SYNDROME

FIELD OF THE INVENTION:

The present invention relates to a method for predicting the risk of developing acute respiratory distress syndrome (ARDS).

BACKGROUND OF THE INVENTION:

Acute respiratory distress syndrome (ARDS) still carries a high mortality rate (1, 2), and a major challenge in targeting the prevention and early treatment of ARDS is the inability to accurately predict which patients will develop the syndrome (3). The Lung Injury Prediction Score (LIPS) was developed to identify patients at high risk of developing ARDS; however, its positive predictive value is limited (0.14-0.23) (4). To improve prediction, one method may be to combine clinical data with plasma biomarkers that reflect the pathogenesis of ARDS such as angiopoietin-2 (Ang-2), a marker of lung endothelial injury (5). In contrast, the soluble receptor for advanced glycation end-products (sRAGE), a marker of lung epithelial injury (6), may predict ARDS more accurately in selected at-risk patients, e.g. after cardiac surgery (7), severe trauma (8), or after major surgery (9). The main soluble forms of RAGE include the extracellular domain of membrane RAGE that is cleaved by proteinases (10) and the endogenous secretory RAGE (esRAGE), which is produced after alternative splicing (1 1). sRAGE has good diagnostic value for ARDS and is associated with lung injury severity, impaired alveolar fluid clearance, and prognosis (12, 13). Although not yet ready for clinical use, genomic applications could facilitate better prediction, diagnosis, disease subclassification, and prognosis for ARDS (14-16). However, the association of single nucleotide polymorphisms (SNPs) flanking the RAGE gene with susceptibility to ARDS remains unknown.

The inventors demonstrated that plasma levels of RAGE iso forms and RAGE SNPs predict the development of ARDS in a high-risk population of patients admitted to intensive care units (ICUs).

SUMMARY OF THE INVENTION:

The present invention relates to a method for predicting the risk of developing acute respiratory distress syndrome (ARDS).

DETAILED DESCRIPTION OF THE INVENTION:

Prediction of acute respiratory distress syndrome (ARDS) remains challenging despite available clinical scores, although identifying molecular endotypes might increase our ability to predict ARDS in at-risk patients. The inventors aimed to assess soluble isoforms and gene variants of the receptor for advanced glycation end-products (RAGE), a marker of lung epithelial injury, as predictors of ARDS in a high-risk population. The soluble receptor for advanced glycation end-products (sRAGE) is a marker of alveolar type I cell injury and correlates with severity and outcome in patients with acute respiratory distress syndrome (ARDS). However, the predictive values of RAGE soluble forms and gene polymorphism for ARDS development in high-risk critically ill patients has never been investigated. The inventors conducted a multicenter, prospective study including adult patients with at least one ARDS risk factor upon admission to one of five intensive care units in France. Plasma soluble RAGE (sRAGE) and endogenous secretory RAGE (esRAGE) were measured at baseline and 24 hours later (day one), and four RAGE single nucleotide polymorphisms (SNPs) were selectively assayed because of previous reports of functionality (rsl800625, rsl800624, rs3134940, and rs2070600). The primary outcome was ARDS development within seven days.

Of 500 patients enrolled, 464 patients who did not develop ARDS within 24 hours of baseline were analyzed, and 59 developed ARDS by day seven. Higher baseline and day one plasma sRAGE, but not esRAGE, were independently associated with an increased rate of ARDS development (OR, 2.25 [95% CI, 1.60-3.16] and 4.33 [95% CI, 2.85-6.56], respectively). The RAGE rs2070600 SNP was associated with increased risk of developing ARDS and higher plasma sRAGE. Among critically ill at-risk patients, higher plasma sRAGE and RAGE SNP rs2070600 identify those who will develop ARDS, thus refining the prediction of ARDS.

Accordingly, the present invention relates to a method for predicting the risk of having or developing acute respiratory distress syndrome (ARDS) in a patient in need thereof, comprising the step of determining the expression level of Plasma soluble RAGE (sRAGE) in a biological sample obtained from said patient.

In a further aspect, the present invention relates to a method for predicting the risk of having or developing acute respiratory distress syndrome (ARDS) in a patient in need thereof, comprising the step of detecting RAGE SNP rs2070600 in a biological sample obtained from said patient.

In a further aspect, the present invention relates to a method for predicting the risk of having or developing acute respiratory distress syndrome (ARDS) in a patient in need thereof, comprising the step of determining the expression level of Plasma soluble RAGE (sRAGE) and/or detecting RAGE SNP rs2070600 in a biological sample obtained from said patient. In a further aspect, the present invention relates to a method for detecting RAGE SNP rs2070600 in a biological sample obtained from a patient in need thereof.

As used herein, the term "patient" denotes a mammal. The term "patient" also refers to any patient (preferably human) admitted to intensive care unit. Typically, a patient according to the invention refers to any at-risk patient having direct insult to the lung such as Pneumonia, Aspiration of gastric contents, Inhalational injury, Pulmonary contusion, Pulmonary vasculitis Drowning. Typically, a patient according to the invention refers to any at-risk patient having indirect insult to the lung such as Non-pulmonary sepsis, Major trauma, High-risk surgery, Pancreatitis, Severe burns, Non-cardiogenic shock, Drug overdose, Multiple transfusions or transfusion-associated acute lung injury (TRALI).

The term "acute respiratory distress syndrome" or "ARDS" has its general meaning in the art and refers to acute respiratory distress syndrome such as revised in the World Health Organisation Classification (ICD-11) CA60. The term "acute respiratory distress syndrome" also refers to acute respiratory distress syndrome such as revised in the World Health Organisation Classification (ICD- 10) J80-J84.

The term "biological sample" refers to any biological sample derived from the patient such as blood sample, plasma sample, or serum sample. Said biological sample is obtained for the purpose of the in vitro evaluation.

The term "Plasma soluble RAGE" or "sRAGE" has its general meaning in the art and refers to plasma soluble receptor for advanced glycation end-products (6).

The term "RAGE SNP rs2070600" has its general meaning in the art and refers to receptor for advanced glycation end-products (RAGE) single nucleotide polymorphisms (SNPs) rs2070600.

In some embodiments, the present invention further comprises the steps of comparing the expression level of the biomarker with a predetermined reference value and concluding that the patient is at risk of having or developing ARDS when the expression level of the biomarker is higher than the predetermined reference value or concluding that the patient is not at risk of having or developing ARDS when the expression level of the biomarker is lower than the predetermined reference value.

Accordingly, the present invention also relates to a method for predicting the risk of having or developing acute respiratory distress syndrome (ARDS) in a patient in need thereof, comprising the steps of: i) determining the expression level of Plasma soluble RAGE (sRAGE) and/or detecting RAGE SNP rs2070600 in a biological sample obtained from said patient, ii) comparing the expression level determined at step i) with a predetermined reference value and concluding that the patient is at risk of having or developing ARDS when the expression level determined at step i) is higher than the predetermined reference value and/or when the RAGE SNP rs2070600 is detected, or concluding that the patient is not at risk of having or developing ARDS when the expression level determined at step i) is lower than the predetermined reference value and/or when the RAGE SNP rs2070600 is not detected.

