U.S. patent application number 14/540165 was filed with the patent office on 2015-10-15 for use of igfbp-7 in the assessment of heart failure.
The applicant listed for this patent is The Governing Council of the University of Toronto, Roche Diagnostics Operations, Inc.. Invention is credited to Dirk Block, Andrew Emili, Vincent Fong, Anthony Gramolini, Georg Hess, Hendrik Huedig, Ruth Isserlin, Thomas Kislinger, Peter Liu, David MacLennan, Herbert von der Eltz, Ursula-Henrike Wienhues-Thelen.
Application Number | 20150293122 14/540165 |
Document ID | / |
Family ID | 37810309 |
Filed Date | 2015-10-15 |
United States Patent
Application |
20150293122 |
Kind Code |
A1 |
Wienhues-Thelen; Ursula-Henrike ;
et al. |
October 15, 2015 |
USE OF IGFBP-7 IN THE ASSESSMENT OF HEART FAILURE
Abstract
The invention relates to a method for assessing heart failure in
vitro comprising the steps of measuring in a sample the
concentration of the marker IGFBP-7, of optionally measuring in the
sample the concentration of one or more other marker(s) of heart
failure, and of assessing heart failure by comparing the
concentration determined in for IGFBP-7 and the concentration(s)
determined for the optionally one or more other marker to the
concentration of this marker or these markers as established in a
reference population. Also disclosed are the use of IGFBP-7 as a
marker protein in the assessment of heart failure, a marker
combination comprising IGFBP-7 and a kit for measuring IGFBP-7.
Inventors: |
Wienhues-Thelen;
Ursula-Henrike; (Krailling, DE) ; Hess; Georg;
(Mainz, DE) ; Huedig; Hendrik; (Penzberg, DE)
; von der Eltz; Herbert; (Weilheim, DE) ; Emili;
Andrew; (Toronto, CA) ; Gramolini; Anthony;
(Toronto, CA) ; Liu; Peter; (Toronto, CA) ;
MacLennan; David; (Toronto, CA) ; Fong; Vincent;
(Auincourt, CA) ; Isserlin; Ruth; (Thornhill,
CA) ; Kislinger; Thomas; (Toronto, CA) ;
Block; Dirk; (Bichl, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Diagnostics Operations, Inc.
The Governing Council of the University of Toronto |
Indianapolis
Toronto |
IN |
US
CA |
|
|
Family ID: |
37810309 |
Appl. No.: |
14/540165 |
Filed: |
November 13, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12504208 |
Jul 16, 2009 |
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14540165 |
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PCT/EP2008/000576 |
Jan 25, 2008 |
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12504208 |
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Current U.S.
Class: |
435/7.92 ;
436/501 |
Current CPC
Class: |
G01N 2333/4745 20130101;
G01N 2800/325 20130101; G01N 2333/5412 20130101; G01N 2333/4737
20130101; G01N 2333/47 20130101; G01N 2333/58 20130101; G01N
33/6893 20130101; G01N 2800/56 20130101; G01N 2333/4727
20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 25, 2007 |
EP |
07001582.1 |
Claims
1. A method for assessing heart failure in an individual comprising
the steps of measuring in a sample obtained from the individual a
concentration of insulin like growth factor binding protein 7
(IGFBP-7), determining a concentration of IGFBP-7 in a control
sample from a healthy individual, optionally measuring in the
sample a concentration of an additional marker of heart failure,
optionally determining a concentration of the additional marker of
heart failure in the control sample, and assessing heart failure by
comparing the concentration(s) determined in the sample from the
individual to the concentration(s) determined in the control
sample.
2. The method of claim 1, wherein the sample is selected from the
group consisting of serum, plasma, and whole blood.
3. The method of claim 1 wherein the additional marker is selected
from the group consisting of a natriuretic peptide marker, a
cardiac troponin marker, and a marker of inflammation.
4. The method of claim 3 wherein the natriuretic peptide marker is
NT-proBNP.
