In vitro method for predicting mortality risk in patients experiencing shock

Abstract

In vitro method for predicting the risk of death in a patient experiencing shock. The present invention relates to the field of medicine. In particular, the present invention relates to an in vitro method for predicting the risk of death in a patient experiencing shock, the method comprising determining the concentration level of a specific protein in a biological sample obtained from the patient.

Description

Technical Field

[0001] The present invention relates to the field of medicine. In particular, the present invention relates to an in vitro method for predicting the risk of death of a patient who is experiencing shock, the method comprising determining the concentration level of a specific protein in a biological sample obtained from the patient. Background Art

[0002] Despite the widespread availability of early revascularization and contemporary intensive care, shock management, especially cardiogenic shock (CS), remains challenging with a mortality rate of approximately 40%. Early and accurate risk stratification is essential for timely identification of the sickest patients who may benefit from advanced therapies. Although clinical predictors of adverse outcomes have been known for decades, they were derived from clinical trials before percutaneous coronary intervention (PCI) and lacked external validation, which precluded their routine use and prompted the development of more modern risk classifiers. Two scores have recently been reported. The CardShock risk score was developed from unselected patients with CS from a large, prospective, multicenter European registry with a wide range of etiologies, two thirds of whom had ST-elevation myocardial infarction (STEMI). The IABP-SHOCK II risk score was developed from participants in the IABP-SHOCK II trial and is highly specific for STEMI-related CS treated with PCI. Both scores were externally validated and included classic clinical and biochemical variables for short-term risk stratification.

[0003] Of note, the laboratory parameters included in these risk scores are basic biochemical tests (glucose and lactate) that have been routinely used in the clinic for decades, but also include some clinical acuity parameters. Recent studies have explored cardiac and extracardiac predictive biomarkers in CS. However, most of these studies were small or not validated in external cohorts or did not assess the incremental predictive value of such biomarkers in combination with current clinical practice. In particular, novel renal biomarkers including cystatin C, plasma neutrophil gelatinase-associated lipocalin, and kidney injury molecule 1 did not outperform the traditional creatinine. In terms of proteomic data in the context of other cardiovascular pathologies, a proteomic approach for stable coronary artery disease was recently reported, in which a nine-protein risk score with a C statistic of 0.74 was reported.

[0004] Thus, until the 21st century, shock in general, and CS in particular, continued to be associated with unacceptably high mortality, substantial morbidity, and resource utilization. Despite the widespread use of early coronary reperfusion, the prevalence of CS in STEMI is approximately 5%, and CS is the leading cause of in-hospital mortality.

[0005] Therefore, although some contemporary risk scores are available, including the CardShock and IABP-SHOCK II risk scores, as mentioned above, more accurate risk stratification strategies are needed in order to effectively predict the risk of mortality in patients with shock.

[0006] On the other hand, there is increasing evidence that CS is not only a pump failure problem but also a systemic inflammatory state in the context of multi-organ failure. Therefore, comprehensive proteomics may enable the unbiased discovery of novel protein biomarkers that can be used to gain pathophysiological insights, improve the accuracy of risk stratification, and identify therapeutic targets.

[0007] The present invention is actually focused on solving the above-mentioned problem, predicting the short-term mortality risk of shock patients, especially CS patients, based on a protein-based score. Therefore, accurate stratification of shock patients, especially patients undergoing CS, according to their short-term mortality risk can be effectively used to foresee or anticipate treatment mainly for cases with a high mortality risk, thereby increasing the probability of successful treatment and thereby increasing the life expectancy of patients undergoing shock. Summary of the invention

[0008] Brief description of the invention

[0009] In the present invention, quantitative proteomic analysis for discovery and validation of CS biomarkers was performed in two independent CS cohorts. In addition, this analysis was also performed on a cohort for discovery and validation of septic shock (SS) biomarkers.

[0010] The 26 proteins listed in Table 1 were initially identified as biomarkers for predicting mortality risk in patients undergoing CS.

[0011] Of the 26 proteins listed above, four proteins described below were identified as preferred candidates in CS and SS, with measured levels significantly improving mortality risk prediction beyond established modern clinical risk scores:

[0012] L-FABP: UniProt reference P07148.

[0013] ·B2MG: UniProt reference P61769.

[0014] ALDOB: UniProt reference P05062.

[0015] IC1: UniProt reference P05155.

[0016] Therefore, based on the results, a protein-based classifier was developed, which was also tested by mass spectrometry and ELISA. This classifier accurately differentiated shock patients according to their short-term risk of death.

[0017] In particular, the inventors have developed an in vitro method for distinguishing patients who are experiencing shock or have been subjected to shock who have a low risk of death from patients who are experiencing shock or have been subjected to shock who have a high risk of death. Specifically, a score based on circulating proteins was developed to predict the short-term risk of death in patients with CS or SS. In a particularly preferred embodiment, the method focuses on CS and includes combined measurement of the concentration levels of four specific proteins (CS4P model), namely: L-FABP, B2MG, ALDOB and IC1. The area under the curve (AUC) of the CS4P model is 0.83 (see Figure 8 ). CardShock (a standard risk score) had an AUC of 0.78. The combination of the two models (CS4P + CardShock) improved the AUC to 0.84 (see Figure 6 ).

[0018] On the other hand, it is important to note that the model based on the combination of the 26 proteins listed in Table 1 improved the AUC of CS to 0.921 when not combined with CardShock (see Fig.10 ). In addition, when the 26 proteins listed in Table 1 were also combined with CardShock, the AUC increased to 0.970 (see Fig. 9 ).

