Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • 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
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
• • 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
  • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
• • 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
  • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
• • 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
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
  • G01N2333/972—Plasminogen activators
  • G01N2333/9723—Urokinase
• • 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
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
  • G01N2333/972—Plasminogen activators
  • G01N2333/9726—Tissue plasminogen activator
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/26—Infectious diseases, e.g. generalised sepsis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

• the present invention relates to the medical field in general, and in particular the field of prognosis in vitro. It relates more specifically to the assessment of the risk of complication in a patient suspected of having an infection with a SOFA score of less than two.
• Sepsis is a complex syndrome involving several clinical phases. It remains associated with high mortality. Early recognition of patients at risk of adverse outcomes is a key determinant of prognosis.
• SIRS is an inflammatory systemic response triggered by a variety of infectious causes or not.
• SIRS conditions triggered by non-infectious causes include traumatic conditions, burns, pancreatitis, acute respiratory syndromes.
• An inflammatory systemic response manifested by at least two of the following signs: a) temperature greater than 38 ° C or less than 36 ° C; b) heart rate greater than 90 beats per minute; c) respiratory rate greater than 20 breaths per minute; d) number of leukocytes greater than 12000 / mm 3 or less than 4000 / mm 3 , - sepsis is an inflammatory systemic response syndrome related to an infection,
• septic shock is a severe sepsis associated with persistent hypotension and can be qualified by:
• SOFA score Sequential Organ Failure Assessment Score
• Sequential Organ Failure Assessment Score is used to track a person's status while in hospital to determine the extent of his or her deficiencies. organs (Vincent JF and Coll, 1996). Fe score is based on six different scores, one for each of the systems following: respiratory, cardiovascular, hepatic, coagulation, renal and neurological. The calculation method for each system is shown in Table 1 below:
• the qSOFA (or quickSOFA) score was introduced in February 2016 as a simplified version of the SOFA score as an initial means of identifying patients at high risk of poor prognosis after infection (Eamon P. Raith, et al. , 2017).
• QSOFA drastically simplifies the SOFA score by including only 3 clinical criteria.
• the qSOFA can easily and quickly be calculated and repeated in patients. In Table 2, below, the calculation mode of qSOFA.
• the qSOFA score therefore varies from 0 to 3 points.
• Identify among patients suspected of having an infection and not serious (with a SOFA score of less than 2), the most at risk of complication, and as soon as possible and in the absence of sepsis, would allow to set up the as soon as possible a strategy to limit their risk of complication.
• antibiotic prophylaxis could be given (Puisieux F et al., 1993, Jensen JU et al., 2011) or preventive treatment (K. Asehnoune et al., 2014) or targeted immunotherapy (Chahin A et al., 2015, Ali YM et al., 2014), or more simply, pathways for pathogens could be limited (ie removal of catheters as soon as possible ).
• markers have already been described for the diagnosis of sepsis and sometimes for the assessment of the risk of complications in patients who already had sepsis.
• markers include pro calcitonin (PCT) or reactive protein C (CRP).
• biomarkers for their use in the diagnosis of sepsis or septic shock but also in the prognosis of evolution of sepsis already proven to be septic shock, according to the old definition.
• biomarkers is the type II receptor for vascular endothelial growth factor (VEGFR2).
• VEGFR2 vascular endothelial growth factor
• the cohorts of patients described in the examples, concerning this marker for the prognosis of progression, are composed of patients admitted for at least 48 hours in an intensive care unit, hospital services specialized in the management of inanimate patients or particularly serious conditions requiring permanent monitoring. These patients have a SOFA score greater than or equal to three.
• Wada and his team describe soluble receptors with angiogenic factors, such as sVEGFR2 or angiopoietin 2, a growth factor implicated in angiogenesis, as predictive of development of acute respiratory distress syndrome (ARDS) in critically ill patients.
• angiogenic factors such as sVEGFR2 or angiopoietin 2, a growth factor implicated in angiogenesis
• ARDS acute respiratory distress syndrome
• the patients included in the cohort of this publication are critically ill patients, already in the hospital setting, all of whom are under ventilatory mechanical respiration and who have either sepsis or severe trauma, or who are resuscitated following cardiac arrest. These patients have very high SOFA scores, ranging from four to almost ten. Again, these ventilated patients are not patients simply suspected of having an infection with a SOFA score of less than two, considered non-serious.
• Fe VEGFR2 is one of the receptors for vascular endothelial growth factor (VEGF).
• VEGF vascular endothelial growth factor
• the family of mammalian VEGF is composed of four glycoproteins referenced from A to D and placental growth factor (P1GF) (Koch and Claesson-Welsh, 2012). Each is expressed in different iso forms due to alternative splicing and proteolitic cleavages.
• P1GF placental growth factor
• These VEGFs bind and activate three types of tyrosine kinase receptors, VEGFR 2, but also VEGFR 1 and 3 respectively called KDR, Flt1 and VEGFR3.
• VEGFs also bind the NP1 and NP2 neuropilins, which act as co-factors for VEGFRs.
• VEGFR2 also called KDR, (subsequently this gene will only be named VEGFR2) encodes the type 2 vascular endothelial growth factor receptor. It is a tyrosine protein kinase that acts as a cell surface receptor for VEGFA, VEGFC and VEGFD. It plays an essential role in the regulation of angiogenesis, vascular development, vascular permeability and embryonic hematopoiesis. This receptor promotes the proliferation, survival, migration and differentiation of endothelial cells but also the reorganization of the actin cytoskeleton. This receptor consists of an extracellular portion consisting of 7 immunoglobulin-like domains, a transmembrane region and an intracellular portion containing a tyrosine kinase domain (Shibuya, 2011).
