Patent Description:
Despite the generalization of early revascularization and modern intensive care, shock management, particularly cardiogenic shock (CS), remains challenging, with mortality rates of ~<NUM>%. Early and accurate risk stratification is crucial for prompt identification of the sickest patients who may benefit from advanced therapies. While clinical predictors of adverse outcome have been well-known for decades, their derivation from pre-PCI (Percutaneous Coronary Intervention) clinical trials and a lack of external validation precluded their routine use and prompted the development of more contemporary risk classifiers. Two scores have been recently reported. The CardShock risk score was developed from a large prospective multicenter European registry of unselected CS patients with a broad spectrum of etiologies, two-thirds with ST-Elevation Myocardial Infarction (STEMI). The IABP-SHOCK II risk score was developed from IABP-SHOCK II trial participants and is highly specific for PCI-treated STEMI-related CS. These two scores are externally validated and include classical clinical and biochemical variables for short-term risk stratification.

Notably, the laboratory parameters included in these risk scores are basic biochemical tests (glucose and lactate) that have been routinely used in the clinic for several decades, but also include some clinical acumen parameters. More recent studies have explored cardiac and extra-cardiac predictive biomarkers in CS. However, most of these studies are small or not validated by external cohorts or did not assess the incremental predictive value of such biomarkers combined with current clinical practice. Particularly, novel renal biomarkers, including cystatin C, plasma neutrophil gelatinase-associated lipocalin, and kidney injury molecule-<NUM>, have not performed better than conventional creatinine. With regards to proteomics data in other cardiovascular pathology contexts, a proteomics approach was recently reported in the setting of stable coronary artery disease, in which a <NUM>-protein risk score was reported with a C-statistic of <NUM>.

<NPL>discloses that adrenomedullin is a marker of impaired hemodynamics, organ dysfunction, and poor prognosis in cardiogenic shock.

Thus, well into the <NUM>st century, shock in general, and CS in particular, remain associated with unacceptable high mortality, substantial morbidity and resource utilization. Despite widespread use of early coronary reperfusion, CS occurs with a prevalence of approximately <NUM>% in STEMI and it is the leading cause of in-hospital death.

Therefore, although, as cited above, some contemporary risk scores are available, including the CardShock and IABP-SHOCK II risk scores, more accurate risk stratification strategies are needed in order to efficiently predict mortality risk in cardiogenic shock patients. On the other hand, accumulating evidence indicates that CS is not only a pump failure problem but is rather a systemic inflammatory status within the context of multiorgan failure. Therefore, comprehensive proteomics may enable the unbiased discovery of novel protein biomarkers that can be used to acquire pathophysiological knowledge, improve risk stratification accuracy, and identify therapeutic targets.

The present invention is actually focused on solving the above cited problem and it is based on a protein-based score to predict short-term mortality risk among patients with CS. Thus, this precise stratification of patients suffering from CS, according to their short-term mortality risk, can be effectively used to foresee or anticipate the treatment mainly in those cases with a high mortality risk, increasing the likelihood of success of the treatment and consequently the life expectancy of the patient suffering from cardiogenic shock.

In the present disclosure, a quantitative proteomics analyses in two independent CS cohorts for the discovery and validation of CS biomarkers was performed. Moreover, a cohort for the discovery and validation of septic sock (SS) biomarkers was performed.

Twenty-six proteins listed in Table <NUM> were initially identified as biomarkers for predicting mortality risk among patients suffering from CS.

Among said twenty-six proteins, the four proteins described below were identified as preferred candidates both in CS and SS, for which the measured levels substantially improved mortality risk prediction beyond established contemporary clinical risk scores:.

Thus, based on the results, a protein-based classifier was developed, which was also tested by mass spectrometry and ELISA. This classifier accurately discriminates shock patients according to their short-term mortality risk.

Particularly, the inventors have developed an in vitro method for discriminating those patients suffering from cardiogenic shock, or who have suffered from cardiogenic shock, with low mortality risk from those patients suffering from cardiogenic shock, or who have suffered from cardiogenic shock, with high mortality risk.