[0019] Furthermore, in the present invention, the twenty-six proteins have been analyzed individually, and more specifically, the four proteins L-FABP, B2MG, ALDOB and IC1 (see Figure 5 , Figure 6 , Figure 7 and Figure 8 ). Non-survivors of CS had higher levels of L-FABP, B2MG, and ALDOB compared to survivors. In contrast, non-survivors of CS had lower levels of IC1 compared to survivors (see Figure 4 ).

[0020] The protein of the present invention is preferably measured within 24 hours after the patient is admitted to the hospital. On the other hand, the method is particularly related to determining the "short-term" risk of death in CS patients. According to the present invention, "short-term" means 90 days. Therefore, in a preferred embodiment, the method of the present invention actually assesses the risk of death in shock patients within 90 days.

[0021] On the other hand, the method of the present invention can be considered a non-invasive or minimally invasive technique since it is preferably performed on a serum or plasma sample obtained from a patient.

[0022] Although, in a particularly preferred embodiment, the method of the present invention is based on determining the concentration level of a combination of the above-mentioned 26 proteins, preferably a combination of the four proteins B2MG, L-FABP, ALDOB and IC1, it is important to consider that the present invention can also be carried out by determining the concentration level of a smaller number of the twenty-six proteins or even a smaller number of the four proteins. In other words, the present invention can be carried out by using any of the 26 individual proteins listed above, in particular one, two, three or four of the proteins B2MG, L-FABP, ALDOB and IC1.

[0023] On the other hand, B2MG is cited herein as the preferred single protein for practicing the present invention because, as can be seen in Table 2, it provides the best single result in CS when B2MG is assayed by ELISA (a method more easily applicable to routine clinical use) without the need for combination with CardShock. However, the present invention provides scientific support for the following (see Figure 5 , Figure 6 , Figure 7 and Figure 8), that is, using any of the 26 proteins listed in Table 1, preferably any of the following proteins B2MG, L-FABP, ALDOB or IC1 as an individual biomarker or using any combination thereof, for predicting the risk of death in patients who are experiencing shock, preferably CS or SS. Therefore, the present invention also relates to the use of any of the 26 proteins listed above or any combination thereof for predicting the risk of death in patients who are experiencing shock, preferably CS or SS. In a preferred embodiment, the combination of proteins comprises at least 2, at least 3 or at least 4 of the proteins B2MG, L-FABP, ALDOB or IC1. Therefore, B2MG or, alternatively, any of the 26 proteins listed above, preferably any of the L-FABP, ALDOB or IC1 proteins, or any combination thereof, is identified in the present invention as a biomarker suitable for predicting the risk of death in patients who are experiencing shock, preferably CS or SS. Therefore, the present invention provides evidence for the use of any of the above proteins or any combination thereof as biomarkers for predicting the risk of death in patients experiencing shock, preferably CS or SS, and showing high sensitivity and sensibility.

[0024] Thus, a first embodiment of the present invention relates to an in vitro method for predicting the risk of death in a patient who is experiencing shock, the method comprising determining the concentration level of at least any of the individual proteins listed above, preferably the concentration level of at least B2MG, or at least L-FABP, or at least ALDOB, or at least IC1, in a biological sample obtained from the patient, wherein an increased level of at least the protein B2MG, or at least L-FABP, or at least ALDOB, or a decreased level of at least IC1 relative to the concentration level of said protein determined in a survivor patient who is experiencing shock is an indication of the risk of death. In a preferred embodiment, the method of the present invention comprises determining the concentration level of the proteins B2MG, L-FABP, ALDOB and IC1, wherein an increased level of the proteins B2MG, L-FABP and ALDOB, and a decreased level of the protein IC1 relative to the concentration level determined in a survivor patient who is experiencing shock are an indication of the risk of death. In a preferred embodiment, the method of the present invention further comprises performing CardShock. CardShock included the following variables: age >75 years, presence of confusion, previous myocardial infarction or coronary artery bypass grafting (CABG), etiology of acute coronary syndrome (ACS), left ventricular ejection fraction <40%, blood lactate, and eGFR CKD-EPI. In a preferred embodiment, the method of the present invention is performed within 24 hours after the patient is admitted to the hospital. In a preferred embodiment, the risk of death within 90 days is assessed. In a preferred embodiment, the biological sample is serum or plasma. In a preferred embodiment, in the present invention, the risk of death is predicted in patients with CS, hypovolemic shock, anaphylactic shock, SS or neurogenic shock, preferably in patients with CS or SS, more preferably in patients with CS. In this regard, please refer to Figures 11 to 13 , which provides ROC curves showing the AUC of individual proteins L-FABP, B2MG, ALDOB and IC1 and their combination for predicting the risk of death in SS patients.

[0025] A second embodiment of the present invention relates to the in vitro use of any of the 26 proteins listed above, preferably at least B2MG, or at least L-FABP, or at least ALDOB, or at least IC1 for predicting the risk of death in patients undergoing shock, preferably CS or SS. In a preferred embodiment, the use comprises determining the concentration levels of B2MG, L-FABP, ALDOB and IC1. In a preferred embodiment, the use further comprises performing CardShock. CardShock includes the following variables: age > 75 years, presentation with confusion, previous myocardial infarction or coronary artery bypass grafting (CABG), acute coronary syndrome (ACS) etiology, left ventricular ejection fraction < 40%, blood lactate, and eGFR CKD-EPI .

[0026] When the method of the present invention comprises measuring the concentration level of a combination of biomarkers, a score value is obtained for marking a signature and the score value is compared with a threshold value defining a diagnostic rule. If a change in the score value is identified relative to the threshold value, the corresponding sample is classified as a positive sample, indicating an increased risk of death in a patient who is undergoing CS. The threshold value has been defined to optimize the sensitivity value and the specificity value. Therefore, in a preferred embodiment, the method of the present invention comprises: a) measuring the concentration level of any of the above-mentioned biomarker combinations in a biological sample obtained from a subject; b) processing the concentration value to obtain a risk score; and c) wherein, if a deviation or change in the risk score value obtained for any of the above-mentioned biomarker combinations compared to a reference value is identified, it indicates an increased risk of death in a patient who is undergoing CS.