In some embodiment, the method of the invention is performed at measured at baseline, the day of admission into the intensive care unit.

In some embodiment, the method of the invention is performed during the 24 hours after admission into the intensive care unit.

In some embodiment, the method of the invention is performed during the 1, 2, 3, 4, 5,

6 and 7 days after admission into the intensive care unit.

As used herein, the "reference value" refers to a threshold value or a cut-off value. The setting of a single "reference value" thus allows discrimination between patient at risk of having or developing acute respiratory distress syndrome (ARDS) and patient not at risk of having or developing acute respiratory distress syndrome (ARDS) with respect to the overall survival (OS) for a patient. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Preferably, the person skilled in the art may compare the expression level (obtained according to the method of the invention) with a defined threshold value. In one embodiment of the present invention, the threshold value is derived from the expression level (or ratio, or score) determined in a biological sample derived from one or more patients at risk of having or developing acute respiratory distress syndrome (ARDS). Furthermore, retrospective measurement of the expression level (or ratio, or scores) in properly banked historical patient samples may be used in establishing these threshold values.

Predetermined reference values used for comparison may comprise "cut-off or "threshold" values that may be determined as described herein. Each reference ("cut-off) value for the biomarker of interest may be predetermined by carrying out a method comprising the steps of a) providing a collection of samples from patients at risk of having or developing acute respiratory distress syndrome (ARDS);

b) determining the expression level of the bio marker for each sample contained in the collection provided at step a);

c) ranking the biological samples according to said expression level;

d) classifying said samples in pairs of subsets of increasing, respectively decreasing, number of members ranked according to their expression level,

e) providing, for each sample provided at step a), information relating to the risk of having or developing acute respiratory distress syndrome (ARDS) or the actual clinical outcome for the corresponding patient (i.e. the duration of the overall survival (OS));

f) for each pair of subsets of samples, obtaining a Kaplan Meier percentage of survival curve;

g) for each pair of subsets of samples calculating the statistical significance (p value) between both subsets;

h) selecting as reference value for the expression level, the value of expression level for which the p value is the smallest.

For example the expression level of a biomarker has been assessed for 100 samples of 100 patients. The 100 samples are ranked according to their expression level. Sample 1 has the best expression level and sample 100 has the worst expression level. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding patient, Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated.

The reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of expression levels.

In routine work, the reference value (cut-off value) may be used in the present method to discriminate samples and therefore the corresponding patients. Kaplan-Meier curves of percentage of survival as a function of time are commonly to measure the fraction of patients living for a certain amount of time after treatment and are well known by the man skilled in the art.

The man skilled in the art also understands that the same technique of assessment of the expression level of a biomarker should of course be used for obtaining the reference value and thereafter for assessment of the expression level of a biomarker of a patient subjected to the method of the invention.

In one embodiment, the reference value may correspond to the expression level of the biomarker determined in a sample associated with patient at risk of having or developing acute respiratory distress syndrome (ARDS). Accordingly, a higher or equal expression level of the biomarker than the reference value is indicative of a patient at risk of having or developing acute respiratory distress syndrome (ARDS), and a lower expression level of the biomarker than the reference value is indicative of a patient not at risk of having or developing acute respiratory distress syndrome (ARDS).

In another embodiment, the reference value may correspond to the expression level of the biomarker determined in a sample associated with patient not at risk of having or developing acute respiratory distress syndrome (ARDS). Accordingly, a higher expression level of the biomarker than the reference value is indicative of a patient at risk of having or developing acute respiratory distress syndrome (ARDS), and a lower or equal expression level of the biomarker than the reference value is indicative of a patient at risk of having or developing acute respiratory distress syndrome (ARDS).

In another embodiment, a score which is a composite of the expression levels of the different biomarkers may also be determined and compared to a reference value wherein a difference between said score and said reference value is indicative of a patient at risk or not at risk of having or developing acute respiratory distress syndrome (ARDS).

In a particular embodiment, the score may be generated by a computer program.

In some embodiment, the reference value is 1033 pg/mL.

In some embodiments, the present invention also relates to a method for predicting the risk of having or developing acute respiratory distress syndrome (ARDS) in a patient in need thereof, comprising the steps of: i) determining the expression level of Plasma soluble RAGE (sRAGE) and/or detecting RAGE SNP rs2070600 in a biological sample obtained from said patient, ii) comparing the expression level determined at step i) with a predetermined reference value and concluding that the patient is at risk of having or developing ARDS when the expression level determined at step i) is higher than the 1033 pg/mL and/or when the RAGE SNP rs2070600 is detected, or concluding that the patient is not at risk of having or developing ARDS when the expression level determined at step i) is lower than the 1033 pg/mL and/or when the RAGE SNP rs2070600 is not detected.

Analyzing the bio marker expression level may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed nucleic acid sequence or translated protein of Plasma soluble RAGE (sRAGE) such as described in the example.

In one embodiment, the biomarker expression level is assessed by analyzing the expression of the protein translated from said gene. Said analysis can be assessed using an antibody (e.g., a radio-labelled, chromophore-labelled, fluorophore-labelled, or enzyme- labelled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin-streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to the protein translated from the gene encoding for the biomarker.

Methods for measuring the expression level of a biomarker in a sample may be assessed by any of a wide variety of well-known methods from one of skill in the art for detecting expression of a protein including, but not limited to, direct methods like mass spectrometry- based quantification methods, protein microarray methods, enzyme immunoassay (EIA), radioimmunoassay (RIA), Immunohistochemistry (IHC), Western blot analysis, ELISA, Luminex, ELISPOT and enzyme linked immunosorbent assay and indirect methods based on detecting expression of corresponding messenger ribonucleic acids (mRNAs). The mRNA expression profile may be determined by any technology known by a man skilled in the art. In particular, each mRNA expression level may be measured using any technology known by a man skilled in the art, including nucleic microarrays, quantitative Polymerase Chain Reaction (qPCR), next generation sequencing and hybridization with a labelled probe.

Said direct analysis can be assessed by contacting the sample with a binding partner capable of selectively interacting with the biomarker present in the sample. The binding partner may be an antibody that may be polyclonal or monoclonal, preferably monoclonal (e.g., a isotope-label, element-label, radio-labelled, chromophore- labelled, fluorophore-labelled, or enzyme-labelled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin-streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to the protein translated from the gene encoding for the biomarker of the invention. In another embodiment, the binding partner may be an aptamer. The binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as an isotope, an element, a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

As used herein, the term "labelled", with regard to the antibody, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as an isotope, an element, a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be produced with a specific isotope or a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited to radioactive atom for scintigraphy studies such as 1123, 1124, Inl l l, Rel86, Rel88, specific isotopes include but are not limited to 13C, 15N, 1261, 79Br, 81 Br.

