Note: Descriptions are shown in the official language in which they were submitted.
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Anti-troponin antibodies and cardiovascular risk
Field and Background of the Invention
This invention relates to the field of myocardial disorders. It discloses that
antibodies to a cardiac troponin found in a sample obtained from an individual
can
be used as a diagnostic marker, especially in the assessment of an
individual's risk of
developing a myocardial disorder. A method aiding in the assessment of an
individual's risk of developing a myocardial disorder, comprising measuring in
vitro antibodies to a cardiac troponin and optionally one or more other marker
useful in assessing an individual's risk of developing a myocardial disorder,
and
correlating the value or the values obtained to the individual's risk of
developing a
myocardial disorder is described.
Despite significant advances in therapy, myocardial disease (CVD) remains the
single most common cause of morbidity and mortality in the developed world.
Thus, prevention of myocardial disorders such as heart failure, myocardial
infarction and stroke is an area of major public health importance. Several
risk
factors for future myocardial disorders have been described and are currently
in
wide clinical use in the detection of individuals at high risk. Such screening
tests
include for example evaluations of total cholesterol level, of LDL cholesterol
level,
of HDL cholesterol level and the level of C-reactive protein. However, a large
number of myocardial disorders occur in individuals with apparently low to
moderate risk profiles, and the diagnostic options to identify such patients
is still
limited.
Many cardiovascular complications will manifest themselves at the heart. These
complications are summarized as coronary heart disease.
Individuals diagnosed as suffering from an underlying coronary heart disease
can
be divided into individuals showing no clinical symptoms and those which
appear
with breathlessness and/or chest pain. The latter group can be divided into
individuals having stable angina pectoris (SAP) and those with acute coronary
syndromes (ACS). ACS patients can show unstable angina pectoris (UAP), or
these
individuals have already suffered from a myocardial infarction (MI). MI can be
a
ST-elevated MI or a non ST-elevated MI. The occurring of a MI can be followed
by
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a left ventricular dysfunction (LVD). LVD patients may undergo congestive
heart
failure (CHF) with a mortality rate of roughly 15 %.
The heart is a unique organ. This is also true for the heart tissue and many
of its
constituents. The release of cardiac specific proteins into the circulation,
e.g., as the
result of a myocardial infarction, is a hall-mark of cardiac necrosis. The
detection of
such cardiac specific markers forms the basis of diagnostic means in the
fields of
myocardial infarction and congestive heart failure. It is well-known and
established
that an acute MI can be diagnosed with high sensitivity and at high
specificity by
measuring the level of a cardiac troponin in the circulation. Severe and acute
stages
of congestive heart failure can be diagnosed by measuring e.g. brain derived
natriuretic peptide (BNP) or its N-terminal propeptide (NT-proBNP).
Whereas quite some progress has been made in the diagnosis of acute stages of
myocardial complications a tremendous need still exists to further improve the
diagnosis in acute stage settings, to differentiate between subsets of
patients that
may require different modes of treatment, and especially to establish and
improve
the assessment of an individual's risk of developing a myocardial disorder.
It has now been found that antibodies to a cardiac troponin can be used as a
diagnostic marker, especially in the field of myocardial disorders. Antibodies
to a
cardiac troponin either alone or optionally in combination with one or more
other
marker of cardiovascular risk are valuable in the assessment of an
individual's risk
of developing a myocardial disorder.
Summary of the Invention
This invention describes new diagnostic tests that determine and utilize the
presence, absence or level of antibodies to a cardiac troponin in the
assessment of a
myocardial disorder.
In one embodiment the present invention relates to method aiding in the
assessment of an individual's risk of developing a myocardial disorder,
comprising
the steps of a) measuring in vitro antibodies to a cardiac troponin and
optionally
one or more other marker useful in assessing an individual's risk of
developing a
myocardial disorder, and b) correlating the value or the values obtained in
(a) to
the individual's risk of developing a myocardial disorder
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These new tests broadly include (1) the assessment of risk of a future
myocardial
disorder such as for example myocardial infarction and congestive heart
failure,
and (2) the determination of the likelihood that certain individuals will
benefit to a
greater or lesser extent from the use of certain treatments designed to
prevent
and/or treat a myocardial disorder.
The present invention also discloses a kit comprising a cardiac troponin and
auxiliary reagents appropriate for measurement of antibodies to said cardiac
troponin.
These and other aspects of the invention will be described in more detail
below in
connection with the detailed description of the invention.
Detailed Description
In a first preferred embodiment the present invention relates to the use of
antibodies to a cardiac troponin as a diagnostic marker.
The skilled artisan is aware of different methods that may be used in the
determination of antibodies as present in an individual's sample. The
diagnostic
field in which antibodies as present in a patient's sample are determined is
called
serology. The detection of an antibody comprised in a patient's sample is for
example very important in the diagnosis of an infectious disease or of an
autoimmune disease.
The release of a cardiac troponin is believed to occur only if cardiac tissue
is
damaged and becomes necrotic. Till the end of the 1990s the gold standard in
detecting cardiac necrosis has been an elevated level of CK-MB (the cardiac-
specific
isoforms of creatinine kinase). At the end of the last decade cardiac
troponins have
emerged as at least as good a marker. The implications of troponin testing
have
been reviewed by Goldmann, B. U., et al. (Curr. Control Trials Cardiovasc.
Med. 2
(2001) 75-84). It is now generally accepted that a positive test for a cardiac
troponin
has a very high sensitivity in the detection of a myocardial infarction.
Dilated cardiomyopathy (DCM) is a myocardial disease characterized by
progressive depression of myocardial contractile function and ventricular
dilation.
Recently Nishimura, H., et al. (Science 291 (2001) 319-322) reported that PD-1
receptor deficient mice develop severe DCM. They further found that these mice
produce antibodies against cardiac troponin I. Okazaki, T. and Honjo, T.
(Trends
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in Molecular Medicine 11 (2005) 322-326) confirm that troponin I is one out of
about fifteen autoantigens recognized by cardiac autoantibodies in patients
with
DCM.
It is known that antibodies to a cardiac troponin may be present in the
circulation
of some patients with acute coronary syndrome. These antibodies have been
identified as the cause of discrepant data between different assays for
measuring the
same type of cardiac troponin. These anti-troponin antibodies lead to a
decrease in
assay sensitivity and thereby may cause a delay in the detection of a cardiac
troponin (Eriksson, S., et al., N. Engl. J. Med. 352 (2005) 98-100), e.g.
after
myocardial infarction. This group of investigators therefore has proposed to
design
novel assays for troponin I that do not suffer from interference from
autoantibodies
against troponin I (Eriksson, S., et al., N. Engl. J. Med. 352 (2005) 98-100).
The inventors of the present invention have now surprisingly found that the
presence and/or level of antibodies against a cardiac troponin as determined
in a
patient's sample is of diagnostic utility. They can e.g. be used as a marker
in the
assessment of an individual's risk of developing a myocardial disorder.
The articles "a" and "an" are used herein to refer to one or to more than one
(i.e. to
at least one) of the grammatical object of the article. By way of example, "a
marker"
means one marker or more than one marker. In this invention "an antibody" and
"antibodies" to a cardiac troponin is considered interchangeable, because, as
the
skilled artisan will appreciate it is always many antibodies which are
detected.