[0027] A third embodiment of the present invention relates to a method for treating a patient who is experiencing shock or has experienced shock, the method comprising providing appropriate treatment to the patient after determining the patient's risk of death by the above method. Therefore, accurate stratification of shock patients (especially patients who are experiencing CS or SS) according to their short-term risk of death can be effectively used to foresee or anticipate treatment mainly for cases with a high risk of death, thereby increasing the likelihood of successful treatment and thereby increasing the life expectancy of patients who are experiencing shock. In a preferred embodiment, the patient is experiencing or has experienced: CS, hypovolemic shock, anaphylactic shock, SS or neurogenic shock, preferably CS or septic shock, more preferably CS.

[0028] In a preferred embodiment, the method of the invention is performed by ELISA or mass spectrometry. In a preferred embodiment, the measurement of proteins is performed on their precursors (if any) or intermediate segments.

[0029] A fourth embodiment of the present invention relates to a kit suitable for predicting the risk of death in a patient who is experiencing cardiogenic shock, the kit comprising: a) a means or medium for obtaining a serum or plasma sample from a patient; and b) a means or medium for measuring the concentration level of at least B2MG. In a preferred embodiment, the kit comprises: a) a means or medium for obtaining a serum or plasma sample from a patient; and b) a means or medium for measuring the concentration levels of B2MG, L-FABP, ALDOB, and IC1.

[0030] On the other hand, established treatment strategies exist when patients are experiencing CS. Early revascularization, primarily by percutaneous coronary intervention (PCI), is currently the most important treatment strategy for CS after myocardial infarction, with a significant reduction in mortality after 6 months, 1 year, and 6 years. In clinical practice, revascularization should be limited to the infarct lesion (culpritlesion), with elective revascularization of other lesions possible at a later time point. Urgent coronary artery bypass grafting (CABG) revascularization may also have a role; however, there is little evidence pointing to surgical versus PCI revascularization. In this regard, antiplatelet and antithrombotic therapy, including but not limited to glycoprotein IIb / IIIa inhibitors and cangrelor, is one of the key features of successful PCI. There are no specific trials targeting antiplatelets or anticoagulation in CS. Enteric absorption is impaired in CS, and opioids are often coadministered, further affecting enteric bioavailability.

[0031] In the intensive care unit, treatment of CS includes initial hemodynamic stabilization with volume expansion, vasopressors, and inotropes plus additional therapies for the prevention or treatment of multiorgan system dysfunction (MODS). Approximately 90% of patients with CS are administered inotropes and vasopressors. In the case of abnormal heart rhythm, synchronized cardioversion or antiarrhythmic drugs, such as adenosine, can be immediately administered. Inotropes that increase the heart's ability to pump blood, such as dobutamine or milrinone, are used to improve contractility and correct hypotension. CS can also be treated with intravenous dobutamine and norepinephrine, which acts on the heart's beta-1 receptors, resulting in increased contractility and heart rate. Other inotropes, such as levosimendan or phosphodiesterase inhibitors, have attracted attention for their potential to improve myocardial contractility and vasodilation without increasing oxygen demand. However, the current evidence for the use of vasodilators in CS is very limited. If this is not enough, it is necessary to apply mechanical circulatory support. Intra-aortic balloon pumps reduce the workload of the heart and improve perfusion of the coronary arteries. Ventricular assist devices (VADs) enhance the pumping function of the heart. Newer developments in VADs include right ventricular support devices such as the Impella RP (Abiomed, Danvers, MA, USA) and the TandemHeart RA-PA (LivaNova, London, UK), which deliver blood from the right atrium or inferior vena cava to the pulmonary artery. Newer left ventricular VADs include the HeartMate PHP (Abbott, Lake Bluff, IL, USA), which is deployed over the aortic valve and delivers blood from the left ventricle to the aorta, similar to the Impella family. Another investigational device is the paracorporeal pulsatile iVAC 2L (PulseCath BV, Arnhem, The Netherlands). Recent advances in miniaturized systems and percutaneous cannulation have led to a wider acceptance among interventional cardiologists of the use of extracorporeal life support systems (ECMO) to treat CS, and recently it has been proposed to help patients with CS. The integral features of ECMO are a blood pump, a heat exchanger, and an oxygenator. As a general reflection on mechanical circulatory support, IABP-SHOCK II showed that a large proportion of CS survivors can survive without any device. Inserting a device into these patients would not have an impact on survival, but on the contrary could result in some complications caused by the device itself, which could lead to death. In the 40% to 50% of cases where there is no survival, there may also be futile cases where even the best available device cannot change the clinical outcome.For patients with severe CS or anoxic brain injury or concomitant severe sepsis, this futility may occur in the range of 25% to 35%. In these regards, mechanical circulatory support can be used as a bridge-to-decision strategy and patient-centered discussions with relatives, but prognostic measurements are needed to validate such decisions, which is currently lacking in this field. Finally, as a last resort, if a person is stable enough and meets other conditions, a heart transplant may be recommended, or, if not eligible for a heart transplant, an artificial heart may be placed. One of the preferred treatment strategies is a ventricular assist device. In this regard, the biomarkers described in the present invention, primarily B2MG, L-FABP, ALDOB and / or IC1, can be used to design companion diagnostic kits or tests to determine the suitability of the above-mentioned treatments (e.g., ventricular assist devices) for specific patients who are experiencing CS. Therefore, these companion diagnostic kits or tests can help clinicians select or exclude patient groups for specific treatments (e.g., use of ventricular assist devices) and determine responders and non-responders to the therapy. Thus, by measuring the concentration levels of B2MG, L-FABP, ALDOB and / or IC1, clinicians can predict whether a particular patient will respond to treatment (e.g., use of a ventricular assist device). On the other hand, clinicians can monitor and track a particular patient's response to treatment (e.g., use of a ventricular assist device) by measuring the concentration levels of B2MG, L-FABP, ALDOB and / or IC1.