Specifically, a circulating protein-based score has been developed to predict short-term mortality risk among patients with CS or SS. In a particularly preferred embodiment the method is focused on CS and comprises measuring, in combination, the concentration level of four specific proteins (CS4P model) namely: L-FABP, B2MG, ALDOB and IC1. The area under the curve (AUC) for this CS4P model is <NUM> (see <FIG>). The AUC for CardShock (which is a standard risk score) is <NUM>. The combination of both models (CS4P + CardShock) gives rise to an improved AUC of <NUM> (see <FIG>).

On the other hand, it is important to note that the model based on the combination of the twenty-six proteins listed in Table <NUM> gives rise to an improved AUC in CS of <NUM> (see <FIG>) when this model is not combined with CardShock. Moreover, an improved AUC of <NUM> was obtained when the twenty-six proteins listed in Table <NUM> was also combined with CardShock (see <FIG>).

Moreover, said twenty-six proteins and, more particularly, said four proteins L-FABP, B2MG, ALDOB and IC1 have been individually analyzed in the present disclosure (see <FIG>, <FIG>, <FIG> and <FIG>). The levels of L-FABP, B2MG and ALDOB are higher in non-survivors CS patients relative to survivors. Conversely, the level of IC1 is lower in non-survivors CS patients relative to survivors (see <FIG>).

The proteins were preferably measured within <NUM> hours of the patient admission On the other hand, the method is particularly directed to determine "short-term" mortality risk among CS patients. According to the present invention, "short term" means <NUM> days. So, in a preferred embodiment, the method of the invention is actually evaluating the mortality risk among cardiogenic shock patients within a period of <NUM> days.

On the other hand, the method of the invention can be considered as a non-invasive or minimally invasive technique because it is preferably performed in serum or plasma samples obtained from the patients.

Although, in a particularly preferred embodiment, the method of the invention is based on determining the concentration level of the four proteins B2MG, L-FABP, ALDOB and IC1 in combination, it is important to consider that the present invention could be also performed by determining the concentration level of a reduced number of said four proteins. In other words, the present invention could be implemented by using one, two, three or four of the proteins: B2MG, L-FABP, ALDOB and IC1, wherein the level of at least B2MG is determined. B2MG is herein cited as an individual protein for implementing the invention because, such as it can be observed in Table <NUM>, it offers the best individual results in CS when it is determined by ELISA (method more readily suitable for routine clinical use) without the need of being combined with CardShock. The present disclosure offers scientific support (see <FIG>, <FIG>, <FIG> and <FIG>), for the use of any of the <NUM> proteins listed in Table <NUM>, preferably any of the following proteins B2MG, L-FABP, ALDOB or IC1 as individual biomarkers, or any combination thereof, for predicting mortality risk among patients suffering from shock, preferably CS or SS. Consequently, the present disclosure also refers to the use of any of the <NUM> proteins listed above, or any combination thereof, for predicting mortality risk among patients suffering from shock, preferably CS or SS. Said combination of proteins comprises at least <NUM>, at least <NUM> or at least <NUM> of the proteins: B2MG, L-FABP, ALDOB or IC1. Thus B2MG or, alternatively, any of the <NUM> proteins listed above, preferably any of the following proteins: L-FABP, ALDOB or IC1, or any combination thereof, are identified in the present disclosure as biomarkers suitable for predicting mortality risk among patients suffering from shock, preferably CS or SS. Consequently, the present disclosure provides evidence for the use of any of the above cited proteins, or any combination thereof, as biomarkers showing a high sensitivity and sensibility, for predicting mortality risk among patients suffering from shock, preferably CS or SS.

So, the first embodiment of the present invention refers to an in vitro method for predicting mortality risk among patients suffering from cardiogenic shock which comprises determining in a biological sample obtained from the patient the concentration level of at least B2MG, preferably in combination with at least L-FABP, or at least ALDOB, or at least IC1, wherein an increased level of at least the protein B2MG or of at least L-FABP, or of at least ALDOB, or a reduced level of at least IC1, with respect to the concentration level of said proteins determined in survivor patients suffering from cardiogenic shock, is an indication of mortality risk.