Cardiac troponins
A "cardiac troponin" is a troponin that is present in heart tissue and not
present at
all or not present to any relevant extend in tissue other than heart. By way
of
example for human beings two cardiac specific troponins have been described.
These human cardiac specific troponins are known as troponin T, and troponin
I,
respectively.
Troponin T has a molecular weight of about 37.000 Da. The troponin T isoform
that is found in cardiac tissue (cTnT) is sufficiently divergent from skeletal
muscle
TnT to allow for the production of antibodies that distinguish both these TnT
isoforms. TnT is considered a marker of acute myocardial damage; cf. Katus,
H.A.,
et al., J. Mol. Cell. Cardiol. 21 (1989) 1349-1353; Hamm, C.W., et al., N.
Engl. J.
Med. 327 (1992) 146-150; Ohman, E.M., et al., N. Engl. J. Med. 335 (1996) 1333-
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1341; Christenson, R.H., et al., Clin. Chem. 44 (1998) 494-501; and
EP 0 394 819.
Troponin I(TnI) is a 25 kDa inhibitory element of the troponin complex, found
in
muscle tissue. TnI binds to actin in the absence of Ca2+, inhibiting the
ATPase
activity of actomyosin. The TnI isoform that is found in cardiac tissue (cTnI)
is
40% divergent from skeletal muscle TnI, allowing both isoforms to be
immunologically distinguished. The normal plasma concentration of cTnI is <0.1
ng/ml (4 pM). cTnl is released into the bloodstream following cardiac cell
death;
thus, the plasma cTnl concentration is elevated in patients with acute
myocardial
infarction (Benamer, H., et al., Am. J. Cardiol. 82 (1998) 845-850).
In one preferred embodiment the present invention relates to a method aiding
in
the assessment of an individual's risk of developing a myocardial disorder,
comprising: a) measuring in vitro antibodies to a cardiac troponin and
optionally
one or more other marker useful in assessing an individual's risk of
developing a
myocardial disorder, and b) correlating the value or the values obtained in
(a) to
the individual's risk of developing a myocardial disorder.
The method according to the present invention will "aid in the assessment" of
an
individual's risk of developing a myocardial disorder. As the skilled artisan
will
appreciate, no biochemical marker is diagnostic with 100% specificity and at
the
same time 100% sensitivity for a given disease. Rather, biochemical markers
are
used to assess with a certain likelihood or predictive value the presence,
absence or
severity of a disease. Therefore, in routine clinical diagnosis various
clinical
symptoms and biological markers are generally considered together in the
diagnosis, treatment, and management of the underlying disease. The
measurement
of antibodies to a cardiac troponin will aid the physician in his task of
establishing
the correct diagnosis or prognosis. The final diagnosis or prognosis is always
made
by the physician.
The terms "myocardial disorder" or "myocardial disorders" relate to a group of
disorders affecting the heart muscle. A preferred group of myocardial
disorders
consists of atherosclerosis, congestive heart failure, acute coronary syndrome
including myocardial infarction and unstable angina. Preferably the myocardial
disorder assessed in a method according to the present invention is selected
from
the group consisting of congestive heart failure and acute coronary syndrome.
Also
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preferred the term myocardial disorder in the sense of the present invention
relates
to the graft rejection in patients after heart transplantation.
In a further preferred embodiment the present invention relates to a method
aiding
in the assessment of the risk for overt heart failure for a patient with
myocardial
infarction, comprising: a) measuring in vitro antibodies to a cardiac troponin
and
optionally one or more other marker useful in assessing an individual's risk
of
developing a myocardial disorder, and b) correlating the value or the values
obtained in (a) to the individual's risk of developing overt heart failure. As
obvious
to the skilled artisan the patient with MI may have suffered from heart
failure
before the MI occurred in this case the method according to the present
invention
may aid in assessing the risk of disease deterioration.
According to the "ACC/AHA 2005 Guideline Update for the Diagnosis and
Management of Chronic Heart Failure in the Adult" (Hunt, S., et al.,
www.acc.org
= the ACC/AHA practice guidelines) the disease continuum in the area of heart
failure is nowadays grouped into four stages. In stages A and B the
individuals at
risk of developing heart failure are found, whereas stages C and D represent
the
groups of patients showing signs and symptoms of heart failure. Details for
defining
the different stages A through D as given in the above reference are hereby
included.
From a clinical perspective, the disease is clinically asymptomatic in the
compensatory and early decompensatory phases (completely asymptomatic in stage
stage A and with structural heart disease but no signs and symptoms of HF in
stage
B, cf. the ACC/AHA practice guidelines). Outward signs of the disease (such as
shortness of breath) do not appear until well into the decompensatory phase
(i.e.,
stages C and D according to the ACC/AHA guidelines). Current diagnosis is
based
on the outward symptoms of patients in stages C and D. Overt heart failure in
the
sense of the present invention is heart failure in the stages C or D as
defined by the
ACC/AHA guidelines.
The antibody to a cardiac troponin is measured "in vitro". This means that a
sample is obtained from an individual for diagnostic purposes. This sample is
used
for one or several in vitro investigations and not for treatment of said
individual.
Preferred samples are cardiac tissue biopsy, whole blood, plasma, or serum,
especially preferred are plasma and serum.
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In a preferred assay set-up for detection of an antibody to a cardiac troponin
the
cardiac troponin antigen is directly or indirectly bound to a solid phase.
Usually the
sample is diluted in a sample buffer. The solid phase bound antigen is
incubated
with the (diluted) sample under investigation. Incubation is performed under
conditions permissive for binding of an antibody comprised in the sample under
investigation to the solid phase bound antigen. The antibody attached to the
solid
phase bound antigen is detected by appropriate means.
In the detection of antibodies against pathogenic agents, such as viral
pathogens,
very frequently and to great advantage antibody detection systems according to
the
double antigen bridge format, e.g., described in US 4,945,042, are used. The
same
assay principle can be used to detect antibodies to a cardiac troponin. The
immunoassays according to this bridge concept require the use of an antigen
directly or indirectly bound to a solid phase and of the same or a cross-
reactive
readily soluble antigen that is directly or indirectly detectable. The
antibodies under
investigation, if present, form a bridge between the solid phase bound antigen
and
the labeled detection antigen. Only if the two antigens are bridged by
specific
antibodies - e.g., by antibodies to a cardiac troponin - a signal is generated
which is
correlated to the concentration of antibodies present in the sample.
The cardiac troponin antigen used in a method according to the present
invention
in one preferred embodiment comprises troponin I and troponin T. It is also
preferred to set up assays for the detection of antibodies to either troponin
I, or
troponin T, respectively. In the latter assays each cardiac troponin is
individually
used as an antigen. In a preferred embodiment the antibodies measured in a
method according to the present invention are antibodies to troponin I.
In a preferred mode of performing the method according to the present
invention a
troponin from skeletal muscle is added to the sample buffer in order to
enhance
specificity of antibody binding to a cardiac troponin by blocking unspecific
antibodies, i.e. antibodies cross-reacting between a muscle and a cardiac
troponin.
As the skilled artisan will appreciate a test result may be recorded in
qualitative and
in quantitative terms.
The invention involves comparing the level of marker for the individual with a
predetermined value. The predetermined value can take a variety of forms. It
can be
single cut-off value, such as a median or mean. It can be established based
upon
comparative groups, such as where the risk in one defined group is double the
risk
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in another defined group. It can be a range, for example, where the tested
population is divided equally (or unequally) into groups, such as-a low-risk
group,
a medium-risk group and a high-risk group, or into quadrants, the lowest
quadrant
being individuals with the lowest risk and the highest quadrant being
individuals
with the highest risk.