5. The method of claim 3 wherein the cardiac troponin marker is
troponin T.
6. The method of claim 1 wherein the individual is at risk for
heart failure.
7. A kit for performing the method according to claim 1, the kit
comprising reagents required to specifically measure IGFBP-7 and
the optional additional marker of heart failure.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/504,208 filed Jul. 16, 2009, which is a continuation of
PCT/EP2008/000576 filed Jan. 25, 2008, and claims priority to EP
07001582.1 filed Jan. 25, 2007.
FIELD OF THE INVENTION
[0002] The present invention relates to a method for assessing
heart failure in an individual comprising the steps of a) measuring
in a sample obtained from the individual the concentration of the
marker insulin like growth factor binding protein 7 (IGFBP-7), of
b) optionally measuring in the sample the concentration of one or
more other marker(s) of heart failure, and of assessing heart
failure by comparing the concentration determined in step (a) and
optionally the concentration(s) determined in step (b) to the
concentration of this marker or these markers as established in a
control sample. Also disclosed are the use of IGFBP-7 as a marker
protein in the assessment of heart failure, a marker combination
comprising IGFBP-7 and a kit for measuring IGFBP-7.
BACKGROUND OF THE INVENTION
[0003] Heart failure (HF) is a major and growing public health
problem. In the United States for example approximately 5 million
patients have HF and over 550 000 patients are diagnosed with HF
for the first time each year (In: American Heart Association, Heart
Disease and Stroke Statistics: 2005 Update, Dallas, Tex., American
Heart Association (2005)). Similarly US-statistics show that HF is
the primary reason for 12 to 15 million office visits and 6.5
million hospital days each year. From 1990 to 1999, the annual
number of hospitalizations has increased from approximately 810 000
to over 1 million for HF as a primary diagnosis and from 2.4 to 3.6
million for HF as a primary or secondary diagnosis. In 2001, nearly
53 000 patients died of HF as a primary cause. Heart failure is
primarily a condition of the elderly, and thus the widely
recognized "aging of the population" also contributes to the
increasing incidence of HF. The incidence of HF approaches 10 per
1000 in the population after age 65. In the US alone, the total
estimated direct and indirect costs for HF in 2005 were
approximately $27.9 billion and approximately $2.9 billion annually
is spent on drugs for the treatment of HF (cf. the above cited
AHA-statistics).
[0004] Heart Failure
[0005] Heart failure is characterized by a loss in the heart's
ability to pump as much blood as the body needs. Failure does not
mean that the heart has stopped pumping but that it is failing to
pump blood as effectively as it should.
[0006] The NYHA (New York Heart Association) and the ACC/AHA
(American Association of Cardiology/American Heart Association)
have both established functional classes of HF to gauge the
progression of the disease. The NYHA classification scheme has four
classes of disease state: Class 1 is asymptomatic at any level of
exertion. Class 2 is symptomatic at heavy exertion and Classes III
and IV are symptomatic at light and no exertion, respectively.
[0007] In the four stage ACC/AHA scheme, Stage A is asymptomatic
but is at risk for developing HF. Stage B there is evidence of
cardiac dysfunction without symptoms. In Stage C there is evidence
of cardiac dysfunction with symptoms. In Stage D, the subject has
symptoms of HF despite maximal therapy.
[0008] Etiology of Heart Failure
[0009] Medically, heart failure (HF) must be appreciated as being a
complex disease. It may be caused by the occurrence of a triggering
event such as a myocardial infarction (heart attack) or be
secondary to other causes such as hypertension, diabetes or cardiac
malformations such as valvular disease. Myocardial infarction or
other causes of HF result in an initial decline in the pumping
capacity of the heart, for example by damaging the heart muscle.
This decline in pumping capacity may not be immediately noticeable,
due to the activation of one or more compensatory mechanisms.