In a preferred embodiment, the method of the 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, with respect to the concentration level determined in survivor patients suffering from cardiogenic shock, is an indication of mortality risk. In a preferred embodiment, the method of the invention further comprises performing CardShock. CardShock includes the following variables: Age > <NUM> years, confusion at presentation, previous myocardial infarction or coronary artery bypass grafting (CABG), acute coronary syndrome (ACS) etiology, left ventricular ejection fraction < <NUM>%, blood lactate, and eGFRCKD-EPI. In a preferred embodiment, the method of the invention is performed within <NUM> hours from the patient admission. In a preferred embodiment, the mortality risk is evaluated within a period of <NUM> days. In a preferred embodiment, the biological sample is serum or plasma. In a preferred embodiment, the mortality risk is predicted in the present invention in patients with CS. <FIG>, provide ROC curves showing the AUC for the individual proteins L-FABP, B2MG, ALDOB and IC1, and combinations thereof, for predicting mortality risk in patients with SS.

The second embodiment of the present invention refers to the in vitro use of at least B2MG, preferably in combination with at least L-FABP, or at least ALDOB, or at least IC1, for predicting mortality risk among patients suffering from CS In a preferred embodiment, said use comprises determining the concentration level of B2MG, L-FABP, ALDOB and IC1. In a preferred embodiment, said use further comprises performing CardShock. CardShock includes the following variables: Age > <NUM> years, confusion at presentation, previous myocardial infarction or coronary artery bypass grafting (CABG), acute coronary syndrome (ACS) etiology, left ventricular ejection fraction < <NUM>%, blood lactate, and eGFRCKD-EPI.

When the method comprises measuring the concentration level of a combinations of biomarkers, a score value is obtained for the signature and this score value is compared with a threshold value which defines the diagnostic rule. If a variation of the score value is identified with respect to the threshold, then the corresponding sample is classified as a positive sample, which is an indication of an increased mortality risk of the patient suffering from CS. The threshold value has been defined in order to optimize sensitivity and specificity values. Consequently, the method comprises: a) Measuring the concentration level of any of the above cited combinations of biomarkers, in a biological sample obtained from the subject, b) processing the concentration values in order to obtain a risk score and c) wherein if a deviation or variation of the risk score value obtained for any of the above cited combinations of biomarkers is identified, as compared with a reference value, this is an indication of an increased mortality risk of the patient suffering from CS.

Further disclosed herein is a method for treating patients suffering from cardiogenic shock, or patients who have suffered from cardiogenic shock, which comprises treating said patients with a suitable treatment after determining, by means of the method described above, their mortality risk. Thus, this precise stratification of patients suffering from CS, according to their short-term mortality risk, can be effectively used to foresee or anticipate the treatment mainly in those cases with a high mortality risk, increasing the likelihood of success of the treatment and consequently the life expectancy of the patient suffering from cardiogenic shock.

In a preferred embodiment the methods of the invention are performed by ELISA or mass spectrometry. In a preferred embodiment, the measurement of the proteins is performed at their precursors (if any), or mid-regional sections.

The third embodiment of the present invention refers to a kit adapted for predicting mortality risk among patients suffering from cardiogenic shock which comprises: a) Tools or media for obtaining a serum or plasma sample from the patient, and b) Tools or media for measuring the concentration level of at least B2MG and at least one of L-FABP and/or ALDOB and/or IC1. In a preferred embodiment, the kit comprises: a) Tools or media for obtaining a serum or plasma sample from the patient, and b) Tools or media for measuring the concentration level of B2MG and L-FABP and ALDOB and IC1.

On the other hand, there are established treatment strategies when a patient is suffering from CS. Early revascularization (mainly through percutaneous coronary intervention (PCI)) is the current, most important, treatment strategy in CS after myocardial infarction, with a significant mortality reduction after <NUM> months, <NUM> year, and <NUM> years. In clinical practice revascularization should be limited to the culprit lesion with possible staged revascularization of other lesions at a later timepoint. There may also be a role for emergent coronary artery bypass grafting (CABG) revascularization; however, there is little evidence to guide surgical vs. 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 for PCI success. There are no specific trials in CS for antiplatelets or anti coagulation. Enteral resorption is impaired in CS and oftentimes opioids are co-administered with further impact on enteral bioavailability.