The predetermined value can depend upon the particular population selected.
For
example, an apparently healthy population will have a different 'normal' range
of
markers than will a population the members of which have had a prior
myocardial
disorder. Accordingly, the predetermined values selected may take into account
the
category in which an individual falls. Appropriate ranges and categories can
be
selected with no more than routine experimentation by those of ordinary skill
in
the art.
A positive result may e.g. be recorded if the antibodies measured are above a
predetermined threshold level. Such threshold level usually is set to the 90 %-
percentile or to the 95%-percentile of a healthy control population. A
threshold
level at the 95%-percentile of a healthy control population is preferred when
practicing this invention. Quantitative values can easily be correlated to a
disease
state by methods that need not to be explained to the man skilled in the art.
At present it is not known what causes the formation of antibodies to a
cardiac
troponin in an individual. It may be that a release of a cardiac troponin into
the
circulation occurs due to one or more necrotic events at the heart. The
cardiac
troponin in the circulation may trigger the formation of antibodies to said
cardiac
troponin.
The autoantibodies or briefly antibodies to a cardiac troponin may be of
different
immunoglobin classes.
In the course of an infection, first antibodies of the immunoglobin class
M(IgM)
are formed. The first humoral immune response in form of IgM is followed by a
second humoral immune response, reflected by a more or less pronounced
formation of antibodies of the immunoglobin class G (IgG). It is generally
accepted
the in the average the IgG-response will be the higher, the longer the
"challenge" to
the immune system lasts, e.g., in case of an infection, the longer the
infection lasts
and/or the more severe the infection is or has been, and/or the more often the
infectious agent has triggered an immune response.
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It has been found that the antibodies to a cardiac troponin as present in a
patient's
sample may comprise antibodies of the IgG as well as of the IgM class of
immunoglobin. It may well be that different classes of antibodies to a cardiac
troponin are indicative for different subsets of patients.
In a preferred embodiment the method according to the present invention is
based
on antibodies to a cardiac troponin that are of both the immunoglobin classes
G,
and M. A high titer in antibodies to a cardiac troponin may be considered
indicative for a higher risk of further cardiac complications.
In a further preferred embodiment the method according to the present
invention
is based on the detection of antibodies to a cardiac troponin that are of
immunoglobin class M(IgM). A high titer in IgM antibodies may be considered
indicative for a more recent necrotic event at the heart muscle. A high titer
of IgM
antibodies may indicate a treatment more suited for acute events at the heart.
In another preferred embodiment the method according to the present invention
is
based on antibodies to a cardiac troponin that are of immunoglobin class G
(IgG).
A high titer in IgG antibodies may be considered indicative for at least one
necrotic
event at the heart muscle in the past. Such event in the past has most likely
occurred
at least four weeks before the sample has been obtained. A high titer of IgG
antibodies may also indicate a severe and/or several necrotic episodes and may
point to a mode of a treatment more suited for past and/or chronic events at
the
heart.
When an individual's sample has been analyzed with the method according to the
present invention for his/her risk of suffering from a future myocardial
disorder
said individual can be stratified for one or more modes of therapeutic
treatment.
These can be selected from antibodies (monoclonal antibodies, polyclonal
antibodies), small molecules, pharmacologically active compounds, i.e. anti-
inflammatory and lipid-lowering drugs (e.g. statins), thrombolytic drugs (e.g.
platelet antagonists), fibrinolytic drugs (e.g. heparin), revascularization
therapy
(e.g. PCTI (percutaneous therapeutic intervention), balloon dilatation,
stenting, by-
pass surgery).
Agents for reducing the risk of a myocardial disorder include those selected
from
the group consisting of anti-inflammatory agents, anti-thrombotic agents
and/or
fibrinolytic agents, anti-platelet agents, lipid reducing agents, direct
thrombin
inhibitors, and glycoprotein 11 b/IIIa receptor inhibitors and agents that
bind to
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cellular adhesion molecules and inhibit the ability of white blood cells to
attach to
such molecules (e.g. anti-cellular adhesion molecule antibodies).
Anti-inflammatory agents include Alclofenac; Alclometasone Dipropionate;
Algestone Acetonide; Alpha Arnylase; Amcinafal; Amcinafide; Amfenac Sodium;
Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone;
Balsalazide
Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains;
Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;
Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate;
Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone;
Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium;
Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate;
Diftalone;
Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam Sodium;
Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen; Fenclofenac;
Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic
Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin
Butyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;
Fluticasone
Propionate; Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate;
Halopredone Acetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen
Piconol; Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole;
Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole
Hydrochloride; Lornoxicam; Loteprednol Etabonate; Meclofenamate Sodium;
Meclofenamic Acid; Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine;
Meseclazone; Methylprednisolone Suleptanate; Morniflumate; Nabumetone;
Naproxen; Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;
Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride; Pentosan
Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone; Piroxicam;
Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen; Prednazate; Prifelone;
Prodolic Acid; Proquazone; Proxazole; Proxazole Citrate; Rimexolone;
Romazarit;
Salcolex; Salnacedin; Salsalate; Salycilates; Sanguinarium Chloride;
Seclazone;
Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate;
Talosalate;
Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide;
Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium;
Triclonide; Triflumidate; Zidometacin; Glucocorticoids; Zomepirac Sodium.
Anti-thrombotic and/or fibrinolytic agents include Plasminogen (to plasmin via
interactions of prekallikrein, kininogens, Factors XII, XIIIa, plasminogen
proactivator, and tissue plasminogen activator[TPA]) Streptokinase; Urokinase:
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Anisoylated Plasminogen-Streptokinase Activator Complex; Pro-Urokinase; (Pro-
UK); rTPA (alteplase or activase; r denotes recombinant), rPro-UK; Abbokinase;
Eminase; Sreptase Anagrelide Hydrochloride; Bivalirudin; Dalteparin Sodium;
Danaparoid Sodium; Dazoxiben Hydrochloride; Efegatran Sulfate=, Enoxaparin
Sodium; Ifetroban; Ifetroban Sodium; Tinzaparin Sodium; retaplase;
Trifenagrel;
Warfarin; Dextrans.
Anti-platelet agents include Clopridogrel; Sulfinpyrazone; Aspirin;
Dipyridamole;
Clofibrate; Pyridinol Carbamate; PGE; Glucagon; Antiserotonin drugs; Caffeine;
Theophyllin Pentoxifyllin; Ticlopidine; Anagrelide.
Lipid reducing agents include gemfibrozil, cholystyramine, colestipol,
nicotinic
acid, probucol lovastatin, fluvastatin, simvastatin, atorvastatin,
pravastatin,
cirivastatin.
Direct thrombin inhibitors include hirudin, hirugen, hirulog, agatroban,
PPACK,
thrombin aptamers.
Glycoprotein Ilb/IIla receptor Inhibitors are both antibodies and non-
antibodies,
and include but are not limited to ReoPro (abciximab), lamifiban, tirofiban.
One preferred agent which may be used to reduce the risk of a future cardiac
disorder in an individual testing positive for antibodies to a cardiac
troponin is
aspirin.
The method according to the present invention also may permit a therapeutic
treatment monitoring of the individual which is treated by said regimen.