• VEGFR2 vascular endothelial growth factor
• the method according to the invention has the advantage of being able to easily evaluate the risk of complication in a patient suspected of having an infection and not presenting the clinical signs of severity (score of SOFA less than two), by having a directly measurable marker, especially by automated analysis, and whose measurement can be made in a local laboratory, upon arrival at an emergency center, at his doctor or patient bed, and so do sorting related to the need to care for the most at-risk patients among all those patients suspected of having an infection.
• the invention firstly relates to a method of in vitro or ex vivo evaluation of the risk of complication in a patient suspected of having an infection having a SOFA score of less than two, including the measurement of the level of expression, in a biological sample from said patient, of at least one expression product of the VEGFR2 gene.
• the invention also relates to a method of stratification and treatment of a patient suspected of having an infection having a SOFA score of less than two, characterized in that it comprises the steps of:
• a last object of the present invention is a kit for the in vitro or ex vivo measurement of the level of expression of at least one expression product of the VEGFR2 gene and at least one expression product of the uPAR gene, in a biological sample, comprising:
• a calibrated control for containing the amounts of said at least one expression product of the VEGFR2 gene which correspond to known quantities of said at least one expression product of the VEGFR2 gene and
• a calibrated control for containing the amounts of said at least one expression product of the uPAR gene which correspond to known quantities of said at least one expression product of the uPAR gene.
• patients are patients suspected of having an infection and having a SOFA score of less than two. In other words, they are patients with no clinical signs of severity.
• Patient suspected of having an infection is defined as a patient with known infection or suspected of being infected by a health care professional in clinical or paraclinical manifestations.
• a health professional is any person who exercises his or her skills and medical judgment, provides a service related to the maintenance, improvement of the health of patients, or the treatment of individuals who are wounded, sick, disabled or disabled. an infirmity by providing them with care.
• urinary infectious focus (low or high): urinary burns, pollakiuria or dysuria, pelvic pain, cough emission in the urinary meatus, unilateral lumbar pains;
• an infectious digestive center abdominal pains, transit disorders
• ALT / SGPT Alignment-Aminotransferase / Serum Glutamopyruvate Transferase
• ASAT / SGOT Aspartate-Aminotransferase / Serum Glutamooxaloacetate Transferase
• the health professional will be able to determine whether the patient is suspected of having an infection or not, using clinical or paraclinical manifestations.
• the patient whose infection is suspected will have one or more clinical and / or paraclinical signs, for example those exemplified above.
• the infection may be caused by the contamination of the patient by any infectious agent, such as a virus, a bacterium, a parasite, a fungus or a protozoan.
• infectious agent such as a virus, a bacterium, a parasite, a fungus or a protozoan.
• viruses such as HIV, SIV, IVF, HCV, HBV, HAV, HEV, VZV, CMV, EBV, HSV1, HSV2, bacteria such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Borrelia burgdorferi stricto sensu, Borrelia afzelii, Borrelia garinii, Borrelia spielmanii, Clostridium difficile, Clostridium botulinum, Salmonella, Klebsiella, Leugionella, Proteus, Klebsiella, Escherichia coli, Shigella, Pseudomonas aeruginosa, Staphylococcus aureus, Treponema pallidum yeasts such as Candida albicans, fungi such as Aspergillus fumigatus, Mucorales, etc. and protozoa such as leishmania, trichomonas vaginalis,
• infections when the patient is suspected of an infection, it can affect any tissue or organ.
• infections By way of examples, mention may be made of infections:
• the infection is of bacterial origin.
• the patient is characterized by his SOFA score which is strictly less than 2, in other words, a patient having a SOFA score of either 0 or 1.
• SOFA score which is strictly less than 2
• these are patients who do not have sepsis.
• the patients according to the invention are therefore patients not presenting a serious clinical condition.
• patients are not in intensive care and / or are not under ventilatory mechanical respiration.
• the patients according to the invention who are not in a serious clinical condition, will be put in contact with health professionals, who will suspect an infection, for example in the following cases: as soon as they arrive at an emergency center, to the doctor himself or to the hospital to his bed, which is another particular embodiment.
• Complication risk assessment means the identification of patients suspected of having an infection and having a SOFA score of less than two, who will see their condition deteriorate in the hours following the measurement of the level of expression of the marker and those who will not see their condition deteriorate during the hours that follow and therefore do not require management by a specialized hospital service.
• organ failure means abnormal functioning of an organ that results in a clinical or biological abnormality of the organ (see below the calculation of the SOFA score).
• organ failure include respiratory, cardiovascular, renal, neurological, hepatic failure or hemato logical failures.
• organ failure definitions (Fagon et al., 1993):
• a patient needs to enter the intensive care unit when his condition requires a continuous monitoring of the vital functions and, where appropriate, the recourse to methods of substitution (transfusion of blood derivatives, vascular filling, mechanical ventilation, catecholamine, hemodialysis, extracorporeal circulation, etc.).
• substitution transfusion of blood derivatives, vascular filling, mechanical ventilation, catecholamine, hemodialysis, extracorporeal circulation, etc.
• the ultimate goal of resuscitation is the restoration of homeostasis.
• the presence of a risk of complication corresponds to the risk that the complication will occur, for example within 5 days, especially within 4 days, especially within 3 days after sample collection and when there are several samples. within 5 days, especially within 4 days, especially within 3 days after the first sample collection.
• the method according to the invention comprises measuring the level of expression, in a biological sample from said patient, of at least one expression product of the VEGFR2 gene.
• sample refers to a part or quantity, more specifically a small part or a small quantity, taken from one or more entities for analysis. This sample may possibly have undergone a prior treatment, involving for example mixing and dilution steps.
• the sample in the context of the method of the invention is a biological sample from the patient suspected of having an infection having a SOFA score of less than two to whom it is desired to evaluate the risk of complication in the hours following the level measurement. expression of the marker (s).