The afore mentioned assays generally involve the binding of the binding partner (ie. antibody or aptamer) to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidene fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, silicon wafers.

In a particular embodiment, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies which recognize said biomarker. A sample containing or suspected of containing said biomarker is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art such as Singulex, Quanterix, MSD, Bioscale, Cytof.

In one embodiment, an Enzyme-linked immunospot (ELISpot) method may be used. Typically, the sample is transferred to a plate which has been coated with the desired anti- biomarker capture antibodies. Revelation is carried out with biotinylated secondary Abs and standard colorimetric or fluorimetric detection methods such as streptavidin-alkaline phosphatase and NBT-BCIP and the spots counted. In one embodiment, when multi-biomarker expression measurement is required, use of beads bearing binding partners of interest may be preferred. In a particular embodiment, the bead may be a cytometric bead for use in flow cytometry. Such beads may for example correspond to BD™ Cytometric Beads commercialized by BD Biosciences (San Jose, California). Typically cytometric beads may be suitable for preparing a multiplexed bead assay. A multiplexed bead assay, such as, for example, the BD(TM) Cytometric Bead Array, is a series of spectrally discrete beads that can be used to capture and quantify soluble antigens. Typically, beads are labelled with one or more spectrally distinct fluorescent dyes, and detection is carried out using a multiplicity of photodetectors, one for each distinct dye to be detected. A number of methods of making and using sets of distinguishable beads have been described in the literature. These include beads distinguishable by size, wherein each size bead is coated with a different target-specific antibody (see e.g. Fulwyler and McHugh, 1990, Methods in Cell Biology 33:613-629), beads with two or more fluorescent dyes at varying concentrations, wherein the beads are identified by the levels of fluorescence dyes (see e.g. European Patent No. 0 126,450), and beads distinguishably labelled with two different dyes, wherein the beads are identified by separately measuring the fluorescence intensity of each of the dyes (see e.g. U.S. patent Nos. 4,499,052 and 4,717,655). Both one-dimensional and two-dimensional arrays for the simultaneous analysis of multiple antigens by flow cytometry are available commercially. Examples of one-dimensional arrays of singly dyed beads distinguishable by the level of fluorescence intensity include the BD(TM) Cytometric Bead Array (CBA) (BD Biosciences, San Jose, Calif.) and Cyto-Plex(TM) Flow Cytometry microspheres (Duke Scientific, Palo Alto, Calif.). An example of a two-dimensional array of beads distinguishable by a combination of fluorescence intensity (five levels) and size (two sizes) is the QuantumPlex(TM) microspheres (Bangs Laboratories, Fisher, Ind.). An example of a two- dimensional array of doubly-dyed beads distinguishable by the levels of fluorescence of each of the two dyes is described in Fulton et al. (1997, Clinical Chemistry 43(9): 1749-1756). The beads may be labelled with any fluorescent compound known in the art such as e.g. FITC (FL1), PE (FL2), fluorophores for use in the blue laser (e.g. PerCP, PE-Cy7, PE-Cy5, FL3 and APC or Cy5, FL4), fluorophores for use in the red, violet or UV laser (e.g. Pacific blue, pacific orange). In another particular embodiment, bead is a magnetic bead for use in magnetic separation. Magnetic beads are known to those of skill in the art. Typically, the magnetic bead is preferably made of a magnetic material selected from the group consisting of metals (e.g. ferrum, cobalt and nickel), an alloy thereof and an oxide thereof. In another particular embodiment, bead is bead that is dyed and magnetized. In one embodiment, protein microarray methods may be used. Typically, at least one antibody or aptamer directed against the biomarker is immobilized or grafted to an array(s), a solid or semi- so lid surface(s). A sample containing or suspected of containing the biomarker is then labelled with at least one isotope or one element or one fluorophore or one colorimetric tag that are not naturally contained in the tested sample. After a period of incubation of said sample with the array sufficient to allow the formation of antibody-antigen complexes, the array is then washed and dried. After all, quantifying said biomarker may be achieved using any appropriate microarray scanner like fluorescence scanner, colorimetric scanner, SIMS (secondary ions mass spectrometry) scanner, maldi scanner, electromagnetic scanner or any technique allowing to quantify said labels.

In another embodiment, the antibody or aptamer grafted on the array is labelled.

In another embodiment, reverse phase arrays may be used. Typically, at least one sample is immobilized or grafted to an array(s), a solid or semi-solid surface(s). An antibody or aptamer against the suspected biomarker is then labelled with at least one isotope or one element or one fluorophore or one colorimetric tag that are not naturally contained in the tested sample. After a period of incubation of said antibody or aptamer with the array sufficient to allow the formation of antibody-antigen complexes, the array is then washed and dried. After all, detecting quantifying and counting by D-SIMS said biomarker containing said isotope or group of isotopes, and a reference natural element, and then calculating the isotopic ratio between the biomarker and the reference natural element, may be achieve using any appropriate microarray scanner like fluorescence scanner, colorimetric scanner, SIMS (secondary ions mass spectrometry) scanner, maldi scanner, electromagnetic scanner or any technique allowing to quantify said labels.

In one embodiment, said direct analysis can also be assessed by mass Spectrometry. Mass spectrometry-based quantification methods may be performed using either labelled or unlabelled approaches (DeSouza and Siu, 2012). Mass spectrometry-based quantification methods may be performed using chemical labeling, metabolic labelingor proteolytic labeling.

Mass spectrometry-based quantification methods may be performed using mass spectrometry label free quantification, LTQ Orbitrap Velos, LTQ-MS/MS, a quantification based on extracted ion chromatogram EIC (progenesis LC-MS, Liquid chromatography-mass spectrometry) and then profile alignment to determine differential expression of the biomarker.

In another embodiment, the biomarker expression level is assessed by analyzing the expression of mR A transcript or mRNA precursors, such as nascent R A, of biomarker gene.

Said analysis can be assessed by preparing mR A/cDNA from cells in a sample from a subject, and hybridizing the mRNA/cDNA with a reference polynucleotide. The prepared mRNA/cDNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses, such as quantitative PCR (TaqMan), and probes arrays such as GeneChip(TM) DNA Arrays (AFFYMETRIX).

Advantageously, the analysis of the expression level of mRNA transcribed from the gene encoding for biomarkers involves the process of nucleic acid amplification, e. g., by RT- PCR (the experimental embodiment set forth in U. S. Patent No. 4,683, 202), ligase chain reaction (Barany, 1991), self- sustained sequence replication (Guatelli et al, 1990), transcriptional amplification system (Kwoh et al., 1989), Q-Beta Replicase (Lizardi et al, 1988), rolling circle replication (U. S. Patent No. 5,854, 033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

In the methods according to the present the invention, the presence or absence of a SNP can be determined by nucleic acid sequencing, PCR analysis or any genotyping method known in the art such as the method described in the example. Examples of such methods include, but are not limited to, chemical assays such as allele specific hybridization, pyrosequencing, primer extension, allele specific oligonucleotide ligation, sequencing, enzymatic cleavage, flap endonuclease discrimination; and detection methods such as fluorescence, chemiluminescence, and mass spectrometry.