In another surprising aspect of the invention, it has been discovered that
antibodies
to a cardiac troponin have a predictive value independent of other markers
used in
assessing an individual's risk of developing a myocardial disorder. In a
further
preferred embodiment the invention relates to a method aiding in the
assessment of
an individual's risk of developing a myocardial disorder, comprising: a)
measuring
in vitro antibodies to a cardiac troponin and one or more other marker useful
in
assessing an individual's risk of developing a myocardial disorder, and b)
correlating the values obtained in (a) to the individual's risk of developing
a
myocardial disorder.
The one or more additional marker used together with an antibody to a cardiac
troponin may be considered to be part of a marker panel for assessing an
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individual's risk of developing a myocardial disorder, i.e., a series of
markers
appropriate to further refine the risk assessment. The total number of markers
in
such marker panel is preferably less than 20 markers, more preferred less than
15
markers, also preferred are less than 10 markers, with 8 or less markers being
even
more preferred. Preferred are marker panels for assessing an individual's risk
of
developing a myocardial disorder comprising 2, 3, 4, 5, or 6 markers in
addition an
antibody to a cardiac troponin.
The skilled artisan is aware of a multitude of markers which have been
described as
useful assessing an individual's risk of developing a myocardial disorder.
Preferably
the one or more other marker will be selected from the group consisting of a
cardiac troponin, a natriuretic peptide or a natriuretic peptide-related
marker, an
inflammation marker, D-dimer, cholesterol, homocysteine, adiponectin, sCD40L,
myeloperoxidase, and ischemia modified albumin.
A "marker" is a molecule or feature whose absence, presence or level can be
correlated to a status of interest, e.g., to a disease.
Natriuretic peptides and natriuretic peptide - related markers
The natriuretic peptide preferably is selected from A-type natriuretic peptide
(ANP)
and/or B-type natriuretic peptide (BNP).
The term "related marker" as used herein refers to one or more polypeptide
fragments having at least 10, 12, 15 or 20 contiguous amino acids of a
particular
marker or its biosynthetic parent. Preferably said fragment is an
immunologically
detectable fragment as present in the circulation. A natriuretic peptide
related
marker preferably is either an ANP-related or a BNP-related marker.
Brain derive natriuretic peptide or B-type natriuretic peptide (BNP)
Human BNP is derived by proteolysis of a 108 amino acid precursor molecule,
referred to hereinafter as BNP 1_108. Mature BNP, or "the BNP natriuretic
peptide,"
or "BNP-32" or simply "BNP" is a 32 amino acid molecule representing amino
acids 77-108 of this precursor, which may also be referred to as BNP77_108.
The
remaining residues 1-76 of the BNP precursor molecule are known in the art as
N-
terminal proBNP (NT-proBNP).
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BNP 1-108 is synthesized as part of an even larger precursor, the pre-pro-BNP,
having 134 amino acids in total of which the N-terminal 26 represent the "pre-
sequence.
Mature BNP itself may be used as an additional marker in the present
invention.
The prepro-BNP, BNP 1-108 and NT-proBNP molecules all represent BNP-related
markers that may be measured either as surrogates for mature BNP or as markers
in
and of themselves. In addition, one or more fragments of these molecules,
including BNP-related polypeptides or markers selected from the group
consisting
of BNP -106, BNP79-106, BNP76-107, BNP69-108, BNP79- i08) BNP80-108) BNP81-
i08) BNP83-
108) BNP39-86, BNP53-85, BNP66-98) BNP30- 103) BNP1-107i BNP9- 1e6, and BNP3-
108 may
also be present in circulation. In addition, natriuretic peptide fragments,
including
BNP fragments, may comprise one or more oxidizable methionines, the oxidation
of which to methionine sulfoxide or methionine sulfone produces additional BNP-
related markers. See, e.g., US Application Serial No. 10/419,059, filed Apr.
17, 2003,
which is hereby incorporated by reference in its entirety.
In a similar fashion, many of the markers described herein below are
synthesized as
larger precursor molecules, which are then processed to provide the mature
molecule or marker; and/or are present in circulation in the form of fragments
and/or a marker molecule carrying secondary modifications. Thus, a "related
markers" to each of the markers described herein below may be identified and
used
in an analogous fashion to that described above for BNP.
A-type natriuretic peptide (ANP)
A-type natriuretic peptide (ANP) (also referred to as atrial natriuretic
peptide or
cardiodilatin Forssmann, W.-G., et al., Histochem. Cell Biol. 110 (1998) 335-
357) is
a 28 amino acid peptide that is synthesized, stored, and released by atrial
myocytes
in response to atrial distension, angiotensin II stimulation, endothelin, and
sympathetic stimulation (beta-adrenoceptor mediated). ANP is synthesized as a
precursor molecule (pro-ANP) that is converted to an active form, ANP, by
proteolytic cleavage and also forming N-terminal ANP (1-98). N-terminal ANP
and
ANP have been reported to increase in patients exhibiting atrial fibrillation
and
heart failure (Rossi, A., et al., J. Am. Coll. Cardiol. 35 (2000) 1256-1262).
As the
skilled artisan will recognize, however, because of its relationship to ANP,
the
concentration of N-terminal ANP molecule can also provide diagnostic or
prognostic information in patients. The phrases "marker related to ANP" or
"ANP
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related peptide" refer to any polypeptide of at least 10, 12, 15 or 20
contiguous
amino acids that originates from the pro-ANP molecule (1-126), other than the
28-
amino acid ANP molecule itself. Proteolytic degradation of ANP and of peptides
related to ANP have also been described in the literature and these naturally
occurring proteolytic fragments are also encompassed it the term "ANP related
peptides."
Cardiac troponin
The two cardiac specific troponins, i.e. troponin I, and troponin T,
respectively,
have been exemplified above. Whereas, above the use of a cardiac troponin as
an
antigen in the detection of anti-troponin antibodies is discussed, the cardiac
troponin used as a further marker or analyte in a marker panel is the molecule
itself.
One skilled in the art recognizes that in measuring a cardiac troponin, one
can
measure the different isoforms of troponin I and troponin T. Thus, one may
preferably measure free cardiac troponin I, free cardiac troponin T, cardiac
troponin I in a complex comprising one or both of troponin T and troponin C,
cardiac troponin T in a complex comprising one or both of troponin I and
troponin C, total cardiac troponin I (meaning free and complexed cardiac
troponin
I), and/or total cardiac troponin T. Preferably cardiac troponin I and/or
cardiac
troponin T are measured according to state of the art procedures and the
values
measured are combined with the result of a measurement for antibodies to a
cardiac troponin and used in the assessment of a cardiac disorder. The
presence of
both of antibodies to a cardiac troponin and of a cardiac troponin may be
further
indicative for a recurring disease with acute coronary complications, like
ACS.
If in an individual's sample elevated values are found for antibodies to a
cardiac
troponin as well as for a natriuretic peptide or for a natriuretic peptide-
related
marker this may be considered indicative of a situation of past myocardial
damage,
like a past myocardial infarction. Elevated levels of antibodies to a cardiac
troponin
have been found to be indicative for severity of disease, and are especially
indicative for an increased risk that such patient will suffer from congestive
heart
failure in the future.
Preferred inflammation markers for use in a marker panel according to the
present
invention together with antibodies to a cardiac troponin are markers of acute
inflammation and so-called proximal inflammatory markers.