[0032] For the purposes of the present invention, the following terms are defined as follows:

[0033] The expression "concentration levels measured in control survivor patients who are experiencing shock" refers to "reference values" of protein concentration levels. If the concentration levels of L-FABP, B2MG and / or ALDOB are higher, and / or the concentration level of IC1 is lower, compared to the "concentration levels measured in control survivor patients who are experiencing shock" used as "reference values", this is an indication of mortality risk.

[0034] "Reference value" can be a threshold value or a cutoff value. Generally, the "threshold value" or "cutoff value" can be determined by experiment, experience or theory. As will be known to those skilled in the art, the threshold value can also be arbitrarily selected according to existing experimental and / or clinical conditions. The threshold value must be determined according to the function of the test and the benefit / risk balance (clinical results of false positives and false negatives) to obtain the best sensitivity and specificity. Preferably, the biomarker level (or score) obtained according to the method of the present invention can be compared with the defined threshold value by a person skilled in the art. Generally, the receiver operating characteristic (ROC) curve based on experimental data can be used to determine the best sensitivity and specificity (as well as the threshold value). For example, after determining the level of the biomarker in a set of references, the measured concentration of the biomarker in the biological sample to be tested can be statistically processed using an algorithm analysis to obtain a classification standard that is meaningful for sample classification. The full name of the ROC curve is the receiver operating characteristic curve, also known as the receiver operating characteristic curve. It is mainly used in clinical biochemical diagnostic tests. The ROC curve is a comprehensive indicator reflecting the true positive rate (sensitivity) and the false positive rate (1-specificity) of continuous variables. It reveals the relationship between sensitivity and specificity through image synthesis methods. A series of different cutoff values ​​(thresholds or critical values, i.e., the boundary values ​​between normal and abnormal results of the diagnostic test) are set as continuous variables to calculate a series of sensitivity values ​​and specificity values. Then a curve is drawn with sensitivity as the ordinate and specificity as the abscissa. The larger the area under the curve (AUC), the higher the accuracy of the diagnosis. On the ROC curve, the point closest to the upper left corner of the coordinate graph is the critical point with both high sensitivity and high specificity. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, as AUC approaches 1, the diagnostic results are getting better and better. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is good. When AUC is higher than 0.9, the accuracy is very high. This calculation method is best done with a computer. The ROC curve can be drawn using existing software or systems in the art, such as: MedCalc 9.2.0.1 medical statistics software, SPSS9.0.

[0035] The term "mortality risk" refers to an estimation of the probability of in-hospital death of a patient. According to the present invention, when the concentration level of L-FABP, B2MG and / or ALDOB is higher and / or the concentration level of IC1 is lower compared to the "concentration level measured in control survivor patients subjected to shock" used as a "reference value", there is a mortality risk.

[0036] The term "shock" refers to a life-threatening condition that occurs when the body does not get enough blood flow. Inadequate blood flow means that cells and organs do not get enough oxygen and nutrients to function properly. Many organs can be damaged as a result. Shock requires immediate treatment and can get worse quickly. The main types of shock include: cardiogenic shock (caused by heart problems), hypovolemic shock (caused by too little blood volume), anaphylactic shock (caused by an allergic reaction), septic shock (caused by infection), and neurogenic shock (caused by damage to the nervous system).

[0037] · “Including” means including but not limited to whatever follows the word “including.” Thus, use of the term “including” indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present.

[0038] • “Consisting of…” means including and limited to whatever follows the phrase “consisting of…” Thus, the phrase “consisting of…” indicates that the listed elements are required or mandatory, and no other elements may be present. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 .The general workflow of this study on CS patients;

[0040] Figure 2 .Targeted proteomic results for CS patients. A) Targeted mass spectrometry chromatograms (PRMs) corresponding to endogenous peptides of proteins L-FABP, B2MG, ALDOB, and IC1 and their isotope-labeled internal standards (or reference MS2 spectra). B) Retention time shifts of endogenous peptides for all analyzed patients shown in Figure A. L-FABP: liver-type fatty acid binding protein; ALDOB: fructose bisphosphate aldolase B; B2MG: beta-2-microglobulin; IC1: SerpinG1;

[0041] Figure 3 In CS patients, the discriminatory power of each model for predicting 90-day mortality risk was improved. CardShock included age >75 years, presentation with confusion, previous MI or CABG, ACS etiology, LVEF <40%, blood lactate, and eGFR CKD-EPI. The CS4P score includes circulating protein abundance measured by parallel reaction monitoring of liver-type fatty acid binding protein (L-FABP), fructose bisphosphate aldolase B (ALDOB), beta-2-microglobulin (B2MG), and SerpinG1 (IC1). CardShock+CS4P includes the CardShock risk score and the CS4P score;

[0042] Figure 4 Box plots of the four proteins of CS4P measured by ELISA in 90-day survivors and non-survivors in CS patients. L-FABP: liver-type fatty acid binding protein; ALDOB: fructose bisphosphate aldolase B; B2MG: beta-2-microglobulin; IC1: SerpinG1;