For example, the presence or absence of said polymorphism may be detected in a DNA sample, preferably after amplification. For instance, the isolated DNA may be subjected to couple reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for the polymorphism or that enable amplification of a region containing the polymorphism. According to a first alternative, conditions for primer annealing may be chosen to ensure specific reverse transcription (where appropriate) and amplification; so that the appearance of an amplification product be a diagnostic of the presence of the polymorphism according to the invention. Otherwise, DNA may be amplified, after which a mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art.

Currently numerous strategies for genotype analysis are available (Antonarakis et al,

1989; Cooper et al, 1991; Grompe, 1993). Briefly, the nucleic acid molecule may be tested for the presence or absence of a restriction site. When a base polymorphism creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR genotype the polymorphism. Further strategies include, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele specific PCR; PCR using mutagenic primers; ligase-PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single-stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et al., 1997). Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method; by enzymatic sequencing, using the Sanger method; mass spectrometry sequencing; pyrosequencing; sequencing using a chip-based technology and real-time quantitative PCR. Preferably, DNA from a patient is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers. However several other methods are available, allowing DNA to be studied independently of PCR, such as the rolling circle amplification (RCA), the InvaderTMassay, or oligonucleotide ligation assay (OLA). OLA may be used for revealing base polymorphisms. According to this method, two oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the polymorphism. DNA ligase will covalently join the two oligonucleotides only if they are perfectly hybridized to one of the allele.

Oligonucleotide probes or primers may contain at least 10, 15, 20 or 30 nucleotides. Their length may be shorter than 400, 300, 200 or 100 nucleotides.

According to the invention, the determination of the presence or absence of said SNP may also be determined by detection or not of the mutated protein by any method known in the art. The presence of the protein of interest may be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith. Labels are known in the art that generally provide (either directly or indirectly) a signal. As used herein, the term "labelled" with regard to the antibody or aptamer, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5), to the antibody or aptamer, as well as indirect labelling of the probe or antibody (e.g., horseradish peroxidise, HRP) by reactivity with a detectable substance. An antibody or aptamer may be also labelled with a radioactive molecule by any method known in the art. For example, radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Inl 11, Rel86 and Rel88. The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which may be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, etc.

More particularly, an ELISA method may be used, wherein the wells of a microtiter plate are coated with an antibody against the protein to be tested. A biological sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate (s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

Alternatively, an immunohistochemistry (IHC) method may be used. IHC specifically provides a method of detecting a target in a biological sample or tissue specimen in situ. The overall cellular integrity of the sample is maintained in IHC, thus allowing detection of both the presence and location of the target of interest. Typically a biological sample is fixed with formalin, embedded in paraffin and cut into sections for staining and subsequent inspection by light microscopy. Current methods of IHC use either direct labeling or secondary antibody- based or hapten-based labeling. Examples of known IHC systems include, for example, En Vision™ (DakoCytomation), Powervision® (Immunovision, Springdale, AZ), the NBA™ kit (Zymed Laboratories Inc., South San Francisco, CA), HistoFine® (Nichirei Corp, Tokyo, Japan).

A further object relates to a kit for performing the methods of the present invention, wherein said kit comprises means for measuring the expression level of Plasma soluble RAGE (sRAGE) and/or detecting RAGE SNP rs2070600 that is indicative of patient at risk of having or developing acute respiratory distress syndrome (ARDS).

Typically the kit may include antibodies, primers, probes, macroarrays or microarrays as above described. For example, the kit may comprise a set of antibodies, primers, or probes as above defined, and optionally pre-labelled. Alternatively, antibodies, primers, or probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers. The kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards. The kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.

Typically the kit may include primers such as described in Table E2.

In a further aspect, the present invention relates to a method of treating acute respiratory distress syndrome (ARDS), wherein the patient has been diagnosed as at risk of having or developing ARDS by performing the method of the invention.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

In some embodiment, the present invention relates to a method of preventing acute respiratory distress syndrome (ARDS), wherein the patient has been diagnosed as at risk of having or developing ARDS by performing the method of the invention.

The method of the invention is particularly suitable for reaching a clinical decision. As used herein the term "clinical decision" refers to any decision to take or not take an action that has an outcome that affects the health or survival of the patient. In particular, in the context of the invention, a clinical decision refers to a decision to use anti-ARDS treatment. Once the patient is at risk of having or developing ARDS, a p2-agonist, a corticoid, and an anti-ARDS treatment may be administered.

The term "anti-ARDS treatment" has its general meaning in the art and refers to β2- agonist such as salbutamol, salmeterol, orciprenaline, terbutaline, and fenoterol; corticosteroids such as 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, anecortave acetate. and any of their derivatives; and pulmonary vasodilator such as nitric oxide.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Flow diagram of the PrediRAGE study. Other reasons for not including patients who were screened included a shortage of personnel, which hampered timely enrolment into the study, and the absence of reason.

Figure 2: Plasma biomarker levels according to ARDS development. Patients who developed ARDS at least 24 hours after the first sample draw (n = 59) had statistically significant increased A) baseline plasma sRAGE, B) plasma sRAGE on day one, C) day one- to-day zero plasma sRAGE ratio, D) baseline plasma sRAGE-to-esRAGE ratio, and E) plasma sRAGE-to-esRAGE ratio on day one than those who did not develop ARDS (n = 405). F) Baseline plasma esRAGE, G) plasma esRAGE on day one, and H) day one-to-day zero plasma esRAGE ratio were similar between groups.

Figure 3: Receiver operating characteristic curves. A) Baseline plasma sRAGE (AUROC, 0.74; 95% CI, 0.68-0.80) and B) plasma sRAGE on day one (AUROC, 0.82; 95% CI, 0.76-0.88) each showed good discrimination between those who developed ARDS and those who did not, but C) the LIPS was poorly discriminative in this population of patients at risk of developing ARDS (AUROC, 0.57; 95% CI, 0.49-0.65).

Figure 4: The cumulative proportion of patients who did not develop ARDS within seven days of admission to the ICU for A) patients with baseline plasma sRAGE above or below 1033 pg/mL (the median value of baseline plasma sRAGE in patients from our cohort) and B) patients with or without homozygous SNP rs2070600 (the Ser/Ser genotype) within the gene coding for RAGE.