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Acute inflammatory markers known to the person skilled in the art include C-
reactive protein (CRP), fibrinogen, D-dimer, serum amyloid A (SAA), pregnancy-
associated polypeptide A (PAPP-A), intercellular adhesion molecules (e.g. ICAM-
1,
VCAM- 1), IL-1-beta, IL-6, IL-18/IL-18b; TNF-alpha; myeloperoxidase (MPO); TF;
monocyte chemoattractant protein 1(MCP-1); P-selectin; E-selectin; platelet
activating factor acetyl hydrolase (PAF-AH); von Willebrand Factor (vWF).
Preferred markers of acute inflammation for use in a method according to the
present invention are CRP, fibrinogen, D-dimer and SAA, of which CRP and D-
dimer are more preferably used.
C-reactive protein (CRP)
C-reactive protein (CRP) is a homopentameric Cazt-binding acute phase protein
with 21 kDa subunits that is involved in host defense. CRP synthesis is
induced by
IL-6, and indirectly by IL-1, since IL-1 can trigger the synthesis of IL-6 by
Kupffer
cells in the hepatic sinusoids. The normal plasma concentration of CRP is < 3
g/m1
(30 nM) in 90% of the healthy population, and < 10 g/m1 (100 nM) in 99% of
healthy individuals. Plasma CRP concentrations can, e.g. be measured by
homogeneous assay formats or ELISA. C-reactive protein is considered a marker
for
ongoing systemic inflammation. Nowadays CRP can be measured with very high
sensitivity and CRP-values in the range of between 1 and 3 mg/1 of blood can
be
reliably detected. A measurement in that range is called a measurement of high-
sensitive CRP or hs-CRP, which also is preferably used in a method according
to
the present invention.
Fibrinogen
Fibrinogen (also called Factor I) is a 340 kD protein encoded on chromosome 4
and
synthesized by hepatocytes. It is composed of two identical subunits, each
containing three dissimilar polypeptide chains (alphaA, betaB, gammaG) which
are
linked by disulphide bonds. Thrombin cleaves fibrinopeptides A and B from
fibrinogen, resulting in the formation of strands of insoluble fibrin monomer
which
consists of three paired alpha, beta and gamma chains. Dysfibrinogenaemia is a
condition associated with production of structurally abnormal fibrinogen. More
than 250 structural variants have been described which are associated with a
bleeding tendency (Ebert, R.F., CRC Press, Boca Raton, 1991). Most of these
variants exhibit impaired thrombin-catalyzed release of fibrinopeptides, or
impaired fibrin polymerization. Some variants of fibrinogen are associated
with a
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thrombotic tendency rather than a bleeding tendency, and this has been
attributed
to impaired binding of plasminogen or tissue plasminogen activator to the
abnormal fibrinogen molecule. Elevated levels of fibrinogen may be indicative
for
an ongoing infection or inflammation.
D-dimer
D-dimer is a crosslinked fibrin degradation product with an approximate
molecular
mass of 200 kDa. The normal plasma concentration of D-dimer is <150 ng/ml (750
pM). The plasma concentration of D-dimer is elevated in patients with acute
myocardial infarction and unstable angina, but not in stable angina
(Hoffmeister,
H.M., et al., Circulation 91 (1995) 2520-2527). The plasma concentration of D-
dimer also will be elevated during any condition associated with coagulation
and
fibrinolysis activation, including stroke, surgery, atherosclerosis, trauma,
and
thrombotic thrombocytopenic purpura. D-dimer is released into the bloodstream
immediately following proteolytic clot dissolution by plasmin. The plasma
concentration of D-dimer can exceed 2 g/ml in patients with unstable angina
(Gurfinkel, E., et al., Br. Heart J. 71 (1994) 151-155). Plasma D-dimer is a
specific
marker of fibrinolysis and indicates the presence of a prothrombotic state
associated with acute myocardial infarction and unstable angina.
Proximal inflammatory markers are macromolecules situated upstream, i.e. close
to
or at the ethiopathogenetic origin of the disease event. In particular, they
are
produced at the site of the coronary heart lesion, preferably at the site of
an arterial
plaque. Proximal inflammatory markers are in particular associated with the
risk
that plaques already present in an individual will undergo inflammation, or
growth,
and with the probability of plaque rupture and thrombus formation.
Proximal inflammatory markers are known to the person skilled in the art, and
non-limiting examples include pregnancy-associated polypeptide A (PAPP-A),
matrix metalloproteinases (MMPs, e.g. MMP-1, -2, -3, -4, -5, -6, -7, -9, -10, -
11)-
12) and lipoprotein-associated phospholipase A2 (Lp-PLA2).
The preferred proximal inflammatory markers are PAPP-A, MMP-9 and Lp-PLA2.
The most preferred proximal inflammatory markers are PAPP-A and Lp-PLA2, in
particular PAPP-A.
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Pregnancy-associated plasma protein-A (PAPP-A)
The pregnancy-associated plasma protein-A (PAPP-A) belongs to the metzincin
superfamily of zinc metalloproteinases. The molecular weight of PAPP-A is
187 kDa. Human pregnancy associated plasma protein A (PAPP-A) cleaves insulin-
like growth factor (IGF) binding protein-4 (IGFBP-4), causing a dramatic
reduction in its affinity for IGF-I and IGF-II. Through this mechanism, PAPP-A
is a
regulator of IGF bioactivity in several systems, including the human ovary and
the
cardiovascular system. A recent study shows that PAPP-A may also be a new
candidate marker of acute coronary syndromes (Bayes-Genis, A., N. Engl. J.
Med.
345 (2001) 1022-1029). The data in this study showed that PAPP-A levels are
significantly elevated in patients with unstable angina or acute myocardial
infarction when compared to patients with stable angina and control subjects.
Lipoprotein-associated phospholipase A2 (Lp-PLA2)
Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a 50 kDa, Ca-insensitive
lipase which is produced predominantly by macrophages. This enzyme resides
mainly on low density lipoprotein (LDL) in human plasma. It is distinct from
secretory phospholipase A2 (sPLA2). The levels of Lp-PLA2 are not affected by
acute
systemic inflammatory conditions. Clinical studies have demonstrated that Lp-
PLA2 is related to atherosclerosis. Elevated plasma levels have been also
found to
correlate with CHD and ischemic stroke risk. In pre-clinical animal studies,
inhibition of the enzyme attenuates the inflammatory process and slows down
atherosclerotic disease progression.
Homocysteine
The concentration of circulating total homocysteine is a sensitive marker of
inadequate folate and vitamin B12 status. Elevated homocysteine concentrations
are associated with an increased risk for vascular disease. Reference ranges
(5th and
95th percentiles) for the total homocysteine concentration have been recently
determined (Selhub, J., et al, Ann. Intern. Med. 131 (1999) 331-339). A high
total
homocysteine concentration was defined as one that exceeded the sex-specific
95th
percentile for the reference sample. Reference ranges for serum total
homocysteine
concentration are age-dependent; these ranges are 4.3 to 9.9 micromole/L for
male
participants and 3.3 to 7.2 micromole/L for female participants 12 to 19 years
of age
and from 5.9 to 15.3 micromole/L for men and 4.9 to 11.6 micromole/L for women
60 years of age or older. A high homocysteine concentration was defined as at
least
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11.4 micromole/L for male participants and at least 10.4 micromole/L for
female
participants.
