WO2020112149A1

WO2020112149A1 - Methods and compositions for the assessment of acute myocardial infarction (ami)

Info

Publication number: WO2020112149A1
Application number: PCT/US2018/063517
Authority: WO
Inventors: Bruce Xuefeng Ling; Linmin CHEN; Shiying Hao
Original assignee: Tianjin Yunjian Medical Technology Co Ltd; Binhai Industrial Technology Research Institute of ZJU; Mprobe Inc
Current assignee: Tianjin Yunjian Medical Technology Co Ltd; Binhai Industrial Technology Research Institute of ZJU; Mprobe Inc
Priority date: 2018-12-01
Filing date: 2018-12-01
Publication date: 2020-06-04
Anticipated expiration: 2021-06-01

Abstract

Acute myocardial infarction (AMI) biomarkers, AMI biomarker panels, and methods obtaining AMI biomarker level representation for a sample are provided. These composition and methods find use in a number of applications, including, for example, stratifying AMI, diagnosing AMI, prognosing AMI, monitoring a subject with AMI, and determining a treatment for AMI. In addition, systems, devices, and kits thereof that find use in practicing the subject methods are provided.

Description

Methods and compositions for the assessment of acute myocardial infarction (AMI)

Field of the invention

The disclosure of current patent application pertains to methods and

compositions for providing an assessment of acute myocardial infarction using four Ceram ides and their ratios as total 6 analytic markers.

Background of the invention

Acute myocardial infarction (AMI) and unstable angina pectoris (UA) are referred to as acute coronary syndrome (ACS), belonging to the most severe categories of coronary artery disease (CAD), which remains a leading cause of mortality in the world. According to the Global Burden of Disease Study 2013, CAD was responsible for an estimated 8.14 million deaths (16.8%) globally. The causes and pathogenesis are still unclear. Multiple, complex molecular events characterize the progression of AMI.

The current clinical diagnosis of CAD and AMI bases on patient symptoms, electrocardiogram (ECG), cardiac markers, stress testing, and coronary angiography. Invasive coronary angiography is the diagnostic“gold standard”, but its specialized technology and high cost limit it to a select population. Abnormal metabolism also characterizes CAD and AMI. Metabolic alterations in the heart result in changes in the metabolome of biofluids. A combination of multiple small-molecule metabolites or their ratios may offer excellent diagnostic values. Thus, there is a need to discover CAD and AMI biomarkers underlying the pathogenetic pathways and construct a more accurate biomarker panels that can provide a definitive early diagnosis, better monitoring, leading to improved outcomes and economic benefits.

Summary of the invention

Acute myocardial infarction (AMI) biomarkers, AMI biomarker panels, and methods obtaining AMI biomarker level representation for a sample are provided. These composition and methods find use in a number of applications, including, for example, diagnosing AMI, prognosing AMI, monitoring a subject with AMI, and determining a therapy for AMI. In addition, systems, devices, and kits thereof that find use in practicing the subject methods are provided.

In some aspects of the invention, a panel of AMI biomarkers is provided, the panel comparing one or more AMI markers selected from the ceramide group consisting of Cer(18:1/18:0), Cer(18:1/16:0), Cer(18:1/24:1 ), Cer(18:1/18:0)/24:0,

Cer(18:1/16:0)/24:0, Cer(18:1/24:1 )/24:0, totally 6 analytes.

In some aspects of the invention, a method is provided for providing an AMI marker level representation for a subject. In some embodiments, the method comprises evaluating a panel of AMI markers in a serum sample from a subject to determine the level of each AMI marker in the serum sample; and obtaining the AMI marker level representation based on the level of each AMI marker in the panel. In some

embodiments, the panel comprises Cer(18: 1/18:0). In some embodiments, the panel comprises Cer(18:1 /16:0) and Cer(18: 1/24:1 ). In some embodiments, the panel further comprises one or more AMI markers selected from the group consisting of

Cer(18:1/18:0)/24:0, Cer(18:1/16:0)/24:0, Cer(18:1/24:1 )/24:0. In some embodiments, the panel comprises all AMI markers froom the group consisting of Cer(18: 1/18:0),

Cer(18:1/16:0), Cer(18: 1/24:1 ), Cer(18:1/18:0)/24:0, Cer(18:1/16:0)/24:0,

Cer(18:1/24:1 )/24:0. In some embodiments, the method further comprises providing a report of the AMI marker level representation. In certain embodiments, the AMI marker representation is an AMI score.

