Field of the Invention

The present invention relates to the detection of myocardial infarction. In particular, the present invention relates to detection using a combination of specific biological markers.

Background to the Invention

The ability to diagnose a disease accurately is of utmost importance. Often, identifying that a patient has a disease is difficult, as the first symptoms of the disease are not specific to that disease but are typical of a number of diseases. Also it is not always possible to determine the extent of the disease based on the outward symptoms, making it difficult for the clinician to prescribe the most appropriate course of action. Biomarkers have long been used to help determine the presence of a disease. However, many biomarkers are present only at one stage of the disease.

Since 2000, the diagnosis of acute myocardial infarction (AMI) has been based on a set of guidelines proposed jointly by the American college of Cardiologists and the Europe School of Cardiology (ACC/ESC; Alpert et al. 2000). These guidelines emphasize the importance of changes in the levels of the biochemical markers cardiac troponin I (cTnl) and creatine kinase MB form (CK-MB) after an infarct, in combination with other diagnostic factors such as electrocardiogram (ECG) results, especially ST segment elevation, but also ST segment depression or T-wave inversion, and typical symptoms of severe chest pain and dyspnoea. The specificity of both troponin I and CK-MB for myocardial injury is high, but both lack sensitivity in the critical period after the event because their blood concentrations do not increase appreciably until 6-8 hours after the onset of AMI (Wu et al. 1996). Such is the specificity of cTnl, that it is now regarded as the 'gold standard' biochemical marker of AMI.

The benefit of thrombolytic therapy is greatest when it is administered as soon as possible after the onset of symptoms. It is therefore highly desirable to identify biomarker proteins that can improve early diagnosis of myocardial infarction, and develop rapid and highly sensitive tests to detect their presence in the blood circulation.

Two early markers are myoglobin and heart fatty acid binding protein (H-FABP). The dynamics of both of these markers are remarkably similar, as levels of both rise in the blood within 2-3 hours of AMI, peak at 5-6 hours and return to normal baseline levels within 24 hours (Mair et al., 1992; Glatz et al., 1997). Myoglobin is already

considered a useful marker and is frequently used. One disadvantage of this marker is a relative lack of specificity, as myoglobin released from skeletal muscle cannot be distinguished from that released from the heart.

H-FABP is a small cytoplasmic protein, involved in lipid homeostasis, which is abundant in heart muscle (Veerkamp ef al., 1991). Although the plasma kinetics of H- FABP closely resemble those of myoglobin, H-FABP has the advantage of higher specificity, in that the heart form is around 10 times more abundant in heart muscle than in skeletal muscle, and basal circulating levels of H-FABP are several fold lower than myoglobin (Ishii et al., 1997). It has also been reported that the magnitude of the increase in plasma H-FABP correlates to the size of the infarction (Glatz et al., 1994). Various studies have proposed the use of H-FABP as an early rule-out marker for AMI (Seino et al., 2003; Glatz et a/., 1998; Pagani et a/., 2002; Okamoto et a/. 2000), but agreement with this concept has not been universal (Ghani et al., 2000; Alansari & Croal, 2004). In their recent article, Alansari and Croal concluded that H-FABP provided little clinical value when measured on admission in patients presenting with chest pain. This was the case in their study, even when they examined different groups of patients, i.e. those who presented less than 4 hours after estimated time of pain onset, those who had normal cTnl on admission, or those whose ECG did not have ST segment elevation. Some groups advocate measuring the dynamics of the markers over time.

Tucker et al. (1994) made serial measurements of myoglobin in blood samples taken from AMI patients at admission and over the first six hours from onset of symptoms, and reported that an increase in the level of myoglobin, even if still within the normal range, was highly specific for AMI (95%). A similar study (Sallach et al., 2004) examined patients presenting with chest pain, whose ECG results were non-diagnostic and whose myoglobin and cTnl levels were not elevated on admission. Measurements were made at presentation and after 90 minutes, 3 hours and 9 hours. In the chosen study population, the authors reported that an increase in myoglobin levels of 20ng/ml or greater provided an accurate diagnosis of AMI within 90 minutes of admission. In contrast, another group showed recently that looking at changes in CK-MB levels ratherthan myoglobin levels 2 hours after admission was a significantly better predictor of AMI in non-ST-segment elevation chest pain patients (Fesmire et al., 2004).