Adiponectin
Adiponectin is a protein of 226 amino acids which is produced mainly by
adipocytes. The level of adiponectin appears to reflect insulin sensitivity
and to link
fat storage and arteriosclerosis. With regard to clinical utility several
different
intended uses are in discussion and/or under investigation. US 6,461,821
describes
and claims the use of adiponectin as a marker for arthrosclerosis.
Soluble CD40 ligand (sCD40L)
The sCD40L has been supposed to be a marker of inflammation (Aukrust, P., et
al.,
Circulation 100 (1999) 614 - 620) and hence to indicate a risk for the
occurrence of
coronary heart events. In WO 03/040691, sCD40L has been described as a
systemic
marker of inflammation. Recently sCD40L has also been discussed and described
as
a candidate marker for myocardial disorders. Heeschen, C., et al., N. Engl. J.
Med.
348 (2003) 1104-1111, indicate that sCD40L might be used as a marker in acute
coronary syndrome. sCD40L is in particular associated with platelet
activation,
platelet aggregation and thrombus propagation, representative of the risks
that
plaque having already become vulnerable will rupture, resulting in reversible
vascular occlusion (UAP) or irreversible vascular occlusion (AMI) which may
lead
to left ventricular dysfunction (LVD), congestive heart failure (CHF) and
death.
Cholesterol
Cholesterol at the same time is a steroid, a lipid, and an alcohol. It is
found in the
cell membranes of all body tissues, and transported in the blood plasma of all
animals. Most cholesterol is not dietary in origin, it is synthesized
internally.
Cholesterol plays a central role in many biochemical processes, but is best
known
for its association with myocardial disease. Cholesterol travels through the
blood in
vesicles wherein it is attached to a protein. This cholesterol-protein package
is called
a lipoprotein. Lipoproteins are either high density or low density, depending
on
how much protein they have in relation to fat. Lipoproteins with more protein
than
fat are called high-density lipoproteins (HDL). Lipoproteins with more fat
than
protein are called low-density lipoproteins (LDL). High-density lipoprotein
cholesterol is sometimes called "good" cholesterol. HDL cholesterol helps to
remove LDL cholesterol from the body by binding with it in the bloodstream and
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carrying it back to the liver for disposal. A high level of HDL cholesterol
appears to
lower your risk of developing heart disease and stroke. Low-density
lipoprotein
cholesterol is sometimes called "bad" cholesterol. LDL cholesterol collects
inside
the walls of the arteries and often contributes to the plaque formation. LDL
cholesterol is calculated from the total cholesterol, HDL, and triglyceride
levels. In a
further preferred embodiment of the present invention LDL cholesterol or the
ratio
of HDL to LDL is determined and used as part of a marker panel in order to
assess
an individual's risk of developing a myocardial disorder.
Myeloperoxidase
Myeloperoxidase is a lysosomal enzyme that is found in white blood cells,
neutrophils. Myeloperoxidase is an enzyme that uses hydrogen peroxide to
convert
chloride to hypochlorous acid. The produced hypochlorous acid reacts with and
destroys bacteria. In many inflammatory pathologies, such as cystic fibrosis
and
rheumatoid arthritis, neutrophils are also causing tissue damage.
Myeloperoxidase
is also produced when arteries are inflamed and have rupture-prone fatty
deposits.
An inflammation in the arteries can lead to a blood clot and eventually to a
heart
attack or stroke. Myeloperoxidase is considered a promising cardiac marker. By
measuring the myeloperoxidase level in blood it is possible to predict whether
a
person is in risk of heart attack or death in the following six months (Baldus
S., et
al., Circulation 108 (2003) 1440-1445).
Placenta growth factor (P1GF)
Placenta growth factor (PIGF) is a polypeptide growth factor and a member of
the
platelet-derived growth factor family but more related to vascular endothelial
growth factor (VEGF). PIGF-1 acts only as a very weak mitogen for some
endothelial cell types and as a potent chemoattractant for monocytes. The
physiological function in vivo is still controversy. In several reports it was
shown
not to be a potent mitogen for endotehlial cells and not angiogenic in vivo by
using
different assays. Very recently it was shown by one investigator, that P1GF-1
from
cell culture supernatants was angiogenic in the CAM assay and in the rabbit
cornea
assay. Two different proteins can be generated by differential splicing of the
human
PIGF gene: PIGF-1 (131 aa native chain) and PIGF-2 (152 aa native chain). Both
mitogens are secretable proteins, but P1GF-2 can bind to heparin with high
affinity.
P1GF-1 is a homodimer, but preparations of P1GF show some heterogeneity on SDS
gels depending of the varying degrees of glycosylation. All dimeric forms
posses a
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similar biological profile. There is good evidence that heterodimeric
molecules
between VEGF and PIGF exists and that they are biological active. A protein
related
of P1GF is VEGF with about 53% homology.
Ischemia modified albumin
The observation that myocardial ischemia produced a lower metal-binding
capacity
for cobalt to albumin (= ischemia modified albumin or IMA) led to the
development of the recently FDA-cleared albumin cobalt binding (ACB) test. The
ACB test is a quantitative assay that measures ischemia-modified albumin (IMA)
in
human serum. In principle, cobalt added to serum does not bind to the NH2
terminus of IMA, leaving more free cobalt to react with dithiothreitol and
form a
darker color in samples from patients with ischemia. At present, the assay is
available on a variety of clinical chemistry platforms. Specific preanalytical
requirements need to be followed, including: avoiding use of collection tubes
with
chelators, performing assay analysis within 2.5 h or freezing at below -20 C,
and
avoiding sample dilutions. In addition, ACB test results should be interpreted
with
caution when serum albumin concentrations are <20 g/L or >55 g/L or in the
presence of increased lactate or ammonia concentrations. Increased IMA values
may be found in patients e.g. with cancer, infections, end-stage renal
disease, liver
disease, and brain ischemia. Several clinical studies have evaluated the
performance
of the ACB assay in cardiac patients, mostly examining the role of IMA in
assessing
ischemia. IMA may be considered as an additional marker to be included into a
marker panel for assessment of an individual's risk of developing a myocardial
disorder.
The method according to the present invention in a preferred embodiment is
practiced in the investigation of apparently healthy individuals. "Apparently
healthy", as used herein, means individuals who have not previously had or at
not
aware of a previous adverse cardiovascular event such as a myocardial
infarction.
Apparently healthy individuals also do not otherwise exhibit symptoms of
disease.
In other words, such individuals, if examined by a medical professional, would
be
characterized as healthy and free of symptoms of disease.
As the skilled artisan will appreciate there are many ways to use the
measurements
of two or more markers in order to improve the diagnostic question under
investigation. In a quite simple, but nonetheless often effective approach, a
positive
result is assumed if a sample is positive for at least one of the markers
investigated.
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This may e.g. be the case when diagnosing an infectious disease, like AIDS, by
either
detecting a nucleic acid or a polypeptide of the infectious agent or by
detecting
antibodies to the infectious agent. Frequently, however, the combination of
markers is mathematically/statistically evaluated. Preferably the values
measured for
markers of a marker panel, e.g. an antibody to a cardiac troponin and the
level of a
cardiac troponin, are mathematically combined and the combined value is
correlated to the underlying diagnostic question. Preferably the diagnostic
question
is the relative risk of developing a myocardial disorder in the future.
Preferably the
relative risk is given in comparison to healthy controls. Preferably healthy
controls
are matched for age and other covariates.