However, the progression of HF has been found to be independent of
the patient's hemodynamic status. Therefore, the damaging changes
caused by the disease are present and ongoing even while the
patient remains asymptomatic. In fact, the compensatory mechanisms
which maintain normal cardiovascular function during the early
phases of HF may actually contribute to progression of the disease
in the long run, for example by exerting deleterious effects on the
heart and its capacity to maintain a sufficient level of blood flow
in the circulation.
[0010] Some of the more important pathophysiological changes which
occur in HF are (i) activation of the
hypothalamic-pituitary-adrenal axis, (ii) systemic endothelial
dysfunction and (iii) myocardial remodeling.
[0011] (i) Therapies specifically directed at counteracting the
activation of the hypothalamic-pituitary-adrenal axis include
beta-adrenergic blocking agents (B-blockers), angiotensin
converting enzyme (ACE) inhibitors, certain calcium channel
blockers, nitrates and endothelin-1 blocking agents. Calcium
channel blockers and nitrates, while producing clinical improvement
have not been clearly shown to prolong survival, whereas B-blockers
and ACE inhibitors have been shown to significantly prolong life,
as have aldosterone antagonists. Experimental studies using
endothelin-1 blocking agents have shown a beneficial effect.
[0012] (ii) Systemic endothelial dysfunction is a well-recognized
feature of HF and is clearly present by the time signs of left
ventricular dysfunction are present. Endothelial dysfunction is
important with respect to the intimate relationship of the
myocardial microcirculation with cardiac myocytes. The evidence
suggests that microvascular dysfunction contributes significantly
to myocyte dysfunction and the morphological changes which lead to
progressive myocardial failure.
[0013] In terms of underlying pathophysiology, evidence suggests
that endothelial dysfunction may be caused by a relative lack of NO
which can be attributed to an increase in vascular
O.sub.2-formation by an NADH-dependent oxidase and subsequent
excess scavenging of NO. Potential contributing factors to
increased O.sub.2-production include increased sympathetic tone,
norepinephrine, angiotensin II, endothelin-1 and TNF-.alpha.. In
addition, levels of IL-10, a key anti-inflammatory cytokine, are
inappropriately low in relation to TNF-.alpha. levels. It is now
believed that elevated levels of TNF-.alpha., with associated
proinflammatory cytokines including IL-6, and soluble TNF-.alpha.
receptors, play a significant role in the evolution of HF by
causing decreased myocardial contractility, biventricular
dilatation, and hypotension and are probably involved in
endothelial activation and dysfunction. It is also believed that
TNF-.alpha. may play a role in the hitherto unexplained muscular
wasting which occurs in severe HF patients. Preliminary studies in
small numbers of patients with soluble TNF-receptor therapy have
indicated improvements in NYHA functional classification and in
patient well-being, as measured by quality of life indices.
[0014] (iii) Myocardial remodeling is a complex process which
accompanies the transition from asymptomatic to symptomatic heart
failure, and may be described as a series of adaptive changes
within the myocardium, like alterations in ventricular shape, mass
and volume (Piano, M. R., et al., J. Cardiovasc. Nurs. 14 (2000)
1-23; Molkentin, J. D., Ann. Rev. Physiol. 63 (2001) 391-426). The
main components of myocardial remodeling are alterations in myocyte
biology, like myocyte hypertrophy, loss of myocytes by necrosis or
apoptosis, alterations in the extracellular matrix and alterations
in left ventricular chamber geometry. It is unclear whether
myocardial remodeling is simply the end-organ response that occurs
following years of exposure to the toxic effects of long-term
neurohormonal stimulation, or whether myocardial remodeling
contributes independently to the progression of heart failure.
Evidence to date suggests that appropriate therapy can slow or halt
progression of myocardial remodeling.
[0015] Markers and Disease State
[0016] As indicated above, myocyte hypertrophy is likely to
represent one of the first steps down the road to HF. Myocyte
hypertrophy is characterized by an increased expression of some
genes encoding contractile proteins, such as p-myosin heavy chain
and troponin T (TnT), and of some non-contractile proteins, such as
A-type and B-type natriuretic peptides, by an increased cell size
and by cytoskeletal alteration (Piano, M. R., et al., J.