It has been proposed that H-FABP may be useful for the identification of patients with unstable angina, based upon detection of myocyte damage. H-FABP is also known to be elevated in unstable angina, but reported to be at lower levels than

in AMI (Tsuji et a/. 1993), however, larger-scale studies would be required to fully substantiate these findings.

For such early tests to be fully effective, the samples need to be collected as soon as possible after the onset of chest pain. Unfortunately, the interval between the initial onset and the arrival of the patient in the emergency department varies, as does their perceived interval since the pain started, which further complicates interpretation of the results. Rapid test results from automated systems and fast laboratory turnaround times are also important to capitalize on any advantage that the measurement of early markers may offer in terms of administration of treatment, whether in the acute stage after admission or for in-hospital monitoring.

Summary of the Invention

The present invention is based on the realisation that the detection of a disease, which can be characterised by different biological markers, can be improved by measuring simultaneously the levels of two or more different biological markers present at elevated levels at different time points during the progression of the disease. Myocardial infarction is a specific disorder where this diagnostic technique can be of benefit.

According to a first aspect of the present invention, a method for the diagnosis of a disease comprises the step of assaying a biological sample from a patient to determine simultaneously the levels of at least three biological markers predictive of the disease, and determining whether the levels are elevated compared to a control, wherein elevated levels indicate the presence of the disease.

According to a second aspect of the present invention, a method for the diagnosis of acute myocardial infarction comprises the step of assaying a patient sample to determine simultaneously the levels of two or more markers predictive of myocardial infarction, and determining whether the levels are elevated compared to a control.

According to a third aspect of the present invention, a biochip comprises at least three biological or chemical agents that bind to the markers defined above.

The simultaneous measurement of markers allows a "snapshot" to be taken of proteins released through the progression of cardiac disease. When combining markers for diagnosis, this snapshot provides a true representation of the relative levels of the markers and allows more reliable diagnostic models to be obtained. This overcomes issues of biomarker instability where one or all the markers may alter with

time and, also, reduces potential interactions between markers or components in a sample. Pre-analytical conditions, such as sample processing, can have an impact on biomarker interaction and therefore interpretation of results. The advantage of the present invention is that simultaneous measurement of markers enables a more accurate assessment of disease status, when compared with markers measured sequentially.

Description of the Drawings

The present invention is described with reference to the accompanying drawings, wherein:

Figure 1 is a graph illustrating the number of patient samples diagnosed correctly as indicating myocardial infarction at 0-4 hours post pain, using various combinations of biological markers;

Figure 2 is a graph showing the number of patient samples correctly diagnosed as not showing myocardial infarction, 0-4 hours post pain onset, using various biological markers; and

Figure 3 is a graph showing the percentage sensitivity and specificity of diagnosis using various combinations of biological markers 0-4 hours post pain onset.

Description of the Invention

The present invention is based on the realisation that a disease may be diagnosed more accurately if at least 2, and preferably 3 biological markers, predictive of the disease, are assayed simultaneously, to determine the levels present in the sample. Elevated levels will indicate the presence of the disease. In many diseases, there are multiple biological markers that may be used as predictors of disease, but which are only present at elevated levels within a specific time frame and which may be missed if the patient sample is taken too early or too late. Selecting two or more different markers, each of which are preferably present at elevated levels at different time points, if the disease is present, significantly improves the detection of the disease, allowing the appropriate therapy to be given to the patient early.

In one aspect of the invention, it has been found that diagnosing acute myocardial infarction can be improved significantly by assaying a patient sample to determine simultaneously the levels of two or more markers predictive of myocardial infarction, and determining whether the levels are elevated compared to a control. Again, the different markers are elevated at different time points during the progression

of the myocardial infarction, but the simultaneous determination of the levels of the markers allows the clinician to determine not only whether a myocardial infarction is present, but also the extent to which the infarction has occurred.

In a preferred embodiment of the invention at least three markers are determined simultaneously. More preferably, four markers are determined simultaneously.

With respect to the diagnosis of acute myocardial infarction, the markers may be selected from the group consisting of: myoglobin, CK-MB, heart fatty acid binding protein, cTNI high sensitivity c-reactive protein, glycogen phosphorylase isoenzyme BB and NT-proBNP/proBNP. Each of these markers are known in the art, and assays to determine the presence and amount of the markers are known to those skilled in the art.