[0043] Figure 5 .ROC curves showing the AUC of individual proteins L-FABP, B2MG, ALDOB and IC1 and their combination to predict the risk of death in patients undergoing CS when the proteins were combined with CardShock and measured by ELISA;

[0044] Figure 6 .ROC curves showing the AUC of the individual proteins L-FABP, B2MG, ALDOB and IC1 and their combination to predict the risk of death in patients undergoing CS when the proteins are combined with CardShock and measured by mass spectrometry;

[0045] Figure 7 .ROC curves showing the AUC of the individual proteins L-FABP, B2MG, ALDOB and IC1 and their combination to predict the risk of death in patients undergoing CS when the proteins are not combined with CardShock and measured by ELISA;

[0046] Figure 8 ROC curves showing the AUC of the individual proteins L-FABP, B2MG, ALDOB and IC1 and their combination to predict the risk of death in patients undergoing CS when the proteins are not combined with CardShock and measured by mass spectrometry;

[0047] Fig. 9 .ROC curve showing the AUC of the model based on the combination of the 26 proteins listed in Table 1 to predict the risk of death in patients undergoing CS when the proteins are combined with CardShock;

[0048] Fig.10.ROC curve showing the AUC of the model based on the combination of the 26 proteins listed in Table 1 to predict the risk of death in patients undergoing CS, when the proteins are not combined with CardShock;

[0049] Fig.11 .ROC curve showing the AUC of individual proteins L-FABP, B2MG, ALDOB and IC1 and their combination to predict the risk of death within 30 days in patients undergoing SS. Experiments were performed by ELISA;

[0050] Fig.12 .ROC curves show the AUC of individual proteins L-FABP, B2MG, ALDOB and IC1 and their combination to predict the risk of death within 90 days in patients undergoing SS. Experiments were performed by ELISA;

[0051] Fig.13 .ROC curves show the AUC of individual proteins L-FABP, B2MG, ALDOB and IC1 and their combination to predict the risk of death within 365 days in patients undergoing SS.The experiment was performed by ELISA. DETAILED DESCRIPTION

[0052] Example 1. Materials and methods.

[0053] Example 1.1. Patient Cohort.

[0054] The Barcelona discovery cohort was a prospective, single-center all-comers study of patients with CS from STEMI between March 2011 and March 2015. STEMI was defined according to the Third Universal Definition of Myocardial Infarction. Patient management was determined by the physician according to guideline recommendations: [Steg PG, James SK, Atar D, et al. ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation. Eur Heart J 2012; 33(20): 2569-619] and [O'Gara PT, Kushner FG, Ascheim DD, et al. 2013ACCF / AHA guideline for the management of ST-elevation myocardial infarction: a report of the American College of Cardiology Foundation / American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol 2013; 61(4): e78-140].

[0055] Two samples (admission and 24 hours) were obtained from each patient (n=48) by venipuncture and stored at -80°C. The clinical endpoint was 90-day mortality. The CardShock validation cohort is a European prospective, multicenter, multinational study of CS of ischemic or non-ischemic origin from October 2010 to December 2012. The clinical characteristics of the cohort and the inclusion and exclusion criteria are reported elsewhere: [Harjola VP, Lassus J, Sionis A, et al; CardShock Study Investigators; GREAT network. Clinical picture and risk prediction of short-term mortality in cardiogenic shock. Eur J Heart Fail 2015; 17(5):501-9].

[0056] In this study, only one sample (n = 97) was used that was removed within 24 hours of admission, immediately frozen, and stored at -80°C. During the follow-up period, vital status was determined by direct contact with the patient or his next of kin, or from population and hospital registers. The clinical endpoint was death at 90 days.

[0057] Both cohorts were approved by the local ethics committees of the participating centers, and the studies were conducted in accordance with the Declaration of Helsinki. Written consent was obtained from the patients or their next of kin.

[0058] The analyzed septic shock cohort consisted of 200 patients provided by the Department of Anesthesiology, Intensive Care, and Burns, GH St-Louis-Lariboisière, Paris, France.

[0059] Serum samples were collected from each patient diagnosed with septic shock, as defined by the Society of Critical Care Medicine (SCCM) and the European Society of Intensive Care Medicine (ESICM), which publish guidelines for the Surviving Sepsis Campaign: septic shock is defined as a subset of sepsis with circulatory and cellular / metabolic dysfunction associated with a higher risk of death [Singer M, Deutschman CS, Seymour CW, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 2016; 315(8):801-810. doi:10.1001 / jama.2016.0287]. Samples were stored at −80°C and sent to Barcelona for further analysis. During follow-up, vital status was determined by direct contact with the patient or his next of kin, or from population and hospital registers. During the follow-up period, 104 of 200 patients (52%) were alive and 96 (48%) died. The clinical endpoint was death at 90 days. The cohort was approved by the local ethics committees of the participating centers, and the study was conducted in accordance with the Declaration of Helsinki. Written consent was obtained from the patients or their next of kin.

[0060] Example 1.2. Biomarker discovery by screening proteomics.

[0061] Quantitative proteomic analysis using mass spectrometry (nLC-MS / MS) was performed to identify potential protein biomarker candidates among proteins that differed in abundance between survivors and non-survivors at day 90. Serum samples (admission and 24 h) from 48 patients from the Barcelona cohort (21 non-survivors and 27 survivors at day 90) were digested into peptides with trypsin and analyzed using label-free screening proteomics (nLC-MS / MS).

[0062] Example 1.3. Validation of biomarkers by targeted proteomics.