Also disclosed is a kit for the analysis of a sample of a bio-fluid of a subject, comprising aliquots of standards of each compound of a panel of metabolite species; an aliquot of an internal standard; and an aliquot of a control bio-fluid. Typically, the control bio-fluid is serum from a control source that is conspecific with the subject. In some embodiments, the internal standard consists of D₇-d18:1 -16:0 Ceramide, D₇-d18:1 -18:0 Ceramide, D₇-d18: 1 -24:0 Ceramide, D₇- d18: 1 -24:1 Ceramide. Typically, the kit includes instructions for use. Brief description of the drawings

The invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

Figure 1. Outline of the study based on discovery and validation of AMI biomarkers.

Figure 2. In AMI and control cohorts, gender, age, and measured high sensitive troponin T were shown.

Figure 3. Boxplot display and scatter plot of biomarker distribution or 3

ceramides analytes: Cer(18: 1 /18:0), Cer(18:1/16:0), Cer(18: 1/24: 1 ), and their ratios to Cer(18: 1/24:0) in case, control, and testing samples in serum.

Figure 4. Ceramide Scoring Matrix distribution (red: case, green: control)

Figure 5. Density plots for 6 ceramide analytes. For each analyte, if testing sample is beyond 90% of the normal distribution, then it scores 1 in the scoring matrix.

Detailed description of the invention

Acute myocardial infarction (AMI) biomarkers, AMI biomarker panels, and methods obtaining AMI biomarker level representation for a sample are provided. These composition and methods find use in a number of applications, including, for example, diagnosing AMI, prognosing AMI, monitoring a subject with AMI, and determining a therapy for AMI. In addition, systems, devices, and kits thereof that find use in practicing the subject methods are provided. These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the compositions and methods as more fully described below.

Before the present methods and compositions are described, it is to be

understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates that may need to be independently confirmed.

As summarized above, aspects of the subject invention include compositions, methods, systems and kits that find use in providing an AMI assessment, e.g.

diagnosing, prognosing, monitoring, and/or treating AMI in a subject. By“AMI” it is meant an acute multisystem complication of partial or complete coronary arteries blockage that may be accompanied by one or more of hypoperfusion of myocardial tissue, brain ischemia, and systemic hypoxia, ischemia, and multiple organs failure.

In describing the subject invention, compositions useful for providing an AMI assessment will be described first, followed by methods, systems and kits for their use.

AMI markers and panels

In some aspects of the invention, AMI biomarkers are provided. By an“AMI biomarker” it is meant a molecular entity whose representation in a sample is

associated with an AMI phenotype. For example, an AMI marker may be differentially represented, i.e. represented at a different level, in a sample from an individual that has developed AMI as compared to a healthy individual. In some instances, an elevated level of marker is associated with the AMI phenotype. For example, the concentration of marker in a sample may be 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 7.5-fold, 10- fold, or greater in a sample associated with the AMI phenotype than in a sample not associated with the AMI phenotype. In other instances, a reduced level of marker is associated with the AMI phenotype. For example, the concentration of marker in a sample may be 10% less, 20% less, 30% less, 40% less, 50% less or more in a sample associated with the AMI phenotype than in a sample not associated with the AMI phenotype.