In the intensive care unit, CS treatment involves initial hemodynamic stabilization by volume expansion, vasopressors, and inotropes plus additional therapy for prevention or treatment of multiorgan system dysfunction (MODS). Inotropes and vasopressors are administered in approximately <NUM>% of patients in CS. In case of an abnormal heart rhythm, immediate synchronized cardioversion or anti-arrhythmic agents may be administered, e.g. adenosine. Positive inotropic agents (such as dobutamine or milrinone), which enhance the heart's pumping capabilities, are used to improve the contractility and correct the low blood pressure. CS may also be treated with intravenous dobutamine simultaneously to norepinephrine, which acts on β1 receptors of the heart leading to increased contractility and heart rate. Other inotropes such as levosimendan or phosphodiesterase-inhibitors are of interest based on their myocardial contractility improvement and potential for vasodilation without increasing oxygen requirements. However, current evidence for inodilators in CS is very limited. Should that not suffice, the application of mechanical circulatory support is necessary. Intra-aortic balloon pump reduces workload for the heart and improves perfusion of the coronary arteries. Ventricular assist devices (VADs) augment the pump-function of the heart. New developments in VADs include right ventricle support devices such as the Impella RP (Abiomed, Danvers, MA, USA) and the TandemHeart RA-PA (LivaNova, London, UK) with blood delivery 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) deployed across the aortic valve and delivering blood from the left ventricle into the aorta similar to the Impella family. Another investigational device is the paracorporeal pulsatile iVAC <NUM> (PulseCath BV, Amhem, The Netherlands). Recent developments with miniaturized systems and percutaneous cannula insertion have led to a wider adoption by interventional cardiologists for the treatment of CS using extracorporeal life support systems (ECMO), which have been proposed lately to help CS patients. Integral features of ECMO are the blood pump, a heat exchanger, and an oxygenator. As a general reflection on mechanical circulatory support, the IABP-SHOCK II has shown that a large percentage of CS survivors, could survive without any device. Inserting a device in these patients will have no impact on survival or may even lead to some complications by the device itself possibly resulting in death. Among the <NUM>-<NUM>% not surviving, there may also be futile situations where even the best available device will not be able to change clinical outcome. This futile situation may occur in the range of <NUM>-<NUM>% for patients with severe CS or those with anoxic brain injury or with concomitant severe sepsis. In these, mechanical circulatory support may be used as a bridge-to-decision strategy and discussion with relatives for a patient-centred decision, but requires a prognostic measurement to validate such decisions, which the field currently lacks. Finally, as a last resort, if the person is stable enough and otherwise qualifies, heart transplantation can be recommended, or, if not eligible for heart transplantation, an artificial heart can be placed. One of the preferred treatment strategies are ventricular assist devices. In this sense, the biomarkers described in the present invention, B2MG, L-FABP, ALDOB and/or IC1, can be used to design companion diagnostic kits or tests to determine the applicability of the above-mentioned treatments to a specific person suffering from CS, for example ventricular assist devices. Consequently, these companion diagnostic kits or tests could help the clinicians in selecting or excluding patient groups for a specific treatment, for example with ventricular assist devices, and to determine responders and non-responders to the therapy. So, by measuring the concentration level of at least B2MG, preferably in combination with L-FABP, ALDOB and/or IC1, the clinicians could predict whether a specific patient would respond to a treatment, for example with ventricular assist device. On the other hand, the clinicians could monitor and follow up the response of a specific patient to the treatment, for example with ventricular assist devices, by measuring the concentration level of at least B2MG, preferably in combination with L-FABP, ALDOB and/or IC1. For the purpose of the present invention the following terms are defined:.

The Barcelona discovery cohort is prospective single-center all-comers study of patients with STEMI-derived CS between March <NUM> and March <NUM>. STEMI was defined according to the Third Universal Definition of Myocardial Infarction. Patient management was determined by the physicians, following guideline recommendations. [<NPL>] and [<NPL>].

Two samples were obtained (admission and <NUM> hours) from every patient (n=<NUM>) by venipuncture and stored at -<NUM>. The clinical end-point was <NUM>-day mortality. The CardShock validation cohort is a European prospective, multicenter, multinational CS study of ischemic or non-ischemic origin between October <NUM> and December <NUM>. Cohort clinical characteristics and inclusion and exclusion criteria are reported elsewhere [<NPL>].