EXAMPLE:

Receptor for advanced glycation end-products and ARDS prediction: a multicenter observational study

Material & Methods

Study design and participants

PrediRAGE (PREDIctive values of plasma soluble RAGE levels and gene polymorphisms for the onset of ARDS in critically ill patients) is an investigator-initiated, multicenter, observational, cohort study undertaken at five ICUs from two hospitals in Clermont-Ferrand, France. The study protocol was approved by the institutional review board Comite de Protection des Personnes Sud-Est-VI (AU10732) and the Comite consultatif sur le traitement de l'information en matiere de recherche (14.017). This study was performed in accordance with the Strengthening the Reporting of Observational studies in Epidemiology (STROBE) statement 17).

Patients aged 18 years or older were eligible if they were admitted to participating ICUs with at least one identified ARDS risk factor (Table El)(l). All participants, or their next of kin, provided written consent. There was no deviation from the approved protocol. This trial was registered with ClinicalTrials.gov, number NCT02070536.

Biological sample collection and measurements

Plasma specimens were obtained within six hours of ICU admission (day zero) and 24 hours later (day one). Plasma sRAGE and esRAGE were measured blindly in duplicate using commercially-available enzyme-linked immunosorbent assay kits (R&D Systems, MN, USA and B-Bridge International, Japan, respectively).

According to the results from previous studies of inflammatory conditions(18-21), four candidate RAGE SNPs were chosen a priori for genotyping: rs 1800625 (the C allele of the 429 T/C polymorphism within the promoter region), rsl 800624 (the A allele of the 374 T/A polymorphism within the promoter region), rs3134940 (the A allele of the 2184 A/G polymorphism within intron 8) and rs2070600 (the A allele of the Gly82Ser polymorphism within exon 3). Their minor allele frequencies were > 0.05 in the European patients. At baseline, a peripheral blood sample was obtained from each participant and DNA was isolated using FlexiGene DNA kit (Qiagen, Venlo, Netherlands). The concentrations of extracted DNAs were measured by NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA.); any sample with a DNA concentration < 50 ng/μΐ was excluded and required another sample. Genotyping of four selected RAGE SNPS was performed by GATC Biotech (Constance, Germany) using the chain termination method (Sanger sequencing) and included bioinformatic analysis. Primers for DNA sequencing and polymerase chain reaction are listed in Table E2. Each of the SNPs was analyzed for the Hardy- Weinberg equilibrium (HWE), and no SNP was excluded from the analysis because it was out of HWE (p < 0.05).

Primary outcome and additional variables

The primary outcome was the development of ARDS within seven days after enrolment into the study. ARDS was defined by physicians (blinded to RAGE levels and RAGE genotype) caring for the patients, based on criteria from the Berlin definition (1). ARDS risk factors were defined as previously described (Table El)(l). Patients who met the criteria for ARDS at baseline, before the sample draw, or within the subsequent 24 hours, were excluded to ensure the removal of ARDS that was present at baseline (Figure 1). Patients were followed for 30 days for mortality and ARDS developing after day seven.

Statistical analysis

Based on previous data(5, 22), we calculated a priori that a sample of 458 patients (with a minimal ARDS incidence of 8%) would be needed to detect a difference in baseline plasma sRAGE (mean ± standard deviation (SD), 500 ± 1000 pg/mL) between the patients with ARDS by day seven and those without, with a statistical power of 80%. Thus, we planned to include 500 patients. Qualitative data were expressed as numbers and percentages, and quantitative data as means, SD, or medians and interquartile ranges [IQR]. A Student's t-test or a Mann- Whitney test were used for quantitative parameters according to t-test assumptions. Categorical data were compared using a chi-square test or Fisher's exact test. Univariate correlations between quantitative outcomes were assessed using Pearson or Spearman correlation coefficients where appropriate. Then, multivariate logistic generalised linear mixed models (logistic for binary endpoint) were adjusted for the potential confounders, which were clinically relevant: sepsis, shock, pneumonia, and the simplified acute physiology score (SAPS II) (23). Both the plasma levels of RAGE iso forms and gene variants were covariate candidates for multivariate prediction. The centre (ICU) effect was considered a random-effect to account for between- and within-centre variability. Results were expressed as odds ratios (ORs, with a 95% confidence interval [CI]) for developing ARDS. Then, ARDS was considered a censored variable upon the last follow-up at day seven, assuming that patients who had been discharged from the ICU before day seven did not develop ARDS at this time-point. The log-rank test was performed for univariate comparisons, and the Cox proportional-hazards regression was used for multivariate analysis. Results were expressed as hazard ratios (HRs, with a 95% CI) for developing ARDS: 1) in patients with plasma sRAGE above or below its median values in the cohort, and 2) in patients with or without SNPs which were eventually found significant in univariate analysis. Finally, the discrimination was tested by calculating the area under a receiver operating characteristic curve (AUROC) and a 95% CI; thresholds were determined according to clinical relevance and usual indexes (e.g. Liu, Youden, efficiency).

Differences in genotype and allele frequencies were compared using a chi-square test or Fisher's exact test where appropriate. Plasma sRAGE and esRAGE were compared across genotypes using linear regression analyses adjusted for the factors previously defined. When appropriate, normality of dependent variables (e.g., plasma sRAGE) was achieved using logarithmic transformation. Genotype groups were analyzed separately and combined together (the homozygous minor allele genotype plus the heterozygous genotype). Sensitivity analyses were planned a priori in patients under invasive mechanical ventilation at baseline. A two-sided p-value < 0.05 was considered significant. Statistical analysis was performed with Stata software (vl4, StataCorp, College Station, TX, USA).

Tables

No ARDS Develop ARDS p-value

(n = 405) (n = 59)

Age (years) 61 ± 16 62 ± 16 0.5

Male sex 267 (66) 46 (78) 0.07

Race/ethnicity 0.2

- White 389 (96) 54 (92)

- Black 12 (3) 4 (7)

- Asian 4 (1) 1 (1)

Body mass index (kg/m ² ) 26.6 ± 6.2 26.9 ± 6.5 0.9

Primary admission diagnosis

- Cardiac 8 (2) 2 (3) 0.6

- Respiratory 251 (62) 38 (65) 0.6

- Gastrointestinal 69 (17) 11 (17) 0.9

- Infectious 117 (29) 22 (37) 0.2

- Neurological 56 (14) 9 (15) 0.8

- Major surgery 101 (25) 14 (23) 0.7

- Other 20 (5) 2 (3) 0.7

Coexisting chronic conditions

- Atherosclerosis 89 (22) 15 (25) 0.6

- Diabetes 73 (18) 8 (14) 0.5

- Hypertension 162 (40) 27 (46) 0.5

- Dyslipidemia 85 (21) 15 (25) 0.5

- Current smoking 101 (25) 1 (32) 0.3

- Asthma 12 (3) 2 (3) 0.9

- COPD 41 (10) 10 (17) 0.2

- Chronic renal failure requiring 12 (3) 1 (2) 0.7 dialysis

- Liver cirrhosis 20 (5) 2 (3) 0.8

- Cancer 77 (19) 14 (22) 0.6

Primary ARDS risk factor

- Shock 101 (25) 16 (27) 0.8

- Sepsis 113 (28) 20 (34) 0.3

- Pneumonia 126 (31) 29 (49) 0.1

- Aspiration 24 (6) 5 (8) 0.9

- Severe trauma 41 (10) 5 (8) 0.9 - Pancreatitis 24 (6) 23 (5) 0.9

- Drug overdose 32 (8) 4 (6) 0.7

- High-risk surgery 89 (22) 9 (14) 0.8

Lung Injury Prediction Score 4.9 ± 2.4 5.7 ± 2.8 0.07 (LIPS)