• a biological sample is chosen from those likely to contain a VEGFR2 expression product or any other marker described later.
• the biological sample according to the present invention may be of different natures.
• this sample is a biological fluid, for example selected from whole blood (as collected from the venous route, that is to say containing white and red cells, platelets and plasma), serum, plasma, bronchoalveolar lavage fluid, cerebrospinal fluid, also called cerebrospinal fluid, and urine.
• the biological sample from the patient is a sample of whole blood, plasma, serum or any derivative.
• the measurement of the expression level of the VEGFR2 gene (SEQ ID No. 1) consists in quantifying at least one expression product of this gene.
• the expression product within the meaning of the invention is any biological molecule resulting from the expression of the VEGFR2 gene.
• the expression product of the gene is an RNA transcript.
• transcript is meant the RNAs, and in particular the messenger RNAs, derived from the transcription of the VEGFR2 gene. More specifically, transcripts are the RNAs produced by the splicing of the gene.
• the measurement of the expression level of one or more RNA transcripts of the VEGFR2 gene can be performed.
• VEGFR2 gene has three transcripts known to date, referenced in the Ensembl database (GRCh38.plO) and identified in Table 3 below.
• transcripts KDR-201 SEQ ID NO: 2
• KDR-202 KDR-202 and KDR-203 and their variants
• variant an RNA having a sequence having at least 99% identity with one of said KDR-201, KDR-202, KDR-203 transcript sequences. Percent identity is determined using sequence alignment software, such as CLUSTALW (Thompson et al., 1994).
• a variant will correspond, in particular, to a polymorphism of the VEGFR2 gene sequence.
• the preferred VEGFR2 gene transcript is the KDR-201 transcript or a variant thereof having at least 99% identity with the sequence of said transcript, the expression of which will be determined alone or in combination with that of the other variants.
• only the expression of the KDR-201 transcript or a variant thereof having at least 99% identity with the sequence of said transcript will be determined.
• measuring the level of expression of an RNA transcript of a target gene may include a preliminary step of extracting total RNAs from a biological sample. This step is followed by a reverse transcription step of these different RNAs to obtain their complementary DNA (cDNA). Then the specific cDNAs of the target gene are amplified and then quantified.
• cDNA complementary DNA
• nucleic acid extraction and purification protocols well known to those skilled in the art.
• the extraction of nucleic acids can be carried out by lysis of the cells present in the biological sample followed by purification or by extraction with phenol, chloroform and alcohol. These steps are well known to those skilled in the art and are described for example by Sambrook J and Russell DW (2017).
• RNAs comprising the ribosomal RNAs, the transfer RNAs and the messenger RNAs.
• a reverse transcription reaction is then carried out using a reverse transcriptase enzyme which makes it possible to obtain, from an RNA fragment, a complementary DNA fragment (cDNA).
• a reverse transcriptase enzyme which makes it possible to obtain, from an RNA fragment, a complementary DNA fragment (cDNA).
• cDNA complementary DNA fragment
• this enzymatic step is carried out in the presence of nucleotide fragments comprising only thymine bases (polyT), which hybridize by complementarity on the polyA sequence of the different mRNAs in order to form a polyT-polyA complex which then serves as a starting point for the reverse transcription reaction carried out by the enzyme reverse transcriptase.
• polyT thymine bases
• an enzymatic amplification reaction is performed.
• An enzymatic amplification reaction is a process that generates multiple copies of a nucleotide fragment by the action of at least one enzyme.
• Such amplification reactions are well known to those skilled in the art and may be mentioned in particular the following techniques:
• the specific reagent comprises at least 2 specific amplification primers in order to amplify a particular region of the DNA complementary to the mRNA derived from the target gene.
• the enzymatic amplification is a PCR carried out after a reverse transcription reaction, it is called RT-PCR.
• the level of expression of the target gene is determined by hybridization using at least one hybridization probe specific for the expression product of this target gene.
• hybridization probe is meant a nucleotide fragment comprising from 5 to 100 nucleotide units, in particular from 6 to 35 nucleotide units, having hybridization specificity under determined conditions to form a hybridization complex with a target nucleotide fragment.
• the target nucleotide fragment can be a nucleotide sequence included in a messenger RNA or a nucleotide sequence included in a complementary DNA obtained by reverse transcription of said messenger RNA.
• Hybridization techniques are well known to those skilled in the art, and mention may in particular be made of the Northern blot technique.
• the quantification of the mRNAs, expression products of the target gene goes through a step of detecting the hybridization reaction between the hybridization probe and the target nucleotide fragment.
• the amount of said at least one transcript of the VEGFR2 gene will be determined by at least one of the following characteristics, taken alone or in combination:
• RT-PCR real-time PCR
• Determining the quantity of several transcripts can be implemented sequentially or simultaneously, according to the methods conventionally known to those skilled in the art, as described above.
• the measured expression product is a protein and / or a polypeptide which is the product of the translation of at least one of the transcripts described above or of a transcript not yet described.
• measurement of the expression level of one or more proteins and / or polypeptides can be performed.
• VEGFR2 derived from the KDR-201 transcript
• iso form 1 SEQ ID No. 3
• No. UniProt P35968-1 which is the transmembrane form
• isoforms 2 SEQ ID No. No. 4
• UniProt No. P35968-2 SEQ ID No. 5
• No. UniProt P35968-3 which are the plasma forms, or soluble, secreted from the receptor.
• All isoforms of VEGFR2 can be used, alone or in combination, as a marker (s) to assess the risk of complication, in a patient suspected of having an infection with a SOFA score of less than two.
• the determination of the amount of one or more isoforms in a biological sample can be done according to techniques widely known to those skilled in the art to determine the amount, or dose, of one or more analytes in a biological sample.
