitro method comprises determining the level of expression of apolipoprotein A-IV, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of apolipoprotein A-IV is lower than a reference value, or alternatively if the level of expression of apolipoprotein A-IV match with the value of a symptomatic carotid stenosis reference. In also another particular embodiment of the method of the first aspect, the in vitro method comprises determining the level of expression of Immunoglobulin heavy constant gamma 3, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of Immunoglobulin heavy constant gamma 3 is higher than a reference value, or alternatively if the level of expression of Immunoglobulin heavy constant gamma 3 match with the value of a symptomatic carotid stenosis reference.
In also another particular embodiment of the method of the first aspect, the in vitro method comprises determining the level of expression of Prothrombin, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of Prothrombin is higher than a reference value, or alternatively if the level of expression of Prothrombin match with the value of a symptomatic carotid stenosis reference.
In also another particular embodiment of the method of the first aspect, the in vitro method comprises determining the level of expression of Serum amyloid P-component, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of Serum amyloid P-component is higher than a reference value, or alternatively if the level of expression of Serum amyloid P-component match with the value of a symptomatic carotid stenosis reference.
CD5 antigen-like is a secretion protein that regulates lipid synthesis and is expressed mainly by macrophages, it appears in inflamed tissues such as atherosclerosis and its target cells are adipocytes in which it inhibits the synthesis of fatty acids and leads lipolysis. This lipolysis induced by CD5 antigen-like occurs in the progression of obesity and participates in the inflammation associated with obesity. It is key in the metabolic activation of Th17 T-helper cells. It inhibits apoptosis in macrophages. It is involved in the early response to infections as a receptor of recognition pattern and promoting autophagy. It is described that CD5 antigen-like is increased its expression in control patients with respect to patients with type 2 diabetes, both groups with an endarterectomy coronary. Human protein is identified in UniProt KB database with accession number 043866, version 1 of June 1 , 1998 of the sequence with 347 amino acids and version 146 of the entry in database.
The C-reactive protein has several functions associated with the defense mechanism: it promotes cell agglutination, capsular inflammation of bacteria, phagocytosis and complement fixation. Human protein is identified in UniProt KB database with accession number P02741 , version 1 of January 1 , 1988 of the sequence with 224 amino acids and version 219 of the entry in database. It is a biomarker of inflammatory reaction and an important mediator of atherosclerosis contributing to the development of the plaque and its instability. Since 2010, theplasma level of C-reactive protein has been used as a prognosis in patients with intermediate risk of cardiovascular diseases.
Fibrinogen alpha chain lysed by thrombin protease forms monomers that polymerize with beta fibrinogen and gamma fibrinogen to form an insoluble fibrin matrix. Human protein is identified in UniProt KB database with accession number P02671 , version 2 of October 1 , 1996 of the sequence with 866 amino acids and version 236 of the entry in database. It has some role in wound repair stabilizing the lesion and guiding cell migration in epithelialization. Mutations of this protein cause hereditary renal amyloidosis and a high percentage of patients with atherosclerosis have been found in carriers of these mutations.
Haptoglobin binds to free hemoglobin in the blood allowing its recycling in the spleen and liver and preventing kidney damage. Human protein is identified in UniProt KB database with accession number P00738, version 1 of July 21 , 1986 of the sequence with 406 amino acids and version 208 of the entry in database. It also acts as an antioxidant, has antibacterial activity and intervenes in the acute response. The hemoglobin / haptoglobin complex is eliminated by the CD163 receptor of Kupffer cells. Variant 2 of haptoglobin is associated with more unstable atheroma plaques, with more iron, with thinner fibrous layers and with a greater probability of bleeding, since a higher incidence of
atherosclerosis in diabetics.
Apolipoprotein A-IV (Uniprot database or other unequivocal identification) is the major component of HDL and chylomicrons. It could be involved in the secretion and catabolism of the latter. It is an atheroprotective and in animal models it has been observed that its administration reduces hemorrhagic disruptions, the fibrous layer and the lipid nucleus in atheromatous plaques, as well as inflammation and associated apoptosis. Human protein is identified in UniProt KB database with accession number P06727, version 3 of March 7, 2006 of the sequence with 396 amino acids and version 206 of the entry in database.
Transthyretin is a thyroid hormone-binding protein and probably transports thyroxine from the bloodstream to the brain. Human protein is identified in UniProt KB database with accession number P02766, version 1 of March 20, 1987 of the sequence with 147 amino acids and version 236 of the entry in database. The dissociation of the tetramer leads to the formation of amyloid aggregates and fibrils. It has been seen that the atheroma plaques of women contain a greater amount of Transthyretin, as well as of Apolipoprotein A-IV. Heparin cofactor 2, activated by glycosaminoglycans, is an inhibitor of thrombin, heparin and dermatan sulfate; and, without the presence of glycosaminoglycans, inhibits chymotrypsin. Its N terminal possesses chemotactic activity in monocytes and neutrophils. The plasma levels of this protein are negatively correlated with the instability of the atheroma plaques. Human protein is identified in UniProt KB database with accession number P05546, version 3 of November 1 , 1991 of the sequence with 499 amino acids and version 202 of the entry in database.
Immunoglobulin heavy constant gamma 2 corresponds to human protein identified in UniProt KB database with accession number P01859, version 2 of December 16, 2008 of the sequence with 326 amino acids and version 169 of the entry in database.
Immunoglobulin heavy constant gamma 4, corresponds to human protein identified in UniProt KB database with accession number P01861 , version 1 of July 21 , 1986 of the sequence with 327 amino acids and version 159 of the entry in database.
Immunoglobulin heavy constant mu corresponds to human protein identified in UniProt KB database with accession number P01871 , version 4 of March 15, 2017 of the sequence with 453 amino acids and version 188 of the entry in database.
Immunoglobulin heavy constant gamma 3 corresponds to human protein identified in UniProt KB database with accession number P01860, version 2 of July 1 , 2008 of the sequence with 377 amino acids, and version 179 of the entry in database. Prothrombin corresponds to human protein identified in UniProt KB database with accession number P00734, version 2 of January 1 , 1990 of the sequence with 622 amino acids, and version 260 of the entry in database.
Serum amyloid P-component corresponds to human protein identified in UniProt KB database with accession number P02743, version 2 of February 1 , 1995 of the sequence with 223 amino acids, and version 218 of the entry in database.
Gelsolin corresponds to human protein identified in UniProt KB database with accession number P06396, version 1 of January 1 , 1988 of the sequence with 782 amino acids for the canonical isoform, and version 238 of the entry in database.
Immunoglobulin kappa variable 4-1 corresponds to human protein identified in UniProt KB database with accession number P06312, version 1 of January 1 , 1988 of the sequence with 121 amino acids, and version 151 of the entry in database. Caveolin-1 corresponds to human protein identified in UniProt KB database with accession number Q03135, version 4 of January 23, 2007 of the sequence with 178 amino acids for the canonical isoform, and version 225 of the entry in database.
The expression“isolated biofluid sample” as use herein relates to any liquid sample, optionally containing cells, which can be obtained from the patient. Particular biofluid samples are selected from plasma, serum, whole blood, saliva, semen, sputum, cerebral spinal fluid (CSF), tears, mucus, sweat and milk. More particular biofluid samples are selected from plasma, serum, and whole blood.
In another particular embodiment of the first aspect, the in vitro method comprises determining the level of expression of CD5 antigen-like and of one or more of of the above-listed proteins; Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Immunoglobulin heavy constant gamma 3, Prothrombin, and Serum amyloid P- component. In another particular embodiment there are determined the level of expression of two or more of the proteins, including CD5 antigen-like, more in particular of three proteins, including CD5 antigen-like; four proteins, including CD5 antigen-like; five proteins, including CD5 antigen-like; six proteins, including CD5 antigen-like; seven proteins, including CD5 antigen-like; eight proteins, including CD5 antigen-like; nine proteins, ten, eleven or twelve, including CD5 antigen-like. If in addition the levels of C- reactive protein are determined, then the method comprises determining at least two proteins being one of them the C-reactive protein and the other CD5 antigen-like. More in particular three proteins, including CD5 antigen-like and C-reactive protein; four proteins, including CD5 antigen-like and C-reactive protein; five proteins, including CD5 antigen-like and C-reactive protein; six proteins, including CD5 antigen-like and C-reactive protein; seven proteins, including CD5 antigen-like and C-reactive protein; eight or nine proteins being one of them the levels of expression of C-reactive protein and another one CD5 antigen-like. More in particular the level of expression of all of CD5 antigen-like, C-reactive protein, Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4,
Immunoglobulin heavy constant mu and Heparin cofactor 2 are determined. If in addition, the level of expression of one or more of Immunoglobulin heavy constant gamma 3, Prothrombin, and Serum amyloid P-component are determined, combinations of two, three, four, five, six seven, eight, nine, ten, eleven, and twelve, always including CD5 antigen-like are determined according to the method of the invention. In a particular embodiment, the thirteen proteins are determined. Determination of more than one of the expression levels of these listed proteins allows improving sensitivity and/or specificity of the methods of the invention. This is also translated to the manufacture of particular kits for carrying out the method including different combinations depending on the accuracy that is desired.
Particular combinations are listed below, wherein the following abbreviations are used: “CD5” for CD5 antigen-like,“Creactive” for C-reactive protein,“Fibrinogen” for Fibrinogen alpha chain,“gamma2” for Immunoglobulin heavy constant gamma 2,“apo4” for
Apolipoprotein A-IV,“Transth” for Transthyretin,“gamma4” for Immunoglobulin heavy constant gamma 4,“mu” for Immunoglobulin heavy constant mu,“heparin” for Heparin cofactor 2,“Haptoglobin” not abbreviated.
Thus, in another particular embodiment of the first aspect the method comprises determining the level of expression of two of the proteins, said two-protein sets (two- membered groups) selected from the group consisting of:1.-CD5, Creactive; 2.-CD5, Fibrinogen; 3.-CD5, Haptoglobin; 4.-CD5, gamma2; 5.-CD5, apo4; 6.-CD5, Transth; 7.- CD5, gamma4; 8.-CD5, mu; 9.-CD5, heparin; 10.- Creactive, Fibrinogen; 11.- Creactive, Haptoglobin; 12.- Creactive, gamma2; 13.- Creactive, apo4; 14.- Creactive, Transth; 15.- Creactive, gamma4; 16.- Creactive, mu; 17.- Creactive, heparin; 18.- Fibrinogen,
Haptoglobin; 19.- Fibrinogen, gamma2; 20.- Fibrinogen, apo4; 21.- Fibrinogen, Transth; 22.- Fibrinogen, gamma4; 23.- Fibrinogen, mu; 24.- Fibrinogen, heparin; 25.- Haptoglobin, gamma2; 26.- Haptoglobin, apo4; 27.- Haptoglobin, Transth
28.- Haptoglobin, gamma4; 29.- Haptoglobin, mu; 30.- Haptoglobin, heparin; 31.- gamma2, apo4; 32.- gamma2, Transth; 33.- gamma2, gamma4; 34.- gamma2, mu; 35.- gamma2, heparin; 36.- apo4, Transth; 37.- apo4, gamma4; 38.- apo4, mu; 39.- apo4, heparin; 40.- Transth, gamma4; 41.- Transth, mu; 42.- Transth, heparin; 43.- gamma4, mu; 44.- gamma4, heparin; and 45.- mu, heparin. In another particular embodiment of the first aspect the method comprises determining the level of expression of three of the proteins, said three-protein sets (three-membered groups) selected from the group consisting of: 1.-CD5, Creactive, Fibrinogen; 2.-CD5, Creactive, Haptoglobin; 3.-CD5, Creactive, gamma2; 4.-CD5, Creactive, apo4; 5.-CD5, Creactive, Transth; 6.-CD5, Creactive, gamma4; 7.-CD5, Creactive, mu; 8.-CD5,
Creactive, heparin; 9.-CD5, Fibrinogen, Haptoglobin; 10.-CD5, Fibrinogen, gamma2; 11.- CD5, Fibrinogen, apo4; 12.-CD5, Fibrinogen, Transth; 13.-CD5, Fibrinogen, gamma4; 14.- CD5, Fibrinogen, mu; 15.-CD5, Fibrinogen, heparin; 16.-CD5, Haptoglobin, gamma2; 17.- CD5, Haptoglobin, apo4; 18.-CD5, Haptoglobin, Transth; 19.-CD5, Haptoglobin, gamma4; 20.-CD5, Haptoglobin, mu; 21.-CD5, Haptoglobin, heparin; 22.-CD5, gamma2, apo4; 23.- CD5, gamma2, Transth; 24.-CD5, gamma2, gamma4; 25.-CD5, gamma2, mu; 26.-CD5, gamma2, heparin; 27.-CD5, apo4, Transth; 28.-CD5, apo4, gamma4; 29.-CD5, apo4, mu; 30.-CD5, apo4, heparin; 31.-CD5, Transth, gamma4; 32.-CD5, Transth, mu; 33.-CD5, Transth, heparin; 34.-CD5, gamma4, mu; 35.-CD5, gamma4, heparin; 36.-CD5, mu, heparin; 37.- Creactive, Fibrinogen, Haptoglobin; 38.- Creactive, Fibrinogen, gamma2;
39.- Creactive, Fibrinogen, apo4; 40.- Creactive, Fibrinogen, Transth; 41.- Creactive, Fibrinogen, gamma4; 42.- Creactive, Fibrinogen, mu; 43.- Creactive, Fibrinogen, heparin; 44.- Creactive, Haptoglobin, gamma2; 45.- Creactive, Haptoglobin, apo4; 46.- Creactive, Haptoglobin, Transth; 47.- Creactive, Haptoglobin, gamma4; 48.- Creactive, Haptoglobin, mu; 49.- Creactive, Haptoglobin, heparin; 50.- Creactive, gamma2, apo4; 51.- Creactive, gamma2, Transth; 52.- Creactive, gamma2, gamma4; 53.- Creactive, gamma2, mu; 54.- Creactive, gamma2, heparin; 55.- Creactive, apo4, Transth; 56.- Creactive, apo4, gamma4; 57.- Creactive, apo4, mu; 58.- Creactive, apo4, heparin; 59.- Creactive, Transth, gamma4; 60.- Creactive, Transth, mu; 61.- Creactive, Transth, heparin; 62.- Creactive, gamma4, mu; 63.- Creactive, gamma4, heparin; 64.- Creactive, mu, heparin; 65.-
Fibrinogen, Haptoglobin, gamma2; 66.- Fibrinogen, Haptoglobin, apo4; 67.- Fibrinogen, Haptoglobin, Transth; 68.- Fibrinogen, Haptoglobin, gamma4; 69.- Fibrinogen,
Haptoglobin, mu; 70.- Fibrinogen, Haptoglobin, heparin; 71.- Fibrinogen, gamma2, apo4; 72.- Fibrinogen, gamma2, Transth; 73.- Fibrinogen, gamma2, gamma4; 74.- Fibrinogen, gamma2, mu; 75.- Fibrinogen, gamma2, heparin; 76.- Fibrinogen, apo4, Transth; 77.- Fibrinogen, apo4, gamma4; 78.- Fibrinogen, apo4, mu; 79.- Fibrinogen, apo4, heparin; 80.- Fibrinogen, Transth, gamma4; 81.- Fibrinogen, Transth, mu; 82.- Fibrinogen, Transth, heparin; 83.- Fibrinogen, gamma4, mu; 84.- Fibrinogen, gamma4, heparin; 85.- Fibrinogen, mu, heparin; 86.- Haptoglobin, gamma2, apo4; 87.- Haptoglobin, gamma2, Transth; 88.- Haptoglobin, gamma2, gamma4; 89.- Haptoglobin, gamma2, mu; 90.-
Haptoglobin, gamma2, heparin; 91.- Haptoglobin, apo4, Transth; 92.- Haptoglobin, apo4, gamma4; 93.- Haptoglobin, apo4, mu; 94.- Haptoglobin, apo4, heparin; 95.- Haptoglobin, Transth, gamma4: 96.- Haptoglobin, Transth, mu; 97.- Haptoglobin, Transth, heparin; 98.- Haptoglobin, gamma4, mu; 99.- Haptoglobin, gamma4, heparin; 100.- Haptoglobin, mu, heparin; 101.- gamma2, apo4, Transth; 102.- gamma2, apo4, gamma4; 103.- gamma2, apo4, mu; 104.- gamma2, apo4, heparin; 105.- gamma2, Transth, gamma4; 106.- gamma2, Transth, mu; 107.- gamma2, Transth, heparin; 108.- gamma2, gamma4, mu; 109.- gamma2, gamma4, heparin; 110.- gamma2, mu, heparin; 111.- apo4, Transth, gamma4; 112.- apo4, Transth, mu; 113.- apo4, Transth, heparin; 114.- apo4, gamma4, mu; 115.- apo4, gamma4, heparin; 116.- apo4, mu, heparin; 117.- Transth, gamma4, mu;
118.- Transth, gamma4, heparin; and 119.- Transth, mu, heparin; and 120.- gamma4, mu, heparin.