Simplified Acute Physiology 43 ± 19 49 ± 18 0.01 Score II

Vasopressor use at admission 93 (23) 17 (29) 0.5

Invasive ventilation at admission 186 (46) 34 (58) 0.09

Noninvasive ventilation at 28 (7) 3 (5) 0.8 admission

30-day mortality 49 (12) 11 (19) 0.003

Table 1. The baseline characteristics of patients who developed acute respiratory distress syndrome (ARDS) (n = 59) or did not develop ARDS (n = 405) by day seven. The data are presented as mean ± standard deviation or n (%). Analyses were performed using the Wilcoxon rank-sum, a chi-square test, or Fisher's exact test, as appropriate. Percentages may not exactly total 100% because of rounding. COPD: chronic obstructive pulmonary disease.

Models OR 95% CI p-value

Baseline sRAGE 2.25 [1.60-3.16] <10 ^"3

SAPS II 1.02 [1.00-1.03] 0.04

Sepsis 1.34 [0.65-2.78] 0.4

Shock 0.87 [0.43-1.74] 0.7

Pneumonia 1.55 [0.67-3.58] 0.3

Plasma sRAGE on day one 4.33 [2.85-6.56] <10 ^"3

SAPS II 1.01 [0.99-1.03] 0.2

Sepsis 1.21 [0.56-2.63] 0.6

Shock 0.80 [0.39-1.65] 0.6

Pneumonia 1.35 [0.56-3.26] 0.5

Day one-to-day zero sRAGE ratio 1.61 [1.17-2.22] 0.004

SAPS II 1.02 [0.99-1.03] 0.07 Sepsis 1.28 [0.63-2.61] 0.5

Shock 0.82 [0.42-1.63] 0.6

Pneumonia 2.10 [0.93-4.74] 0.07

Baseline sRAGE 3.21 [2.17-4.75] <10 ^"3

Day one-to-day zero sRAGE ratio 2.52 [1.73-3.67] <10 ^"3

SAPS II 1.01 [0.99-1.03] 0.2

Sepsis 1.32 [0.62-2.81] 0.5

Shock 0.82 [0.40-1.69] 0.6

Pneumonia 1.58 [0.67-3.73] 0.3

Table 2. The associations between soluble RAGE levels and the prediction of ARDS by day seven in multivariate analyses. The analyses were adjusted for baseline severity (as assessed by SAPS II) and the presence of sepsis, shock, or pneumonia at baseline. Plasma sRAGE levels (in pg/mL) are natural log-transformed in the logistic regression model to meet the assumption of linearity with log-odds of outcome; the ORs presented here are for each log increase in the level of plasma sRAGE.

Table El. The common risk factors for ARDS used to select at-risk patients in the PrediRAGE study.

SNP Region SNP sequence Primers for PCR (5'·3') Tm Sequencing Primer rsl800625 promoter AAAAAAATGAT

(_429 T/C) TTTCTTTCACGA

AG[C/T]TCCAAA Forward: Forward:

CAGGTTTCTCTC TCCTCACTTGTAAACT TGTAAACTTGTGTA TGTGTAG (23b) (SEQ GTTTCAC (21b) (SEQ CTGTTCC (SEQ

ID NO: 5) 64°C ID NO: 7) ID NO: 1)

Reverse:

TCTAGGGTCTCATTCC Reverse:

CTCAG (21b) (SEQ ID TCAGTCCATCAGGG NO: 6) CTGCCTG (21b) (SEQ

ID NO: 8) rsl800624 promoter GCCTTCATGATG

(J74 T/A) CAGGCCCAA[T/

A] TGC ACCCTTG

CAGACAACA

(SEQ ID NO: 2) rs3134940 intron 8 TTCTTCCCTCTG Forward: Forward:

CAGCACAGGCTCTAA TCTGGCCTTATCCC

( 184 AGCTAAAAAAA

TTTCCTG (22b) (SEQ TAACAG (20b) (SEQ A/G) GG[A/G]ACAGAC ID NO: 9) 66°C ID NO: 11)

GGCTGGGCGCG

Reverse: Reverse:

GTGGCTCA (SEQ GGCATGTGCCACCAT TAGTAGAAATGGG

GCCTG (20b) (SEQ ID GTTTCTC (20b) (SEQ ID NO: 3)

NO: 10) ID NO: 12) rs2070600 exon 3 CAGTGTGGCTCG Forward: Forward:

AGACCAGCAATGATT 64°C AGGCCTTGCACTGT

(Gly82Ser) TGTCCTTCCCAA

TGGATCC (22b) (SEQ TTAGGC (20b) (SEQ C[A/G]GCTCCCT ID NO: 13) ID NO: 15) CTTCCTTCCGGC

Reverse: 66°C Reverse:

TGTCGG (SEQ ID

ATGGGCCAAGGCTGG AGACACGGACTCG NO: 4) GGTTG (20b) (SEQ ID GTAGTTG (20b) (SEQ

NO: 14) ID NO: 16)

Table E2. The primers of the four variants of the RAGE gene and their PCR and sequencing conditions. SNP: single nucleotide polymorphism. PCR: polymerase chain reaction. Tm: temperature of melting. A: adenine. C: cytosine. G: guanine. T: thymine.

Models OR 95% CI P value 2.9 [1.65-3.45] <10 ^"3

Baseline sRAGE

SNP rs2070600A (Ser/Ser genotype) 124.59 [14.89-1043.34] <10 ^"3

SAPS II 1.01 [0.99-1.03] 0.1

Sepsis 1.68 [0.78-3.64] 0.2

Shock 0.93 [0.44-1.99] 0.9

Pneumonia 1.53 [0.62-3.79] 0.4

Table E3. The associations between plasma sRAGE, the RAGE gene variant rs2070600A (the Ser/Ser genotype), and the prediction of ARDS by day seven in multivariate analyses. Analyses were adjusted for baseline severity (as assessed by SAPS II) and the presence of sepsis, shock, or pneumonia at baseline. Plasma sRAGE levels (in pg/mL) are natural log-transformed in the logistic regression model to meet the assumption of linearity with log-odds of outcome; the ORs presented here are for each log increase in the level of plasma sRAGE.