Cardiovasc. Nurs. 14 (2000) 1-23; Molkentin, J. D., Ann. Rev.
Physiol. 63 (2001) 391-426).
[0017] Studies of human and animal models of heart failure suggest
depressed myocyte function in the later stages of cardiac failure.
The mechanisms that underlie myocyte dysfunction have been
suggested to involve alterations in the calcium-handling network,
myofilament and cytoskeleton (de Tombe, P. P., Cardiovasc. Res. 37
(1998) 367-380). For example, in human and animal models of heart
failure, sarcoplasmic reticulum calcium-ATPase enzyme activity is
reduced, while both mRNA and protein levels of the sarcolemmal
Na+/Ca2+ exchanger are increased. Moreover, there is
isoform-switching of TnT, reduced phosphorylation of troponin I
(TnI), decreased myofibrillar actomyosin ATPase activity and
enhanced microtubule formation in both human and animal models of
heart failure.
[0018] Initially the changes to the heart, leading to myocardial
remodeling are meant to compensate for the diseased parts of the
myocardium in order to sustain the body's demand for oxygen and
nutrients. However, the compensatory phase of heart failure is
limited, and, ultimately, the failing heart is unable to maintain
cardiac output adequate to meet the body's needs. Thus, there is a
transition from a compensatory phase to a decompensatory phase. In
the decompensatory phase, the cascade of changes in the heart
continues but is no longer beneficial, moving the patient down the
progression of heart failure to a chronic state and eventual
death.
[0019] According to the "ACC/AHA 2005 Guideline Update for the
Diagnosis and Management of Chronic Heart Failure in the Adult",
the disease continuum in the area of heart failure is nowadays
grouped into four stages as noted above. 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 by reference.
[0020] Diagnostic Methods in Heart Failure
[0021] The single most useful diagnostic test in the evaluation of
patients with HF is the comprehensive 2-dimensional echocardiogram
coupled with Doppler flow studies to determine whether
abnormalities of myocardium, heart valves, or pericardium are
present and which chambers are involved. Three fundamental
questions must be addressed: 1) is the LVEF preserved or reduced,
2) is the structure of the LV normal or abnormal, and 3) are there
other structural abnormalities such as valvular, pericardial, or
right ventricular abnormalities that could account for the clinical
presentation? This information should be quantified with a
numerical estimate of EF, measurement of ventricular dimensions
and/or volumes, measurement of wall thickness, and evaluation of
chamber geometry and regional wall motion. Right ventricular size
and systolic performance should be assessed. Atrial size should
also be determined semiquantitatively and left atrial dimensions
and/or volumes measured.
[0022] Noninvasive hemodynamic data acquired at the time of
echocardiography are an important additional correlate for patients
with preserved or reduced EF. Combined quantification of the mitral
valve inflow pattern, pulmonary venous inflow pattern, and mitral
annular velocity provides data about characteristics of LV filling
and left atrial pressure. Evaluation of the tricuspid valve
regurgitant gradient coupled with measurement of inferior vena
caval dimension and its response during respiration provides an
estimate of systolic pulmonary artery pressure and central venous
pressure.
[0023] Stroke volume may be determined with combined dimension
measurement and pulsed Doppler in the LV outflow tract. However,
abnormalities can be present in any of these parameters in the
absence of HF. No one of these necessarily correlates specifically
with HF; however, a totally normal filling pattern argues against
clinical HF.
[0024] From a clinical perspective, the disease is clinically
asymptomatic in the compensatory and early decompensatory phases
(completely asymptomatic in 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.