In a preferred embodiment, the biological markers are CK-MB, heart fatty acid binding protein and cTNI. In a preferred embodiment, the assay is carried out using a biochip having immobilized thereon different analytes specific for the biological markers to be determined. The analytes for each marker may be immobilized on the biochip in specific arrays at predetermined concentration, allowing the user to determine the extent to which they are present in the sample. A suitable apparatus for carrying out the analysis of the patient samples is the Evidence ^® biochip array analyser (Randox Laboratories Ltd).

Any suitable assay may be used to determine the presence or concentration of the biological markers. Typically, the biological markers will be identified using an ELISA assay, which utilises antibodies specific to the markers, to determine the presence and concentration of the markers in the biological sample. Alternative assays are known, including sandwich assays, western blots and the like.

The term "antibody" refers broadly to any immunological binding agent such as IgG, IgM, IgA, IgD and IgE. Antibody is also used to refer to any antibody-like molecule that has an antigen-binding region and includes antibody fragments such as single domain antibodies (DABS), Fv, scFv, aptomers, etc. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterising antibodies are also well known in the art.

The antibodies may be immobilized on a suitable substrate, for example a biochip, using methods known in the art.

The patient sample will be any suitable sample that is expected to contain the biological markers. For example, the sample may be a blood sample, urine or tissue sample. The sample is preferably isolated from the patient and the assay is preferably performed ex vivo, more preferably in vitro. The present invention is used typically to protect the presence and extent of a disease which has a rapid onset. For example, the invention may be used preferably to detect the presence and extent of a myocardial infarcation. The biological markers to be detected are chosen on the basis that they are present at elevated levels at different time points during the progression of the disease. In this way, it is possible to not only detect the disease, but to make a determination on the extent of progression of the disease, based on the levels of the markers in the patient sample. It is preferred to choose at least three markers, the first of which may present at the early onset of the disease, with the latter two markers being present at elevated levels later in the disease progression. The invention is now exemplified in the following Example, with reference to the accompanying drawings.

Example

The experiment described below was carried out to determine whether the inclusion of measurements for H-FABP or myoglobin, or both, as early stage markers into the emergency department diagnosis of AMI offered any advantages over established methods of diagnosing AMI in our patient cohort. In particular, we aimed to ascertain whether multiplex analysis of two, three, or all four biomarkers gave improved diagnostic sensitivity over any single marker. The sensitivity and specificity of various combinations of markers was examined, to define the best combination of parameters for diagnosis of AMI in this patient cohort. A panel of rapid immunoassay- based tests covering the four biomarkers cTnl, CK-MB, myoglobin and FABP is offered by the Evidence ^® biochip array analyzer (Randox Laboratories Ltd.), which was used for analysis in this study. Samples for this investigation were obtained from patients presenting at the

Department of Emergency Medicine, St. James' Hospital, Dublin, Ireland, with chest pain. The samples analysed in this study were collected between April 2000 and June 2003. Consent for participation in the study was obtained by a research nurse, and an initial sample was taken (TO) at time of admission. Because a combination of markers was examined, whose plasma profiles vary over a period of hours to days, the protocol

required subsequent samples to be provided between 0 and 4h, 4 and 8h, 8 and 12h, 12 and 24h, & 12-48 hours estimated time of pain onset, and this was followed wherever possible. The samples were processed to separate the serum fractions, which were frozen at -2O ⁰ C and subsequently transported in batches every two to three weeks to Randox Laboratories Ltd. for analysis.

In addition to collecting the blood samples, a range of clinical information was also recorded by the research nurse including risk factors, symptoms and time of onset estimated by the patient, ECG results, the hospital biomarker results and the final diagnosis (see table 1). A thirty and sixty day follow-up check was made where possible.

The median age of the patients examined in the study was 55, 33% were female and 67% male. Approximately 12% of the patients in the study were confirmed as having AMI (n=46). 34 AMI patients were male and 12 female. The analysis presented here includes all of these AMI patients and 249 non-MI patients. Final diagnosis of AMI was made according to the ACC/ESC guidelines (Alpert et al. 2000). Analysis of samples

Analysis of the samples was performed in large batches using the Evidence ^® biochip array analyzer and the cardiac markers immunoassay biochip array. This array comprises a set of 4 markers, cTNI, CK-MB, myoglobin and H-FABP, for which results are obtained simultaneously on the same sample. The cut off levels recommended in the Randox kit insert were used for CK-MB 8ng/ml; myoglobin 90ng/ml; FABP 9ng/ml and cTnl 0.74ng/ml (95 ^th percentile).