Table E4. The associations between plasma sRAGE, the RAGE gene variant rs2070600A (the Ser/Ser genotype), and the prediction of ARDS by day seven in multivariate analyses. Analyses were adjusted for baseline severity (as assessed by SAPS II) and the presence of sepsis, shock, or pneumonia at baseline. The cutoff value of 1,033 pg/mL was chosen because it was the median value of baseline plasma sRAGE (in pg/mL) in our cohort.

Models OR 95% CI P value

Baseline sRAGE 2.25 [1.16-4.37] 0.02 SNP rs2070600A (Ser/Ser genotype) 15.52 [1.48-162.40] 0.02

Tidal volume 0.79 [0.48-1.30] 0.4

PEEP 0.96 [0.71-1.29] 0.8

Pplat 1.04 [0.91-1.19] 0.6

Pa0 ₂ /Fi0 ₂ 0.99 [0.98-0.99] 0.03

SAPS II 0.98 [0.94-1.02] 0.3

Sepsis 1.44 [0.25-8.40] 0.7

Shock 1.57 [0.42-5.91] 0.5

Pneumonia 1.14 [0.12-10.45] 0.9

Table E5. The associations between plasma sRAGE, the RAGE gene variant rs2070600A (the Ser/Ser genotype), and the prediction of ARDS by day seven after multivariate analyses in patients under invasive mechanical ventilation (n = 222). Analyses were adjusted for tidal volume (in mL/kg predicted body weight), positive end-expiratory pressure (PEEP), inspiratory plateau pressure (Pplat), arterial oxygenation (the partial pressure of arterial oxygen [Pa02] to the fraction of inspired oxygen [Fi02] ratio), baseline severity (as assessed by SAPS II), and the presence of sepsis, shock, or pneumonia at baseline. Plasma sRAGE levels (in pg/mL) are natural log-transformed in the logistic regression model to meet the assumption of linearity with log-odds of outcome; the ORs presented here are for each log increase in the level of plasma sRAGE.

Results

Baseline characteristics

Between June 2014 and January 2015, 1,967 patients were screened among the five ICUs. A total of 500 patients were enrolled in the study and were analyzed, among whom 464 patients did not meet the criteria for ARDS within 24 hours (Figure 1, Table 1). Patients who developed ARDS by day seven (n = 59, 13%) were more severely ill, with higher SAPS II and increased vasopressor use at baseline than patients who did not develop ARDS (n = 405). Plasma samples were drawn a median of 4.2 ± 1.8 hours after ICU admission. 18 patients (4%) developed ARDS between days seven and 30, and had similar plasma sRAGE and esRAGE than those who did not.

The predictive value of the soluble forms of RAGE for developing ARDS

Plasma sRAGE at baseline and on day one were significantly higher among patients who developed ARDS by day seven than for those who did not (Figures 2A-2E). In contrast, esRAGE at baseline and on day one were similar in both groups (Figures 2F-2H). To evaluate the discrimination of the models based on RAGE isoforms, we calculated AUROC. Plasma sRAGE at baseline and on day one had good discrimination, with AUROC of 0.74 (95% CI, 0.68-0.80; Figure 3A) and 0.82 (95% CI, 0.76-0.88; Figure 3B), respectively. The day one-to-day zero plasma sRAGE ratio, the baseline plasma sRAGE-to-esRAGE ratio, and the day one plasma sRAGE-to-esRAGE ratio had AUROCs of 0.66 (95% CI, 0.58-0.73), 0.62 (95% CI, 0.54-0.71), and 0.69 (95% CI, 0.61-0.78), respectively. The LIPS showed poor discrimination (AUROC, 0.57; 95%> CI, 0.49-0.65; Figure 3C). Using conventional methods, thresholds for sRAGE were set at 1,340 pg/mL at baseline and 1,096 pg/mL on day one. At these thresholds, the plasma sRAGE at baseline and on day one had sensitivities, specificities, positive (PPV) and negative predictive values (NPV) of 75%, 68%, 24%, and 95% and 86%, 64%o, 25%o, and 97%>, respectively, for the development of ARDS. In our cohort, plasma sRAGE, at baseline or on day one, outperformed LIPS (cutpoint > four points: PPV, 14%>; NPV, 89%)(4).

The predictive value of RAGE gene polymorphisms for developing ARDS

The RAGE SNPs rs 1800625, rs 1800624 and rs3134940 (either homozygous or heterozygous) were distributed similarly between patients who developed ARDS by day seven and those who did not (p = 0.6 for all). In contrast, ARDS was more frequent with than without SNP rs2070600 (32% vs. 12%, respectively, p < 10-3). In particular, homozygous rs2070600 (the Ser/Ser genotype) was associated with a higher risk of developing ARDS (92%>) than heterozygous rs2070600 (the Gly/Ser genotype) (7%) or without the SNP (13%, p < 10-3). Therefore, only the Ser/Ser genotype was included in subsequent multivariate analyses. Patients with the Ser/Ser genotype had higher baseline plasma sRAGE than those without the SNP (median [IQR], 1370 [846-2059] vs. 1013 [627-1788] pg/mL, p = 0.035). The frequency of rs 1800625, rs 1800624, rs3134940, and rs2070600 did not differ between patients who developed ARDS after day seven and those who did not (p = 0.9, 0.5, 0.8, and 0.4, respectively).

Multivariate adjustments of the predictor models

Baseline plasma sRAGE, the plasma sRAGE on day one, and the day one-to-day zero plasma sRAGE ratio predicted ARDS development (OR, 2.25 [95% CI, 1.60-3.16], 4.33 [95% CI, 2.85-6.56], and 1.61 [95% CI, 1.17-2.22], respectively), even after adjustment for SAPS II and the presence of sepsis, shock, or pneumonia at baseline (Table 2). Both baseline plasma sRAGE and the day one-to-day zero plasma sRAGE ratio were independent predictors of ARDS development (OR, 3.21 [95% CI, 2.17-2.22] and 2.52 [95% CI, 1.73-3.67], respectively) (Table 2). Both higher baseline plasma sRAGE and SNP rs2070600 were associated with an increased risk of developing ARDS (OR, 2.39 [95% CI, 1.65-3.45] and 124.59 [95% CI, 14.89- 1043.34], respectively) after multivariate adjustment (Table E3, appendix). Using this model, 90%) of patients were correctly classified as being at risk of developing ARDS or not (AUROC, 0.81 (95% CI, 0.75-0.88); sensitivity, 20%; specificity, 100%; PPV, 92%; NPV, 90%).

Analysis of ARDS development as a censored variable

Using the same previously described covariates, the HR for developing ARDS within seven days, when baseline plasma sRAGE was above 1,033 pg/mL (the median value of baseline plasma sRAGE in our cohort), was 4.59 (95% CI, 1.47-14.37, p = 0.01) (Figure 4A). The rs2070600 SNP was associated with a HR for developing ARDS of 15.09 (95% CI, 6.33- 35.99, p < 10-3) (Figure 4B) (Table E5, appendix).