For the present study, only one sample withdrawn within <NUM> hours of admission, immediately frozen and stored at -<NUM>, was used (n=<NUM>). During follow-up, vital status was determined by direct contact with the patients or their next of kin, or from population and hospital registers. The clinical end-point was <NUM>-day mortality.

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

The septic shock cohort analyzed was composed of <NUM> patients, provided by the Department of Anesthesiology and Critical Care and Burn Unit, GH St-Louis-Lariboisi&re, Paris, France.

Serum samples were collected for 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 launched the Surviving Sepsis Campaign Guidelines: Septic shock is defined as a subset of sepsis with circulatory and cellular/metabolic dysfunction associated with higher risk of mortality [<NPL>. doi:<NUM>/jama. Samples were stored at -<NUM> and sent to Barcelona for further analysis. During follow-up, vital status was determined by direct contact with the patients or their next of kin, or from population and hospital registers. Out of <NUM> patients, <NUM> (<NUM>%) survived and <NUM> (<NUM>%) deceased during the follow up. The clinical end-point was <NUM>-day mortality. This cohort was approved by local ethics committees at the participating centers, and studies were conducted in accordance with the Declaration of Helsinki. Written consent was obtained from the patients or their next of kin.

Quantitative proteomics analysis was performed using mass spectrometry (nLC-MS/MS) to identify potential protein biomarker candidates among those proteins differing in abundance between <NUM>-day survivors and non-survivors. Serum samples (admission and <NUM> hours) from <NUM> patients of the Barcelona cohort (<NUM> non-survivors and <NUM> survivors at <NUM> days) were trypsin digested to peptides and analyzed using label-free screening proteomics (nLC-MS/MS).

Candidate biomarker proteins identified in the discovery phase, were evaluated in terms of classification power in the CardShock validation cohort with targeted proteomics quantification using parallel reaction monitoring (PRM). Plasma samples corresponding to <NUM> patients from the CardShock cohort (<NUM> non-survivors and <NUM> survivors at <NUM> days), were trypsin digested and analyzed using targeted nLC-PRM and isotopically-labeled standard peptides as internal references. Fragment ion chromatographic traces for all targeted precursor peptides were evaluated, logarithmic transformed and normalized using the internal reference peptides. Protein abundances were estimated, and protein relative quantification was assessed between survivors and non-survivors.

Four commercially available ELISA kits were used, following the instructions of the manufacturer, for each validated protein.

L-FABP was quantitative determined by Human L-FABP ELISA Kit. The minimum concentration which can be measured was <NUM> pg/mL and the measurable concentration range was <NUM>-<NUM>,<NUM> pg/ml. Samples had to be diluted at least 20x before use and they were diluted <NUM>/<NUM> in dilution buffer 1x prior measurement. The method of analysis was according to the supplier's manual. There is not cross-reactivity with human H-FABP and human I-FABP.

ALDOB was measured by an Enzyme-linked Immunosorbent Assay Kit. The standard curve range used was <NUM>-<NUM> ng/ml and the minimun detectable concentration was <NUM> ng/ml. Intra-assay and inter assay precision was <<NUM>% and <<NUM>%, respectively. Samples were diluted <NUM>/<NUM> in Phosphate Buffered Saline (PBS) 1x prior measurement. No significant cross-reactivity or interference between ALDOB and analogues was observed.

B2MG was determined by an Enzyme Immunoassay. The calculation range of this ELISA was <NUM>-<NUM>µg/ml, where the functional sensitivity was determined in <NUM>µg/ml. Intra-assay and inter assay precision was <<NUM>% and <<NUM>%, respectively. Samples were diluted <NUM>/<NUM> in sample buffer PU 1x prior measurement. No interference has been observed with haemolytic (up to <NUM>/dl) or lipemic (up to <NUM>/dl) serum. The analysis was performed according to the manual.

Serpin G1 was measured by the Human SERPING1 ELISA. The sensitivity or the minimum detectable dose was <NUM> pg/mL and the detection range was <NUM>-<NUM>,<NUM> pg/mL. Intra-assay and inter assay precision were <<NUM>% and <<NUM>%, respectively. Samples were diluted <NUM>/<NUM>,<NUM> in sample diluent prior measurement. This assay has high specificity for natural and recombinant human SerpinG1 and there is no detectable cross-reactivity with other relevant proteins.