• immunoassay assays such as ELISA (Enzyme Linked Immuno Sorbent Assay), ELFA (Enzyme Linked Fluorescent Assay) and RIA (Radio Immunoassay), and mass spectrometric assays. which constitutes an embodiment of the invention.
• the immunoassay assay is a method well known to those skilled in the art and widely used in the field of the analysis of biological samples. It allows to to quantify analytes in samples in particular form of proteins (antigens / antibodies), peptides and haptens, for example steroids or vitamins, involving immunological reactions between the analyte to be detected, in this case the one of the VEGFR2 isoforms, and one or more binding partner (s) to this analyte.
• proteins antigens / antibodies
• peptides and haptens for example steroids or vitamins
• These immunoassay methods are based on measurements to quantify the signals emitted during the analysis of the biological sample.
• the amount of signal detected is generally proportional to the quantity, or dose, of analyte to be measured (for example during a sandwich assay) or inversely proportional to the quantity or dose of analyte to be measured (for example assay in competition).
• the term "immuno" in "immunoassay” for example is not to be considered in this application as strictly indicating that the binding partner is an immunological partner, such as an antibody. Indeed, those skilled in the art also use this term widely when the binding partner, also called ligand, is not an immunological partner but is for example a receptor for the analyte that we want to quantify.
• VEGFR2 isoform binding partner mention may be made of antibodies, antibody fractions, nanofitins, aptamers (Ochsner UA et al., 2014) or any other molecule which is known to have an interaction with VEGFR2 to be investigated, such as lipopolysaccharides (Bucki R. et al., 2005)
• the binding partner antibodies are, for example, either polyclonal antibodies or monoclonal antibodies whose production is widely known to those skilled in the art.
• Such antibodies for isoforms of VGEFR2 are commercially available, such as the following polyclonal antibodies: Human VEGF R2 / KDR / Flk the Biotinylated Antibody ref: BAF357 biothechne ® and the following monoclonal antibodies: Human VEGF R2 / KDR / Flk-l Antibody ref: MAB3573 biothechne ® .
• antibody fragments By way of example of antibody fragments, mention may be made of the Fab, Fab ', F (ab') 2 fragments as well as the scFv (Single chain variable fragment) and dsFv (Double-stranded) chains. fragment variable). These functional fragments can in particular be obtained by genetic engineering.
• the immunoassay for determining the amount of the isoform (s) of VEFGR2 preferably implements two binding partners of the VEGFR2 isoforms.
• One of the two partners may be coupled to a label to form a conjugate or a tracer.
• the other link partner can be captured on a solid support. This is called capture partner for the latter and detection partner for the first.
• the measured signal emitted during the immunoassay is then proportional to the quantity, or dose, of the VEGFR2 isoform in the biological sample.
• a mathematical model pre-established from a standard range should be used. This standard range will be obtained previously in a known manner. In a few words, obtaining a standard range consists in measuring the signal generated by increasing or known quantities or concentrations of the VEGFR2 isoform, in plotting the curve giving the signal as a function of the quantity or concentration, and in find a mathematical model that represents this relationship as faithfully as possible. The mathematical model will be used to extrapolate the amounts, or concentrations, of the unknown VEGFR2 isoform contained in the biological sample to be tested.
• label used to form the conjugate is meant, in particular, any molecule containing a group reactive with a group of the binding partner, directly without chemical modification, or after chemical modification to include such a group, which molecule is capable of generating directly or indirectly a detectable signal.
• a non-limiting list of these direct detection markers consists of:
• enzymes that produce a detectable signal for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose-6-phosphate dehydrogenase;
• chromophores such as fluorescent compounds, luminescent compounds, dyes
• radioactive molecules such as 32P, 35S or 1251;
• fluorescent molecules such as Alexa or phycocyanines
• electrochemiluminescent salts such as organometallic derivatives based on acridinium or ruthenium.
• Indirect detection systems can also be used, such as, for example, ligands capable of reacting with an anti-ligand. The ligand then corresponds to the marker to form, with the binding partner, the conjugate.
• the ligand / anti-ligand couples are well known to those skilled in the art, which is the case, for example, in the following pairs: biotin / streptavidin, hapten / antibody, antigen / antibody, peptide / antibody, sugar / lectin, polynucleotide / complement of polynucleotide.
• the anti-ligand can then be detectable directly by the direct detection markers described above or be itself detectable by another ligand / antiligand pair, and so on.
• reagents allowing the visualization of the marking or the emission of a detectable signal by any type of suitable measuring device, such as for example a spectrophotometer, a spectrofluorimeter, a densitometer or a high definition camera.
• the immunoassay may also include other steps known to those skilled in the art, such as washing steps and incubation steps.
• the immunoassay may be a one-step or two-step assay, as is widely known to those skilled in the art.
• a one-step immunoassay involves bringing the test sample simultaneously with the two binding partners
• a two-step immunoassay involves placing the test sample in the presence of the two binding partners. part with the first binding partner, then the analyte complex-first bonding partner thus formed is brought into contact with the second binding partner.
• Mass spectrometry which can be substituted for previously developed techniques such as immunoassays. It is implemented in a mass spectrometer. It is a powerful tool increasingly used for the analysis and quantification of different types of molecules in biological samples. In general, any type of molecule that can be ionized can be quantified according to its molecular mass using a mass spectrometer. Depending on the nature of the molecule Quantified, of protein or metabolic origin, some mass spectrometry technologies may be more suitable. Nevertheless, whatever the mass spectrometry method used for the quantification, the latter comprises a step of ionization of the target molecule into so-called molecular ions and a step of separation of the molecular ions obtained according to their mass. A mass spectrometer measures the ratio of mass to charge (m / z) of ionized molecules that is correlated to the target molecule to be analyzed.