As demonstrated in the examples of the present disclosure, the inventors have identified 6 ceramides or their ratios that are associated with AMI and that find use in combination (ie. as a panel) in providing an AMI assessment, e.g. diagnosing AMI, prognosing AMI, monitoring a subject with AMI, determining a treatment for a subject affected with AMI, and the like. These include, but are not limited to: Cer(18:1/18:0), Cer(18:1/16:0), Cer(18: 1/24:1 ), Cer(18:1/18:0)/24:0, Cer(18:1/16:0)/24:0,

Cer(18:1/24:1 )/24:0, that are represented at elevated levels in blood samples of AMI, and thus, that find use as biomarkers in providing an AMI assessment, e.g. diagnosing an AMI, prognosing an AMI, determining a treatment for a subject affected with AMI, monitoring a subject with AMI, and the like.

As mentioned above, also provided herein are AMI panels. By a“panel” of AMI markers it is meant two or more AMI markers, e.g. 2 or more, 3 or more, 4 or more, or 5 or more markers, whose levels, when considered in combination, find use in providing AMI assessment, e.g. making AMI diagnosis, prognosis, monitoring, and/or treatment. Of particular interest are ceramide panels that comprise the AMI marker Cer(18:1/18:0), Cer(18:1/16:0), Cer(18: 1/24:1 ), Cer(18:1/18:0)/24:0, Cer(18:1/16:0)/24:0, and Cer(18:1/24:1 )/24:0. For example, in some embodiments, the AMI panel may comprise Cer(18: 1 /18:0) and Cer(18:1/16:0), or Cer(18:1/18:0) and Cer(18: 1/24:1 ), or

Cer(18:1/18:0) and Cer(18:1/18:0)/24:0, etc.

Other combinations of AMI markers that find use as AMI panels in the subject methods may be readily identified by the ordinarily skilled artisan using any convenient statistical methodology, e.g. as known in the art or described in the working examples herein. Methods

In some aspects of the invention, methods are provided for obtaining an AMI marker level representation for a subject. By an AMI marker level representation, it is meant a representation of the levels of one or more of the subject AMI marker(s), e.g. a panel of AMI markers, in a biological sample from a subject. The term "biological sample" encompasses a variety of sample types obtained from an organism and can be used in a diagnostic, prognostic, or monitoring assay. The term encompasses blood and other liquid samples of biological origin or cells derived therefrom and the progeny thereof. The term encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components. The term encompasses a clinical sample, and also includes cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. Clinical samples for use in the methods of the invention may be obtained from a variety of sources, particularly blood samples. The subject AMI biomarkers find use in making AMI assessment for a patient, or "subject". By a " AMI assessment", it is generally meant an estimation of a subject's susceptibility to AMI, a determination as to whether a subject is presently affected by AMI, a prognosis of a subject affected by AMI (e.g., identification of AMI states), and the use of therapeutics (e.g., monitoring a subject's condition to provide information as to the effect or efficacy of therapy on the AMI). Thus, for example, the subject AMI biomarkers and biomarker panels may be used to diagnose AMI, to provide a prognosis to a patient having AMI, to provide a prediction of the responsiveness of a patient with AMI to a medical therapy, to monitor a patient having AMI, to treat a patient having AMI, etc. In practicing the subject methods, an AMI biomarker signature for a patient is obtained. By an " AMI biomarker signature" or more simply, " AMI signature", it is meant a representation of the measured level/activity of an AMI biomarker or biomarker panel of interest. A biomarker signature typically comprises the quantitative data on the biomarker levels/activity of these one or more biomarkers of interest.

Examples of biomarker signatures include collections of measured small molecular metabolites levels. As used herein, the term "biomarker signature" means metabolites signature. Examples of biomarker signatures include biomarker profiles and biomarker scores. By a "biomarker profile" it is meant the normalized representation of one or more biomarkers of interest, i.e. a panel of biomarkers of interest, in a patient sample. By a "biomarker score" it is meant a single metric value that represents the sum of the weighted representations of one or more biomarkers of interest, more usually two or more biomarkers of interest, i.e. a panel of biomarkers of interest, in a patient sample. Biomarker profiles and scores are discussed in greater detail below. For example, in some embodiments, the subject methods may be used to obtain an AMI signature. That is, the subject methods may be used to obtain a representation of the metabolite, e.g. Cer(18:1/18:0), Cer(18:1/16:0), Cer(18: 1/24:1 ),

Cer(18:1/18:0)/24:0, Cer(18:1/16:0)/24:0, Cer(18:1/24:1 )/24:0, that are all up-regulated (i.e. , expressed at a higher level, exhibits a higher level of activity, etc.), in patients with

AMI.