In another particular embodiment of the first aspect the method comprises determining the level of expression of four of the proteins, said four-protein sets (four-membered groups) selected from the group consisting of: 1.-CD5, Creactive, Fibrinogen, Haptoglobin; 2.- CD5, Creactive, Fibrinogen, gamma2; 3.-CD5, Creactive, Fibrinogen, apo4; 4.-CD5, Creactive, Fibrinogen, Transth; 5.-CD5, Creactive, Fibrinogen, gamma4; 6.-CD5,
Creactive, Fibrinogen, u; 7.-CD5, Creactive, Fibrinogen, heparin; 8.-CD5, Creactive, Haptoglobin, gamma2; 9.-CD5, Creactive, Haptoglobin, apo4; 10.-CD5, Creactive, Haptoglobin, Transth; 11.-CD5, Creactive, Haptoglobin, gamma4; 12.-CD5, Creactive, Haptoglobin, mu; 13.-CD5, Creactive, Haptoglobin, heparin; 14.-CD5, Creactive, gamma2, apo4; 15.-CD5, Creactive, gamma2, Transth; 16.-CD5, Creactive, gamma2, gamma4; 17.- CD5, Creactive, gamma2, mu; 18.-CD5, Creactive, gamma2, heparin; 19.-CD5, Creactive, apo4, Transth; 20.-CD5, Creactive, apo4, gamma4; 21.-CD5, Creactive, apo4, mu; 22.- CD5, Creactive, apo4, heparin; 23.-CD5, Creactive, Transth, gamma4; 24.-CD5,
Creactive, Transth, mu; 25.-CD5, Creactive, Transth, heparin; 26.-CD5, Creactive, gamma4, mu; 27.-CD5, Creactive, gamma4, heparin; 28.-CD5, Creactive, mu, heparin; 29.-CD5, Fibrinogen, Haptoglobin, gamma2; 30.-CD5, Fibrinogen, Haptoglobin, apo4; 31.-
CD5, Fibrinogen, Haptoglobin, Transth; 32.-CD5, Fibrinogen, Haptoglobin, gamma4; 33.- CD5, Fibrinogen, Haptoglobin, mu; 34.-CD5, Fibrinogen, Haptoglobin, heparin; 35.-CD5, Fibrinogen, gamma2, apo4; 36.-CD5, Fibrinogen, gamma2, Transth; 37.-CD5, Fibrinogen, gamma2, gamma4; 38.-CD5, Fibrinogen, gamma2, mu; 39.-CD5, Fibrinogen, gamma2, heparin; 40.-CD5, Fibrinogen, apo4, Transth; 41.-CD5, Fibrinogen, apo4, gamma4; 42.- CD5, Fibrinogen, apo4, mu; 43.-CD5, Fibrinogen, apo4, heparin; 44.-CD5, Fibrinogen, Transth, gamma4; 45.-CD5, Fibrinogen, Transth, mu; 46.-CD5, Fibrinogen, Transth, heparin; 47.-CD5, Fibrinogen, gamma4, mu; 48.-CD5, Fibrinogen, gamma4, heparin; 49.- CD5, Fibrinogen, mu, heparin; 50.-CD5, Haptoglobin, gamma2, apo4; 51.-CD5,
Haptoglobin, gamma2, Transth; 52.-CD5, Haptoglobin, gamma2, gamma4; 53.-CD5, Haptoglobin, gamma2, mu; 54.-CD5, Haptoglobin, gamma2, heparin; 55.-CD5,
Haptoglobin, apo4, Transth; 56.-CD5, Haptoglobin, apo4, gamma4; 57.-CD5, Haptoglobin, apo4, mu; 58.-CD5, Haptoglobin, apo4, heparin; 59.-CD5, Haptoglobin, Transth, gamma4; 60.-CD5, Haptoglobin, Transth, mu; 61.-CD5, Haptoglobin, Transth, heparin; 62.-CD5, Haptoglobin, gamma4, mu; 63.-CD5, Haptoglobin, gamma4, heparin; 64.-CD5,
Haptoglobin, mu, heparin; 65.-CD5, gamma2, apo4, Transth; 66.-CD5, gamma2, apo4, gamma4; 67.-CD5, gamma2, apo4, mu; 68.-CD5, gamma2, apo4, heparin; 69.-CD5, gamma2, Transth, gamma4; 70.-CD5, gamma2, Transth, mu; 71.-CD5, gamma2, Transth, heparin; 72.-CD5, gamma2, gamma4, mu; 73.-CD5, gamma2, gamma4, heparin; 74.- CD5, gamma2, mu, heparin; 75.-CD5, apo4, Transth, gamma4; 76.-CD5, apo4, Transth, mu; 77.-CD5, apo4, Transth, heparin; 78.-CD5, apo4, gamma4, mu; 79.-CD5, apo4, gamma4, heparin; 80.-CD5, apo4, mu, heparin; 81.-CD5, Transth, gamma4, mu; 82.-CD5, Transth, gamma4, heparin; 83.-CD5, Transth, mu, heparin; 84.-CD5, gamma4, mu, heparin; 85.- Creactive, Fibrinogen, Haptoglobin, gamma2; 86.- Creactive, Fibrinogen, Haptoglobin, apo4; 87.- Creactive, Fibrinogen, Haptoglobin, Transth; 88.- Creactive, Fibrinogen, Haptoglobin, gamma4; 89.- Creactive, Fibrinogen, Haptoglobin, mu; 90.- Creactive, Fibrinogen, Haptoglobin, heparin; 91.- Creactive, Fibrinogen, gamma2, apo4; 92.- Creactive, Fibrinogen, gamma2, Transth; 93.- Creactive, Fibrinogen, gamma2, gamma4; 94.- Creactive, Fibrinogen, gamma2, u; 95.- Creactive, Fibrinogen, gamma2, heparin; 96.- Creactive, Fibrinogen, apo4, Transth; 97.- Creactive, Fibrinogen, apo4, gamma4; 98.- Creactive, Fibrinogen, apo4, mu; 99.- Creactive, Fibrinogen, apo4, heparin; 100.- Creactive, Fibrinogen, Transth, gamma4; 101.- Creactive, Fibrinogen, Transth, mu; 102.- Creactive, Fibrinogen, Transth, heparin; 103.- Creactive, Fibrinogen, gamma4, mu; 104.- Creactive, Fibrinogen, gamma4, heparin; 105.- Creactive, Fibrinogen, mu, heparin;
106.- Creactive, Haptoglobin, gamma2, apo4; 107.- Creactive, Haptoglobin, gamma2, Transth; 108.- Creactive, Haptoglobin, gamma2, gamma4; 109.- Creactive, Haptoglobin, gamma2, mu; 110.- Creactive, Haptoglobin, gamma2, heparin; 111.- Creactive,
Haptoglobin, apo4, Transth; 112.- Creactive, Haptoglobin, apo4, gamma4; 113.- Creactive, Haptoglobin, apo4, mu; 114.- Creactive, Haptoglobin, apo4, heparin; 115.- Creactive, Haptoglobin, Transth, gamma4; 116.- Creactive, Haptoglobin, Transth, mu; 117.- Creactive, Haptoglobin, Transth, heparin; 118.- Creactive, Haptoglobin, gamma4, mu; 119.- Creactive, Haptoglobin, gamma4, heparin; 120.- Creactive, Haptoglobin, mu, heparin; 121.- Creactive, gamma2, apo4, Transth; 122.- Creactive, gamma2, apo4, gamma4; 123.- Creactive, gamma2, apo4, mu; 124.- Creactive, gamma2, apo4, heparin; 125.- Creactive, gamma2, Transth, gamma4; 126.- Creactive, gamma2, Transth, mu;
127.- Creactive, gamma2, Transth, heparin; 128.- Creactive, gamma2, gamma4, mu;
129.- Creactive, gamma2, gamma4, heparin; 130.- Creactive, gamma2, mu, heparin;
131.- Creactive, apo4, Transth, gamma4; 132.- Creactive, apo4, Transth, mu; 133.- Creactive, apo4, Transth, heparin; 134.- Creactive, apo4, gamma4, mu; 135.- Creactive, apo4, gamma4, heparin; 136.- Creactive, apo4, mu, heparin; 137.- Creactive, Transth, gamma4, mu; 138.- Creactive, Transth, gamma4, heparin; 139.- Creactive, Transth, mu, heparin; 140.- Creactive, gamma4, mu, heparin; 141.- Fibrinogen, Haptoglobin, gamma2, apo4; 142.- Fibrinogen, Haptoglobin, gamma2, Transth; 143.- Fibrinogen, Haptoglobin, gamma2, gamma4; 144.- Fibrinogen, Haptoglobin, gamma2, mu; 145.- Fibrinogen, Haptoglobin, gamma2, heparin; 146.- Fibrinogen, Haptoglobin, apo4, Transth; 147.- Fibrinogen, Haptoglobin, apo4, gamma4; 148.- Fibrinogen, Haptoglobin, apo4, mu; 149.- Fibrinogen, Haptoglobin, apo4, heparin; 150.- Fibrinogen, Haptoglobin, Transth, gamma4; 151.- Fibrinogen, Haptoglobin, Transth, mu; 152.- Fibrinogen, Haptoglobin, Transth, heparin; 153.- Fibrinogen, Haptoglobin, gamma4, mu; 154.- Fibrinogen, Haptoglobin, gamma4, heparin; 155.- Fibrinogen, Haptoglobin, mu, heparin; 156.- Fibrinogen, gamma2, apo4, Transth; 157.- Fibrinogen, gamma2, apo4, gamma4; 158.- Fibrinogen, gamma2, apo4, mu; 159.- Fibrinogen, gamma2, apo4, heparin; 160.- Fibrinogen, gamma2, Transth, gamma4; 161.- Fibrinogen, gamma2, Transth, mu; 162.- Fibrinogen, gamma2, Transth, heparin; 163.- Fibrinogen, gamma2, gamma4, mu; 164.- Fibrinogen, gamma2, gamma4, heparin; 165.- Fibrinogen, gamma2, u, heparin; 166.- Fibrinogen, apo4, Transth, gamma4; 167.- Fibrinogen, apo4, Transth, mu; 168.- Fibrinogen, apo4, Transth, heparin; 169.- Fibrinogen, apo4, gamma4, mu; 170.- Fibrinogen, apo4, gamma4, heparin; 171.- Fibrinogen, apo4, mu, heparin; 172.- Fibrinogen, Transth, gamma4, mu; 173.- Fibrinogen, Transth, gamma4, heparin; 174.- Fibrinogen, Transth, mu, heparin; 175.- Fibrinogen, gamma4, mu, heparin; 176.- Haptoglobin, gamma2, apo4, Transth; 177.- Haptoglobin, gamma2, apo4, gamma4; 178.- Haptoglobin, gamma2, apo4, mu; 179.- Haptoglobin, gamma2, apo4, heparin; 180.- Haptoglobin, gamma2, Transth, gamma4; 181.- Haptoglobin, gamma2, Transth, mu; 182.- Haptoglobin, gamma2, Transth, heparin; 183.- Haptoglobin, gamma2, gamma4, mu; 184.- Haptoglobin, gamma2, gamma4, heparin;
185.- Haptoglobin, gamma2, mu, heparin; 186.- Haptoglobin, apo4, Transth, gamma4; 187.- Haptoglobin, apo4, Transth, mu; 188.- Haptoglobin, apo4, Transth, heparin; 189.- Haptoglobin, apo4, gamma4, mu; 190.- Haptoglobin, apo4, gamma4, heparin; 191.- Haptoglobin, apo4, mu, heparin; 192.- Haptoglobin, Transth, gamma4, mu; 193.-
Haptoglobin, Transth, gamma4, heparin; 194.- Haptoglobin, Transth, mu, heparin; 195.- Haptoglobin, gamma4, mu, heparin; 196.- gamma2, apo4, Transth, gamma4; 197.- gamma2, apo4, Transth, mu; 198.- gamma2, apo4, Transth, heparin; 199.- gamma2, apo4, gamma4, mu; 200.- gamma2, apo4, gamma4, heparin; 201.- gamma2, apo4, mu, heparin; 202.- gamma2, Transth, gamma4, mu; 203.- gamma2, Transth, gamma4, heparin; 204.- gamma2, Transth, mu, heparin; 205.- gamma2, gamma4, mu, heparin; 206.- apo4, Transth, gamma4, mu; 207.- apo4, Transth, gamma4, heparin; 208.- apo4, Transth, mu, heparin; 209.- apo4, gamma4, mu, heparin; and 210.- Transth, gamma4, mu, heparin.