• RNA transcript when there are several measured expression products, they can be of the same nature but also of different natures. In other words, they can be of a molecular nature (RNA transcript, mRNA) and / or of a protein nature (protein, polypeptide).
• the method of the invention may comprise or consist in the implementation of the steps of:
• the measurement of the quantity of at least one expression product of the VEGLR2 gene is carried out as described above. For convenience, we will call biological sample from a patient for whom it is desired to evaluate the risk of complication, sample to be tested.
• the amount of said at least one expression product or a value derived from that amount will be compared to a predetermined reference value, in order to evaluate the risk of complication.
• a reference value is widely known to those skilled in the art. It consists in particular in implementing a method of assay identical or at least comparable to that used in the method of the invention, in biological samples of the two populations studied, and in determining the value of the test. (quantity) to discriminate between these two populations, in this case between that which will be complicated and that which will not get complicated.
• a value derived from the quantity may for example be the absolute concentration, calculated by means of a calibration curve.
• the predetermined reference value used to compare the quantity measured in the context of the invention will be determined from the same VEGFR2 gene expression product (s) as those quantified in the biological sample to be tested. .
• the samples from which the reference values are determined may be of different types, and in particular of a biological nature as mentioned above with respect to the sample to be tested (biological fluids).
• these reference biological samples are of the same nature as that of the biological sample to be tested or at least of a compatible nature to constitute a reference for the quantification of the expression product (s) of the selected VEGFR2 gene (s). ).
• they will be biological samples corresponding to the same biological fluid as that of the test sample, such as whole blood, serum or plasma samples.
• the reference sample (s) used are preferably from persons having the same characteristics or a majority of common characteristics, in particular of the same sex and / or of a similar or identical age and / or of the same ethnic origin, with those the subject or patient suspected of having an infection without sepsis where it is desired to evaluate the risk of complications.
• the reference sample (s) used will be from patients suspected of having an infection and having a SOFA score of less than two at the time of biological sample collection. Their evolution towards a complication or not will nevertheless have been documented a posteriori to know if they belong to the population which was complicated or that which did not become complicated.
• samples taken at the same time that is to say from the moment of characterization.
• the patient is suspected of having an infection with a SOFA score of less than two, or at the latest 24 hours later.
• the results of the analyte measurement tests depend to a large extent on the characteristics of the binding partner (s) used.
• the results depend in particular on the characteristics of the partners used, such as the nature, the degree of affinity with the analyte or the size, the characteristics of the compositions, etc., and that these characteristics influence the measured values. It is therefore conceivable that it is not possible to give precise reference values and that the reference value (s) adapted to the test used can be determined in each case by simple routine experiments. The same goes for molecular detection.
• the determination of the reference value according to the invention is done on a significant sample of patients, that is to say on a minimum number of samples to obtain statistically relevant results and therefore representative of the population studied.
• reference value is either a discrete value or an interval of values corresponding to a zone of indeterminacy.
• reference value is either a discrete value or an interval of values corresponding to a zone of indeterminacy.
• ROC Receiver Operating Characteristic Curve
• the values of the area under the ROC curve vary between 0.5 (no difference in the distribution of the dosage values between the two subgroups, the ROC curve corresponds to the bisector) and 1 (perfect separation of the dosage values of the two subgroups, the ROC curve passes through the point (0, 1)).
• the area under the ROC curve is a quantitative expression of the position of the curve OCR relative to (0, 1) (Hanley, JA and McNeil, B.J., 1982, Zweig, MH and Campbell G., 1993).
• Sensitivity represents the percentage of True Positives among all Positives, recognized as such. It expresses the aptitude of the test to detect really positive biological samples, which correspond to the pathology. In a "probabilistic" language, it corresponds to the probability of observing a Positive result knowing the positive sample.
• Specificity represents the percentage of True Negatives among all Negatives recognized as such. It expresses the ability of the test not to diagnose positive really negative samples, which correspond to a healthy individual. In a "probabilistic" language, it corresponds to the probability of observing a Negative result knowing the Negative sample.
• the patient suspected of having a tested infection is a patient at increased risk of complication.
• the value of the amount of said at least one expression product of the VEGFR2 gene is greater than said reference value, it means that the patient suspected of having a tested infection is not a patient at increased risk of complication.
• An at-risk patient is as defined above.
• the determination of the risk of complication can also be implemented by measuring the amount of at least one expression product of the VEGFR2 gene in a biological sample to be tested at two different times.
• the method may comprise or consist in the implementation of the steps consisting in:
• the first Tl sampling will take place within 12 hours after the identification of the patient as suspected of having an infection without sepsis and the second sampling at time T2 will take place within 24 hours (T24) that follow the first sample at the instant Tl.
• this particular embodiment of the invention comprises a step of calculating the variation between the quantity of the product of expression of the VEGFR2 gene at T2 and that at T1, giving a value D.
• the calculation of the value D can be carried out by any calculation known to those skilled in the art to show a difference between a quantity at T1 and a quantity at T2.
• the value D has the same magnitude as the quantity (VEGFR2 to T1) or (VEGFR2 to T2) determined.
• the value D may correspond to the relative rate of variation and is calculated according to one of the following formulas (II) or (II):
• VEGFR2 to T1-VEGFR2 to T2 TT VEGFR2 to T1-VEGFR2 to T2 "_ _ TT .
• the value D is either a rate (II) or a percentage (II) '.
• the value D can correspond to the difference in quantity per unit of time and is calculated according to the following formula (III):
• the value D has as magnitude one unit of quantity per unit of time.
• the method comprises a step of comparing the value D, obtained in the previous step, with a reference value determined from two populations of patients suspected of having an infection with a SOFA score of less than two, one being complicated and the other not.