[0025] Typically patients with heart failure receive a standard
treatment with drugs that interact with specific mechanisms
involved in heart failure. There are no diagnostic tests that
reflect those specific mechanisms reliably and help the physician
to choose the right drug (and dose) for the right patient (e.g.,
ACE inhibitor, AT II, .beta.-blockers, etc).
[0026] Prior Diagnosis of Heart Failure with Markers
[0027] Early assessment of patients at risk for heart failure
appears to be possible only by biochemical markers since the
individual at risk of developing heart failure at that stage is
still free of clinical HF symptoms. There are no established
biochemical markers currently available for the reliable
pre-symptomatic assessment of the disease. By the time the
diagnosis HF is established nowadays, the disease is already well
underway.
[0028] The natriuretic peptide family, especially the atrial
natriuretic peptide family and the brain natriuretic peptide family
have in recent years proven to be of significant value in the
assessment of HF.
[0029] Heart Failure Prognosis and Need
[0030] At least partially due to the late diagnosis, 50% of
patients with HF die within two years of diagnosis. The 5-year
survival rate is less than 30%. There is a significant need for new
biochemical markers aiding in the early diagnosis of heart
failure.
[0031] An improvement in the early assessment of individuals at
risk for heart failure, i.e., of individuals that are clinically
asymptomatic for heart failure is warranted.
[0032] It has been established in recent years that B-type
natriuretic peptide markers represent an excellent tool to monitor
disease progression in patients with HF and to assess their risk of
cardiovascular complications, like heart attack.
[0033] However, as for many other diagnostic areas a single marker
is not sufficient.
[0034] Whereas a low value of NT-proBNP has a very high negative
predictive value for ruling out HF or LVD, the positive predictive
value for heart failure in the above and other studies (cf.
Triepels R. H., et al., Clin. Chem. 49, Suppl. A (2003) 37-38) has
been found to be in the range of 50-60%. Thus a marker useful in
assessing individuals at risk for heart failure that on its own
e.g., has a high, or in combination with NT-proBNP, and as compared
to NT-proBNP alone has an improved positive predictive value for HF
is of high clinical/practical importance.
[0035] A marker aiding in the assessment of a patient with heart
failure also is of high importance to achieve further technical
progress in this clinically very important and demanding diagnostic
area.
SUMMARY OF THE INVENTION
[0036] It has now been found and established that the marker
insulin like growth factor binding protein 7 (IGFBP-7) can aid in
the assessment of heart failure. In one embodiment it can help to
assess whether an individual is at risk of developing heart
failure. In a further aspect it can aid in the assessment of
disease progression. In another embodiment, it can aid in
predicting the onset of heart failure. In another embodiment it can
aid in assessing and selecting an appropriate treatment regimen to
prevent or treat heart failure.
[0037] Disclosed herein is a method for assessing heart failure in
an individual comprising the steps of measuring in a sample
obtained from the individual the concentration of the marker
IGFBP-7, of optionally measuring in the sample the concentration of
one or more other marker(s) of heart failure, and of assessing
heart failure by comparing the concentration of IGFBP-7 and
optionally the concentration(s) of the one or more other marker to
the concentration of this marker or these markers as established in
a control sample.
[0038] The invention also relates to the use of protein IGFBP-7 as
a marker molecule in the assessment of heart failure.
[0039] Further disclosed is the use of a marker combination
comprising IGFBP-7 and one or more other marker of heart failure in
the assessment of heart failure.
[0040] Also provided is a kit for performing the method for
assessing heart failure in vitro comprising the steps of measuring
in a sample the concentration of the marker IGFBP-7, of optionally
measuring in the sample the concentration of one or more other
marker(s) of heart failure, and of assessing heart failure by
comparing the concentration of IGFBP-7 and optionally the
concentration(s) of the one or more other marker to the
concentration of this marker or these markers as established in a
reference population, the kit comprising the reagents required to
specifically measure IGFBP-7 and the optionally one or more other
marker of heart failure.