In another particular embodiment of the first aspect the method comprises determining the level of expression of five of the proteins, said five-protein sets (five-membered groups) selected from the group consisting of: 1.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2; 2.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4; 3.-CD5, Creactive,
Fibrinogen, Haptoglobin, Transth; 4.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma4; 5.-CD5, Creactive, Fibrinogen, Haptoglobin, mu; 6.-CD5, Creactive, Fibrinogen,
Haptoglobin, heparin; 7.-CD5, Creactive, Fibrinogen, gamma2, apo4; 8.-CD5, Creactive, Fibrinogen, gamma2, Transth; 9.-CD5, Creactive, Fibrinogen, gamma2, gamma4; 10.- CD5, Creactive, Fibrinogen, gamma2, mu; 11.-CD5, Creactive, Fibrinogen, gamma2, heparin; 12.-CD5, Creactive, Fibrinogen, apo4, Transth; 13.-CD5, Creactive, Fibrinogen, apo4, gamma4; 14.-CD5, Creactive, Fibrinogen, apo4, mu; 15.-CD5, Creactive,
Fibrinogen, apo4, heparin; 16.-CD5, Creactive, Fibrinogen, Transth, gamma4; 17.-CD5, Creactive, Fibrinogen, Transth, mu; 18.-CD5, Creactive, Fibrinogen, Transth, heparin; 19.- CD5, Creactive, Fibrinogen, gamma4, mu; 20.-CD5, Creactive, Fibrinogen, gamma4, heparin; 21.-CD5, Creactive, Fibrinogen, mu, heparin; 22.-CD5, Creactive, Haptoglobin, gamma2, apo4; 23.-CD5, Creactive, Haptoglobin, gamma2, Transth; 24.-CD5, Creactive, Haptoglobin, gamma2, gamma4; 25.-CD5, Creactive, Haptoglobin, gamma2, u; 26.- CD5, Creactive, Haptoglobin, gamma2, heparin; 27.-CD5, Creactive, Haptoglobin, apo4, Transth; 28.-CD5, Creactive, Haptoglobin, apo4, gamma4; 29.-CD5, Creactive,
Haptoglobin, apo4, mu; 30.-CD5, Creactive, Haptoglobin, apo4, heparin; 31.-CD5, Creactive, Haptoglobin, Transth, gamma4; 32.-CD5, Creactive, Haptoglobin, Transth, mu; 33.-CD5, Creactive, Haptoglobin, Transth, heparin; 34.-CD5, Creactive, Haptoglobin, gamma4, mu; 35.-CD5, Creactive, Haptoglobin, gamma4, heparin; 36.-CD5, Creactive, Haptoglobin, mu, heparin; 37.-CD5, Creactive, gamma2, apo4, Transth; 38.-CD5,
Creactive, gamma2, apo4, gamma4; 39.-CD5, Creactive, gamma2, apo4, mu; 40.-CD5, Creactive, gamma2, apo4, heparin; 41.-CD5, Creactive, gamma2, Transth, gamma4; 42.- CD5, Creactive, gamma2, Transth, mu; 43.-CD5, Creactive, gamma2, Transth, heparin; 44.-CD5, Creactive, gamma2, gamma4, mu; 45.-CD5, Creactive, gamma2, gamma4, heparin; 46.-CD5, Creactive, gamma2, mu, heparin; 47.-CD5, Creactive, apo4, Transth, gamma4; 48.-CD5, Creactive, apo4, Transth, mu; 49.-CD5, Creactive, apo4, Transth, heparin; 50.-CD5, Creactive, apo4, gamma4, mu; 51.-CD5, Creactive, apo4, gamma4, heparin; 52.-CD5, Creactive, apo4, mu, heparin; 53.-CD5, Creactive, Transth, gamma4, mu; 54.-CD5, Creactive, Transth, gamma4, heparin; 55.-CD5, Creactive, Transth, mu, heparin; 56.-CD5, Creactive, gamma4, mu, heparin; 57.-CD5, Fibrinogen, Haptoglobin, gamma2, apo4; 58.-CD5, Fibrinogen, Haptoglobin, gamma2, Transth; 59.-CD5,
Fibrinogen, Haptoglobin, gamma2, gamma4; 60.-CD5, Fibrinogen, Haptoglobin, gamma2, mu; 61.-CD5, Fibrinogen, Haptoglobin, gamma2, heparin; 62.-CD5, Fibrinogen,
Haptoglobin, apo4, Transth; 63.-CD5, Fibrinogen, Haptoglobin, apo4, gamma4; 64.-CD5, Fibrinogen, Haptoglobin, apo4, mu; 65.-CD5, Fibrinogen, Haptoglobin, apo4, heparin; 66.- CD5, Fibrinogen, Haptoglobin, Transth, gamma4; 67.-CD5, Fibrinogen, Haptoglobin, Transth, mu; 68.-CD5, Fibrinogen, Haptoglobin, Transth, heparin; 69.-CD5, Fibrinogen, Haptoglobin, gamma4, mu; 70.-CD5, Fibrinogen, Haptoglobin, gamma4, heparin; 71.- CD5, Fibrinogen, Haptoglobin, mu, heparin; 72.-CD5, Fibrinogen, gamma2, apo4, Transth; 73.-CD5, Fibrinogen, gamma2, apo4, gamma4; 74.-CD5, Fibrinogen, gamma2, apo4, mu; 75.-CD5, Fibrinogen, gamma2, apo4, heparin; 76.-CD5, Fibrinogen, gamma2, Transth, gamma4; 77.-CD5, Fibrinogen, gamma2, Transth, mu; 78.-CD5, Fibrinogen, gamma2, Transth, heparin; 79.-CD5, Fibrinogen, gamma2, gamma4, mu; 80.-CD5, Fibrinogen, gamma2, gamma4, heparin; 81.-CD5, Fibrinogen, gamma2, mu, heparin; 82.- CD5, Fibrinogen, apo4, Transth, gamma4; 83.-CD5, Fibrinogen, apo4, Transth, mu; 84.- CD5, Fibrinogen, apo4, Transth, heparin; 85.-CD5, Fibrinogen, apo4, gamma4, mu; 86.- CD5, Fibrinogen, apo4, gamma4, heparin; 87.-CD5, Fibrinogen, apo4, mu, heparin; 88.- CD5, Fibrinogen, Transth, gamma4, mu; 89.-CD5, Fibrinogen, Transth, gamma4, heparin; 90.-CD5, Fibrinogen, Transth, mu, heparin; 91.-CD5, Fibrinogen, gamma4, mu, heparin; 92.-CD5, Haptoglobin, gamma2, apo4, Transth; 93.-CD5, Haptoglobin, gamma2, apo4, gamma4; 94.-CD5, Haptoglobin, gamma2, apo4, mu; 95.-CD5, Haptoglobin, gamma2, apo4, heparin; 96.-CD5, Haptoglobin, gamma2, Transth, ga a4; 97.-CD5, Haptoglobin, gamma2, Transth, u; 98.-CD5, Haptoglobin, gamma2, Transth, heparin; 99.-CD5, Haptoglobin, gamma2, gamma4, mu; 100.-CD5, Haptoglobin, gamma2, gamma4, heparin 101.-CD5, Haptoglobin, gamma2, mu, heparin; 102.-CD5, Haptoglobin, apo4, Transth, gamma4; 103.-CD5, Haptoglobin, apo4, Transth, mu; 104.-CD5, Haptoglobin, apo4, Transth, heparin; 105.-CD5, Haptoglobin, apo4, gamma4, mu; 106.-CD5, Haptoglobin, apo4, gamma4, heparin; 107.-CD5, Haptoglobin, apo4, mu, heparin; 108.-CD5,
Haptoglobin, Transth, gamma4, mu; 109.-CD5, Haptoglobin, Transth, gamma4, heparin; 110.-CD5, Haptoglobin, Transth, mu, heparin; 111.-CD5, Haptoglobin, gamma4, mu, heparin; 112.-CD5, gamma2, apo4, Transth, gamma4; 113.-CD5, gamma2, apo4,
Transth, mu; 114.-CD5, gamma2, apo4, Transth, heparin; 115.-CD5, gamma2, apo4, gamma4, mu; 116.-CD5, gamma2, apo4, gamma4, heparin; 117.-CD5, gamma2, apo4, mu, heparin; 118.-CD5, gamma2, Transth, gamma4, mu; 119.-CD5, gamma2, Transth, gamma4, heparin; 120.-CD5, gamma2, Transth, mu, heparin; 121.-CD5, gamma2, gamma4, mu, heparin; 122.-CD5, apo4, Transth, gamma4, mu; 123.-CD5, apo4, Transth, gamma4, heparin; 124.-CD5, apo4, Transth, mu, heparin; 125.-CD5, apo4, gamma4, mu, heparin; 126.-CD5, Transth, gamma4, mu, heparin; 127.- Creactive, Fibrinogen,
Haptoglobin, gamma2, apo4; 128.- Creactive, Fibrinogen, Haptoglobin, gamma2, Transth; 129.- Creactive, Fibrinogen, Haptoglobin, gamma2, gamma4; 130.- Creactive, Fibrinogen, Haptoglobin, gamma2, mu; 131.- Creactive, Fibrinogen, Haptoglobin, gamma2, heparin; 132.- Creactive, Fibrinogen, Haptoglobin, apo4, Transth; 133.- Creactive, Fibrinogen, Haptoglobin, apo4, gamma4; 134.- Creactive, Fibrinogen, Haptoglobin, apo4, mu; 135.- Creactive, Fibrinogen, Haptoglobin, apo4, heparin; 136.- Creactive, Fibrinogen,
Haptoglobin, Transth, gamma4; 137.- Creactive, Fibrinogen, Haptoglobin, Transth, mu; 138.- Creactive, Fibrinogen, Haptoglobin, Transth, heparin; 139.- Creactive, Fibrinogen, Haptoglobin, gamma4, mu; 140.- Creactive, Fibrinogen, Haptoglobin, gamma4, heparin; 141.- Creactive, Fibrinogen, Haptoglobin, mu, heparin; 142.- Creactive, Fibrinogen, gamma2, apo4, Transth; 143.- Creactive, Fibrinogen, gamma2, apo4, gamma4; 144.- Creactive, Fibrinogen, gamma2, apo4, mu; 145.- Creactive, Fibrinogen, gamma2, apo4, heparin; 146.- Creactive, Fibrinogen, gamma2, Transth, gamma4; 147.- Creactive, Fibrinogen, gamma2, Transth, mu; 148.- Creactive, Fibrinogen, gamma2, Transth, heparin; 149.- Creactive, Fibrinogen, gamma2, gamma4, mu; 150.- Creactive, Fibrinogen, gamma2, gamma4, heparin; 151.- Creactive, Fibrinogen, gamma2, mu, heparin; 152.- Creactive, Fibrinogen, apo4, Transth, gamma4; 153.- Creactive, Fibrinogen, apo4, Transth, mu; 154.- Creactive, Fibrinogen, apo4, Transth, heparin; 155.- Creactive, Fibrinogen, apo4, gamma4, mu; 156.- Creactive, Fibrinogen, apo4, gamma4, heparin; 157.- Creactive, Fibrinogen, apo4, mu, heparin; 158.- Creactive, Fibrinogen, Transth, gamma4, mu; 159.- Creactive, Fibrinogen, Transth, gamma4, heparin; 160.- Creactive, Fibrinogen, Transth, u, heparin; 161.- Creactive, Fibrinogen, gamma4, mu, heparin; 162.- Creactive, Haptoglobin, gamma2, apo4, Transth; 163.- Creactive, Haptoglobin, gamma2, apo4, gamma4; 164.- Creactive, Haptoglobin, gamma2, apo4, mu; 165.- Creactive, Haptoglobin, gamma2, apo4, heparin; 166.- Creactive, Haptoglobin, gamma2, Transth, gamma4; 167.- Creactive, Haptoglobin, gamma2, Transth, mu; 168.- Creactive, Haptoglobin, gamma2, Transth, heparin; 169.- Creactive, Haptoglobin, gamma2, gamma4, mu; 170.- Creactive, Haptoglobin, gamma2, gamma4, heparin; 171.- Creactive, Haptoglobin, gamma2, mu, heparin; 172.- Creactive, Haptoglobin, apo4, Transth, gamma4; 173.- Creactive, Haptoglobin, apo4, Transth, mu; 174.- Creactive, Haptoglobin, apo4, Transth, heparin; 175.- Creactive, Haptoglobin, apo4, gamma4, mu; 176.- Creactive, Haptoglobin, apo4, gamma4, heparin; 177.- Creactive, Haptoglobin, apo4, mu, heparin; 178.- Creactive, Haptoglobin, Transth, gamma4, mu; 179.- Creactive,
Haptoglobin, Transth, gamma4, heparin; 180.- Creactive, Haptoglobin, Transth, mu, heparin; 181.- Creactive, Haptoglobin, gamma4, mu, heparin; 182.- Creactive, gamma2, apo4, Transth, gamma4; 183.- Creactive, gamma2, apo4, Transth, mu; 184.- Creactive, gamma2, apo4, Transth, heparin; 185.- Creactive, gamma2, apo4, gamma4, mu; 186.- Creactive, gamma2, apo4, gamma4, heparin; 187.- Creactive, gamma2, apo4, mu, heparin; 188.- Creactive, gamma2, Transth, gamma4, mu; 189.- Creactive, gamma2, Transth, gamma4, heparin; 190.- Creactive, gamma2, Transth, mu, heparin; 191.-
Creactive, gamma2, gamma4, mu, heparin; 192.- Creactive, apo4, Transth, gamma4, mu; 193.- Creactive, apo4, Transth, gamma4, heparin; 194.- Creactive, apo4, Transth, mu, heparin; 195.- Creactive, apo4, gamma4, mu, heparin; 196.- Creactive, Transth, gamma4, mu, heparin; 197.- Fibrinogen, Haptoglobin, gamma2, apo4, Transth; 198.- Fibrinogen, Haptoglobin, gamma2, apo4, gamma4; 199.- Fibrinogen, Haptoglobin, gamma2, apo4, mu; 200.- Fibrinogen, Haptoglobin, gamma2, apo4, heparin; 201.- Fibrinogen,
Haptoglobin, gamma2, Transth, gamma4; 202.- Fibrinogen, Haptoglobin, gamma2, Transth, mu; 203.- Fibrinogen, Haptoglobin, gamma2, Transth, heparin; 204.- Fibrinogen, Haptoglobin, gamma2, gamma4, mu; 205.- Fibrinogen, Haptoglobin, gamma2, gamma4, heparin; 206.- Fibrinogen, Haptoglobin, gamma2, mu, heparin; 207.- Fibrinogen,
Haptoglobin, apo4, Transth, gamma4; 208.- Fibrinogen, Haptoglobin, apo4, Transth, mu; 209.- Fibrinogen, Haptoglobin, apo4, Transth, heparin; 210.- Fibrinogen, Haptoglobin, apo4, gamma4, mu; 211.- Fibrinogen, Haptoglobin, apo4, gamma4, heparin; 212.- Fibrinogen, Haptoglobin, apo4, mu, heparin; 213.- Fibrinogen, Haptoglobin, Transth, gamma4, mu; 214.- Fibrinogen, Haptoglobin, Transth, gamma4, heparin; 215.-
Fibrinogen, Haptoglobin, Transth, mu, heparin; 216.- Fibrinogen, Haptoglobin, gamma4, mu, heparin; 217.- Fibrinogen, gamma2, apo4, Transth, gamma4; 218.- Fibrinogen, gamma2, apo4, Transth, mu; 219.- Fibrinogen, gamma2, apo4, Transth, heparin; 220.- Fibrinogen, gamma2, apo4, gamma4, mu; 221.- Fibrinogen, gamma2, apo4, gamma4, heparin; 222.- Fibrinogen, gamma2, apo4, mu, heparin; 223.- Fibrinogen, gamma2, Transth, gamma4, u; 224.- Fibrinogen, gamma2, Transth, gamma4, heparin; 225.- Fibrinogen, gamma2, Transth, mu, heparin; 226.- Fibrinogen, gamma2, gamma4, mu, heparin; 227.- Fibrinogen, apo4, Transth, gamma4, mu; 228.- Fibrinogen, apo4, Transth, gamma4, heparin; 229.- Fibrinogen, apo4, Transth, mu, heparin; 230.- Fibrinogen, apo4, gamma4, mu, heparin; 231.- Fibrinogen, Transth, gamma4, mu, heparin; 232.- Haptoglobin, gamma2, apo4, Transth, gamma4; 233.- Haptoglobin, gamma2, apo4, Transth, mu; 234.- Haptoglobin, gamma2, apo4, Transth, heparin; 235.- Haptoglobin, gamma2, apo4, gamma4, mu; 236.- Haptoglobin, gamma2, apo4, gamma4, heparin; 237.- Haptoglobin, gamma2, apo4, mu, heparin; 238.- Haptoglobin, gamma2, Transth, gamma4, mu; 239.- Haptoglobin, gamma2, Transth, gamma4, heparin; 240.- Haptoglobin, gamma2, Transth, mu, heparin; 241.- Haptoglobin, gamma2, gamma4, mu, heparin; 242.- Haptoglobin, apo4, Transth, gamma4, mu; 243.- Haptoglobin, apo4, Transth, gamma4, heparin; 244.- Haptoglobin, apo4, Transth, mu, heparin; 245.- Haptoglobin, apo4, gamma4, mu, heparin; 246.- Haptoglobin, Transth, gamma4, mu, heparin; 247.- gamma2, apo4, Transth, gamma4, mu; 248.- gamma2, apo4, Transth, gamma4, heparin; 249.- gamma2, apo4, Transth, mu, heparin; 250.- gamma2, apo4, gamma4, mu, heparin; 251.- gamma2, Transth, gamma4, mu, heparin; and252.- apo4, Transth, gamma4, mu, heparin. In another particular embodiment of the first aspect the method comprises determining the level of expression of six of the proteins, said six-protein sets (six-membered groups) selected from the group consisting of: 1.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4; 2.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, Transth; 3.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, gamma4; 4.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, mu; 5.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, heparin; 6.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, Transth; 7.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, gamma4; 8.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, mu; 9.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, heparin; 10.-CD5, Creactive, Fibrinogen, Haptoglobin, Transth, gamma4; 11.-CD5, Creactive, Fibrinogen, Haptoglobin, Transth, mu; 12.-CD5, Creactive, Fibrinogen, Haptoglobin, Transth, heparin; 13.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma4, mu; 14.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma4, heparin; 15.-CD5, Creactive, Fibrinogen, Haptoglobin, mu, heparin; 16.-CD5, Creactive, Fibrinogen, gamma2, apo4, Transth; 17.-CD5, Creactive, Fibrinogen, gamma2, apo4, gamma4; 18.-CD5, Creactive, Fibrinogen, gamma2, apo4, mu; 19.-CD5, Creactive, Fibrinogen, gamma2, apo4, heparin; 20.-CD5, Creactive, Fibrinogen, gamma2, Transth, gamma4; 21.-CD5, Creactive, Fibrinogen, gamma2, Transth, mu; 22.-CD5, Creactive, Fibrinogen, gamma2, Transth, heparin; 23.-CD5, Creactive, Fibrinogen, gamma2, gamma4, mu; 24.-CD5, Creactive, Fibrinogen, gamma2, gamma4, heparin; 25.-CD5, Creactive, Fibrinogen, gamma2, mu, heparin; 26.-CD5, Creactive, Fibrinogen, apo4, Transth, gamma4; 27.-CD5, Creactive, Fibrinogen, apo4, Transth, mu; 28.-CD5, Creactive, Fibrinogen, apo4, Transth, heparin
29.-CD5, Creactive, Fibrinogen, apo4, gamma4, u; 30.-CD5, Creactive, Fibrinogen, apo4, gamma4, heparin; 31.-CD5, Creactive, Fibrinogen, apo4, mu, heparin; 32.-CD5, Creactive, Fibrinogen, Transth, gamma4, mu; 33.-CD5, Creactive, Fibrinogen, Transth, gamma4, heparin; 34.-CD5, Creactive, Fibrinogen, Transth, mu, heparin; 35.-CD5, Creactive, Fibrinogen, gamma4, mu, heparin; 36.-CD5, Creactive, Haptoglobin, gamma2, apo4, Transth; 37.-CD5, Creactive, Haptoglobin, gamma2, apo4, gamma4; 38.-CD5, Creactive, Haptoglobin, gamma2, apo4, mu; 39.-CD5, Creactive, Haptoglobin, gamma2, apo4, heparin; 40.-CD5, Creactive, Haptoglobin, gamma2, Transth, gamma4; 41.-CD5, Creactive, Haptoglobin, gamma2, Transth, mu; 42.-CD5, Creactive, Haptoglobin, gamma2, Transth, heparin; 43.-CD5, Creactive, Haptoglobin, gamma2, gamma4, mu; 44.- CD5, Creactive, Haptoglobin, gamma2, gamma4, heparin; 45.-CD5, Creactive,
Haptoglobin, gamma2, mu, heparin; 46.-CD5, Creactive, Haptoglobin, apo4, Transth, gamma4; 47.-CD5, Creactive, Haptoglobin, apo4, Transth, mu; 48.-CD5, Creactive, Haptoglobin, apo4, Transth, heparin; 49.-CD5, Creactive, Haptoglobin, apo4, gamma4, mu; 50.-CD5, Creactive, Haptoglobin, apo4, gamma4, heparin; 51.-CD5, Creactive, Haptoglobin, apo4, mu, heparin; 52.-CD5, Creactive, Haptoglobin, Transth, gamma4, mu; 53.-CD5, Creactive, Haptoglobin, Transth, gamma4, heparin; 54.-CD5, Creactive, Haptoglobin, Transth, mu, heparin; 55.-CD5, Creactive, Haptoglobin, gamma4, mu, heparin; 56.-CD5, Creactive, gamma2, apo4, Transth, gamma4; 57.-CD5, Creactive, gamma2, apo4, Transth, mu; 58.-CD5, Creactive, gamma2, apo4, Transth, heparin; 59.- CD5, Creactive, gamma2, apo4, gamma4, mu; 60.-CD5, Creactive, gamma2, apo4, gamma4, heparin; 61.-CD5, Creactive, gamma2, apo4, mu, heparin; 62.-CD5, Creactive, gamma2, Transth, gamma4, mu; 63.-CD5, Creactive, gamma2, Transth, gamma4, heparin; 64.-CD5, Creactive, gamma2, Transth, mu, heparin; 65.-CD5, Creactive, gamma2, gamma4, mu, heparin; 66.-CD5, Creactive, apo4, Transth, gamma4, mu; 67.- CD5, Creactive, apo4, Transth, gamma4, heparin; 68.-CD5, Creactive, apo4, Transth, mu, heparin; 69.-CD5, Creactive, apo4, gamma4, mu, heparin; 70.-CD5, Creactive, Transth, gamma4, mu, heparin; 71.-CD5, Fibrinogen, Haptoglobin, gamma2, apo4, Transth; 72.- CD5, Fibrinogen, Haptoglobin, gamma2, apo4, gamma4; 73.-CD5, Fibrinogen,