Clinical variables are presented as number (n) and percentage (%) for categorical variables, mean and standard deviation (SD) for normally distributed variables, or median and interquartile range (IQR) for skewed variables. Comparisons between groups were performed using the chi-square test, Student's t-test, or Mann-Whitney U-test as appropriate.

Protein abundance estimates and relative protein quantification between groups (survivors vs. non-survivors) from proteomics data were performed with the software packages Skyline <NUM> and MSstats <NUM>. The best protein combinations for classifying <NUM>-day mortality risk in patients with CS were challenged within the CardShock cohort, who was divided into training (<NUM>/<NUM>) and a validation set (<NUM>/<NUM>). Within the training set the abundance of each protein was fitted in a logistic regression model between survivors and non-survivors, and the classification ability of each protein and protein combinations was evaluated by the area under the curve (AUC) of a receiver operating characteristic (ROC) curve as previously described. The four identified proteins were tested as continuous variables.

The CardShock risk score, used as baseline model, includes age > <NUM> years, confusion at presentation, previous myocardial infarction or coronary artery bypass grafting (CABG), acute coronary syndrome (ACS) etiology, left ventricular ejection fraction < <NUM>%, blood lactate, and eGFRCKD-EPI. Model calibrations were calculated using the Hosmer-Lemeshow (HL) test, and patient discrimination and reclassification were evaluated using the Harrell C-statistic (AUC) and continuous net reclassification improvement (cNRI). Confidence intervals for the C-statistic and the NRI were obtained by <NUM>-fold bootstrap resampling.

Analyses were performed using STATA V. <NUM> (StataCorp, College Station, TX), PredictABEL R package v1. <NUM>, and SPSS V. <NUM> (IBM Corp, Armonk, NY).

Table <NUM> shows the clinical, biochemical, and follow-up data from the Barcelona discovery cohort. The mean age was <NUM> ± <NUM> years, <NUM>% were women, and <NUM>% were treated with primary percutaneous coronary intervention. <NUM>-day mortality was <NUM>%.

A total of <NUM> proteins were identified in the dataset, of which <NUM> were present in over <NUM> % of the patients. After protein relative quantification among the different variables: patient outcome (survivors, non-survivors), and sampling time (admission, 24hours), a total of <NUM> proteins were selected for the validation phase in the independent CardShock cohort. Briefly, <NUM> proteins that changed in abundance either between survivor and non-survivor patients or within the first <NUM> hours after admission (admission, <NUM>) were considered for further validation. Additionally, <NUM> proteins were included in the study based on previous knowledge and clinical relevance.

The classification power of <NUM> of the <NUM> selected proteins was further validated in the CardShock cohort using targeted proteomics quantification by parallel reaction monitoring. Table <NUM> shows the characteristics of the CardShock validation cohort. The mean age was <NUM> ± <NUM> years, <NUM>% were women, and <NUM>-day mortality was <NUM>%. The most common cause of CS was ACS (<NUM>%), mainly driven by STEMI (<NUM>%). Compared to the Barcelona Cohort, CardShock patients exhibited higher hemoglobin and lower creatinine, lactate, and glucose levels.

Targeted mass spectrometry chromatographic profiles were obtained for all measured proteins and compared to the corresponding internal references for relative protein quantification. See <FIG> as an example for the obtained mass spectrometric signals for proteins Liver-type fatty acid-binding protein (L-FABP), the Fructose-bisphosphate aldolase B (ALDOB), the Beta-<NUM>-microglobulin (B2MG), and the SerpinG1 (IC1), measured in all patients. The best protein combinations for classifying <NUM>-day survivors and non-survivors in CS patients were identified by performing a predictor selection combined with cross-validation as previously described [<NPL>]. According to the <FIG>, the following sequences were used for obtaining the mass spectrometry chromatographic profile for each protein: SEQ ID NO: <NUM> and SEQ ID NO: <NUM> for B2MG, SEQ ID NO: <NUM> and SEQ ID NO: <NUM> for ALDOB, SEQ ID NO: <NUM> for FABPL and SEQ ID NO: <NUM> for ID1. Please note that although this method has been used in the present invention for the identification of the protein, any other part of the protein, or even the entire protein could be used for this purpose.