[0063] Candidate biomarker proteins identified during the discovery phase were evaluated for their classification ability in the CardShock validation cohort using targeted proteomic quantification using parallel reaction monitoring (PRM). Plasma samples from 97 patients corresponding to the CardShock cohort (36 nonsurvivors and 61 survivors at 90 days) were trypsin digested and analyzed using targeted nLC-PRM and isotope-labeled standard peptides as internal references. Fragment ion chromatographic traces of all targeted precursor peptides were evaluated, log-transformed, and normalized using the internal reference peptide. Protein abundance was estimated, and relative protein quantification between survivors and nonsurvivors was assessed.

[0064] Example 1.4. Validated protein ELISA.

[0065] Four commercially available ELISA kits were used for each validated protein following the manufacturer's instructions.

[0066] L-FABP was quantitatively determined by human L-FABP ELISA kit. The lowest measurable concentration was 102pg / mL, and the measurable concentration range was 102pg / ml to 25000pg / ml. Samples had to be diluted at least 20 times before use, and they were diluted 1 / 100 in 1× dilution buffer before measurement. The analytical method was performed according to the supplier's manual. There was no cross-reactivity between human H-FABP and human I-FABP.

[0067] ALDOB was measured by an enzyme-linked immunosorbent assay kit. The standard curve used ranged from 2.5 ng / ml to 160 ng / ml, with a minimum detectable concentration of 0.9 ng / ml. Intra- and inter-assay precision was <10% and <12%, respectively. Before measurement, samples were diluted 1 / 8 in 1× phosphate buffered saline (PBS). No significant cross-reaction or interference between ALDOB and analogs was observed.

[0068] B2MG was determined by enzyme immunoassay. The calculated range of this ELISA was 0 μg / ml to 12 μg / ml, with a functional sensitivity determined to be 0.1 μg / ml. Intra- and inter-assay precision were <3.5% and <4.5%, respectively. Before measurement, samples were diluted 1 / 100 in 1× sample buffer PU. No interference was observed with hemolytic serum (up to 1000 mg / dl) or lipemic serum (up to 3 g / dl). The analysis was performed according to the manual.

[0069] SerpinG1 is measured by human SERPING1 ELISA. The sensitivity or minimum detectable dose is 10pg / mL, and the detection range is 156pg / mL to 10000pg / mL. Intra-assay and inter-assay precision are <5.6% and <5.9%, respectively. Samples are diluted 1 / 50000 in sample diluent before measurement. The assay is highly specific for native and recombinant human SerpinG1, and has no detectable cross-reactivity with other related proteins.

[0070] Example 1.5. Statistical analysis.

[0071] Clinical variables were expressed as numbers (n) and percentages (%) for categorical variables, as means and standard deviations (SD) for normally distributed variables, or as medians and interquartile ranges (IQR) for skewed variables. Chi-square test, Student's t-test, or Mann-Whitney U-test were used for comparisons between groups, as appropriate.

[0072] Protein abundance estimation in proteomics data and relative protein quantification between groups (survivors vs. non-survivors) were performed using the software packages Skyline 3.7 and MSstats 3.8.2. The optimal protein combination for classifying the 90-day mortality risk of CS patients was challenged in the CardShock cohort divided into a training set (2 / 3) and a validation set (1 / 3). In the training set, the abundance of each protein was fitted to a logistic regression model between survivors and non-survivors, and the classification ability of each protein and protein combination was evaluated by the area under the curve (AUC) of the receiver operating characteristic (ROC) curve as described previously. The four identified proteins were tested as continuous variables.

[0073] The CardShock risk score used as a baseline model included: age >75 years, presenting with confusion, previous myocardial infarction or coronary artery bypass graft (CABG), acute coronary syndrome (ACS) etiology, left ventricular ejection fraction <40%, blood lactate, and eGFR CKD-EPIModel calibration was calculated using the Hosmer-Lemeshow (HL) test, and patient partitioning and reclassification were assessed using the Harrell C-statistic (AUC) and continuous net reclassification improvement (cNRI). Confidence intervals for the C-statistic and NRI were obtained by 1000-fold bootstrap resampling.

[0074] Analyses were performed using STATA v.13.0 (StataCorp, College Station, TX), PredictABEL R package v1.2, and SPSS v.20.0 (IBM Corp, Armonk, NY).

[0075] Example 2. Results.

[0076] Example 2.1. Unbiased Discovery of Protein Biomarker Candidates (Barcelona Cohort).

[0077] Table 3 shows clinical, biochemical and follow-up data from the Barcelona Discovery Cohort. Mean age was 69 ± 13 years, 35% were female, and 94% had undergone percutaneous coronary intervention. The 90-day mortality rate was 45.8%.

[0078] A total of 2662 proteins were identified in the dataset, of which 488 proteins were present in more than 30% of the patients. A total of 51 proteins were selected for the validation phase in each CardShock cohort after relative protein quantification between different variables: patient outcome (survivors, non-survivors) and sampling time (admission, 24 hours). In brief, 32 proteins that changed in abundance between survivors and non-survivors patients or within the first 24 hours after admission (admission, 24 hours) were considered for further validation. In addition, 19 proteins were included in the study based on previous knowledge and clinical relevance.

[0079] Example 2.2. Targeted proteomic validation of circulating biomarker candidates (CardShock cohort).

[0080] The classification ability of 26 of the 51 selected proteins was further validated in the CardShock cohort using targeted proteomic quantification by parallel reaction monitoring. Table 3 shows the characteristics of the CardShock validation cohort. The mean age was 66 ± 14 years, 25% were female, and the 90-day mortality rate was 37.1%. The most common cause of CS was ACS (71%), mainly triggered by STEMI (52%). Compared with the Barcelona cohort, CardShock patients showed higher hemoglobin and lower creatinine, lactate, and glucose levels.