Haptoglobin, gamma2, apo4, mu; 74.-CD5, Fibrinogen, Haptoglobin, gamma2, apo4, heparin; 75.-CD5, Fibrinogen, Haptoglobin, gamma2, Transth, gamma4; 76.-CD5, Fibrinogen, Haptoglobin, gamma2, Transth, mu; 77.-CD5, Fibrinogen, Haptoglobin, gamma2, Transth, heparin; 78.-CD5, Fibrinogen, Haptoglobin, gamma2, gamma4, mu; 79.-CD5, Fibrinogen, Haptoglobin, gamma2, gamma4, heparin; 80.-CD5, Fibrinogen, Haptoglobin, gamma2, mu, heparin; 81.-CD5, Fibrinogen, Haptoglobin, apo4, Transth, gamma4; 82.-CD5, Fibrinogen, Haptoglobin, apo4, Transth, mu; 83.-CD5, Fibrinogen, Haptoglobin, apo4, Transth, heparin; 84.-CD5, Fibrinogen, Haptoglobin, apo4, gamma4, mu; 85.-CD5, Fibrinogen, Haptoglobin, apo4, gamma4, heparin; 86.-CD5, Fibrinogen, Haptoglobin, apo4, u, heparin; 87.-CD5, Fibrinogen, Haptoglobin, Transth, gamma4, mu; 88.-CD5, Fibrinogen, Haptoglobin, Transth, gamma4, heparin; 89.-CD5, Fibrinogen, Haptoglobin, Transth, mu, heparin; 90.-CD5, Fibrinogen, Haptoglobin, gamma4, mu, heparin; 91.-CD5, Fibrinogen, gamma2, apo4, Transth, gamma4; 92.-CD5, Fibrinogen, gamma2, apo4, Transth, mu; 93.-CD5, Fibrinogen, gamma2, apo4, Transth, heparin; 94.- CD5, Fibrinogen, gamma2, apo4, gamma4, mu; 95.-CD5, Fibrinogen, gamma2, apo4, gamma4, heparin; 96.-CD5, Fibrinogen, gamma2, apo4, mu, heparin; 97.-CD5,
Fibrinogen, gamma2, Transth, gamma4, mu; 98.-CD5, Fibrinogen, gamma2, Transth, gamma4, heparin; 99.-CD5, Fibrinogen, gamma2, Transth, mu, heparin; 100.-CD5, Fibrinogen, gamma2, gamma4, mu, heparin; 101.-CD5, Fibrinogen, apo4, Transth, gamma4, mu; 102.-CD5, Fibrinogen, apo4, Transth, gamma4, heparin; 103.-CD5, Fibrinogen, apo4, Transth, mu, heparin; 104.-CD5, Fibrinogen, apo4, gamma4, mu, heparin; 105.-CD5, Fibrinogen, Transth, gamma4, mu, heparin; 106.-CD5, Haptoglobin, gamma2, apo4, Transth, gamma4; 107.-CD5, Haptoglobin, gamma2, apo4, Transth, mu; 108.-CD5, Haptoglobin, gamma2, apo4, Transth, heparin; 109.-CD5, Haptoglobin, gamma2, apo4, gamma4, mu; 110.-CD5, Haptoglobin, gamma2, apo4, gamma4, heparin; 111.-CD5, Haptoglobin, gamma2, apo4, mu, heparin; 112.-CD5, Haptoglobin, gamma2, Transth, gamma4, mu; 113.-CD5, Haptoglobin, gamma2, Transth, gamma4, heparin; 114.-CD5, Haptoglobin, gamma2, Transth, mu, heparin; 115.-CD5, Haptoglobin, gamma2, gamma4, mu, heparin; 116.-CD5, Haptoglobin, apo4, Transth, gamma4, mu; 117.-CD5, Haptoglobin, apo4, Transth, gamma4, heparin; 118.-CD5, Haptoglobin, apo4, Transth, mu, heparin; 119.-CD5, Haptoglobin, apo4, gamma4, mu, heparin; 120.-CD5,
Haptoglobin, Transth, gamma4, mu, heparin; 121.-CD5, gamma2, apo4, Transth, gamma4, mu; 122.-CD5, gamma2, apo4, Transth, gamma4, heparin; 123.-CD5, gamma2, apo4, Transth, mu, heparin; 124.-CD5, gamma2, apo4, gamma4, mu, heparin; 125.-CD5, gamma2, Transth, gamma4, mu, heparin; 126.-CD5, apo4, Transth, gamma4, mu, heparin; 127.- Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, Transth; 128.- Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, gamma4; 129.- Creactive,
Fibrinogen, Haptoglobin, gamma2, apo4, mu; 130.- Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, heparin; 131.- Creactive, Fibrinogen, Haptoglobin, gamma2, Transth, gamma4; 132.- Creactive, Fibrinogen, Haptoglobin, gamma2, Transth, mu; 133.- Creactive, Fibrinogen, Haptoglobin, gamma2, Transth, heparin; 134.- Creactive,
Fibrinogen, Haptoglobin, gamma2, gamma4, mu; 135.- Creactive, Fibrinogen,
Haptoglobin, gamma2, gamma4, heparin; 136.- Creactive, Fibrinogen, Haptoglobin, gamma2, mu, heparin; 137.- Creactive, Fibrinogen, Haptoglobin, apo4, Transth, gamma4; 138.- Creactive, Fibrinogen, Haptoglobin, apo4, Transth, mu; 139.- Creactive, Fibrinogen, Haptoglobin, apo4, Transth, heparin; 140.- Creactive, Fibrinogen, Haptoglobin, apo4, gamma4, mu; 141.- Creactive, Fibrinogen, Haptoglobin, apo4, gamma4, heparin; 142.- Creactive, Fibrinogen, Haptoglobin, apo4, mu, heparin; 143.- Creactive, Fibrinogen, Haptoglobin, Transth, gamma4, u; 144.- Creactive, Fibrinogen, Haptoglobin, Transth, gamma4, heparin; 145.- Creactive, Fibrinogen, Haptoglobin, Transth, mu, heparin; 146.- Creactive, Fibrinogen, Haptoglobin, gamma4, mu, heparin; 147.- Creactive, Fibrinogen, gamma2, apo4, Transth, gamma4; 148.- Creactive, Fibrinogen, gamma2, apo4, Transth, mu; 149.- Creactive, Fibrinogen, gamma2, apo4, Transth, heparin; 150.- Creactive, Fibrinogen, gamma2, apo4, gamma4, mu; 151.- Creactive, Fibrinogen, gamma2, apo4, gamma4, heparin; 152.- Creactive, Fibrinogen, gamma2, apo4, mu, heparin; 153.- Creactive, Fibrinogen, gamma2, Transth, gamma4, mu; 154.- Creactive, Fibrinogen, gamma2, Transth, gamma4, heparin; 155.- Creactive, Fibrinogen, gamma2, Transth, mu, heparin; 156.- Creactive, Fibrinogen, gamma2, gamma4, mu, heparin; 157.- Creactive, Fibrinogen, apo4, Transth, gamma4, mu; 158.- Creactive, Fibrinogen, apo4, Transth, gamma4, heparin; 159.- Creactive, Fibrinogen, apo4, Transth, mu, heparin; 160.- Creactive, Fibrinogen, apo4, gamma4, mu, heparin; 161.- Creactive, Fibrinogen, Transth, gamma4, mu, heparin; 162.- Creactive, Haptoglobin, gamma2, apo4, Transth, gamma4; 163.- Creactive, Haptoglobin, gamma2, apo4, Transth, mu; 164.- Creactive, Haptoglobin, gamma2, apo4, Transth, heparin; 165.- Creactive, Haptoglobin, gamma2, apo4, gamma4, mu; 166.- Creactive, Haptoglobin, gamma2, apo4, gamma4, heparin; 167.- Creactive, Haptoglobin, gamma2, apo4, mu, heparin; 168.- Creactive, Haptoglobin, gamma2, Transth, gamma4, mu; 169.- Creactive, Haptoglobin, gamma2, Transth, gamma4, heparin; 170.- Creactive, Haptoglobin, gamma2, Transth, mu, heparin; 171.- Creactive, Haptoglobin, gamma2, gamma4, mu, heparin; 172.- Creactive, Haptoglobin, apo4, Transth, gamma4, mu; 173.- Creactive, Haptoglobin, apo4, Transth, gamma4, heparin; 174.- Creactive, Haptoglobin, apo4, Transth, mu, heparin; 175.- Creactive, Haptoglobin, apo4, gamma4, mu, heparin; 176.- Creactive, Haptoglobin, Transth, gamma4, mu, heparin; 177.- Creactive, gamma2, apo4, Transth, gamma4, mu; 178.- Creactive, gamma2, apo4, Transth, gamma4, heparin; 179.- Creactive, gamma2, apo4, Transth, mu, heparin; 180.- Creactive, gamma2, apo4, gamma4, mu, heparin; 181.- Creactive, gamma2, Transth, gamma4, mu, heparin; 182.- Creactive, apo4, Transth, gamma4, mu, heparin; 183.- Fibrinogen, Haptoglobin, gamma2, apo4, Transth, gamma4; 184.-
Fibrinogen, Haptoglobin, gamma2, apo4, Transth, mu; 185.- Fibrinogen, Haptoglobin, gamma2, apo4, Transth, heparin; 186.- Fibrinogen, Haptoglobin, gamma2, apo4, gamma4, mu; 187.- Fibrinogen, Haptoglobin, gamma2, apo4, gamma4, heparin; 188.- Fibrinogen, Haptoglobin, gamma2, apo4, mu, heparin; 189.- Fibrinogen, Haptoglobin, gamma2, Transth, gamma4, mu; 190.- Fibrinogen, Haptoglobin, gamma2, Transth, gamma4, heparin; 191.- Fibrinogen, Haptoglobin, gamma2, Transth, mu, heparin; 192.- Fibrinogen, Haptoglobin, gamma2, gamma4, mu, heparin; 193.- Fibrinogen, Haptoglobin, apo4, Transth, gamma4, mu; 194.- Fibrinogen, Haptoglobin, apo4, Transth, gamma4, heparin; 195.- Fibrinogen, Haptoglobin, apo4, Transth, mu, heparin; 196.- Fibrinogen, Haptoglobin, apo4, gamma4, mu, heparin; 197.- Fibrinogen, Haptoglobin, Transth, gamma4, u, heparin; 198.- Fibrinogen, gamma2, apo4, Transth, gamma4, mu; 199.- Fibrinogen, gamma2, apo4, Transth, gamma4, heparin; 200.- Fibrinogen, gamma2, apo4, Transth, mu, heparin; 201.- Fibrinogen, gamma2, apo4, gamma4, mu, heparin; 202.- Fibrinogen, gamma2, Transth, gamma4, mu, heparin; 203.- Fibrinogen, apo4, Transth, gamma4, mu, heparin; 204.- Haptoglobin, gamma2, apo4, Transth, gamma4, mu; 205.- Haptoglobin, gamma2, apo4, Transth, gamma4, heparin; 206.- Haptoglobin, gamma2, apo4, Transth, mu, heparin; 207.- Haptoglobin, gamma2, apo4, gamma4, mu, heparin; 208.- Haptoglobin, gamma2, Transth, gamma4, mu, heparin; 209.- Haptoglobin, apo4, Transth, gamma4, mu, heparin; and 210.- gamma2, apo4, Transth, gamma4, mu, heparin
In another particular embodiment of the first aspect, the method comprises determining the level of expression of seven of the proteins, said seven-protein sets (seven-membered groups) selected from the group consisting of:1.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, Transth; 2.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, gamma4; 3.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, mu; 4.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, heparin; 5.-CD5, Creactive,
Fibrinogen, Haptoglobin, gamma2, Transth, gamma4; 6.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, Transth, mu; 7.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, Transth, heparin; 8.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, gamma4, mu; 9.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, gamma4, heparin; 10.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, mu, heparin; 11.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, Transth, gamma4; 12.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, Transth, mu; 13.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, Transth, heparin; 14.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, gamma4, mu; 15.- CD5, Creactive, Fibrinogen, Haptoglobin, apo4, gamma4, heparin; 16.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, mu, heparin; 17.-CD5, Creactive, Fibrinogen, Haptoglobin, Transth, gamma4, mu; 18.-CD5, Creactive, Fibrinogen, Haptoglobin, Transth, gamma4, heparin; 19.-CD5, Creactive, Fibrinogen, Haptoglobin, Transth, mu, heparin; 20.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma4, mu, heparin; 21.-CD5, Creactive,
Fibrinogen, gamma2, apo4, Transth, gamma4; 22.-CD5, Creactive, Fibrinogen, gamma2, apo4, Transth, mu; 23.-CD5, Creactive, Fibrinogen, gamma2, apo4, Transth, heparin; 24.- CD5, Creactive, Fibrinogen, gamma2, apo4, gamma4, mu; 25.-CD5, Creactive,
Fibrinogen, gamma2, apo4, gamma4, heparin; 26.-CD5, Creactive, Fibrinogen, gamma2, apo4, mu, heparin; 27.-CD5, Creactive, Fibrinogen, gamma2, Transth, gamma4, mu; 28.- CD5, Creactive, Fibrinogen, gamma2, Transth, gamma4, heparin; 29.-CD5, Creactive, Fibrinogen, gamma2, Transth, mu, heparin; 30.-CD5, Creactive, Fibrinogen, gamma2, gamma4, mu, heparin; 31.-CD5, Creactive, Fibrinogen, apo4, Transth, gamma4, mu; 32.- CD5, Creactive, Fibrinogen, apo4, Transth, gamma4, heparin; 33.-CD5, Creactive, Fibrinogen, apo4, Transth, mu, heparin; 34.-CD5, Creactive, Fibrinogen, apo4, gamma4, u, heparin; 35.-CD5, Creactive, Fibrinogen, Transth, gamma4, mu, heparin; 36.-CD5, Creactive, Haptoglobin, gamma2, apo4, Transth, gamma4; 37.-CD5, Creactive,
Haptoglobin, gamma2, apo4, Transth, mu; 38.-CD5, Creactive, Haptoglobin, gamma2, apo4, Transth, heparin; 39.-CD5, Creactive, Haptoglobin, gamma2, apo4, gamma4, mu; 40.-CD5, Creactive, Haptoglobin, gamma2, apo4, gamma4, heparin; 41.-CD5, Creactive, Haptoglobin, gamma2, apo4, mu, heparin; 42.-CD5, Creactive, Haptoglobin, gamma2, Transth, gamma4, mu; 43.-CD5, Creactive, Haptoglobin, gamma2, Transth, gamma4, heparin; 44.-CD5, Creactive, Haptoglobin, gamma2, Transth, mu, heparin; 45.-CD5, Creactive, Haptoglobin, gamma2, gamma4, mu, heparin; 46.-CD5, Creactive,
Haptoglobin, apo4, Transth, gamma4, mu; 47.-CD5, Creactive, Haptoglobin, apo4, Transth, gamma4, heparin; 48.-CD5, Creactive, Haptoglobin, apo4, Transth, mu, heparin; 49.-CD5, Creactive, Haptoglobin, apo4, gamma4, mu, heparin; 50.-CD5, Creactive, Haptoglobin, Transth, gamma4, mu, heparin; 51.-CD5, Creactive, gamma2, apo4, Transth, gamma4, mu; 52.-CD5, Creactive, gamma2, apo4, Transth, gamma4, heparin; 53.-CD5, Creactive, gamma2, apo4, Transth, mu, heparin; 54.-CD5, Creactive, gamma2, apo4, gamma4, mu, heparin; 55.-CD5, Creactive, gamma2, Transth, gamma4, mu, heparin; 56.-CD5, Creactive, apo4, Transth, gamma4, mu, heparin; 57.-CD5, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, gamma4; 58.-CD5, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, mu; 59.-CD5, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, heparin; 60.-CD5, Fibrinogen, Haptoglobin, gamma2, apo4, gamma4, mu; 61.-CD5, Fibrinogen, Haptoglobin, gamma2, apo4, gamma4, heparin; 62.-CD5, Fibrinogen, Haptoglobin, gamma2, apo4, mu, heparin; 63.-CD5, Fibrinogen, Haptoglobin, gamma2, Transth, gamma4, mu; 64.-CD5, Fibrinogen, Haptoglobin, gamma2, Transth, gamma4, heparin; 65.-CD5, Fibrinogen, Haptoglobin, gamma2, Transth, mu, heparin; 66.-CD5, Fibrinogen, Haptoglobin, gamma2, gamma4, mu, heparin; 67.-CD5, Fibrinogen,
Haptoglobin, apo4, Transth, gamma4, mu; 68.-CD5, Fibrinogen, Haptoglobin, apo4, Transth, gamma4, heparin; 69.-CD5, Fibrinogen, Haptoglobin, apo4, Transth, mu, heparin; 70.-CD5, Fibrinogen, Haptoglobin, apo4, gamma4, mu, heparin; 71.-CD5, Fibrinogen, Haptoglobin, Transth, gamma4, mu, heparin; 72.-CD5, Fibrinogen, gamma2, apo4, Transth, gamma4, mu; 73.-CD5, Fibrinogen, gamma2, apo4, Transth, gamma4, heparin; 74.-CD5, Fibrinogen, gamma2, apo4, Transth, mu, heparin; 75.-CD5, Fibrinogen, gamma2, apo4, gamma4, mu, heparin; 76.-CD5, Fibrinogen, gamma2, Transth, gamma4, mu, heparin; 77.-CD5, Fibrinogen, apo4, Transth, gamma4, mu, heparin; 78.-CD5, Haptoglobin, gamma2, apo4, Transth, gamma4, mu; 79.-CD5, Haptoglobin, gamma2, apo4, Transth, gamma4, heparin; 80.-CD5, Haptoglobin, gamma2, apo4, Transth, mu, heparin; 81.-CD5, Haptoglobin, gamma2, apo4, gamma4, mu, heparin; 82.-CD5,
Haptoglobin, gamma2, Transth, gamma4, mu, heparin; 83.-CD5, Haptoglobin, apo4, Transth, gamma4, mu, heparin; 84.-CD5, gamma2, apo4, Transth, gamma4, mu, heparin; 85.- Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, gamma4; 86.- Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, mu; 87.- Creactive,
Fibrinogen, Haptoglobin, gamma2, apo4, Transth, heparin; 88.- Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, ga a4, u; 89.- Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, gamma4, heparin; 90.- Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, mu, heparin; 91.- Creactive, Fibrinogen, Haptoglobin, gamma2, Transth, gamma4, mu; 92.- Creactive, Fibrinogen, Haptoglobin, gamma2, Transth, gamma4, heparin; 93.- Creactive, Fibrinogen, Haptoglobin, gamma2, Transth, mu, heparin; 94.- Creactive, Fibrinogen, Haptoglobin, gamma2, gamma4, mu, heparin; 95.- Creactive, Fibrinogen, Haptoglobin, apo4, Transth, gamma4, mu; 96.- Creactive, Fibrinogen, Haptoglobin, apo4, Transth, gamma4, heparin; 97.- Creactive, Fibrinogen, Haptoglobin, apo4, Transth, mu, heparin; 98.- Creactive, Fibrinogen, Haptoglobin, apo4, gamma4, mu, heparin; 99.- Creactive, Fibrinogen, Haptoglobin, Transth, gamma4, mu, heparin; 100.- Creactive, Fibrinogen, gamma2, apo4, Transth, gamma4, mu; 101.- Creactive, Fibrinogen, gamma2, apo4, Transth, gamma4, heparin; 102.- Creactive, Fibrinogen, gamma2, apo4, Transth, mu, heparin; 103.- Creactive, Fibrinogen, gamma2, apo4, gamma4, mu, heparin; 104.- Creactive, Fibrinogen, gamma2, Transth, gamma4, mu, heparin; 105.- Creactive, Fibrinogen, apo4, Transth, gamma4, mu, heparin; 106.- Creactive, Haptoglobin, gamma2, apo4, Transth, gamma4, mu; 107.- Creactive, Haptoglobin, gamma2, apo4, Transth, gamma4, heparin; 108.- Creactive, Haptoglobin, gamma2, apo4, Transth, mu, heparin; 109.- Creactive, Haptoglobin, gamma2, apo4, gamma4, mu, heparin; 110.- Creactive, Haptoglobin, gamma2, Transth, gamma4, mu, heparin; 111.- Creactive, Haptoglobin, apo4, Transth, gamma4, mu, heparin; 112.- Creactive, gamma2, apo4, Transth, gamma4, mu, heparin; 113.- Fibrinogen, Haptoglobin, gamma2, apo4, Transth, gamma4, mu; 114.- Fibrinogen, Haptoglobin, gamma2, apo4, Transth, gamma4, heparin; 115.- Fibrinogen, Haptoglobin, gamma2, apo4, Transth, mu, heparin; 116.- Fibrinogen, Haptoglobin, gamma2, apo4, gamma4, mu, heparin; 117.- Fibrinogen, Haptoglobin, gamma2, Transth, gamma4, mu, heparin; 118.- Fibrinogen, Haptoglobin, apo4, Transth, gamma4, mu, heparin; 119.- Fibrinogen, gamma2, apo4, Transth, gamma4, mu, heparin; and 120.- Haptoglobin, gamma2, apo4, Transth, gamma4, mu, heparin.