This evaluation resulted in the identification of a <NUM>-protein combination with proteins L-FABP, B2MG, ALDOB and IC1 as the best protein classifier to identify short-term mortality risk with an AUC of <NUM> (<NUM>% CI <NUM>-<NUM>) (Table <NUM> and <FIG>).

An additional model was constructed by combining the CardShock risk score with the new CS4P model (CardShock+CS4P). The CardShock+CS4P significantly improved the C-statistics for mortality prediction compared with the CardShock risk score alone (AUC <NUM> vs. AUC <NUM>; P=<NUM>; Table <NUM> and <FIG>). Furthermore, the CardShock+CS4P showed a marked benefit in patient reclassification, with an NRI of <NUM> (P=<NUM>) (Table <NUM>). Overall, CardShock+CS4P resulted in an improved reclassification of <NUM>% of patients compared to CardShock risk score.

In an exploratory analysis, we also combined the CS4P model with another contemporary risk score, the IABP-SHOCK II, generating the IABP-SHOCK II+CS4P. The IABP-SHOCK II+CS4P also provided better prediction metrics compared to IABP-SHOCK II, with an NRI of <NUM> (P=<NUM>).

The CS4P model defined by targeted proteomics was tested by ELISA to support its prompt translation into routine clinical practice. Median (IQR) circulating concentrations of the studied proteins were: L-FABP, <NUM> pg/mL (<NUM>-<NUM> pg/mL); B2MG, <NUM>µg/mL (<NUM>-<NUM>µg/mL); ALDOB, <NUM> ng/mL (<NUM>-<NUM> ng/mL); and IC1, <NUM> pg/mL (<NUM>-<NUM> pg/mL), respectively. Circulating L-FABP (<NUM> vs. <NUM> pg/mL, p=<NUM>), B2MG (<NUM> vs. <NUM>µg/mL, p<<NUM>), and ALDOB (<NUM> vs. <NUM> ng/mL, p=<NUM>) were higher in non-survivors relative to survivors. By contrast, IC1 concentration was significantly lower in non-survivors relative to survivors (<NUM> vs. <NUM> pg/mL, p=<NUM>) (<FIG>).

Protein concentrations of the CS4P obtained by ELISA combined with the CardShock risk score provided an AUC of <NUM> (<NUM>% CI <NUM>-<NUM>), not significantly different from that obtained by targeted proteomics (p=<NUM>).

The CS4P model defined by targeted proteomics was tested by ELISA to support its prompt translation into routine clinical practice. Median (IQR) circulating concentrations of the studied proteins were: L-FABP, <NUM> pg/mL (<NUM>-<NUM> pg/mL); B2MG, <NUM>µg/mL (<NUM>-<NUM>µg/mL); ALDOB, <NUM> ng/mL (<NUM>-<NUM> ng/mL); and IC1, <NUM> pg/mL (<NUM>-<NUM> pg/mL), respectively. Circulating L-FABP (p=<NUM>, OR <NUM>, <NUM>% CI <NUM>-<NUM>), B2MG (p<<NUM>, OR <NUM>, <NUM>% CI <NUM>-<NUM>), and ALDOB (p=<NUM>, OR <NUM>, <NUM>% CI <NUM>-<NUM>) were higher in non-survivors relative to survivors after analysing all-cause of death at <NUM> days in a univariate analysis. In the multivariable analysis, L-FABP (p=<NUM>, HR <NUM>, <NUM>%CI <NUM>-<NUM>) and B2MG (p=<NUM>, HR <NUM>, <NUM>% CI <NUM>-<NUM>) were higher in non-survivors relative to survivors after analysing all-cause of death at <NUM> days.

Protein concentrations of the CS4P obtained by ELISA provided an AUC of <NUM> (<NUM>% CI <NUM>-<NUM>).

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

Claim 1:
In vitro method for predicting mortality risk among patients suffering from cardiogenic shock which comprises determining in a biological sample which has been obtained from the patient the concentration level of at least B2MG, wherein an increased level of at least the protein B2MG with respect to the concentration level determined in control survivor patients suffering from cardiogenic shock, is an indication of mortality risk.