Receiver operator characteristic (ROC) analysis and calculation of specificity and sensitivity, based on the cut-off values above the threshold for each marker, was performed, to compare the diagnostic performance of individual markers and combinations of markers amongst Ml and Non-MI patient groups.

As our study set out to examine the value of the addition of tests for early markers to the process of diagnosis of AMI in the emergency department, all analysis was performed using time intervals since the onset of chest pain, and ECG analysis was always included as part of the diagnostic process, unless otherwise stated. Results

At the time of recruitment, 34% of patients had hypertension, 26% with hyperlipidemia, 33% with coronary heart disease, 11 % with diabetes, 43% were smokers, 46% had a family history of cardiac complications and 35% were obese.

Cardiac complications of the patient cohort were 18% with Unstable Angina, 17% with Stable Angina, 12% with Myocardial Infarction (Ml) and 1% with heart failure. The study cohort consisted of 295 patients, 67% were male and 33% were female, 46 diagnosed with myocardial infarction (Ml) and 249 as Non-myocardial infarction (Non- Ml).

When referring to post admission results, the troponin I (TnI) AUC value was higher than all other markers at the early time points up to 8 hours. At 8-12 hours the AUC value for TnI decreases and then increases again at 12-24hours. AUC values for FABP remain consistently high at all time points studied. Myoglobin was the least accurate of all biomarkers studied at all time points.

When using time points with reference to post pain, the results show similar AUC values for TnI and FABP at 0-4hrs, however the AUC value for FABP was higher than all other biomarkers at 4-8 hours. TnI values were highest for all time points thereafter. Myoglobin was the least accurate of all the biomarkers studied, in diagnosing patients at all time points. Biomarker combinations

We examined the percentage sensitivity, specificity and diagnostic accuracy of four biomarkers, cTnl, CKMB, myoglobin and H-FABP, measured alone and in combination with other markers in each of the time windows 0-4h, 4-8h, 8-12h, 12-24h and 24-48h post estimated time of pain onset. This was a sub-study of a larger patient cohort and the number of patients at each data point is limited. For the purposes of the study we have considered the diagnostic accuracy for individual patient samples rather than patients. As early detection of Ml is the aim of this study, only the data for the 0-4 hours time point post pain onset is presented (see Figure 1). The percentage sensitivity denotes the percentage of the total number of Ml samples correctly diagnosed, the percentage specificity denotes the percentage of the total non-MI samples correctly diagnosed, while diagnostic accuracy denotes the percentage of correctly diagnosed samples overall. For ECG, the criteria used to identify patients likely to be suffering AMI in this study was ST elevation in 3 contiguous leads of the 12-lead electrocardiogram.

Patients presenting within 4 hours of estimated time of pain onset (n=12MI, n=35 NMI)

The total number of 47 patients presented within 4 hours of pain onset. Some patients had more than one blood sample taken within this time period and the total number analysed were n=16 Ml patient samples and n=41 Non-MI patient samples.

The number of patient samples correctly diagnosed using individual or a combination of biomarkers with Ml is shown in Figure 1 and with Non-MI in Figure 2.

Results show that of the 16 Ml samples analysed, FABP correctly diagnosed more patients with Ml (n=13) than any other single marker, including Troponin I (n=9) and myoglobin (n=5), CK-MB (n=9).

Any biomarker combination that included FABP diagnosed more Ml samples correctly than combinations without FABP. A panel combination of CK-MB, Troponin I and FABP correctly diagnosed all Ml patient samples in the study group. Results in Figure 2 show that of the 41 Non-MI samples analysed, Troponin I diagnosed all samples correctly, myoglobin (n=39) whereas FABP correctly diagnosed patients (n=35). Six patient samples were FABP positive despite being diagnosed as Non-MI. These patient samples were followed up with regards to patient history and blood sample results at later time points.

Biomarker combinations that included FAB P showed lower specificity of diagnosis than panels without the marker.

In Figure 3, the sensitivity, specificity and accuracy of diagnosis at 0-4hours were calculated for both the Ml and Non-MI patients. The performance of H-FABP was better than any other marker in terms of sensitivity, either alone, or in combination with cTnl and CK-MB. A combination of CKMB, Troponin I and FABP showed 100% sensitivity for Ml samples. However the performance of FABP in terms of specificity was worse than any other combination of markers.