In another particular embodiment of the first aspect, the method comprises determining the level of expression of eight of the proteins, said eight-protein sets (eight-membered groups) selected from the group consisting of: 1.-CD5, Creactive, Fibrinogen,
Haptoglobin, gamma2, apo4, Transth, gamma4; 2.-CD5, Creactive, Fibrinogen,
Haptoglobin, gamma2, apo4, Transth, mu; 3.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, heparin; 4.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, gamma4, mu; 5.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, gamma4, heparin; 6.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, mu, heparin; 7.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, Transth, gamma4, mu; 8.- CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, Transth, gamma4, heparin; 9.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, Transth, u, heparin; 10.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, gamma4, mu, heparin; 11.-CD5, Creactive,
Fibrinogen, Haptoglobin, apo4, Transth, gamma4, mu; 12.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, Transth, gamma4, heparin; 13.-CD5, Creactive, Fibrinogen,
Haptoglobin, apo4, Transth, mu, heparin; 14.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, gamma4, mu, heparin; 15.-CD5, Creactive, Fibrinogen, Haptoglobin, Transth, gamma4, mu, heparin; 16.-CD5, Creactive, Fibrinogen, gamma2, apo4, Transth, gamma4, mu; 17.-CD5, Creactive, Fibrinogen, gamma2, apo4, Transth, gamma4, heparin; 18.-CD5, Creactive, Fibrinogen, gamma2, apo4, Transth, mu, heparin; 19.-CD5, Creactive, Fibrinogen, gamma2, apo4, gamma4, mu, heparin; 20.-CD5, Creactive, Fibrinogen, gamma2, Transth, gamma4, mu, heparin; 21.-CD5, Creactive, Fibrinogen, apo4, Transth, gamma4, mu, heparin; 22.-CD5, Creactive, Haptoglobin, gamma2, apo4, Transth, gamma4, mu; 23.-CD5, Creactive, Haptoglobin, gamma2, apo4, Transth, gamma4, heparin; 24.-CD5, Creactive, Haptoglobin, gamma2, apo4, Transth, mu, heparin; 25.-CD5, Creactive, Haptoglobin, gamma2, apo4, gamma4, mu, heparin; 26.- CD5, Creactive, Haptoglobin, gamma2, Transth, gamma4, mu, heparin; 27.-CD5, Creactive, Haptoglobin, apo4, Transth, gamma4, mu, heparin; 28.-CD5, Creactive, gamma2, apo4, Transth, gamma4, mu, heparin; 29.-CD5, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, gamma4, mu; 30.-CD5, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, gamma4, heparin; 31.-CD5, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, mu, heparin; 32.-CD5, Fibrinogen, Haptoglobin, gamma2, apo4, gamma4, mu, heparin; 33.-CD5, Fibrinogen, Haptoglobin, gamma2, Transth, gamma4, mu, heparin; 34.- CD5, Fibrinogen, Haptoglobin, apo4, Transth, gamma4, mu, heparin; 35.-CD5,
Fibrinogen, gamma2, apo4, Transth, gamma4, mu, heparin; 36.-CD5, Haptoglobin, gamma2, apo4, Transth, gamma4, mu, heparin; 37.- Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, gamma4, mu; 38.- Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, gamma4, heparin; 39.- Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, mu, heparin; 40.- Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, gamma4, mu, heparin; 41.- Creactive, Fibrinogen, Haptoglobin, gamma2, Transth, gamma4, mu, heparin; 42.- Creactive, Fibrinogen, Haptoglobin, apo4, Transth, gamma4, mu, heparin; 43.- Creactive, Fibrinogen, gamma2, apo4, Transth, gamma4, mu, heparin; 44.- Creactive, Haptoglobin, gamma2, apo4, Transth, gamma4, mu, heparin; and 45.- Fibrinogen, Haptoglobin, gamma2, apo4, Transth, gamma4, mu, heparin
In another particular embodiment of the first aspect, the method comprises determining the level of expression of nine of the proteins, said nine-protein sets (nine-membered groups) selected from the group consisting of: 1.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, gamma4, mu; 2.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, ga a4, heparin; 3.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, u, heparin; 4.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, apo4, gamma4, mu, heparin; 5.-CD5, Creactive, Fibrinogen, Haptoglobin, gamma2, Transth, gamma4, mu, heparin; 6.-CD5, Creactive, Fibrinogen, Haptoglobin, apo4, Transth, gamma4, mu, heparin; 7.-CD5, Creactive, Fibrinogen, gamma2, apo4, Transth, gamma4, mu, heparin; 8.-CD5, Creactive, Haptoglobin, gamma2, apo4, Transth, gamma4, mu, heparin; 9.-CD5, Fibrinogen, Haptoglobin, gamma2, apo4, Transth, gamma4, mu, heparin; and 10.- Creactive, Fibrinogen,
Haptoglobin, gamma2, apo4, Transth, gamma4, mu, heparin
Once the level of expression of one or more of the proteins have been determined, the subject suffering from carotid stenosis, previously diagnosed by methods known for the skilled man in the art, can be correctly classified as an asymptomatic if the detected values correspond to asymptomatic reference values or as symptomatic if the detected in the sample values (also called test values) correspond to symptomatic reference values.
In addition, and as will be shown below, if the subject is classified as an asymptomatic patient, the level of expression of additional proteins allow the correct classification among asymptomatic stable patients and asymptomatic progressive patients. This further classification is also of particular interest in order to decide if surgical therapy is finally applied as asymptomatic progressive stenosis have higher stroke risk in comparison with stable stenosis. In a more particular embodiment, the reference value is selected from the level of expression of the one or more proteins in a symptomatic subject, the level of expression of the one or more proteins in an asymptomatic subject, and a control non-suffering carotid stenosis. Said reference value can be a discrete value or a range of values in which one of the conditions to be discriminated are included.
In another particular embodiment of the method of the invention according to the first and second aspects, if the subject is diagnosed as suffering from asymptomatic carotid stenosis, the method further comprises the step of determining in the isolated sample the level of expression of one or more of the proteins selected from the group consisting of Immunoglobulin lambda variable 2-18, Immunoglobulin heavy constant delta, Insulin-like growth factor-binding protein 3, Haptoglobin, Alpha-2-HS-glycoprotein, Immunoglobulin kappa variable 3-15, Antithrombin-Ill, Immunoglobulin kappa variable 4-1 , Gelsolin, Immunoglobulin heavy constant gamma 3 and Caveolin-1. In a more particular embodiment, of the method of the invention according to the first and second aspects, if the subject is diagnosed as suffering from asymptomatic carotid stenosis, the method further comprises the step of determining in the isolated sample the level of expression of one or more of the proteins selected from the group consisting of Immunoglobulin kappa variable 4-1 , Gelsolin, Immunoglobulin heavy constant gamma 3 and Caveolin-1.AII these additional proteins are reliable individual markers distinguishing among progressive asymptomatic and stable asymptomatic subjects. Differential diagnosis among asymptomatic subjects allows accommodating a proper therapy regimen recommendation. For example, in some progressive asymptomatic subjects CEA or stenting can be recommended if other additional physical parameters (imaging) or clinical parameters (age, gender, etc.) are in favor of these therapies. The proteins
Immunoglobulin kappa variable 4-1 , Gelsolin, Immunoglobulin heavy constant gamma 3 are of particular interest because their levels in the isolated biofluid samples correlate with the levels in atheromatous plaque.
In another particular embodiment of the method of the invention according to the first and second aspects, if the subject is diagnosed as suffering from asymptomatic carotid stenosis, the method further comprises the step of determining in the isolated sample the level of expression of one or more of the proteins selected from the group consisting of Immunoglobulin lambda variable 2-18, Immunoglobulin heavy constant delta, Insulin-like growth factor-binding protein 3, Haptoglobin, Alpha-2-HS-glycoprotein, Immunoglobulin kappa variable 3-15, Antithrombin-Ill, and Immunoglobulin kappa variable 4-1.
In a more particular embodiment of the method of the invention according to the first and second aspects, if the subject is diagnosed as suffering from asymptomatic carotid stenosis, the method further comprises the step of determining in the isolated sample the level of expression of one or more of Immunoglobulin kappa variable 4-1 and Gelsolin. Levels of both proteins determined in combination allow a very good classification.
Gelsolin levels are elevated in asymptomatic stable subjects in relation with progressive (1.225 fold change, p=0.021), and the levels of Immunoglobulin kappa variable 4-1 are reduced in asymptomatic stable subjects in relation with progressive (0.774 fold change, p=0.035). This reduction of the levels is also corroborated in atheroma plaques, thus, again representing a real image in biofluid samples of the atheroma plaque. Thus, these proteins are, in a particular embodiment, of the second aspect, used in combination with the levels of CD5 antigen-like to select a patient as candidate for surgical therapy.
In a more particular embodiment of the method of the invention according to the first and second aspects, if the subject is diagnosed as suffering from asymptomatic carotid stenosis, the method further comprises the step of determining in the isolated sample the level of expression of Immunoglobulin heavy constant gamma 3. The levels of expression of this protein are lower in stable asymptomatic carotid stenosis subjects in relation with progressive asymptomatic subjects and symptomatic subjects (0.662 fold change, p=0.016). Thus, this protein is, in a particular embodiment of the second aspect, used in combination with the levels of CD5 antigen-like to select a patient as candidate for surgical therapy.
In yet another more particular embodiment of the method of the invention according to the first and second aspects, if the subject is diagnosed as suffering from asymptomatic carotid stenosis, the method further comprises the step of determining in the isolated sample the level of expression of Caveolin-1. In a similar way than Immunoglobulin heavy constant gamma 3, Caveolin-1 levels in isolated biofluid samples are lower in stable asymptomatic subjects than in progressive asymptomatic and in symptomatic patients (0.517 fold change, p=0.000). In a cohort analysing only serum samples, Caveolin-1 differentially distinguished asymptomatic stable patients from asymptomatic progressive and symptomatic patients with an accuracy (AUC, 95% Cl) of 0.745 (0.621-0.869), using a cutoff of 0.484 ng/ml. Correspondent sensitivity was of 0.61 , the specificity was of 0.81 , being the positive predictive value (PPV) of 0.97 and the negative predictive value (NVP) of 0.19.