To obtain an AMI signature, the metabolite level of the one or more AMI biomarkers of interest is detected in a patient sample. That is, the representation of one or more AMI biomarkers, e.g., Cer(18:1/18:0), Cer(18:1/16:0), Cer(18: 1/24:1 ),

Cer(18:1/18:0)/24:0, Cer(18:1/16:0)/24:0, Cer(18:1/24:1 )/24:0, and in some instances other AMI biomarkers in the art, e.g. a panel of biomarkers, is determined for a patient sample.

The term“sample” with respect to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived or isolated therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations.

The definition also includes samples that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc. The term "biological sample" encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like. The term "blood sample" encompasses a blood sample (e.g., peripheral blood sample) and any derivative thereof (e.g., fractionated blood, plasma, serum, etc.).

In performing the subject methods, the biomarker level is typically assessed in a body fluid sample (e.g., a sample of blood, e.g., whole blood, fractionated blood, plasma, serum, etc.) that is obtained from an individual. The sample that is collected may be freshly assayed or it may be stored and assayed at a later time. If the latter, the sample may be stored by any convenient means that will preserve the sample so that gene expression may be assayed at a later date. For example, the sample may freshly cryopreserved, that is, cryopreserved without impregnation with fixative, e.g. at 4°C, at - 20°C, at -60°C, at -80°C, or under liquid nitrogen. Alternatively, the sample may be fixed and preserved, e.g. at room temperature, at 4°C, at -20°C, at -60°C, at -80°C, or under liquid nitrogen, using any of a number of fixatives known in the art, e.g. alcohol, methanol, acetone, formalin, paraformaldehyde, etc.

The resultant data provides information regarding activity for each of the AMI biomarkers that have been measured, wherein the information is in terms of whether or not the biomarker is present (e.g. expressed and/or active) and, typically, at what level, and wherein the data may be both qualitative and quantitative.

Once the representation of the one or more biomarkers has been determined, the measurement(s) may be analyzed in any of a number of ways to obtain a biomarker signature.

For example, the representation of the one or more AMI biomarkers may be analyzed individually to develop a biomarker profile. As used herein, a "biomarker profile" is the normalized representation of one or more biomarkers in a patient sample, for example, the normalized level of serological metabolite concentrations in a patient sample, the normalized activity of a biomarker in the sample, etc. A profile may be generated by any of a number of methods known in the art. Other methods of calculating a biomarker signature will be readily known to the ordinarily skilled artisan.

As another example, the measurement of an AMI biomarker or biomarker panel may be analyzed collectively to arrive at an AMI biomarker score, and the AMI biomarker signature is therefore a single score. By "biomarker assessment score" it is meant a single metric value that represents the sum of the weighted representations of each of the biomarkers of interest, more usually two or more biomarkers of interest, in a biomarker panel. As such, in some embodiments, the subject method comprises detecting the amounts of markers of an AMI biomarker panel in the sample, and calculating an AMI biomarker score based on the weighted levels of the biomarkers. In certain embodiments, the biomarker score is based on the weighted levels of the biomarkers. In certain embodiments, the biomarker score may be a“metabolite biomarker score”, or simply“metabolite score”, i.e. it comprises the weighted expression level(s) of the one or more biomarkers, e.g. each biomarker in a panel of biomarkers.