Accuracy of diagnosis was comparable for all biomarker combinations with values between 0.82-0.87 for all combinations except myoglobin alone, when the accuracy value was markedly lower at a value of 0.77. FABP alone, and in combination with other markers, appears to present a better balance between sensitivity and specificity when compared with any other combinations. Without FABP, good specificity was evident for all markers at 0-4hours post pain, but this is at the expense of sensitivity at this time. Results clearly show that FABP is a more accurate early marker than myoglobin. Non-AMI patients with FABP elevated on admission

The inclusion of H-FABP testing may be of most value in detecting Ml patients and it appears a better early marker than myoglobin. We looked at the patient history and marker profile for each of the 6 FABP positive samples (representing 5 patients) that were diagnosed as Non-MI. Table 1 presents the data.

Table 1

Patient history of Non-MI patients with a positive FABP within 0-4hours.

LBBB- Left bundle branch block

On examination of patient data it is evident that although these patients were diagnosed as Non-MI, three of the five had positive ECGs, and two of the patients with positive ECGs had FABP levels that remained elevated after 1 hour. FABP levels for three of the five patients decreased after 1 hour. Despite the diagnosis of Non-MI on this visit, all the patients with a positive FABP had a previous medical history that could be associated with cardiac complications. Discussion The current methods for diagnosis of AMI (cTnl or CK-MB + the 12-lead ECG) still suffer from a lack of sensitivity in the most critical early hours after the ischaemic event, during which time the administration of thrombolytic therapy would be of most benefit. This study set out to determine whether the addition of either H-FABP or myoglobin or both as early stage biomarkers of AMI could improve the sensitivity of current methods of diagnosis, especially during the early hours after the onset of pain.

In our patient cohort, we demonstrated the importance of assessing biomarker levels with reference to time post pain onset rather than time on a post admission basis. Troponin is known as the gold standard in diagnosis of acute myocardial

infarction (Ml) however it lacks sensitivity in the early hours of Ml ₁ suggesting the need for a marker released within shorter time periods. Some studies have reported the use of FABP as an earlier marker of Ml, however not all studies agree with these findings (Alansari & Croal, 2004). Our study shows that with reference to post pain onset, FABP is the most sensitive marker of Ml at 4-8 hours, whereas troponin performed best on a post admission basis. Biomarkers are released into the bloodstream after a cardiac event and levels should be more closely correlated with pain onset rather than admission time, as a patient's pain threshold may prevent presentation for up to 12 hours after pain onset. We demonstrated that testing for H-FABP alone or in combination with other markers post pain onset, was of benefit in the sensitive early diagnosis of patients with AMI. FABP was the most sensitive single marker for diagnosis of Ml, identifying four more Ml patients, than any other single marker, including troponin, at 0-4hours post pain onset. When combined with troponin and CK-MB, FABP correctly diagnosed all Ml patients within 4 hours, indicating that a panel approach to Ml diagnosis may be more effective in the identification of Ml patients.

Differentiating between Ml and non-MI patients at this early time point is important and the study showed that FABP was least effective of all the markers either alone or in combination. After examination of medical history, 60% of the non-MI patients with elevated FABP had positive ECGs and all had pre-existing conditions that may be associated with cardiac complications. H-FABP levels decreased in 3 of the non-MI samples after 1 hour of the initial measurement, indicating a decline and no further risk of infarct. This may indicate that FABP can identify patients at risk of further cardiac complications, despite the absence of an Ml. Troponin, although known as the most specific marker for Ml diagnosis, lacks sensitivity in early detection of Ml. This is the case for most of cardiac biomarkers currently used, which offer excellent specificity but poor sensitivity in the early hours after the infarct.

This study indicates that FABP may offer the best all-round marker for myocardial infarction, combining good specificity with excellent sensitivity for myocardial infarction. Combination of FABP, troponin and CK-MB effectively identified all Ml patients in this study cohort and may offer a more accurate approach to early diagnosis.

FABP was especially valuable in patients admitted within 4 hours of the estimated time of pain onset, and was of more benefit than adding a test for myoglobin.

In summary, the data presented in this study show that the measurement of H- FABP in patients admitted to the emergency department with chest pain can be a sensitive early indicator of AMI. Adding the H-FABP biomarkertest to the cTnl and/or CK-MB test has the potential to improve the sensitivity of diagnosis. H-FABP would be especially useful in identifying the small percentage of AMI patients whose clinical picture is normal in the first hours after admission to the emergency department with chest pain. This can be achieved rapidly and effectively using the Evidence ^® cardiac markers biochip array.

The content of each publication referred to herein is incorporated herein by reference.

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