[0081] Targeted mass spectrometry chromatograms are obtained for all measured proteins and compared to corresponding internal standards for relative protein quantification. Figure 2 , Figure 2 is an example of mass spectrometry signals obtained by measuring the proteins liver fatty acid binding protein (L-FABP), fructose bisphosphate aldolase B (ALDOB), beta-2-microglobulin (B2MG) and SerpinG1 (IC1) in all patients. The optimal protein combination for classifying 90-day survivors and non-survivors of CS patients was determined by performing predictor selection as described previously combined with cross-validation [Borràs E, Cantó E, Choi M, et al. Protein-Based Classifier to Predict Conversion from Clinically Isolated Syndrome to Multiple Sclerosis. Mol Cell Proteomics. 2016; 15(1): 318-28]. Figure 2 , the following sequences were used to obtain the mass spectrometry chromatograms for each protein: SEQ ID NO: 1 and SEQ ID NO: 2 for B2MG; SEQ ID NO: 3 and SEQ ID NO: 4 for ALDOB; SEQ ID NO: 5 for FABPL; and SEQ ID NO: 6 for ID1. Please note that although this method was used in the present invention to identify proteins, any other part of a protein, or even the entire protein, can be used for this purpose.

[0082] This evaluation identified the combination of 4 proteins with proteins L-FABP, B2MG, ALDOB, and IC1 as the best protein classifier for determining short-term mortality risk, with an AUC of 0.83 (95% CI 0.74–0.89) ( Table 4 and Figure 3 ).

[0083] An additional model (CardShock+CS4P) was constructed by combining the CardShock risk score with the novel CS4P model. CardShock+CS4P significantly improved the C statistic for mortality prediction compared with the CardShock risk score alone (AUC 0.84 vs AUC 0.78; P = 0.033; Table 4 and Figure 3 ). In addition, CardShock+CS4P showed a significant benefit in patient reclassification, with an NRI of 0.49 (P=0.020) (Table 4). Overall, CardShock+CS4P improved the reclassification of 32% of patients compared with the CardShock risk score.

[0084] In an exploratory analysis, we also combined the CS4P model with another modern risk score, IABP-SHOCK II, to generate IABP-SHOCK II + CS4P. IABP-SHOCK II + CS4P also provided a better predictive index compared with IABP-SHOCK II, with an NRI of 0.57 (P = 0.032).

[0085] Example 2.3. Conversion of CS4P to an enzyme-linked immunosorbent assay (ELISA) in CS.

[0086] The CS4P model defined by targeted proteomics was tested by ELISA to support its rapid translation into routine clinical practice. The median (IQR) circulating concentrations of the proteins studied were: L-FABP: 160 pg / mL (42 pg / mL to 1720 pg / mL); B2MG: 482 μg / mL (276 μg / mL to 752 μg / mL); ALDOB: 101 ng / mL (70 ng / mL to 209 ng / mL); and IC1: 218 pg / mL (169 pg / mL to 259 pg / mL). Circulating L-FABP (453 pg / mL vs. 94 pg / mL, p=0.02), B2MG (709 μg / mL vs. 344 μg / mL, p<0.001), and ALDOB (156 ng / mL vs. 84 ng / mL, p=0.05) were higher in non-survivors relative to survivors. In contrast, IC1 concentrations in non-survivors were significantly lower than those in survivors (205 pg / mL vs. 226 pg / mL, p=0.02) ( Figure 4 ).

[0087] The protein concentration of CS4P obtained by ELISA combined with the CardShock risk score provided an AUC of 0.82 (95% CI 0.73-0.90), which was not significantly different from the AUC obtained by targeted proteomics (p=0.123).

[0088] Example 2.4. Conversion of CS4P to an enzyme-linked immunosorbent assay (ELISA) in SS.

[0089] The CS4P model defined by targeted proteomics was tested by ELISA to support its rapid translation into routine clinical practice. The median (IQR) circulating concentrations of the investigated proteins were: L-FABP: 34.29 pg / mL (0.6 pg / mL to 2500 pg / mL); B2MG: 1034 μg / mL (204 μg / mL to 4637 μg / mL); ALDOB: 91.43 ng / mL (38 ng / mL to 637 ng / mL); and IC1: 217 pg / mL (19 pg / mL to 500 pg / mL). After analyzing all-cause mortality at 30 days in univariate analysis, nonsurvivors had higher circulating L-FABP (p=0.002, OR 1.47, 95% CI 1.18-1.44), B2MG (p<0.001, OR 1.42, 95% CI 1.14-1.77), and ALDOB (p=0.007, OR 1.34, 95% CI 1.08-1.65) relative to survivors. In multivariate analysis, after analyzing all-cause mortality at 30 days, nonsurvivors had higher L-FABP (p=0.004, HR 1.38, 95% CI 1.10-1.69) and B2MG (p=0.001, HR 1.44, 95% CI 1.16-1.80) relative to survivors.

[0090] The protein concentration of CS4P obtained by ELISA provided an AUC of 0.68 (95% CI 0.60–0.76).

[0091] sheet

[0092] Table 1. 26 protein biomarkers used for patient classification.

[0093]

[0094]

[0095] Table 2. Four proteins were identified as reliable single biomarkers for predicting mortality risk in patients undergoing CS (+CS: in combination with CardSock. -CS: not in combination with CardSock. E: ELISA technique. MS: mass spectrometry technique).

[0096]

[0097] Table 3. Comparison of baseline characteristics, clinical presentation, management, analysis parameters, and outcomes between the discovery and validation cohorts.