In some instances, the weighting factor, or simply "weight" for each marker in a panel may be a reflection of the change in analyte level in the sample. For example, the analyte level of each biomarker may be log2 transformed and weighted either as 1 (for those markers that are increased in level in a group of AMI of interest, etc.) or -1 (for those markers that are decreased in level in a group of AMI of interest, etc.), and the ratio between the sum of increased markers as compared to decreased markers determined to arrive at an AMI biomarker signature. In other instances, the weights may be reflective of the importance of each marker to the specificity, sensitivity and/or accuracy of the marker panel in making the diagnostic, prognostic, or monitoring assessment. Such weights may be determined by any convenient statistical machine learning methodology, e.g. Principle Component Analysis (PCA), linear regression, support vector machines (SVMs), and/or random forests of the dataset from which the sample was obtained may be used. In some instances, weights for each marker are defined by the dataset from which the patient sample was obtained. In other instances, weights for each marker may be defined based on a reference dataset, or“training dataset”. Methods of analysis may be readily performed by one of ordinary skill in the art by employing a computer-based system, e.g. using any hardware, software and data storage medium as is known in the art, and employing any algorithms convenient for such analysis. For example, data mining algorithms can be applied through“cloud computing”, smartphone based or client-server based platforms, and the like.

Thus, in some instances, an AMI biomarker signature may be expressed as a series of values that are each reflective of the level of a different biomarker (e.g., as a biomarker profile, i.e. the normalized expression values for multiple biomarkers), while in other instances, the AMI biomarker signature may be expressed as a single value (e.g., an AMI biomarker score).

In some instances, the subject methods of obtaining or providing an AMI biomarker signature for a subject further comprise providing the AMI biomarker signature as a report. Thus, in some instances, the subject methods may further include a step of generating or outputting a report providing the results of an AMI biomarker evaluation in the sample, which report can be provided in the form of an electronic medium (e.g., an electronic display on a computer monitor), or in the form of a tangible medium (e.g., a report printed on paper or other tangible medium). Any form of report may be provided, e.g. as known in the art or as described in greater detail below.

The AMI signature that is so obtained may be employed to make an

AMI assessment. Typically, in making the subject AMI assessment, the AMI signature is employed by comparing it to a reference or control, and using the results of that comparison (a“comparison result”) to make the AMI assessment, e.g. diagnosis, prognosis, prediction of responsiveness to treatment, etc. The terms“reference” or “control”, e.t.“reference signature” or“control signature”,“reference profile” or“control profile”, and“reference score” or“control score” as used herein mean a standardized biomarker signature, e.g. biomarker profile or biomarker score, that may be used to interpret the AMI biomarker signature of a given patient and assign a diagnostic, prognostic, and/or responsiveness class thereto. The reference or normal control is typically an AMI biomarker signature that is obtained from a sample (e.g., a body fluid, e.g. blood) with a known association with a particular phenotype, Typically, the comparison between the AMI signature and reference will determine whether the AMI signature correlates more closely with the positive reference or the negative reference, and the correlation employed to make the assessment. By "correlates closely", it is meant is within about 40% of the reference, e.g. 40%, 35%, or 30%, in some

embodiments within 25%, 20%, or 15%, sometimes within 10%, 8%, 5%, or less.

In certain embodiments, the obtained AMI signature for a subject is compared to a single reference/control biomarker signature to obtain information regarding the phenotype. In other embodiments, the obtained biomarker signature for the subject is compared to two or more different reference/control biomarker signatures to obtain more in-depth information regarding the phenotype of the assayed tissue. For example, a biomarker profile, or a biomarker score to obtain confirmed information regarding whether the tissue has the phenotype of interest. As another example, a biomarker profile or score may be compared to multiple biomarker profiles or scores, each correlating with a particular diagnosis, prognosis or therapeutic responsiveness.

Reports

A“report,” as described herein, is an electronic or tangible document which includes report elements that provide information of interest relating to a diagnosis assessment, a prognosis assessment, a treatment assessment, a monitoring

assessment, etc. and its results. A subject report can be completely or partially electronically generated. A subject report includes at least an AMI assessment, e.g., a diagnosis as to whether a subject has a high likelihood of having an AMI. A subject report can further include one or more of: 1 ) information regarding the testing facility; 2) service provider information; 3) patient data; 4) sample data; 5) an assessment report, which can include various information: a) reference values employed, and b) test data, where test data can include: i) the biomarker levels of one or more AMI biomarkers, and/or ii) the biomarker signatures for one or more AMI biomarkers; 6) other features.