• the determination of the reference value is done as indicated above. In this context, it also requires two sampling times of the reference samples.
• the method according to this embodiment makes it possible to conclude on the level of complication risk in the patient from which the biological sample is derived, a value D less than said reference value meaning that the patient tested is a patient at increased risk of complication. , and a value D greater than said reference value meaning that the patient being tested is not a patient at increased risk of complication.
• An at-risk patient is as defined above.
• the method of the invention can be improved by also measuring the level of expression of at least one expression product of at least one other gene, in addition to measuring the level of expression of at least one product of expression of the VEGFR2 gene.
• the combination of at least two markers makes it possible to improve the specificity and the sensitivity of the method of evaluating the risk of complication.
• an embodiment of the invention comprises or also consists of measuring the level of expression of at least one expression product of the uPAR gene.
• the expression product (s) of uPAR within the meaning of the invention may or may be any biological molecule resulting from the expression of one of this gene.
• the uPAR gene (urokinase-type plasminogen activator), also called PLAUR (it will only be named uPAR thereafter), encodes the urokinase plasminogen activator receptor and, given its role in the localization and promotion of The formation of plasmin probably influences many processes in the healthy and sick patient related to cell surface plasminogen activation and localized degradation of the extracellular matrix. It binds to both the pro-protein and mature forms of the urokinase plasminogen activator and allows the activation of the pro-enzyme associated with receptor by plasmin. Several transcription variants result from the alternative splicing of this gene (NCBI Reference Sequence Database, July 2008).
• the Ensembl base (GRCh38.pl0) identified 16 transcripts derived from the transcription of the uPAR gene. These transcripts are identified in Table 2 below.
• isoform 1 SEQ ID No. 11
• No. UniProt Q03405-1 also called uPAR1 or GPI-anchored, which is the membrane form
• isoforms 2 SEQ ID N 12
• UniProt No. Q03405-2 and 3 (SEQ ID NO: 13)
• UniProt No. Q03405-3 which are the plasma forms, or soluble forms, secreted from the receptor.
• the soluble forms of uPAR are called suPAR.
• said at least one transcript of the uPAR gene which is measured is chosen from the transcripts mentioned in Table 2 and their variants, the sequence of a variant having at least 99% identity with one of the sequences of said transcripts.
• the percentage of identity is determined using sequence alignment software, such as CLUSTALW.
• a variant will correspond, in particular, to a polymorphism of the sequence of the gene chosen.
• all the isoforms, from the uPAR gene, referenced above, or not yet identified can be used as a marker to assess the risk of complications, in a patient suspected of have an infection with a SOFA score of less than two.
• the iso-form binding partners of this marker are of the same nature as those described for VEGFR2.
• Such antibodies for uPAR isoforms are commercially available, for example the following polyclonal antibody: Human biotinylated Antibody ref: BAF807 biotechne ® , and the following monoclonal antibodies: Human upar Antibody ref: MAB807 biotechne ® .
• RNA transcript RNA transcript
• mRNA protein
• protein protein
• the method of the present invention comprises or consists of performing the steps of:
• the different steps of quantity measurements can be implemented sequentially or simultaneously.
• the different steps of comparisons to the reference values can be implemented sequentially or simultaneously.
• the method in the context of this embodiment makes it possible to conclude that there is an increased risk of complication in a patient suspected of having an infection having a SOFA score of less than two, when the value of the quantity of said at least one product of Expression of the VEGFR2 gene is less than the SVEGFR2 reference value and the value of the amount of said at least one expression product of the uPAR gene is greater than the reference value S U PAR.
• the determination of the risk of complication can also be implemented by measuring the quantity of the expression products of the different markers in a biological sample to be tested at two different times.
• another embodiment is a method which comprises or consists of the implementation of the steps of:
• the different stages of calculation of variations and the different stages of comparisons with reference values with respect to the different markers can be implemented sequentially or simultaneously, insofar as, of course, the chronology T1, measure at T2, comparison to a reference value and establish a conclusion is respected for each marker.
• the measurement of the amount uPAR can be carried out from a first sample taken at the time of identification of the patient as suspected of having an infection and measurement of the amounts of VEGFR2 from a starting point of one first sample taken 6 hours after the patient's identification as suspected of having an infection.
• the first samples at time T1 can take place within 12 hours of identifying the patient as suspected of having an infection and the second samples at time T2 can take place within 24 hours (T24) following the first samples at the moment Tl.
• sampling times, T1 and T2, for the uPAR marker may be identical to those of VEGFR2 but may also be different.
• the method in the context of this embodiment makes it possible to conclude that there is an increased risk of complication in a patient suspected of having an infection having a SOFA score of less than two when the A V EGFR2 value is lower than said reference value.
• AS U PAR the reference value for the patient suspected of having an infection having a SOFA score of less than two is not a patient at increased risk of complication, when the value A V EGFR2 is greater than said reference value ASVEGFR2 and when the AS U reference value is lower than the AS U reference value PAR.
• the determination of the risk of complication can also be implemented by using the calculation of a score related to the different markers used.
• the method according to this particular embodiment comprises or consists of the implementation of the steps of:
• the steps of measuring the quantities of the expression products of these markers are implemented as described above and can be implemented sequentially or simultaneously.
• the score may be a combination of multiplication, ratio or threshold with different weightings of at least two markers.
• the different quantized markers can also be combined by means of various mathematical algorithms well known to those skilled in the art.
• the method according to this particular embodiment makes it possible to conclude that there is an increased risk of complication in a patient suspected of having an infection having a SOFA score of less than two, when the score of the combination is greater than the reference score and makes it possible to conclude that the patient suspected of having an infection with a SOFA score of less than two is not a patient at increased risk of complication, when the score of the combination is lower than the baseline score.