Sensitivity analyses

Analyses were repeated only in patients under invasive mechanical ventilation at baseline (n = 222), of whom 34 (15%>) developed ARDS within seven days. Plasma sRAGE was still higher in patients who developed ARDS than in those who did not (p < 10-4 at baseline and on day one). Baseline plasma sRAGE (AUROC, 0.71 [95% CI, 0.63-0.79]) and sRAGE on day one (AUROC, 0.79 [95% CI, 0.70-0.87]) had good discrimination in this subset, whereas the LIPS showed poor discrimination (AUROC, 0.57 [95% CI, 0.46-0.69]). The AUROC for the day one-to-day zero plasma sRAGE ratio was 0.63 [95% CI, 0.53-0.73].

After adjusting for respiratory parameters (tidal volume, level of positive end-expiratory pressure, and inspiratory plateau pressure), SAPS II, and Pa02/Fi02 and the presence of sepsis, shock, or pneumonia at baseline, higher baseline sRAGE and SNP rs2070600 remained associated with an increased risk of developing ARDS (Table E5, appendix).

Discussion

In this sample of critically ill at-risk patients, plasma sRAGE could predict the development of ARDS within seven days, and RAGE SNP rs2070600 (the Ser/Ser genotype) was associated with a higher risk of developing ARDS and higher plasma sRAGE. In contrast, plasma esRAGE did not predict ARDS.

Because ARDS mortality remains high(2), current initiatives should include primary prevention(24). However, a key challenge is to identify at-risk patients in whom ARDS is likely to develop and who would benefit if ARDS were prevented. Although clinical scores can identify patients who are more likely to develop ARDS(4), biomarkers may improve the predictive value of the clinical-only LIPS in pre-identified at-risk populations(5). In our high- risk cohort (mean LIPS, 5 ± 2.5), the plasma sRAGE was particularly adept at ARDS prediction, with better test characteristics than LIPS or a biomarker (Ang-2) previously validated in unselected patients(5). Because personalised approaches can decrease the incidence of ARDS(25), the identification of predictive clinical or biological variables is of major importance to developing preventive strategies, early detection, and treatment. Our findings may be important not only because they provide an approach to ARDS prediction but also because they provide new insights into a molecular endotype of RAGE activation that may be implicated in the early pathogenesis of ARDS: the increased plasma levels of sRAGE and the genetic susceptibility driven by rs2070600. Because epithelial injury is a major mechanism of lung injury, this hypothesis is further supported by the good predictive value of plasma sRAGE on day one, suggesting that the kinetics of sRAGE itself may better identify patients at greatest risk. Moreover, evidence is mounting to support the role of both sRAGE and esRAGE as biomarkers of ligand-RAGE activity in diseases or endogenous protection factors against RAGE-mediated pathogenesis(26). In our cohort, the predictive value of plasma esRAGE was poor, although the sRAGE-to-esRAGE ratio remained informative, in line with previous findings in other lung diseases(27). sRAGE comprises a heterogeneous population including esRAGE, a soluble splice variant, and its proteolytically cleaved forms shed into the bloodstream by metalloproteinases(lO). In general, plasma esRAGE is between two- and fivefold lower than plasma sRAGE, and plasma esRAGE only explains about one-third of the variation in plasma sRAGE, suggesting isoform specific kinetics(28). In addition, the balance in the respective levels of membrane-bound RAGE, esRAGE, and sRAGE, may be of primary importance in controlling RAGE-dependent actions, thus reinforcing the value of sRAGE as a biomarker in ARDS(12, 13, 22, 29).

Our findings support a novel association between rs2070600 (the Ser/Ser genotype) and an increased risk of developing ARDS. The somewhat more common (G) allele encodes for glycine (Gly), while the minor (A) allele encodes for serine (Ser). Although the exchange of glycine for serine amino acids within RAGE protein is unlikely to impact its secondary or three- dimensional structure (data not shown), it may modify cell surface expression of RAGE, ligand- affinity, and pro -inflammatory signalling(18, 30). Future studies are needed to better understand the precise mechanisms through which rs2070600 predisposes at-risk patients for developing ARDS. In addition, because rs2070600 results in a coding change in the V-type ligand-binding domain, it may directly influence the expression of sRAGE itself, e.g. through changes in sites of proteolytic cleavage. Previous studies have found an association of RAGE gene variants with enhanced susceptibility to RAGE-mediated pathogenesis(20), and preliminary results suggest that plasma sRAGE may be, at least in part, under genetic control. In our study, rs2070600 was associated with plasma sRAGE, thus contrasting with the previous reports of lower sRAGE in nondiabetic, nonobese patients(31).

Our study had some limitations. First, our findings may only hold true in patients with a high-risk of developing ARDS, as assessed by elevated LIPS, but not in all critically ill patients(5). Therefore, the identification of clinical risk factors remains a cornerstone in the prediction of ARDS(4). Second, we cannot say whether elevated plasma sRAGE may be a better ARDS predictor in patients with pulmonary risk factors (e.g. pneumonia and aspiration, which were predominant in our cohort) than in those with non-pulmonary risk factors.

However, direct injury to the lung is the primary cause of ARDS in most patients and our results may further support a molecular endotype consistent with more severe lung epithelial injury in direct ARDS(32). Third, there is no point-of-care test for sRAGE at this time, and genomic applications are not yet ready for clinical use, which currently limits the application of our findings in clinical medicine. If sRAGE is confirmed as a useful ARDS predictor, further research will be needed to make rapid assays more immediately available to clinicians. Last, because we only investigated only four selected RAGE SNPs, a lot of variations in the gene remains unexplored. Of note, our study also lacked a validation cohort in which to verify the use of RAGE iso forms and gene variants for the prediction of ARDS.

Our study also had several unique strengths. First, it was designed specifically for the early collection of blood samples, allowing us to capture many at-risk patients before they developed ARDS. This is a difficult population to recruit, because most patients present with

ARDS, but it is also the population that is most likely to benefit from such predictions(24).

Second, we rigorously excluded patients with ARDS at admission and those who developed

ARDS within the first 24 hours of the study to reduce the likelihood of enrolling patients with established early disease. Third, our study included both intubated and nonintubated patients, with few exclusion criteria, which allowed us to analyze a broad range of critically ill patients with ARDS risk factors; these patients represent a clinically relevant population and their breadth increases the generalisability of our study.

In conclusion, our study is the first to report the predictive value of plasma sRAGE and

RAGE SNP rs2070600 for developing ARDS in a high-risk population. Although further confirmatory studies are warranted, such a molecular endotype might open new avenues for the research stratification of at-risk patients in future clinical trials of early therapeutic or preventive approaches to ARDS.

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