In some embodiments, providing an AMI signature or providing an AMI assessment, e.g., a diagnosis of AMI, includes generating a written report that includes AMI signature and/or the AMI assessment e.g. a "diagnosis assessment", a suggestion of possible treatment regimens (a "treatment assessment") and the like. Thus, the subject methods may further include a step of generating or outputting a report providing the results of an analysis of an AMI biomarker or biomarker panel, a diagnosis assessment, a prognosis assessment, or a treatment assessment, which report can be provided in the form of an electronic medium (e.g., an electronic display on a computer monitor), or in the form of a tangible medium (e.g., a report printed on paper or other tangible medium).

The report may include information about the testing facility, which information is relevant to the hospital, clinic, or laboratory in which sample gathering and/or data generation was conducted. This information can include one or more details relating to, for example, the name and location of the testing facility, the identity of the lab technician who conducted and/or analyzed, the location where the sample and/or result data is stored, the lot number of the reagents (e.g., kit, etc.) used in the assay, and the like. Report fields with this information can generally be populated using information provided by the user.

The report may include information about the service provider, which may be located outside the healthcare facility at which the user is located, or within the healthcare facility. Examples of such information can include the name and location of the service provider, the name of the reviewer, and where necessary or desired the name of the individual who conducted sample gathering and/or data generation. Report fields with this information can generally be populated using data entered by the user, which can be selected from among pre-scripted selections (e.g., using a drop-down menu). Other service provider information in the report can include contact information for technical information about the result and/or about the interpretive report. The report may include a patient data section, including patient medical history as well as administrative patient data such as information to identify the patient (e.g., name, patient date of birth (DOB), gender, mailing and/or residence address, medical record number (MRN), room and/or bed number in a healthcare facility), insurance information, and the like), the name of the patient's physician or other health

professional who ordered the monitoring assessment and, if different from the ordering physician, the name of a staff physician who is responsible for the patient's care (e.g., primary care physician).

The report may include a sample data section, which may provide information about the biological sample analyzed in the monitoring assessment, such as the source of biological sample obtained from the patient (e.g. blood, saliva, or type of tissue, etc.), how the sample was handled (e.g. storage temperature, preparatory protocols) and the date and time collected. Report fields with this information can generally be populated using data entered by the user, some of which may be provided as pre-scripted selections (e.g., using a drop-down menu).

It will also be readily appreciated that the reports can include additional elements or modified elements. For example, where electronic, the report can contain hyperlinks which point to internal or external databases which provide more detailed information about selected elements of the report. For example, the patient data element of the report can include a hyperlink to an electronic patient record, or a site for accessing such a patient record, which patient record is maintained in a confidential database.

This latter embodiment may be of interest in an in-hospital system or in-clinic setting. When in electronic format, the report is recorded on a suitable physical medium, such as a computer readable medium, e.g., in a computer memory, zip drive, CD, DVD, etc.

It will be readily appreciated that the report can include all or some of the elements above, with the proviso that the report generally includes at least the elements sufficient to provide the analysis requested by the user (e.g. a diagnosis, a prognosis).

Reagents, systems and kits

Also provided are reagents, devices and kits thereof for practicing one or more of the above-described methods. The subject reagents, devices and kits thereof may vary greatly. Reagents and devices of interest include those mentioned above with respect to the methods of assaying metabolites levels, where such reagents may include stable isotope labeled internal standards D₇-d18: 1 -16:0 Ceramide, D₇-d18: 1 -18:0 Ceramide, D₇-d18: 1-24:0 Ceramide, D₇- d18: 1 -24:1 Ceramide. The subject kits may also comprise one or more biomarker signature references, e.g. a reference for an AMI signature, for use in employing the biomarker signature obtained from a patient sample. For example, the reference may be a sample of a known phenotype, e.g. an unaffected individual, or an affected individual, e.g. from a particular risk group that can be assayed alongside the patient sample, or the reference may be a report of disease diagnosis, disease prognosis, or responsiveness to therapy that is known to correlate with one or more of the subject AMI biomarker signatures.