• the steps of calculation and comparison of the score can be replaced by the establishment of a decision tree.
• the decision tree is a decision support tool representing a set of choices in the graphical form of a tree.
• the various possible decisions are located at the ends of the branches (the "leaves" of the tree), and are reached according to decisions taken at each stage.
• transcript KDR-201 VEGFR2 ⁇ X picoM
• a subject of the invention is a method of treating a patient suspected of having an infection having a SOFA score of less than two, characterized in that it comprises or consists, moreover, in the following stages:
• identifying the patients present a risk of complication by implementing a method of in vitro or ex vivo evaluation of the risk of complication in a patient suspected of having an infection having a SOFA score of less than two according to the invention and
• a patient identified as being at increased risk of complication may have appropriate health care management to reduce the risk of complication and, for example, to reduce the risk of developing sepsis, septic shock or risk. of death.
• Examples of care management include an immunostimulatory treatment or a prophylactic antibiotic treatment, the two treatments can be combined and / or refer to a continuing care or resuscitation service to reduce the risk of complications, for example, to reduce the risk of developing sepsis, septic shock or even the risk of death in the days following the measurement of the level of expression of the marker (s).
• immunostimulatory treatments suitable for preventing the risk of complications are, without limitation, treatment with GM-CSF, IL7, IFNy or anti-PD1.
• prophylactic antibiotic treatments suitable for preventing pneumonia are described in particular in the Annales conses d'Aesthésie and Reanimation (30, 2011; 168-190).
• a patient who is not at risk of complication can be referred quickly to a day-care service, such as an infectious disease department, rather than staying in a service with close monitoring that he will not need. .
• another object of the invention is a kit for to predict the risk of complication in a patient suspected of having an infection with a SOFA score below two, comprising at least one specific binding partner of at least one expression product of the VEGFR2 gene and at least one specific binding partner of at least one expression product of the uPAR gene.
• kits for measuring the level of expression, in vitro or ex vivo, of at least one expression product of the VEGFR2 gene and at least one expression product of the uPAR gene, in a biological sample comprising:
• a calibrated control for containing the amounts of said at least one expression product of the VEGFR2 gene which correspond to known quantities of said at least one expression product of the VEGFR2 gene and
• a calibrated control for containing the amounts of said at least one expression product of the uPAR gene which correspond to known quantities of said at least one expression product of the uPAR gene.
• a control contains a known quantity of one or more expression products of the marker (s) cited in the present application.
• the control contains a known quantity of one or more expression products of only one of the markers mentioned in the present application.
• This control may be either a synthetic sample containing a calibrated quantity of expression product (s) of the gene (s) of interest, or a biological sample whose known quantity (s) of expression product (s) of the genes of interest.
• said specific binding partner of an expression product of the kit according to the invention is at least one hybridization probe and / or at least one amplification primer, or at least one antibody, or at least one antibody fragment, or at least one affinity protein, or at least one aptamer.
• the invention also covers the use of a kit according to the invention for carrying out the method of the invention, and in particular for predicting the risk of complication, in a patient suspected of having an infection having a SOFA score of less than two.
• said specific binding partner of an expression product of the kit according to the invention is at least one hybridization probe and / or at least one amplification primer, or at least one antibody, or at least one antibody fragment, or at least one affinity protein, or at least one aptamer.
• Figure 1 represents box plots (or box diagrams) which correspond to graphical representations of the expression levels of the sVEGFR2 and suPAR proteins in a sample taken immediately after admission to the emergency department (T0) according to the occurrence or not of complications in patients: A. Levels of expression of sVEGFR2 to T0; B. Levels of expressions of suPAR at T0; Figure 2 represents box plots (or box diagrams) which correspond to graphic representations of the expression levels of the sVEGFR2 and suPAR proteins in a sample taken six hours after the first sampling (T6) according to the occurrence or not of complications in patients: A. Levels of expression of sVEGFR2 to T6; B. Expression levels of suPAR to T6;
• Figure 3 represents box plots (or box diagrams) that represent graphical representations of the variation in expression level of the sVEGFR2 marker between the first blood sample immediately after admission to the emergency department (T0) and the second six blood sample. hours after the first sampling (T6) according to the occurrence or not of complications in patients.
• FIG. 4 represents the apparent ROC curve of the association between the combination sVEGFR2 and suPAR at T0 (first blood sample immediately after admission to the emergency department) and the probability of complication of the patients during the 72h following the first sampling T0.
• Example 1 Obtaining and Preparing Blood Samples This retrospective observational study was conducted in patients aged 19 to 101 years (111 men, 122 women, median age: 53 years) newly admitted to the emergency department of 14 French and Belgian hospitals between 2015 and 2017 for the burden of a suspected infection. These patients are all hospitalized for suspected infection. The inclusion criteria were as follows:
• o respiratory rate greater than 20 breaths per minute or PaCO 2 ⁇ 32 mmHg
• o leukocyte count greater than 12000 / mm3 or less than 4000 / mm3.
• the clinical exclusion criteria were:
• the first blood sample was taken within the first 12 hours after the arrival of the patient in the emergency department (T0);
• the second blood sample was taken between 4 and 8 hours (T6 ⁇ 2h) following the first blood sample (T0);
• the mortality is evaluated at 28 days following the arrival of the patient in the emergency department.