Thus, it is also disclosed a method for the differential diagnosis of asymptomatic stable carotid stenosis patients from asymptomatic progressive and symptomatics that comprises determining in isolated serum the levels of caveolin-1. Also an in vitro method for selecting a subject suffering from carotid stenosis as candidate for surgical therapy, comprising determining in an isolated biofluid sample of a subject the level of expression of Caveolin-1 , wherein the level is optionally compared with a reference value or interval of values, and the subject is selected as candidate if the levels in the sample match with those of symptomatic or of asymptomaic progressive patients. Correct discrimination of stable asymptomatic subjects from progressive asymptomatic patiens, as well as symptomatic subjects, is of interest, in order to further value other clinical parameters to finally decide to recommend a therapeutic surgery in progressive asymptomatic subjects as in symptomatic subjects, as previously indicated. In even a more particular embodiment of the method of the invention according to the first and second aspects, if the subject is diagnosed as suffering from asymptomatic carotid stenosis, the method further comprises the step of determining in the isolated sample the level of expression of two or more of the proteins Immunoglobulin lambda variable 2-18, Immunoglobulin heavy constant delta, Insulin-like growth factor-binding protein 3, Haptoglobin, Alpha-2-HS-glycoprotein, Immunoglobulin kappa variable 3-15,
Antithrombin-Ill, Immunoglobulin kappa variable 4-1 , Gelsolin, Immunoglobulin heavy constant gamma 3 and Caveolin-1. More in particular of three, four, five proteins, six, seven, eight, nine, ten and the eleven proteins. More in particular the level of expression of all of Immunoglobulin lambda variable 2-18, Immunoglobulin heavy constant delta, Insulin-like growth factor-binding protein 3, Haptoglobin, Alpha-2-HS-glycoprotein, Immunoglobulin kappa variable 3-15, Antithrombin-Ill, Immunoglobulin kappa variable 4-1 , Gelsolin, Immunoglobulin heavy constant gamma 3 and Caveolin-1. Inventors also detected the differential expression pattern of other proteins between biofluid samples of symptomatic and asymptomatic carotid stenosis, including stable asymptomatic carotid stenosis and progressive asymptomatic carotid stenosis. Thus, in another particular embodiment of the methods of the invention, optionally in combination with any embodiment above or below, the method comprise further determining the level of expression of one or more proteins selected from the group consisting of
immunoglobulin kappa variable 1-13, Immunoglobulin heavy variable 2-26,
Immunoglobulin lambda variable 2-18, Immunoglobulin heavy variable 3-72, Fetuin-B, Immunoglobulin kappa variable 6D-21 , Immunoglobulin heavy variable3-64D, Actin cytoplasmic 2, Immunoglobulin kappa variable 3-20, Serum amyloid A-1 protein,
Apolipoprotein C-lll, Immunoglobulin heavy variable 1-18, C4b-binding protein alpha chain, Serum amyloid A-4 protein, Immunoglobulin lambda variable 1-40, Immunoglobulin lambda variable 4-69, Immunoglobulin heavy variable 4-34, Immunoglobulin kappa variable 1-5, Immunoglobulin heavy constant delta, Monocyte differentiation antigen CD14, Complement factor B, Haptoglobin-related protein, Immunoglobulin heavy variable 3-64, , Immunoglobulin kappa variable 3-15, Immunoglobulin lambda variable 1-47,
Immunoglobulin lambda variable 7-43, Apolipoprotein(a), Immunoglobulin lambda variable 9-49, Immunoglobulin lambda-like polypeptide 5, Apolipoprotein A-ll, Immunoglobulin kappa variable 3-11 , Immunoglobulin heavy constant alpha 1 , Galectin-3-binding protein, Immunoglobulin lambda variable 3-25, Alpha-1 -antichymotrypsin, Vitamin K-dependent protein S, Immunoglobulin heavy variable 3-43D, Carboxypeptidase N catalytic chain, Immunoglobulin heavy variable 3-30-5, Immunoglobulin lambda constant 2,
Immunoglobulin lambda variable 3-19, Triple functional domain protein, Immunoglobulin lambda variable 6-57, Immunoglobulin heavy variable 1-46, Immunoglobulin kappa variable 1-27, Complement component C8 gamma chain, Complement C1 r
subcomponent , Hemoglobin subunit beta, Biotinidase, Immunoglobulin kappa variable 1 D-12, Apolipoprotein F, Complement C4-B, Hemoglobin subunit alpha, Immunoglobulin kappa variable 2D-28, Uncharacterized protein ZSWIM9, Coagulation factor XIII B chain, , Keratin type I cytoskeletal 10, Complement C1q subcomponent subunit C , Alpha-2- macroglobulin, Immunoglobulin heavy variable 6-1 , Complement factor H, Transcription factor E2F8, Apolipoprotein A-l , Ficolin-3, Immunoglobulin lambda variable 8-61 , Fibronectin, Immunoglobulin heavy variable 3-7, Lumican, Thyroxine-binding globulin, Inter-alpha-trypsin inhibitor heavy chain H4, Vitronectin, Retinol-binding protein 4, , and Phosphatidylcholine-sterol acyltransferase.
All these proteins had a fold change higher than 1.5 between symptomatic and asymptomatic carotid stenosis. In examples below particular change folds are indicated, as well as the particular discrimination among carotid stenosis subtypes.
Various species of mammals are known to suffer from carotid stenosis. The method of the invention can be applied to all of them. Thus, in another particular embodiment of the methods and the uses defined herewith, optionally in combination with any of the embodiments provided above or below, the subject is a mammal. In a more particular embodiment, the mammal is a domestic animal mammal. In another particular embodiment, optionally in combination with any of the embodiments provided above or below, the mammal is a human.
A second aspect of the invention is an in vitro method for selecting a subject suffering from carotid stenosis as candidate for surgical therapy comprising determining in an isolated biofluid sample of a subject the level of expression of CD5 antigen-like and comparing the level of expression with a corresponding reference value.]
In other words, this second aspect is an in vitro method for selecting a subject suffering from carotid stenosis as candidate for surgical therapy, comprising the following steps:
(a) determining in an isolated biofluid sample of a subject the level of expression of CD5 antigen-like; and
(b) comparing the level of expression of CD5 antigen-like with a corresponding reference value, and wherein the subject is selected as a candidate for surgical therapy if:
the levels of expression of CD5 antigen-like match with corresponding reference values of symptomatic carotid stenosis; or alternatively, the level of expression of CD5 antigen-like, is higher than a reference value.
In a particular embodiment of the second aspect, the in vitro method further comprises determining the level of expression of one or more proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein,
Immunoglobulin heavy constant gamma 3, Prothrombin, Serum amyloid P-component, Immunoglobulin kappa variable 4-1 , Gelsolin, and Caveolin-1. In another particular embodiment of the second aspect, the in vitro method for selecting a subject suffering from carotid stenosis as candidate for surgical therapy, comprises the following steps:
(a) determining in an isolated biofluid sample of a subject the level of expression of CD5 antigen-like, and further the level of expression of one or more proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein,
Immunoglobulin heavy constant gamma 3, Prothrombin, Serum amyloid P-component, Immunoglobulin kappa variable 4-1 , Gelsolin, and Caveolin-1 , and
(b) comparing the level of expression of CD5 antigen-like, and of the one or more proteins with a corresponding reference value, and wherein the subject is selected as a candidate for surgical therapy if:
- the levels of expression of CD5 antigen-like, and of one of the one or more of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Immunoglobulin heavy constant gamma 3,
Prothrombin, Serum amyloid P-component, Immunoglobulin kappa variable 4-1 , Gelsolin, and Caveolin-1 match with corresponding reference values of symptomatic carotid stenosis; or alternatively,
the level of expression of CD5 antigen-like, and of one of the one or more Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Prothrombin, Serum amyloid P-component, Immunoglobulin kappa variable 4-1 , and Gelsolin, are determined, and the level of CD5 antigen-like and of one of the one or more determined are higher than a reference value, and/or the level of expression of CD5 antigen-like and of one or more of apolipoprotein A-IV, transthyretin, Immunoglobulin heavy constant gamma 3 and Caveolin-1 are determined, and the level of expression of CD5 antigen-like is higher than a reference value and the levels of expression of one of the A-IV, transthyretin, Immunoglobulin heavy constant gamma 3 and caveolin-1 are lower than a reference value.
In another particular embodiment of this second aspect, , the subject is selected as candidate for additional determination of the level of expression of one or more proteins in the isolated sample and/or as candidate for intensive medical treatment if:
the level of expression of CD5 antigen-like, and of one or more of
Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Immunoglobulin heavy constant gamma 3,
Prothrombin, Serum amyloid P-component, Immunoglobulin kappa variable 4-1 , Gelsolin, and Caveolin-1 match with corresponding reference values of asymptomatic carotid stenosis; or alternatively,
the level of expression of CD5 antigen-like, and of one or more of
Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Prothrombin, Serum amyloid P-component, Immunoglobulin kappa variable 4-1 , and Gelsolin, are determined, and the levels of one of them are lower than a reference value, and/or the level of expression of CD5 antigen-like and of one or more of apolipoprotein A-IV, transthyretin, Immunoglobulin heavy constant gamma 3 and Caveolin-1 are determined, and the level of expression of CD5 antigen-like is lower than a reference value and the levels of expression of the one of apolipoprotein A-IV, transthyretin
Immunoglobulin heavy constant gamma 3 and Caveolin-1 are higher than a reference value.
This previous embodiment can also be worded as a method for selecting and/or recommending a therapy regimen for a subject suffering from carotid stenosis, wherein the method comprises determining the level of expression of CD5 antige-like, and of the one or more of all above listed proteins.
In a particlar embodiment is a method for selecting and/or recommending a therapy regimen for a subject suffering from carotid stenosis, wherein the method comprises:
(a) determining in an isolated biofluid sample of the subject the level of expression of CD5 antigen-like and of one or more proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2; and
(b) comparing the level of expression of CD5 antigen-like and of the one or more proteins with a corresponding reference value, and wherein:
(i) the subject is diagnosed as symptomatic carotid stenosis and an effective treatment comprising surgical therapy is selected or recommended if: the levels of expression of CD5 antigen-like and of one or more of fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2 apolipoprotein A-IV and transthyretin match with corresponding reference values of symptomatic carotid stenosis; or alternatively,
the level of expression of CD5 antigen-like and of one or more of fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2 are determined, and the levels of CD5 antigen like and of one of the one or more proteins are higher than a reference value, and/or if the level of expression of CD5 antigen-like and of one or more of apolipoprotein A-IV and transthyretin are determined and the levels of expression of CD5 antigen-like and of one of apolipoprotein A-IV and transthyretinare lower than a reference value; and
(i) the subject is diagnosed as asymptomatic carotid stenosis and determination of additional level of expression of one or more proteins in the isolated sample and/or an intensive medical treatment is selected or recommended if: the level of expression of CD5 antigen-like and of one or more of fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, apolipoprotein A-IV and transthyretin match with corresponding reference values of asymptomatic carotid stenosis; or alternatively,
the level of expression of CD5 antigen-like and of one or more of fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2 are determined, and the levels of CD5 antigen like and of one of the one or more proteins are lower than a reference value, and/or the level of expression of CD5 antigen-like and of one or more of apolipoprotein A-IV and transthyretin are determined and the levels of expression of CD5 antigen-like and of one of apolipoprotein A-IV and transthyretin are higher than a reference value. In a more particular embodiment of the second aspect, it comprises determining the level of expression of C-reactive protein in the isolated biofluid sample, and wherein the subject is selected as a candidate for surgical therapy if the level of expression of the protein is higher than a reference value or match with a value of a symptomatic carotid stenosis subject.
In another more particular embodiment of the in vitro method of this second aspect, it comprises determining the level of expression of Apolipoprotein A- IV in the isolated biofluid sample, and wherein the subject is selected as a candidate for surgical therapy if the level of expression of the Apolipoprotein A- IV is lower than a reference value, or alternatively, if the level of expression of Apolipoprotein A- IV match with the value of a symptomatic carotid stenosis reference.
In also another particular embodiment of the method of the second aspect, the in vitro method comprises determining the level of expression of Immunoglobulin heavy constant gamma 3, and the subject is is selected as a candidate for surgical therapy if the level of expression of Immunoglobulin heavy constant gamma 3 is higher than a reference value, or alternatively if the level of expression of Immunoglobulin heavy constant gamma 3 match with the value of a symptomatic carotid stenosis reference. In also another particular embodiment of the method of the seconf aspect, the in vitro method comprises determining the level of expression of Prothrombin, and the subject is selected as a candidate for surgical therapy if the level of expression of Prothrombin is higher than a reference value, or alternatively if the level of expression of Prothrombin match with the value of a symptomatic carotid stenosis reference.
In also another particular embodiment of the method of the second aspect, the in vitro method comprises determining the level of expression of Serum amyloid P-component, and the subject is selected as a candidate for surgical therapy if the level of expression of Serum amyloid P-component is higher than a reference value, or alternatively if the level of expression of Serum amyloid P-component match with the value of a symptomatic carotid stenosis reference.
In another particular embodiment of the second aspect, the recommended surgical therapy is selected from the group consisting of CEA, stenting and combinations thereof. Yet another particular embodiment of the second aspect, optionally in combination with any embodiment above or below, is that the method further comprises determining one or more clinical parameters of the subject. Particular clinical parameters include, without being a limitative listgender, age, echography determination of plaque features, and vascular risk profile, among others. The combination of determining the level of expression of above-listed proteins in an isolated biofluid sample, together with additional clinical parameters is, in another particular embodiment, carried out by means of decision algorithms allowing improvement of accuracy of patient classification. On the alternative, values detected for each of the proteins can be discretized and/or applied to them particular weight factors by means of mathematical formula, optionally considering these additional clinical parameters, in order to give a cutoff or value allowing classification.
In another particular embodiment of the first and second aspects, the methods further comprise the step of treating by surgical therapy the selected candidate patients if the level of expression of the one or more proteins match with a value of a symptomatic carotid stenosis subject, or if they are higher or lower than a reference level depending on the protein as above indicated. Particular surgical therapies include CEA, stenting and combinations thereof. In another particular embodiment, if the subject is classified as asymptomatic carotid stenosis, methods include the step of determining additional proteins allowing
classification between progressive and stable patients and also other physical parameters (imaging) and/or clinical parameters (age, gender, etc.); and the step of treating progressive asymptomatic subjects with CEA, stenting and combinations thereof.
All these particular embodiments including treatment, can be formulated as a method of treatment by surgical therapy of a patient suffering carotid stenosis, said method of treatment comprising the steps of carrying out any of the methods as defined in the first and/or second aspects of the invention or any of their particular embodiments.
Particular embodiments regarding biofluid sample and the number of proteins whose levels are determined indicated for the first aspect do also apply to this second aspect. Thus, particular listed combinations for the first aspect also apply to this second and additional aspects of the invention or to reformulations of these aspects.
In another particular embodiment of the methods of the first and second aspects, the level of expression of one or more of the proteins is carried out at protein level by means for detecting and quantifying proteins, which are selected from the group consisting of immunoassays, protein migration, chromatography, mass spectrometry, turbidimetry, nephelometry and combination thereof.
On the alternative, the level of expression of one or more of the proteins is carried out at nucleic acid level by means of polymerase chain reaction (PCR).This is in particular used when the biofluid sample comprises cells or in case mRNA coding for the proteins can be detected outside cells.
By way of a non-limiting illustration, the expression levels are determined by means of the quantification of the levels of mRNA encoded by said genes. The latter can be quantified by means of using conventional methods, for example, methods comprising the amplification of mRNA and the quantification of the amplification product of said mRNA, such as electrophoresis and staining, or alternatively, by means of Northern blot and the use of suitable probes, Northern blot and use of specific probes of the mRNA of the genes of interest or of their corresponding cDNA/cRNA, mapping with the SI nuclease, RT-PCR, hybridization, microarrays, etc. Similarly, the levels of the cDNA/cRNA corresponding to said mRNA encoded by the marker genes can also be quantified by means of using conventional techniques; in this event, the method of the invention includes a step of synthesis of the corresponding cDNA by means of reverse transcription (RT) of the corresponding mRNA followed by the synthesis (RNA polymerase) and amplification of the cRNA complementary to said cDNA. Conventional methods of quantifying the expression levels can be found in laboratory manuals.
In order to normalize the values of mRNA expression among the different samples, it is possible to compare the expression levels of the mRNA of interest in the test samples with the expression of a control RNA. A "control RNA" as used herein, relates to RNA whose expression levels do not change or change only in limited amounts. Preferably, the control RNA is mRNA derived from housekeeping genes and which code for proteins which are constitutively expressed and carry out essential cellular functions. Preferred housekeeping genes for use in the present invention include 18-S ribosomal protein, b-2-microglobulin, ubiquitin, cyclophilin, GAPDH, PSMB4, tubulin and b-actin.
Alternatively, it is also possible to determine the expression levels of the marker genes by means of the determination of the expression levels of the proteins encoded by said genes, since if the expression of genes is increased, an increase of the amount of corresponding protein should occur and if the expression of genes is decreased, a decrease of the amount of corresponding protein should occur.
The determination of the expression levels of the proteins can be carried out by tests selected from the group consisting of an immunological test, bioluminescence, fluorescence, chemiluminescence, electrochemistry and mass spectrometry. Particular tests that can be implemented in a point of care test format (POCT) are recommended to make easy and fast the determining of marker levels. Independently of the test format, particular quantitative tests are selected from the group consisting of an immunological test, bioluminescence, fluorescence, chemiluminescence, electrochemistry and mass spectrometry.
In one embodiment, the level of expression is determined by immunological techniques such as enzyme-linked immunosorbent assay (ELISA), enzyme immunodot assay, agglutination assay, antibody-antigen-antibody sandwich assay, antigen-antibody-antigen sandwich assay, immunocromatography, or other immunoassay formats well-known to the ordinarily skilled artisan, such as radioimmunoassay, as well as protein microarray formats, such as single molecular assay (SIMOA), Western Blot or immunofluorescence. In all of these immunological techniques an antibody or fragment thereof specifically binds to the protein of interest, if present in the isolated sample.
Western blot is based on the detection of proteins previously resolved by gel
electrophoreses under denaturing conditions and immobilized on a membrane, generally nitrocellulose by the incubation with an antibody specific and a developing system (e.g. chemiluminescent). The analysis by immunofluorescence requires the use of an antibody specific for the target protein for the analysis of the expression. ELISA is based on the use of antigens or antibodies labelled with enzymes so that the conjugates formed between the target antigen and the labelled antibody results in the formation of enzymatically-active complexes. Since one of the components (the antigen or the labelled antibody) are immobilized on a support, the antibody-antigen complexes are immobilized on the support and thus, it can be detected by the addition of a substrate which is converted by the enzyme to a product which is detectable by, e.g. spectrophotometry .fluorometry, mass spectrometry or tandem mass tags (TMT). SIMOA is a type of assay more sensitive than an ELISA, since it uses arrays of femtoliter-sized reaction chambers, which are termed single-molecule arrays (SimoaTM) that can isolate and detect single enzyme molecules. Because the array volumes are approximately 2 billion times smaller than a conventional ELISA, a rapid build-up of fluorescent product is generated if a labeled protein is present. With diffusion defeated, this high local concentration of product can be readily observed. Only a single molecule is needed to reach the detection limit. Using the same reagents as a conventional ELISA, this method has been used to measureproteins in a variety of different matrices (serum, plasma, cerebrospinal fluid, urine, cell extracts, etc.) at femtomolar (fg/mL) concentrations, offering aroughly 1000-fold improvement in sensitivity In another particular embodiments of the in vitro methods of the invention that provide a differential diagnostic andinformation for selecting a therapy, they further comprise the steps of (i) collecting the diagnostic information, and (ii) saving the information in a data carrier.