In addition to the above components, the subject kits may further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, DVD, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.

The following examples are offered by way of illustration and not by way of limitation.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1

Study design The study for AMI metabolomics experiment was comprised of the analysis of an independent AMI cohort (n=50) and normal control cohort (n=50) (Figure 1 ). Specific panels for AMI identification were constructed. AMI and normal control cohorts were constructed to match age, ethnicity, and parity (Figure 2).

Hypothesis generation

A biological analysis of pathway related to the development and progression of AMI was conducted through a metabolomics approach integrated with statistical learning technologies. A literature review was conducted in parallel to identify markers associated with AMI. The results of the two approaches were integrated to generate a hypothesis that Cer(18: 1/18:0), Cer(18: 1/16:0), Cer(18:1/24:1 ), Cer(18:1/18:0)/24:0, Cer(18:1/16:0)/24:0, Cer(18:1/24:1 )/24:0 can be used for AMI assessment.

Validation of PE marker candidates using mass spectrometry.

A LC-MS method was developed to quantify serum level of Cer(18:1/18:0), Cer(18:1/16:0), Cer(18: 1/24:1 ), Cer(18:1/24:0). Sample characteristics

The AMI and normal control subjects were used for serological protein biomarker discovery and validation. Internal standard preparation

Dilute to 200 mL with 70%MeOH in a 200-mL volumetric flask to obtain concentrations of different analytes (Table 1 ). Vortex vigorously and stored at 4 °C prior to use.

5 Table 1. Concentrations of different internal standard analytes

Concentration Concentration

Labeled Analyte Labeled Analyte

(mM) (mM)

²D₇-Dehydroepiandrosterone

2.220 2 D₇-d18:1-16:0 Ceramide 18.300

Sulfate

¹³C₆-T riiodothyronine 1.000 ²D₇-d18:1-18:0 Ceramide 17.400

13 C₆-Thyroxine 1 .000 2 D₇-d18:1-24:0 Ceramide 15.200

2 Dy-Cholesterol 1.000 2 D₇-d18:1-24: 1 Ceramide 15.000

Sample preparation

Preparation of serum sample: Serum sample was taken from -80 °C freezer and thawed on ice.10 pL of each serum sample was transferred into a new tube, and 90 pL 10 extraction buffer was added for extraction. The samples were vortexed vigorously for 1 min and subjected to high-speed centrifuge at 12,000 g for 5 min under room

temperature. The supernatant from each sample was collected for analysis

Preparation of quality control samples (Serum): 1 mL of serum samples from 3-5 normal control individuals were pooled and vortexed for 1 min, then centrifuge at 3,000 15 g for 1 min. The pooled serum sample was divided into 10-uL aliquots and stored at -80 °C before use. 6 of 10-uL aliquots from pooled serum were unambiguously processed as internal quality controls for 90 unknown samples in a 96-well plate during the sample prep.

MS/MS detection

1. Liquid Chromatography

Pump: Thermo Scientific™ Dionex™ UltiMate™ HPG-3200 RS

Autosampler: UltiMate WPS-3000 TRS

Mobile phase 50:50:1 acetonitrile/water/formic acid

LC flow gradient (Table 2) Table 2. LC flow gradient parameter characteristics

Time (min) Flow Rate (mL/min) %Mobile phase

0.000 0.100 Too

1 .200 0.100 100

1 .201 0.500 100

1 .450 0.500 100

1 .451 0.100 100

1 .500 0.100

2. Mass spectrometer

Machine: TSQ Quantiva triple quadrupole mass spectrometer.