[0098]

[0099]

[0100]

[0101] TIA: transient ischemic attack, PCI: percutaneous coronary intervention, CABG: coronary artery bypass grafting, STEMI: ST-segment elevation myocardial infarction, LVEF: left ventricular ejection fraction, TIMI: thrombolysis in myocardial infarction, IABP: intra-aortic balloon pump, eGFR CKD-EPI : glomerular filtration rate estimated by the formula of the Chronic Kidney Disease Epidemiology Collaboration, hsTnT: high-sensitivity troponin T, and NT-proBNP: N-terminal pro-B-type natriuretic peptide.

[0102] Table 4. Comparison of model performance for predicting 90-day mortality in patients with CS.

[0103]

[0104] *P value, compared with CardShock. AUC: area under the curve, HL: Hosmer-Lemeshow, NRI: net reclassification improvement. CardShock included: age > 75 years, presenting with confusion, previous MI or CABG, ACS etiology, LVEF < 40%, blood lactate and eGFR CKD-EPI . CS4P score includes: liver-type fatty acid binding protein (L-FABP); fructose bisphosphate aldolase B (ALDOB); beta-2-microglobulin (B2MG); and SerpinG1 (IC1). CardShock+CS4P includes CardShock and CS4P scores. Sequence Listing <110> German Trias Appleton Health Sciences Institute Foundation Gene Regulation Center Foundation Pompeu Fabra University <120> In vitro method for predicting mortality risk in patients experiencing shock <130> 904 958 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> ARTIFICIAL SEQUENCE <220> <223> ARTIFICIAL SEQUENCE <400> 1 Val Glu His Ser Asp Leu Ser Phe Ser Lys 1 5 10 <210> 2 <211> twenty two <212> PRT <213> ARTIFICIAL SEQUENCE <220> <223> ARTIFICIAL SEQUENCE <400> 2 Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile 1 5 10 15 Glu Val Asp Leu Leu Lys 20 <210> 3 <211> 13 <212> PRT <213> ARTIFICIAL SEQUENCE <220> <223> ARTIFICIAL SEQUENCE <400> 3 Leu Asp Gln Gly Gly Ala Pro Leu Ala Gly Thr Asn Lys 1 5 10 <210> 4 <211> 13 <212> PRT <213> ARTIFICIAL SEQUENCE <220> <223> ARTIFICIAL SEQUENCE <400> 4 Glu Thr Thr Ile Gln Gly Leu Asp Gly Leu Ser Glu Arg 1 5 10 <210> 5 <211> 8 <212> PRT <213> ARTIFICIAL SEQUENCE <220> <223> ARTIFICIAL SEQUENCE <400> 5 Phe Thr Ile Thr Ala Gly Ser Lys 1 5 <210> 6 <211> 12 <212> PRT <213> ARTIFICIAL SEQUENCE <220> <223> ARTIFICIAL SEQUENCE <400> 6 Leu Val Leu Leu Asn Ala Ile Tyr Leu Ser Ala Lys 1 5 10

Claims

1. Use of B2MG in the preparation of a kit for predicting the risk of death in a patient suffering from cardiogenic shock, wherein: The kit is for use in an in vitro method for predicting the risk of death in a patient experiencing cardiogenic shock, the in vitro method comprising determining the concentration level of at least B2MG in a biological sample obtained from the patient, wherein an increased level of at least protein B2MG relative to the concentration level determined in a survivor patient control experiencing cardiogenic shock is indicative of the risk of death.

2. The use according to claim 1, wherein The method further comprises determining the concentration level of protein L-FABP, and / or ALDOB, and / or IC1, wherein an increased level of the protein L-FABP or ALDOB, or a decreased level of the protein IC1 relative to the concentration level determined in a survivor patient experiencing shock is indicative of a risk of mortality.

3. The use according to any one of claims 1 or 2, wherein The method comprises determining the concentration levels of proteins B2MG and L-FABP and ALDOB and IC1, wherein elevated levels of proteins B2MG and L-FABP and ALDOB relative to concentration levels determined in survivor patients experiencing shock and decreased levels of protein IC1 are indicative of risk of mortality.

4. The use according to any one of claims 1 or 2, wherein The method also includes determining the concentration levels of the following proteins: FABPL, and / or ALDOB, and / or IC1, and / or F13A, and / or HPTR, and / or PLMN, and / or AACT, and / or ANGT, and / or FIBA, and / or FIBB, and / or FIBG, and / or CRP, and / or RET4, and / or S10A8, and / or HEP2, and / or IBP2, and / or MUC18, and / or SEPP1, and / or ILRL1, and / or FHR4, and / or CELR1, and / or MYO5A, and / or CATA, and / or ALDOA, and / or APOB.

5. The use according to claim 1, further comprising measuring lactate and estimated glomerular filtration rate eGFRCKD-EPI in patients over 75 years old, presenting with confusion, previous myocardial infarction or coronary artery bypass grafting, acute coronary syndrome etiology, and left ventricular ejection fraction less than 40%.

6. The use according to claim 1, wherein The method is performed within 24 hours after the patient is admitted to the hospital.

7. The use according to claim 1, wherein The risk of death within 90 days was assessed.

8. The use according to claim 1, wherein The biological sample is serum or plasma.

9. The use according to claim 1, wherein Determination of said concentration level of said protein is performed by ELISA or by mass spectrometry.

10. A kit for predicting the risk of death in a patient suffering from cardiogenic shock, the kit comprising: a. a means or medium for obtaining a serum or plasma sample from said patient, and b. A means or medium for measuring the concentration level of B2MG and a means or medium for measuring the concentration level of protein L-FABP, and / or ALDOB, and / or IC1.

11. The kit according to claim 10, comprising: a. a means or medium for obtaining a serum or plasma sample from said patient, and b. Instruments or media for measuring the concentration levels of B2MG, L-FABP, ALDOB, and IC1.

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