Marker values may be combined by any appropriate state of the art mathematical
method. Well-known mathematical methods for correlating a marker combination
to a disease or to the risk of developing a disease employ methods like,
Discriminant analysis (DA) (i.e. linear-, quadratic-, regularized-DA), Kernel
Methods (i.e. SVM), Nonparametric Methods (i.e. k-Nearest-Neighbor
Classifiers),
PLS (Partial Least Squares), Tree-Based Methods (i.e. Logic Regression, CART,
Random Forest Methods, Boosting/Bagging Methods), Generalized Linear Models
(i.e. Logistic Regression), Principal Components based Methods (i.e. SIMCA),
Generalized Additive Models, Fuzzy Logic based Methods, Neural Networks and
Genetic Algorithms based Methods. The skilled artisan will have no problem in
selecting an appropriate method to evaluate a marker combination of the
present
invention. Preferably the method used in correlating the marker combination of
the
invention e.g. to the absence or presence of myocardial disease and/or to the
risk of
developing a cardial disorder is selected from DA (i.e. Linear-, Quadratic-,
Regularized Discriminant Analysis), Kernel Methods (i.e. SVM), Nonparametric
Methods (i.e. k-Nearest-Neighbor Classifiers), PLS (Partial Least Squares),
Tree-
Based Methods (i.e. Logic Regression, CART, Random Forest Methods, Boosting
Methods), or Generalized Linear Models (i.e. Logistic Regression). Details
relating
to these statistical methods are found in the following references: Ruczinski,
I., J. of
Computational and Graphical Statistics 12 (2003) 475-511; Friedman, J. H.,
Regularized Discriminant Analysis, JASA 84 (1989) 165-175; Hastie, T., et al.,
The
Elements of Statistical Learning, Springer Series in Statistics, 2001;
Breiman, L., et
al., Classification and regression trees, Wadsworth International Group,
Belmont/California (1984); Breiman, L., Machine Learning 45 (2001) 5-32; Pepe,
M.S., The Statistical Evaluation of Medical Tests for Classification and
Prediction,
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Oxford Statistical Science Series, 28 (2003); and Duda, R.O., et al., Pattern
Classification, Wiley Interscience, 2nd edition (2001).
It is a preferred embodiment of the invention to use an optimized multivariate
cut-
off for the underlying combination of biological markers and to e. g.
discriminate
patients with low, intermediate and high risk of developing a myocardial
disorder.
In this type of multivariate analysis the markers are no longer independent
but
form a marker panel.
Accuracy of a diagnostic method is best described by its receiver-operating
characteristics (ROC) (see especially Zweig, M.H., and Campbell, G., Clin.
Chem.
39 (1993) 561-577). The ROC graph is a plot of all of the
sensitivity/specificity pairs
resulting from continuously varying the decision thresh-hold over the entire
range
of data observed.
The clinical performance of a laboratory test depends on its diagnostic
accuracy, or
the ability to correctly classify subjects into clinically relevant subgroups.
Diagnostic
accuracy measures the test's ability to correctly distinguish two different
conditions
of the subjects investigated. Such conditions are for example health and
disease or
benign versus malignant disease, respectively.
In each case, the ROC plot depicts the overlap between the two distributions
by
plotting the sensitivity versus 1- specificity for the complete range of
decision
thresholds. On the y-axis is sensitivity, or the true-positive fraction
[defined as
(number of true-positive test results)/(number of true-positive + number of
false-
negative test results)]. This has also been referred to as positivity in the
presence of
a disease or condition. It is calculated solely from the affected subgroup. On
the x-
axis is the false-positive fraction, or 1- specificity [defined as (number of
false-
positive results)/(number of true-negative + number of false-positive
results)]. It is
an index of specificity and is calculated entirely from the unaffected
subgroup.
Because the true- and false-positive fractions are calculated entirely
separately, by
using the test results from two different subgroups, the ROC plot is
independent of
the prevalence of disease in the sample. Each point on the ROC plot represents
a
sensitivity/1-specificity pair corresponding to a particular decision
threshold. A test
with perfect discrimination (no overlap in the two distributions of results)
has an
ROC plot that passes through the upper left corner, where the true-positive
fraction
is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0
(perfect
specificity). The theoretical plot for a test with no discrimination
(identical
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distributions of results for the two groups) is a 45 diagonal line from the
lower left
corner to the upper right corner. Most plots fall in between these two
extremes. (If
the ROC plot falls completely below the 45 diagonal, this is easily remedied
by
reversing the criterion for "positivity" from "greater than" to "less than" or
vice
versa.) Qualitatively, the closer the plot is to the upper left corner, the
higher the
overall accuracy of the test.
One convenient goal to quantify the diagnostic accuracy of a laboratory test
is to
express its performance by a single number. The most common global measure is
the area under the ROC plot. By convention, this area is always > 0.5 (if it
is not,
one can reverse the decision rule to make it so). ROC-values range between 1.0
(perfect separation of the test values of the two groups) and 0.5 (no apparent
distributional difference between the two groups of test values). The area
does not
depend only on a particular portion of the plot such as the point closest to
the
diagonal or the sensitivity at 90% specificity, but on the entire plot. This
is a
quantitative, descriptive expression of how close the ROC plot is to the
perfect one
(area = 1.0).
In a preferred embodiment the present invention relates to a method for
improving
the assessment of an individual's risk of developing a myocardial disorder by
measuring in a sample the concentration of an antibody to a cardiac troponin
and
the level of cardiac troponin and correlating the concentrations determined to
the
risk of developing a myocardial disorder.
In a preferred embodiment the present invention relates to a method for
improving
the assessment of an individual's risk of developing a myocardial disorder by
measuring in a sample the concentration of an antibody to a cardiac troponin
and
the level of cholesterol and correlating the concentrations determined to the
risk of
developing a myocardial disorder.
In a preferred embodiment the present invention relates to a method for
improving
the assessment of an individual's risk of developing a myocardial disorder by
measuring in a sample the concentration of at an antibody to a cardiac
troponin
and the level of CRP and correlating the concentrations determined to the risk
of
developing a myocardial disorder.
In a preferred embodiment the present invention relates to a method for
improving
the assessment of an individual's risk of developing a myocardial disorder by
measuring in a sample the concentration of at an antibody to a cardiac
troponin
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and the level of natriuretic peptide or a natriuretic peptide-related marker
and
correlating the concentrations determined to the risk of developing a
myocardial
disorder.
In preferred embodiments the invention provides novel kits or assays which are
specific for, and have appropriate sensitivity with respect to antibodies to a
cardiac
troponin. A preferred kit accordingly to the present invention comprises a
cardiac
troponin and auxiliary reagents appropriate for measurement of antibodies to
said
cardiac troponin.
As discussed above the invention provides methods for evaluating the
likelihood
that an individual will benefit from treatment with an agent for reducing risk
of a
future myocardial disorder. This method may have important implications for
patient treatment and also for clinical development of new therapeutics.
Physicians
select therapeutic regimens for patient treatment based upon the expected net
benefit to the patient. The net benefit is derived from the risk to benefit
ratio. The
present invention may permit selection of individuals who are more likely to
benefit by intervention, thereby aiding the physician in selecting a
therapeutic
regimen. This might include using drugs with a higher risk profile where the
likelihood of expected benefit has increased. Likewise, clinical investigators
desire to
select for clinical trials a population with a high likelihood of obtaining a
net
benefit. The present invention can help clinical investigators select such
individuals.