In the sense of the invention a“data carrier” is to be understood as any means that contain meaningful information data for the differential diagnosis and/or prognosis of carotid stenosis, such as paper. The carrier may also be any entity or device capable of carrying the differential diagnosis data or information for selecting a therapy. For example, the carrier may comprise a storage medium, such as a ROM, for example a CD ROM or a semiconductor ROM, or a magnetic recording medium, for example a floppy disc or hard disk. Further, the carrier may be a transmissible carrier such as an electrical or optical signal, which may be conveyed via electrical or optical cable or by radio or other means. When the diagnosis/prognosis data are embodied in a signal that may be conveyed directly by a cable or other device or means, the carrier may be constituted by such cable or other device or means. Other carriers relate to USB devices and computer archives. Examples of suitable data carrier are paper, CDs, USB, computer archives in PCs, or sound registration with the same information.
The invention also proposes using means for detecting the level of expression of CD5 antigen-like, and optionally of one or more proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2,
Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4,
Immunoglobulin heavy constant mu, Heparin cofactor 2 detecting C-reactive protein, Prothrombin, Serum amyloid P-component, and Immunoglobulin kappa variable 4-1 in an isolated sample of a subject in the in vitro method of any one of the first and second aspects.
In a particular embodiment, said means comprise a corresponding antibody or a fragment thereof able to specifically bind to a corresponding protein of the list.
In another particular embodiment, said means form part of a kit.
Present invention provides also a kit of parts comprising reagent means for detecting the level of expression of CD5 antigen-like, and optionally reagent means for detecting one or more of the proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV,
Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Prothrombin, Serum amyloid P-component, and Immunoglobulin kappa variable 4-1. The kit of parts according to the invention further comprises reagent means for detecting the level of expression of one or more of the proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4,
Immunoglobulin heavy constant mu and Heparin cofactor 2, and optionally reagent means for detecting C-reactive protein, Prothrombin, Serum amyloid P-component, and mmunoglobulin kappa variable 4-1. In a more particular embodiment of the kit of parts according to the invention, it comprises reagent means for detecting the level of expression of CD5 antigen-like and reagent means for detecting the level of expression of one two, three, four, five, six, seven, eight nine, ten, eleven, twelve proteins selected from Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin,
Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Prothrombin, Serum amyloid P-component, and
Immunoglobulin kappa variable 4-1.
In even a more particular embodiment it comprises reagents means for detecting the level of expression of CD5 antigen-like and Apolipoprotein A-IV, and optionally C-reactive protein, Prothrombin, Serum amyloid P-component and Immunoglobulin kappa variable 4- 1.
In also another particular embodiment, the kit of parts accodting to the invention comprises reagent means for detecting the level of expression of one or more of the proteins selected from the group consisting of Immunoglobulin lambda variable 2-18, Immunoglobulin heavy constant delta, Insulin-like growth factor-binding protein 3, Haptoglobin, Alpha-2-HS-glycoprotein, Immunoglobulin kappa variable 3-15,
Antithrombin-Ill, Immunoglobulin kappa variable 4-1 , Gelsolin, Immunoglobulin heavy constant gamma 3 and Caveolin-1.
The term "kit", as used herein, refers to a product containing the different reagents (or reagent means) necessary for carrying out the methods of the invention packed so as to allow their transport and storage. Materials suitable for packing the components of the kit include crystal, plastic (e.g. polyethylene, polypropylene, polycarbonate), bottles, vials, paper, or envelopes.
Additionally, the kits of the invention can contain instructions for the simultaneous, sequential or separate use of the different components which are in the kit. Said instructions can be in the form of printed material or in the form of an electronic support capable of storing instructions susceptible of being read or understood, such as, for example, electronic storage media (e.g. magnetic disks, tapes), or optical media (e.g. CD- ROM, DVD), or audio materials. Additionally or alternatively, the media can contain internet addresses that provide said instructions.
As above indicated, the reagent means (or simply reagents) of the kit include compounds that bind specifically to the marker proteins. Preferably, said compounds are antibodies, aptamers or fragments thereof.
In a preferred embodiment of the kits, the reagent is an antibody or fragments thereof. Thus, the reagent means are one or more antibodies that specifically recognize the proteins of interest. The antibodies of the kit of the invention can be used according to techniques known in art for determining the protein expression levels, such as, for example, flow cytometry, Western blot, ELISA, radioimmune assay (RIA), competitive Enzymatic immuno assay (EIA), double antibody sandwich RLISA (DAS-ELISA), techniques based on the use of biochips, protein microarrays, or assays of colloidal precipitation in reactive strips. The antibodies can be fixed to a solid support such as a membrane, a plastic or a glass, optionally treated to facilitate the fixation of said antibodies to the support. Said solid support comprises, at least, a set of antibodies which specifically recognize the marker (i.e. the protein of interest), and which can be used for detecting the levels of expression of said marker.
Additionally, the kits of the invention comprise reagents for detecting a protein encoded by a constitutive gene. The availability of said additional reagents allows normalizing the measurements performed in different samples (for example, the sample to be analyzed and the control sample) to rule out that the differences in the expression of the biomarkers are due to a different quantity of total protein amount in the sample more than the real differences in the relative levels of expression. The constitutive genes in the present invention are genes that are always active or being transcribed constantly and which encode for proteins that are expressed constitutively and carry out essential cellular functions. Proteins that are expressed constitutively and can be used in the present invention include, without limitation, b-2-microglobulin (B2M), ubiquitin , 18-S ribosomal protein, cyclophilin, GAPDH, PSMB4, tubulin and actin.
In a preferred embodiment, the reagent means for assaying the levels of the different biomarkers comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the total amount of reagents for assaying biomarkers forming the kit. Thus, in the particular case of kits comprising reagents for assaying the levels of the thirteen proteins (i.e. CD5 antigen-like, Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2,
Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4,
Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Prothrombin, Serum amyloid P-component, and Immunoglobulin kappa variable 4-1), the reagents specific for said biomarkers (i.e. antibodies which bind specifically to the proteins) comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the antibodies present in the kit. These kits are, thus, simplified kits including mainly the reagent means for detecting the levels of CD5 antigen like or of the thirteen proteins.
In another aspect, the invention relates to the use of said kit of the invention for differentiating symptomatic carotid stenosis from asymptomatic carotid stenosis or for selecting a patient suffering carotid stenosis for surgical therapy (CEA or stenting).
Thus, in a particular embodiment, the invention relates to the use of the kit of the invention in any of the methods of the invention.
The invention also includes as an aspect the use of CD5 antigen-like as biomarker of differential diagnosis of symptomatic versus asymptomatic carotid stenosis or as biomarker for selecting a subject suffering from carotid stenosis as candidate for surgical therapy.
In a more particular embodiment, this use is in combination with one or more of
Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2,
Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4,
Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Prothrombin, Serum amyloid P-component, and Immunoglobulin kappa variable 4-1 as biomarkers of differential diagnosis of symptomatic versus asymptomatic carotid stenosis or as biomarkers for selecting a subject suffering from carotid stenosis as candidate for surgical therapy.
Thus, present invention relates to the capability of distinguishing symptomatic carotid stenosis subjects from asymptomatic carotid stenosis subjects by determining the level of expression of one or more of the proteins selected from CD5 antigen-like, Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, and Heparin cofactor 2, and optionally in combination with the levels of expression of C-reactive protein, Prothrombin, Serum amyloid P-component, and Immunoglobulin kappa variable 4-1. In particular the capability of distinguishing symptomatic carotid stenosis subjects from asymptomatic carotid stenosis subjects by determining the level of expression of two or more of the proteins. More in particular three, four, five, six, seven, eight or nine proteins, or ten, eleven, telwe or thireen if protein levels of C-reactive protein, Prothrombin, Serum amyloid P-component, and Immunoglobulin kappa variable 4-1 are also determined.
Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps.
Furthermore, the word“comprise” encompasses the case of“consisting of’. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.
Example 1.
Prospective study of consecutively admitted patients with symptomatic carotid stenosis of >70% and with asymptomatic progressive carotid stenosis >70%.
1.- Materials and methods
Patients were selected as is common clinical practice in Hospital Universitari Dr. Josep Trueta de Girona and they gave their signed informed consent for the clinical study.
Prospective study of consecutively admitted patients was performed with symptomatic carotid stenosis of >70% (Nr of patients 7) and with asymptomatic progressive carotid stenosis >70% (Nr of patients 7).
Inclusion criteria were: Patients aged >18 with a first episode of minor stroke or transient ischemic attack (TIA) of a secondary atherothrombotic mechanism due to ipsilateral carotid stenosis >70% and treated >30 days after first symptoms. Patients with
asymptomatic carotid stenosis >70% with progression to carotid stenosis (10-15% of the total). Exclusion criteria were: Patients treated >30 days of last symptomatology, treated with carotid angioplasty or with a disease affecting inflammatory molecules. Patients with symptomatic carotid stenosis and 2 potential causes of stroke. Endarterectomy samples were collected and serum samples (2 vials of 4.5 ml for biochemistry analysis of serum and 2 vials of BD Vacutainer PPTTM Plasma for the analysis of markersin plasma) were obtained immediately before carotid endarterectomy. As controls, serum samples were collected from patients with asymptomatic stable carotid stenosis >70% (Nr of patients 5) whose were stable in at least two separate controls.
Proteomic Cualitative analysis in both, surgical pieces of atheromatous plaques and serum samples, was performed by liquid cromatography-mass spectrometry (LC-MS/MS) by means of data-dependent adquisition (DDA) (Triple TOF 6600 ABSciex). Those proteins with differential expression between symptomatic and asymptomatic patients (in plaques and in serum) and those that were coincident in both, plaque and serum samples in the same patients, were identified by label-free quantitive proteomics using a data- independent acquisition (DIA) methods (SWATH-SM) (see references 1-3, below) and Draw Venn Diagram software (http://bioinformatics.psb.ugent.be/webtools/Venn/). The diferenciated proteins were selected using a p-value<0.05 and a fold change>1.5.
References:
1. Ortea, I., Ruiz-Sanchez, I., Canete, R., Caballero-Villarraso, J. & Canete, M. D. Identification of candidate serum biomarkers of childhood-onset growth hormone deficiency using SWATH-MS and feature selection. J. Proteomics 175, 105-113 (2018).
2. Garrido-Rodhguez, M., Ortea, I., Calzado, M. A., Munoz, E. & Garcia, V. SWATH proteomic profiling of prostate cancer cells identifies NUSAP1 as a potential molecular target for Galiellalactone. J. Proteomics 193, 217-229 (2018).
3. Ortea, I., Rodhguez-Ariza, A., Chicano-Galvez, E., Arenas Vacas, M. S. & Jurado
Gamez, B. Discovery of potential protein biomarkers of lung adenocarcinoma in bronchoalveolar lavage fluid by SWATH MS data-independent acquisition and targeted data extraction. J. Proteomics 138, 106-114 (2016).
Blood extraction was also done 3-months post-endarterectomy.
Statistical analysis: Continuous variables are disclosed with the mean and standard deviation (SD) or with the media depending on the probability distribution. Categorical variables are disclosed as percentages. For bivariate analysis T-Student test or U- Mann. Whitney test is used for continuous variables and Fisher or X2 for categorical variables. ANOVA and F-Fischer tests are used for multivariate analysis.
2.- Results Coincident differentially expressed proteins in serum and in plaques between symptomatic and progressive asymptomatic subjects were detected using software of Bioinformatics & Evolutionary Genomics accessible at http://bioinformatics.psb.ugent.be/webtools/Venn/ at filing date, which generates Venn diagrams stablishing the coincident elements among different data sets. Next Table 1 shows the 10 proteins differentially diagnosis the subjects and that were simultaneously detected in plaque and in serum:
Table 1. Common proteins among the comparatives between the symptomatic and asymptomatic progressive groups.
Figure imgf000049_0001
Bold values indicate that the fold change is symptomatic vs asymptomatic and the underwritten and cursive values indicates that the fold change is asymptomatic progressive vs. symptomatic
In addition, inventors detected that also a differential expression can be observed at protein level between asymptomatic progressive and asymptomatic stable subjects.
These additional proteins are valuable markers to further decide what medical regimen is finally recommended to the subject. Data are depicted in Table 2 below.
Table 2. Proteins with a fold change greater than or equal to 1.5 between asymptomatic progressive and asymptomatic stable.
Figure imgf000050_0001
Proteins with a fold change greater than or equal to 1.5 between asymptomatic progressive and asymptomatic stable. Bold values indicate fold change for asymptomatic progressive vs. asymptomatic stable and underwritten and cursive values fold change for asymptomatic stable vs. asymptomatic progressive.
Beside proteins indicated above, differential expression of additional proteins were also detected in isolated serum samples of symptomatic subjects in relation with progressive asymptomatic and stable asymptomatic subjects. They are indicated in Tables 3 and 4 below.
Table 3. Proteins with a fold change greater than or equal to 1.5 between symptomatic and progressive asymptomatic.
Figure imgf000050_0002
Figure imgf000051_0001
Cells with bold values indicate fold change for symptomatic vs. asymptomatic progressive and cells with underwritten and cursive values fold change for asymptomatic progressive vs. symptomatic
Table 4. Proteins with a fold change greater than or equal to 1.5 between symptomatic and asymptomatic stable.
Figure imgf000052_0001
Figure imgf000053_0001
Cells with bold values indicate fold change for symptomatic vs. asymptomatic stable and cells with underwritten and cursive values fold change for asymptomatic stable vs.
symptomatic
Example 2.
With the same protocol as for Example 1 , including the same materials and methods, and bioinformatic tools, a new cohort of patients was studied including symptomatic carotid stenosis, asymptomatic stable and asymptomatic progressive (n=7-9 per group). Next table 5.1 to 5.4 show the fold change between groups.
Table 5.1. Symptomatic vs progressive asymptomatic:
Figure imgf000053_0002
Table 5.2. Symptomatic vs progressive and stable asymptomatic:
Figure imgf000054_0001
Table 5.3. Progressive vs stable asymptomatic:
Figure imgf000054_0002
Table 5.4. Stable asymptomatic vs progressive asymptomatic and symptomatic:
Figure imgf000054_0003
Further aspects/embodiments of the present invention can be found in the following clauses:
Clause 1.- An in vitro method for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis, comprising the steps of:
(a) determining in an isolated biofluid sample of a subject the level of expression of one or more proteins selected from the group consisting of CD5 antigen-like, Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant muand Heparin cofactor 2; and
(b) comparing the level of expression of the one or more proteins with a corresponding reference value, and:
(b1) diagnosing the subject of symptomatic carotid stenosis or of asymptomatic carotid stenosis if the levels of expression of one or more of CD5 antigen-like, fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2 match with corresponding reference values of symptomatic carotid stenosis or asymptomatic carotid stenosis; or, alternatively,
(b2) diagnosing the subject of symptomatic carotid stenosis if the level of expression of one or more of CD5 antigen-like, fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2 are determined, and the levels of one of them are higher than a reference value, and/or one or more of apolipoprotein A- IV and transthyretin are determined and the levels of expression of one of them are lower than a reference value; and
diagnosing the subject of asymptomatic carotid stenosis if the level of expression of one or more of CD5 antigen-like, fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2 are determined, and the levels of one of them are lower than a reference value, and/or one or more of apolipoprotein A-IV and transthyretin are determined and the levels of expression of one of them are higher than a reference value.
Clause 2.- The in vitro method according to clause 1 , which further comprises determining the level of expression of C-reactive protein, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of C-reactive protein is higher than a reference value, or alternatively if the level of expression of C-reactive protein match with the value of a symptomatic carotid stenosis reference.
Clause 3.- The in vitro method according to any one of clauses 1-2, wherein the isolated biofluid sample is selected from plasma, serum, whole blood, saliva, semen, sputum, cerebral spinal fluid (CSF), tears, mucus, sweat and milk. Clause 4. -The in vitro method according to any one of clauses 1-3, wherein the isolated biofluid sample is selected from plasma, serum and whole blood.
Clause 5. -The in vitro method according to any one of clauses 1-4, wherein if the subject is diagnosed as suffering from asymptomatic carotid stenosis, the method further comprises the step of determining in the isolated sample the level of expression of one or more of the proteins selected from the group consisting oflmmunoglobulin lambda variable 2-18, Immunoglobulin heavy constant delta, Insulin-like growth factor-binding protein 3, Haptoglobin, Alpha-2-HS-glycoprotein, Immunoglobulin kappa variable 3-15,
Antithrombin-Ill, and Immunoglobulin kappa variable 4-1.
Clause 6.- An in vitro method for selecting a subject suffering from carotid stenosis as candidate for surgical therapy, comprising the following steps: (a) determining in an isolated biofluid sample of a subject the level of expression of one or more proteins selected from the group consisting of CD5 antigen-like, Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2; and
(b) comparing the level of expression of the one or more proteins with a corresponding reference value, and wherein the subject is selected as a candidate for surgical therapy if:
the levels of expression of one or more of CD5 antigen-like, fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2 apolipoprotein A-IV and transthyretin match with corresponding reference values of symptomatic carotid stenosis; or alternatively,
the level of expression of one or more of CD5 antigen-like, fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2,
Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2 are determined, and the levels of one of them are higher than a reference value, and/or if one or more of apolipoprotein A-IV and transthyretin are determined and the levels of expression of one of them are lower than a reference value.