• the complication was determined by a adjudication committee composed of 3 independent physicians under study. This committee determines the complication according to several criteria; in particular, the appearance of new organ failures (increase of the score of SOFA), the death or the necessity of entering the intensive care unit. Of the 233 patients in this cohort, 36 patients (21%) will get complicated within 72 hours of admission (“COMPLICATION”) and 185 patients (79%) will not get complicated (“NON COMPLICATION”)
• SVEGFR2 protein was assayed using antibody marketed by Biotechne ® (mAb anti Human VEGFR2 (KDR) ref: MAB3573, and Human VEGF R2 / KDR / Flk-l Antibody Antigen Affinity-Purified Goat IgG Polyclonal ref: AF357 ) and an ELISA test using the Vidas ® controller (bioMérieux). To do this, the ELISA test was constructed using the reagents of the Vidas ® BRAHMS PCT TM kit cartridge (bioMérieux, Cat No.30450) without the use of antibodies and control calibrators.
• VIDAS ® is a multiparametric automaton of immunoanalyses. It is a closed system for unit testing, offering great flexibility. This machine is characterized by its robustness, flexibility, ease of use and is intended for small and medium size laboratories. It allows routine tests, confirmation and tests with high medical value.
• the detection is done by the Enzyme Linked Fluorescent Assay (ELFA) technique in serum or plasma.
• ELFA assay principle is the combination of immunoenzymatic reactions with fluorescence endpoint detection.
• the enzyme used is alkaline phosphatase which catalyzes the hydrolysis reaction of the substrate, 4-methylumbelliferyl phosphate into a product; 4-methyl-umbellierone.
• the product emits at a wavelength of 450 nm after excitation at 370 nm.
• the results are analyzed automatically by VIDAS® and expressed in relative fluorescence intensity or RFV (Relative Fluorescent Value). This RFV value is determined by subtracting the background noise value (BKG) from the raw value obtained.
• RFV Relative Fluorescent Value
• the cones were sensitized with monoclonal antibody MAB3573 at a concentration of 2.5 ⁇ g / ml. (Indirect coating with a first anti-mouse mAb at 10 ⁇ g / ml then anti-sVEGFR2 Ab at 2.5 mg / ml); 2. The contents of the fourth well of the Vidas ® BRAHMS PCT TM Kit Cartridge were replaced with 400 m ⁇ of revealing antibody (ref .: AF357), coupled with biotin, diluted to 1 ⁇ g / ml;
• the ELISA reaction was performed using the Vidas ® controller and the Vidas ® BRAHMS PCT TM kit protocol;
• the results were obtained as raw values after subtraction of the background noise (reading of the substrate before reaction).
• a standard curve was established by assaying a range of concentrations of the marker in the form of recombinant protein (Recombinant Human VEGF R2 / KDR / Flk-l Fc Chimera Biotechne ® ref. 357-KD-050).
• the standard curve was plotted by plotting the concentration of the marker on the abscissa and the signal read by Vidas ® (RFV or Relative Fluorescence Value).
• the marker concentration present in the serum was calculated by plotting the concentration corresponding to the RFV signal read by Vidas ® .
• suPAR Blood levels of suPAR were measured using frozen sera (samples stored at -80 ° C). The samples were analyzed using the CE / IVD labeled AUTO / CE marked suPARnostic ® ELISA kit, according to the manufacturer's instructions (Virogates, Birkeroed, Denmark).
• the suPARnostic ® ELISA is based on a simplified monoclonal double-antibody sandwich ELISA, in which serum samples and peroxidase-conjugated anti-suPARs are first mixed and then incubated in anti-suPAR pre-coated micro-wells. .
• the recombinant suPAR standards of the kit are calibrated and make it possible to calculate a standard curve.
• the concentrations of suPAR are determined in ng / ml of plasma. The test was validated to measure suPAR levels between 0.6 and 22 ng / ml.
• the predictive ability to measure the level of expression of markers was studied in relation to the occurrence or non-occurrence of complications in patients within 72 hours after the first T0 blood sample.
• the Wilcoxon-Mann-Whitney test was used to characterize this association.
• VEGFR2 and suPAR at T0 and T6 were measured as described above in blood samples of 233 patients suspected of having an infection with a SOFA score of less than two. The results are shown in Table 5 and Figures 1 and 2.
• the level of expression of the markers studied makes it possible to discriminate the patients who will become complicated within 72 hours after the first blood sample at T0 of those who will not complicate.
• patients with complications will have lower levels of SVEGFR2 expression than patients who do not suffer from any complication.
• patients with complications they have higher levels of expression than patients who do not suffer from any complication.
• the level of expression of sVEGFR2 at T0 and T6 was measured as described above in the plasma samples of 233 patients suspected of having an infection with a SOFA score of less than two. For each patient the variation was calculated according to the following formula:
• OR 1: no association
• OR ⁇ 1 an increase from 1st to 3rd quartile is associated with a decrease in the risk of complication
• OR> 1 an increase from 1st to 3rd quartile is associated with an increased risk of complication
• the IQR is the quartile gap.
• the IQR is a measure of dispersion that is obtained by differentiating between the third and the first quartile.
• IQR.OR was measured for blood samples from 233 patients suspected of having an infection with a SOFA score of less than two.
• the logistic regression model was also performed to analyze the performance ratio performance of sVEGFR2 and suPAR.
• the objective is to show that the ratio of markers is significantly associated with the risk of complication within 72 hours after the first sampling.
• the value of the IQR.OR for the suPAR marker on the studied population is 1.49 with a p-value equal to 0.02.
• patients with a high suPAR expression level (3rd quartile) have a significantly higher probability of complication (1.49 times) than patients with a low expression level (1st quartile).
• the value of the IQR.OR for the ratio between sVEGFR2 and suPAR is 1.74 with a p-value equal to 0.0005.
• patients with an expression ratio between sVEGFR2 and high suPAR (3rd quartile) have a significantly higher likelihood of complication (1.74 times) than patients with a low expression ratio (1st quartile).
• RT-PCR Trends and Problems Journal of Molecular Endocrinology, 29: 23-39
• VEGF Vascular Endothelial Growth Factor
• VEGFR Vascular Endothelial Growth Factor

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