The mass spectrometer conditions were as follows:

Ionization: Heated electrospray ionization (HESI)

Spray voltage: Positive, 3500 V

Sheath gas: 40 Arb Aux gas: 10 Arb

Sweep gas: 1 Arb

Ion transfer tube temperature: 350 °C

Vaporizer temperature: 250 °C

Data acquisition mode: Selected-reaction monitoring (SRM)

Cycle time: 1 s

Q1 resolution (FWHM): 0.7

Q3 resolution (FWHM): 0.7

CID gas: 1.5 mTorr

Source fragmentation: 0 V

Chrome filter: 3 s

Data analysis and statistics for metabolomics panel construction

Software and packages used in the data analysis included: random forest modeling analysis was performed using R random Forest package (http://www.r- project.org/). Biomarker feature selection and panel optimization was performed using a genetic algorithm (R genalg package). The predictive performance of each biomarker panel analysis was evaluated by ROC curve analysis (Zweig et al. Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clinical chemistry 1993;39:561 -77; Sing et al. ROCR: visualizing classifier performance in R. Bioinformatics 2005;21 :3940-1 ).

Results: Metabolomics-based discovery revealing AMI marker candidates

Claims

This study identified Cer(18: 1 /18:0), Cer(18: 1 /16:0), Cer(18: 1/24:1 ),Cer(18:1/18:0)/24:0, Cer(18:1/16:0)/24:0, Cer(18: 1/24:1 )/24:0 as differential biomarkers for AMI. Claims What is claimed is:

1. A method comprising: measuring the concentration of 1 to 6 metabolite species in a sample of a bio-fluid from a subject to be tested for AMI, wherein the 1 to 6 metabolite species is a component of a panel of a plurality of metabolite species, wherein a change in the concentration of the metabolite species is a characteristic that is associated with AMI, which providing an AMI marker level representation for a subject, the method comprising: evaluating a panel of AMI markers in a blood sample from a subject to determine the level of each AMI marker in the blood sample; and obtaining the AMI marker level representation based on the level of each AMI marker in the panel, wherein the panel of AMI markers comprise Ceramide(Cer).

2. The method according to claim 1 , wherein the panel of AMI markers further comprise one or more AMI markers or their ratios selected from the group consisting of

Cer(18:1/18:0), Cer(18: 1 /16:0), Cer(18: 1/24:1 ), Cer(18:1/18:0)/24:0,

Cer(18:1/16:0)/24:0, and Cer(18:1/24:1 )/24:0

3. The method according to claim 1 , wherein the panel of AMI markers comprises metabolite species that have been identified by liquid chromatography-mass spectrometry (LC-MS): Cer(18:1/18:0), Cer(18:1/16:0), Cer(18:1/24:1 ),

Cer(18:1/18:0)/24:0, Cer(18:1/16:0)/24:0, and Cer(18:1/24:1 )/24:0.

4. The method according to claim 2, wherein the panel of AMI markers comprises of Cer(18:1/18:0), and Cer(18:1/18:0)/24:0.

5. The method of claim 1 , wherein the bio-fluid is selected from the group consisting of blood, plasma, serum, sweat, saliva, sputum, and urine.

6. The method of claim 1 , wherein the bio-fluid is serum.

7. A panel of metabolite species, the metabolite species are selected from a group consisting of Cer(18: 1/18:0), Cer(18:1/16:0), Cer(18: 1/24:1 ), Cer(18:1/18:0)/24:0, Cer(18:1/16:0)/24:0, and Cer(18:1/24:1 )/24:0.

8. The panel of claim 7, wherein the panel is provided in a diagnostic cassette.

9. The diagnostic cassette of claim 8, further comprising reagents for the detection of the metabolite species of the panel.

10. A kit for the analysis of a sample of a biofluid of a subject, comprising: a. Aliquots of standards of each compound of a panel of metabolite species; b. An aliquot of an internal standard; and c. An aliquot of a control biofluid.

11. The kit of claim 10, wherein the control biofluid is serum from a control source that is conspecific with the subject.

12. The kit of claim 11 , wherein the panel consists of D₇-d18:1 -16:0 Ceramide, D₇- d18: 1 -18:0 Ceramide, D₇-d18: 1 -24:0 Ceramide, D₇- d18: 1 -24:1 Ceramide 13. The kit of claim 10, further comprising instructions for use.