Clause 7.- The in vitro method according to clause 6, in which the subject is selected as candidate for additional determination of the level of expression of one or more proteins in the isolated sample and/or as candidate for intensive medical treatment if: the level of expression of one or more of CD5 antigen-like, fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, apolipoprotein A-IV and transthyretin match with corresponding reference values of asymptomatic carotid stenosis; or alternatively,
the level of expression of one or more of CD5 antigen-like, fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2 are determined, and the levels of one of them are lower than a reference value, and/or one or more of apolipoprotein A-IV and transthyretin are determined and the levels of expression of one of them are higher than a reference value. Clause 8.- The in vitro method according to any one of clauses 6-7, which further comprises determining the level of expression of C-reactive protein in the isolated biofluid sample, and wherein the subject is selected as a candidate for surgical therapy if the level of expression of the protein is higher than a reference value, or alternatively, if the level of expression of C-reactive protein match with the value of a symptomatic carotid stenosis reference.
Clause 9. -The method according to any of clauses 1- 8 further comprising determining one or more clinical parameters. Clause 10. -The in vitro method according to any one of clauses 1-9, wherein determining the level of expression of one or more of the proteins is carried out at protein level by means for detecting and quantifying proteins selected from the group consisting of immunoassays, protein migration, chromatography, mass spectrometry, turbidimetry, nephelometry and combination thereof.
Clause 11.- The in vitro method according to any one of clauses 1-9, wherein determining the level of expression of one or more of the proteins is carried out at nucleic acid level by means of polymerase chain reaction (PCR). Clause 12. Use of one or more of CD5 antigen-like, Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin,
Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2, optionally in combination with C-reactive protein, as biomarkers of differential diagnosis of symptomatic versus asymptomatic carotid stenosis or as biomarkers for selecting a subject suffering from carotid stenosis as candidate for surgical therapy.
Clause 13.- A kit of parts comprising reagent means for detecting the level of expression of one or more of the proteins selected from the group consisting of CD5 antigen-like, Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2,
Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4,
Immunoglobulin heavy constant mu and Heparin cofactor 2, and optionally reagent means for detecting C-reactive protein.
Citation List
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Rekasem et al.,“C-Reactive Protein is Elevated in Symptomatic Compared with Asymptomatic Patients with Carotid Artery Disease”, Eur. J. Vase Endovasc Surg 2002, vol. no. 23, pp.: 505-509
Burtis C. A. et al., 2008, Chapter 14, section“Statistical Treatment of Reference Values”
Ortea, I., Ruiz-Sanchez, I., Canete, R., Caballero-Villarraso, J. & Canete, M. D. Identification of candidate serum biomarkers of childhood-onset growth hormone deficiency using SWATH-MS and feature selection. J. Proteomics 175, 105-113
(2018).
Garrido-Rodhguez, M., Ortea, I., Calzado, M. A., Munoz, E. & Garcia, V. SWATH proteomic profiling of prostate cancer cells identifies NUSAP1 as a potential molecular target for Galiellalactone. J. Proteomics 193, 217-229 (2018).
- Ortea, I., Rodriguez-Ariza, A., Chicano-Galvez, E., Arenas Vacas, M. S. & Jurado
Gamez, B. Discovery of potential protein biomarkers of lung adenocarcinoma in bronchoalveolar lavage fluid by SWATH MS data-independent acquisition and targeted data extraction. J. Proteomics 138, 106-114 (2016).

Claims

Claims 1 An in vitro method for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis, comprising the steps of: (a) determining in an isolated biofluid sample of a subject the level of expression of CD5 antigen-like; and (b) comparing the level of expression of CD5 antigen-like with a corresponding reference value, and:
(b1) diagnosing the subject of symptomatic carotid stenosis or of asymptomatic carotid stenosis if the levels of expression of CD5 antigen-like match with corresponding reference values of symptomatic carotid stenosis or asymptomatic carotid stenosis; or, alternatively,
(b2) diagnosing the subject of symptomatic carotid stenosis if the level of expression of CD5 antigen-like is higher than a reference value; and
diagnosing the subject of asymptomatic carotid stenosis if the level of expression of CD5 antigen-like is lower than a reference value.
2 - The in vitro method for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis according to claim 1 , which further comprises determining the level of expression of one or more proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV,
Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Immunoglobulin heavy constant gamma 3, Prothrombin, and Serum amyloid P-component. 3.- The in vitro method for the differential diagnosis of symptomatic versus asymptomatic carotid stenosis according to any one of claims 1-2, comprising the steps of:
(a) determining in an isolated biofluid sample of a subject the level of expression of CD5 antigen-like, and further the level of expression of one or more proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein,
Immunoglobulin heavy constant gamma 3, Prothrombin, and Serum amyloid P- component; and (b) comparing the level of expression of CD5 antigen-like, and of the one or more proteins with a corresponding reference value, and: (b1) diagnosing the subject of symptomatic carotid stenosis or of asymptomatic carotid stenosis if the levels of expression of CD5 antigen-like, and of the one or more of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2,
Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Immunoglobulin heavy constant gamma 3, Prothrombin, and Serum amyloid P-componentmatch with corresponding reference values of symptomatic carotid stenosis or asymptomatic carotid stenosis; or, alternatively,
(b2) diagnosing the subject of symptomatic carotid stenosis if the level of expression of CD5 antigen-like, and the level of expression of one or more of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C- reactive protein, Immunoglobulin heavy constant gamma 3, Prothrombin, and Serum amyloid P-component are determined, and the levels of CD5 antigen-like and of one of the one or more determined are higher than a reference value, and/or the level of expression of CD5 antigen-like and of one or more of Apolipoprotein A-IV and
Transthyretin are determined and the level of expression of CD5 antigen-like is higher than a reference value and the levels of expression of one of Apolipoprotein A-IV and Transthyretin are lower than a reference value; and
diagnosing the subject of asymptomatic carotid stenosis if the level of expression of CD5 antigen-like, and the level of expression of one or more of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Immunoglobulin heavy constant gamma 3, Prothrombin, and Serum amyloid P-component are determined, and the levels of CD5 antigen-like and of one of the one or more determined are lower than a reference value, and/or the level of expression of CD5 antigen-like and the level of expression of one or more of Apolipoprotein A-IV and Transthyretin are determined and the level of expression of CD5 antigen-like is lower than a reference value and the levels of expression of one of the apolipoprotein A-IV and transthyretin are higher than a reference value.
4 - The in vitro method according to any one of claims 1-3, which comprises determining the level of expression of C-reactive protein, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of C-reactive protein is higher than a reference value, or alternatively if the level of expression of C-reactive protein match with the value of a symptomatic carotid stenosis reference.
5.- The in vitro method according to any one of claims 1-4, which comprises determining the level of expression of Apolipoprotein A-IV, and the subject is diagnosed of
symptomatic carotid stenosis if the level of expression of Apolipoprotein A-IV is lower than a reference value, or alternatively if the level of expression of Apolipoprotein A-IV match with the value of a symptomatic carotid stenosis reference.
6.- The in vitro method according to any one of claims 1-5, which comprises determining the level of expression of Immunoglobulin heavy constant gamma 3, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of Immunoglobulin heavy constant gamma 3 is higher than a reference value, or alternatively if the level of expression of Immunoglobulin heavy constant gamma 3 match with the value of a symptomatic carotid stenosis reference.
7.- The in vitro method according to any one of claims 1-6, which comprises determining the level of expression of Prothrombin, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of Prothrombin is higher than a reference value, or alternatively if the level of expression of Prothrombin match with the value of a symptomatic carotid stenosis reference.
8.- The in vitro method according to any one of claims 1-7, which comprises determining the level of expression of Serum amyloid P-component, and the subject is diagnosed of symptomatic carotid stenosis if the level of expression of Serum amyloid P-component is higher than a reference value, or alternatively if the level of expression of Serum amyloid P-component match with the value of a symptomatic carotid stenosis reference.
9.- The in vitro method according to any one of claims 1-8, wherein the isolated biofluid sample is selected from plasma, serum, whole blood, saliva, semen, sputum, cerebral spinal fluid (CSF), tears, mucus, sweat and milk.
10. -The in vitro method according to any one of claims 1-9, wherein the isolated biofluid sample is selected from plasma, serum and whole blood.
11.-The in vitro method according to any one of claims 1-10, wherein if the subject is diagnosed as suffering from asymptomatic carotid stenosis, the method further comprises the step of determining in the isolated sample the level of expression of one or more of the proteins selected from the group consisting of Immunoglobulin lambda variable 2-18, Immunoglobulin heavy constant delta, Insulin-like growth factor-binding protein 3,
Haptoglobin, Alpha-2-HS-glycoprotein, Immunoglobulin kappa variable 3-15,
Antithrombin-Ill, Immunoglobulin kappa variable 4-1 , Gelsolin, Immunoglobulin heavy constant gamma 3 and Caveolin-1.
12.- The in vitro method according to any one of claims 1-11 , wherein if the subject is diagnosed as suffering from asymptomatic carotid stenosis, the method further comprises the step of determining in the isolated sample the level of expression of one or more of the proteins selected from the group consisting oflmmunoglobulin lambda variable 2-18, Immunoglobulin heavy constant delta, Insulin-like growth factor-binding protein 3,
Haptoglobin, Alpha-2-HS-glycoprotein, Immunoglobulin kappa variable 3-15,
Antithrombin-Ill, and Immunoglobulin kappa variable 4-1.
13.- An in vitro method for selecting a subject suffering from carotid stenosis as candidate for surgical therapy, comprising the following steps:
(a) determining in an isolated biofluid sample of a subject the level of expression of CD5 antigen-like; and
(b) comparing the level of expression of CD5 antigen-like with a corresponding reference value, and wherein the subject is selected as a candidate for surgical therapy if:
the levels of expression of CD5 antigen-like match with corresponding reference values of symptomatic carotid stenosis; or alternatively, the level of expression of CD5 antigen-like, is higher than a reference value.
14.- The in vitro method according to claim 13, which further comprises determining the level of expression of one or more proteins selected from the group consisting of
Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2,
Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4,
Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein,
Immunoglobulin heavy constant gamma 3, Prothrombin, Serum amyloid P-component, Immunoglobulin kappa variable 4-1 , Gelsolin, and Caveolin-1.
15.- The in vitro method according to any one of claims 13-14, which further comprises determining the level of expression of one or more proteins selected from the group consisting of Immunoglobulin lambda variable 2-18, Immunoglobulin heavy constant delta, Insulin-like growth factor-binding protein 3, Haptoglobin, Alpha-2-HS-glycoprotein, Immunoglobulin kappa variable 3-15, and Antithrombin-Ill.
16.- The in vitro method for selecting a subject suffering from carotid stenosis as candidate for surgical therapy according to any one of claims 13-15, comprising the following steps:
(a) determining in an isolated biofluid sample of a subject the level of expression of CD5 antigen-like, and further the level of expression of one or more proteins selected from the group consisting Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu Heparin cofactor 2, C-reactive protein,
Immunoglobulin heavy constant gamma 3, Prothrombin, Serum amyloid P-component, Immunoglobulin kappa variable 4-1 , Gelsolin, and Caveolin-1 ; and
(b) comparing the level of expression of CD5 antigen-like, and of the one or more proteins with a corresponding reference value, and wherein the subject is selected as a candidate for surgical therapy if:
the levels of expression of CD5 antigen-like, and of one of the one or more of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, Apolipoprotein A-IV Transthyretin, C-reactive protein, Immunoglobulin heavy constant gamma 3, Prothrombin, Serum amyloid P-component, Immunoglobulin kappa variable 4-1 ,
Gelsolin, and Caveolin-1 match with corresponding reference values of symptomatic carotid stenosis or asymptomatic progressive carotid stenosis; or alternatively,
the level of expression of CD5 antigen-like, and of one of the one or more of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, C-reactive protein, Prothrombin, Serum amyloid P-component, Immunoglobulin kappa variable 4-1 , and Gelsolin, are determined, and the level of CD5 antigen-like and of one of the one or more determined are higher than a reference value, and/or the level of expression of CD5 antigen-like andof one or more of Apolipoprotein A-IV Transthyretin, caveolin-1 and Immunoglobulin heavy constant gamma 3 are determined, and the level of expression of CD5 antigen-like is higher than a reference value and the levels of expression of one of the
Apolipoprotein A-IV, Transthyretin, Caveolin-1 and Immunoglobulin heavy constant gamma 3 are lower than a reference value.
17.- The in vitro method for selecting a subject suffering from carotid stenosis as candidate for surgical therapy, comprising the following steps: (a) determining in an isolated biofluid sample of a subject the level of expression of CD5 antigen-like, and further the level of expression of one or more proteins selected from the group consisting Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2; and
(b) comparing the level of expression of CD5 antigen-like, and of the one or more proteins with a corresponding reference value, and wherein the subject is selected as a candidate for surgical therapy if:
the levels of expression of CD5 antigen-like, and of one of the one or more of fibrinogen alpha chain, haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, Apolipoprotein A-IV and
Transthyretin match with corresponding reference values of symptomatic carotid stenosis; or alternatively,
- the level of expression of CD5 antigen-like, and of one of the one or more of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2 are determined, and the level of CD5 antigen-like and of one of the one or more determined are higher than a reference value, and/or the level of expression of CD5 antigen-like andof one or more of Apolipoprotein A-IV and transthyretin are determined, and the level of expression of CD5 antigen-like is higher than a reference value and the levels of expression of one of the Apolipoprotein A-IV and
Transthyretin are lower than a reference value.
18.- The in vitro method according to any one of claims 13-17, in which the subject is selected as candidate for additional determination of the level of expression of one or more proteins in the isolated sample and/or as candidate for intensive medical treatment if:
- the level of expression of CD5 antigen-like, and of one or more of
Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu, Heparin cofactor 2, Apolipoprotein A-IV and transthyretin match with corresponding reference values of asymptomatic carotid stenosis; or alternatively,
the level of expression of CD5 antigen-like, and of one or more of
Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Immunoglobulin heavy constant gamma 4, Immunoglobulin heavy constant mu and Heparin cofactor 2 are determined, and the levels of one of them are lower than a reference value, and/or the level of expression of CD5 antigen-like and of one or more of Apolipoprotein A-IV and transthyretin are determined and the level of expression of CD5 antigen-like is lower than a reference value and the levels of expression of the one of apolipoprotein A-IV and transthyretin are higher than a reference value.
19.- The in vitro method according to any one of claims 13-18, which comprises determining the level of expression of C-reactive protein in the isolated biofluid sample, and wherein the subject is selected as a candidate for surgical therapy if the level of expression of the protein is higher than a reference value, or alternatively, if the level of expression of C-reactive protein match with the value of a symptomatic carotid stenosis reference.
20.- The in vitro method according to any one of claims 13-19, which comprises determining the level of expression of Apolipoprotein A- IV in the isolated biofluid sample, and wherein the subject is selected as a candidate for surgical therapy if the level of expression of the Apolipoprotein A- IV is lower than a reference value, or alternatively, if the level of expression of Apolipoprotein A- IV match with the value of a symptomatic carotid stenosis reference.
21. -The method according to any of claims 1- 20 further comprising determining one or more clinical parameters.
22. -The in vitro method according to any one of claims 1-21 , wherein determining the level of expression of one or more of the proteins is carried out at protein level by means for detecting and quantifying proteins selected from the group consisting of immunoassays, protein migration, chromatography, mass spectrometry, turbidimetry, nephelometry and combination thereof.
23.- The in vitro method according to any one of claims 1-22, wherein determining the level of expression of one or more of the proteins is carried out at nucleic acid level by means of polymerase chain reaction (PCR).
24.- Use of CD5 antigen-like as biomarker of differential diagnosis of symptomatic versus asymptomatic carotid stenosis or as biomarker for selecting a subject suffering from carotid stenosis as candidate for surgical therapy.
25.- The use of CD5 antigen-like according to claim 24, in combination with one or more of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4,
Immunoglobulin heavy constant mu Heparin cofactor 2, C-reactive protein,
Immunoglobulin heavy constant gamma 3, Prothrombin, Serum amyloid P-component, Immunoglobulin kappa variable 4-1 , Gelsolin, and Caveolin-1 as biomarkers of differential diagnosis of symptomatic versus asymptomatic carotid stenosis or as biomarkers for selecting a subject suffering from carotid stenosis as candidate for surgical therapy.,
26.- The use of CD5 antigen-like according to claim 23, in combination with one or more of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4,
Immunoglobulin heavy constant mu and Heparin cofactor 2, optionally in combination with C-reactive protein, as biomarkers of differential diagnosis of symptomatic versus asymptomatic carotid stenosis or as biomarkers for selecting a subject suffering from carotid stenosis as candidate for surgical therapy.
27.- A kit of parts comprising reagent means for detecting the level of expression of CD5 antigen-like
28.- The kit of parts according to claim 27, further comprising reagent means for detecting the level of expression of one or more of the proteins selected from the group consisting of Fibrinogen alpha chain, Haptoglobin, Immunoglobulin heavy constant gamma 2, Apolipoprotein A-IV, Transthyretin, Immunoglobulin heavy constant gamma 4,
Immunoglobulin heavy constant mu Heparin cofactor 2, C-reactive protein,
Immunoglobulin heavy constant gamma 3, Prothrombin, and Serum amyloid P- component.
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