It is expected that clinical investigators now will use the present invention
for
determining entry criteria for clinical trials.
The presence of an antibody to a cardiac troponin in an individual's sample
may
implicate that inflammatory processes are going on, which might lead to
further
damage of heart tissue. Anti-inflammatory therapy may be especially important
for
patients testing positive for antibodies to cardiac troponin I.
A cardiac troponin may be released into the circulation during a surgical
intervention at the heart. This may be specially the case for patients
undergoing
surgery for heart transplantation. The release of a cardiac troponin during
cardiac
surgery may or may not trigger the formation of autoantibodies. Anti-troponin
autoantibodies, however, once induced may well have a negative impact on the
patient and may e.g. become relevant in rejection of the transplanted heart.
In a
further preferred embodiment, autoantibodies to a cardiac troponin will be of
aid
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in the follow-up of patients after heart surgery, especially and preferably in
the
follow-up of heart transplantations.
Heart transplant patients that develop anti-troponin antibodies may require
additional or different treatment as compared to patients not testing positive
for
such autoantibodies.
The following examples are provided to aid the understanding of the present
invention, the true scope of which is set forth in the appended claims. It is
understood that modifications can be made in the procedures set forth without
departing from the spirit of the invention.
Example 1
General procedure for detection of antibodies to a cardiac troponin
In order to detect serum anti-cardiac troponin T, or troponin I antibodies the
following assay can be used: Wells of a microtiter plate are first coated with
a mouse
monoclonal antibody to cardiac troponin T or I. In a second step the
corresponding
antigen, either cardiac troponin T or I is bound to the antibodies. By
incubating an
appropriately diluted serum sample with the bound troponin antigen the serum
antibodies capable of binding to the troponin in the well will bind thereto.
The
bound serum antibodies can then be detected by an appropriate detection
antibody,
e.g. an anti-human IgG peroxidase conjugate. The skilled artisan is familiar
with
appropriate blocking and washing steps.
ExamRIe 2
Detection of antibodies to cardiac troponin I in human serum samples
Wells of a microtiter plate MaxiSorp flat-bottom 96 well plate, Nunc order
number 44-2404 were coated with an antibody to cardiac troponin I. Coating was
performed at an antibody concentration of 0.5 g/ml in coating buffer (= 0.1 M
NaHCO3 Sigma order number S-51761, 34 mM Na2CO3; Sigma order number S-
7795 pH 9.5) with 100 l/well at 4 C over night. Wells were washed thrice (300
1
per well and wash) with PBS/Tw (phosphate buffered saline NaCI Sigma order
number S-5886, potassium chloride Sigma order number P-4504, sodium
phosphate, Sigma order number S-5136, potassium phosphate monobasic, Sigma
P-5655 with 0.05 % Tween 20 Roth order number 9127.1). To block non-specific
binding all wells received 300 l of 1% gelatin (cold water fish skin, Sigma
order
no. G-7765) in PBS. Incubation was performed at RT for two hours. Wells were
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washed with PBS/Tw as above. Test wells received 100 l of troponin I solution
(3
g/ml in sample diluent = PBS with 0.1 % Tween 20 and 1% bovine serum
albumin (BSA Sigma order number A-9647)). Control wells received 100 l of
sample diluent. Incubation was performed at room temperature (RT) for two
hours. Wells were washed with PBS/Tw as above. Human sera were diluted 1:20
and further down in steps of 2 in sample diluent. Duplicates of 100 l diluted
serum
per well were incubated at RT for 90 min in both test wells as well as control
wells,
respectively. Wells were washed with PBS/Tw as above. Per well 100 1 of
detection
antibody (Horseradish Peroxidase (HRP) conjugated anti-human IgG Monoclonal
Antibody- BD Pharmingen product-no. 555788) diluted 1:10,000 in sample diluent
were then added to each well and incubated for one hour, followed by washing
as
described above. Peroxidase activity bound to the wells was detected by use of
100
l/well Blue Star TMB-HRP-Substrate (Diarect AG, product-no. DIA91000) as
recommended by the supplier. Reaction was stopped after 45 min by adding 100
l/well of 0.3 M H2SO4 J.T. Baker order number 6057. Extinction was recorded
450
nm using 550 nm as a reference wave length by SLT Spectra II, Tecan.
Median values were calculated for both the double determinations in test wells
as
well as for the double determinations in control wells. Corrected OD-results
were
calculated by subtracting the median of control wells from median of the
corresponding test wells in order to compensate for non-specific binding. A
corrected OD-value of more than 0.2 optical densities was considered as
positive.
Results are summarized in Table 1.
Table 1: Anti-troponin I antibody test results
origin of samples Number of positive samples/
total number of samples
HOCM (hypertrophic obstructive 5/7
cardiomyopathy)
DCM (dilated cardiomyopathy) 6/13
ICM (ischemic cardiomyopathy) 5/13
control (no known cardiac dysfunction) 0/4
As can be seen, a significant number of patients suffering from
cardiomyopathies of
various kind has been found to have antibodies to cardiac troponin I in their
sample. This is a clear indication that the presence of antibodies to a
cardiac
troponin may represent a hallmark of myocardial disease. Since (IgG) antibody
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production by the human body is not a matter of days theses antibody very
likely
may serve as a marker of risk for suffering from a cardiovascular disease,
especially
from a cardiomyopathy.
Example
Assessment of patients with AMI with and without antibodies to troponin I
Patients with AMI have been subgrouped into a group with autoantibodies to
cTNI
and into a subgroup without autoantibodies to TNI. The course of disease has
been
assessed by measuring the left ventricular ejection fraction (LVEF) 6-24 h
after MI
and 6-9 months later, respectively. The LVEF is indicative for the pumping
function
of the heart. A decreasing LVEF is indicative for the development of heart
failure,
whereas an increase in LVEF during follow-up is indicative for a regain in
pumping
function and a patient's recovery. The results of a pilot study are shown in
Table 2.
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Table 2: Change in LVEF for AMI patients with and without antibodies to TNI
TNI-positive TNI-negative
Sample LVEF LVEF Change Sample LVEF LVEF Change
No. (6-24 h) (6-9 m) in % No. (6-24 h) 6-9 m) in %
1 73.4 66.7 -9.1 1 54.2 53.5 -1.3
2 64.3 47.9 -25.5 2 65.7 69.3 5.4
3 71.6 72.4 1.1 3 62.2 59.1 -5.0
4 59.5 59.2 -0.5 4 58.9 61.6 4.6
54.5 56.7 4.0
6 60.0 66.9 11.5
7 65.1 59.8 -8.1
8 65.2 61.0 -6.4
9 67.9 69.2 1.9
46.0 61.5 55.4
11 55.1 50.2 -8.9
12 47.5 52.6 10.7
13 50.9 60.5 18.9
14 40.6 56.5 39.2
61.1 57.5 -5.9
16 62.5 62.0 -0.1
17 68.1 70.2 3.1
18 60.2 76.9 27.7
19 48.6 48.9 0.1
Mean 53.8 49.2 -8.5 Mean 57.6 60.7 7.7
value: value:
As is obvious from Table 2 the mean change in LVEF is negative for patients
with
anti-troponin antibodies in their blood, whereas the mean change is positive
5 (improvement of cardiac function) for patients not having a detectable level
of
autoantibodies to troponin in the circulation.