[0041] Additional aspects and advantages of the present invention
will be apparent in view of the description which follows. It
should be understood, however, that the detailed description and
the specific examples, while indicating preferred embodiments of
the invention, are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0042] FIG. 1A: Phenotypic analyses of wildtype and R9C mice. (A)
Survival curves for wildtype mice (n=79) and R9C mice (n=44) are
generated following a 24 week period
[0043] FIG. 1B: (B) Cardiac shortening assessed by echocardiography
(=fractional shortening). Significant functional impairment in the
R9C transgenic animals begin as early as 8 weeks of age.
[0044] FIG. 2: Echocardiographic and hemodynamic parameters in
wildtype and AB mice. (A) Changes in maximum pressure in mmHg at 2,
4, and 8 weeks post surgery. (B) Change in % left ventricular
ejection fraction (LVEF) at 2, 4, and 8 weeks after surgery.
(Closed circles indicate the data from sham operated mice and open
circles indicate the data from mice with aortic binding (AB).
[0045] FIG. 3: Western Blotting data as obtained with cardiac
tissue from R9C and control mice, respectively. A strong
overexpression of IGFBP-7 is observed in tissue samples derived
from experimental (R9C) animals suffering from heart failure versus
tissue samples derived from healthy mice (=+/+). Numbers underneath
the stained bands indicate relative expression levels determined by
the numbers of mass spectra recorded.
[0046] FIG. 4: IGFBP-7 measured in 10 HF and control samples,
respectively. Optical densities (ODs) in the IGFBP-7 assay are
given for samples derived from patients with heart failure are
labeled (HF=rhombi), and for healthy controls (normal human
serum=NHS=squares), respectively.
[0047] FIG. 5: IGFB-7 values as detected in HF samples from
clinical routine and in an extended control panel, respectively.
Calculated concentrations are given for IGFBP-7 as measured in
samples derived from patients with heart failure are labeled (HF)
and in samples from healthy controls (normal human serum=NHS),
respectively. The box-and-whisker-blots show the lower and upper
quartiles (boxes) as well as the highest and lowest values
(whiskers).
DETAILED DESCRIPTION OF THE INVENTION
[0048] In a first embodiment the present invention relates to a
method for assessing heart failure in an individual comprising the
steps of a) measuring in a sample obtained from the individual the
concentration of the marker IGFBP-7, b) optionally measuring in the
sample the concentration of one or more other marker(s) of heart
failure, and c) assessing heart failure by comparing the
concentration determined in step (a) and optionally the
concentration(s) determined in step (b) to the concentration of
this marker or these markers as established in a control
sample.
[0049] As used herein, each of the following terms has the meaning
associated with it in this section.
[0050] 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, "an antibody" means one antibody
or more than one antibody.
[0051] The expression "one or more" denotes 1 to 50, preferably 1
to 20 also preferred 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15.
[0052] The term "marker" or "biochemical marker" as used herein
refers to a molecule to be used as a target for analyzing a
patient's test sample. In one embodiment examples of such molecular
targets are proteins or polypeptides. Proteins or polypeptides used
as a marker in the present invention are contemplated to include
naturally occurring fragments of said protein in particular,
immunologically detectable fragments. Immunologically detectable
fragments preferably comprise at least 6, 7, 8, 10, 12, 15 or 20
contiguous amino acids of said marker polypeptide. One of skill in
the art would recognize that proteins which are released by cells
or present in the extracellular matrix may be damaged, e.g., during
inflammation, and could become degraded or cleaved into such
fragments. Certain markers are synthesized in an inactive form,
which may be subsequently activated by proteolysis. As the skilled
artisan will appreciate, proteins or fragments thereof may also be
present as part of a complex. Such complex also may be used as a
marker in the sense of the present invention. In addition, or in
the alternative a marker polypeptide may carry a post-translational
modification. Examples of posttranslational modifications amongst
others are glycosylation, acylation, and/or phosphorylation.
[0053] The term "assessing heart failure" is used to indicate that
the method according to the present invention will aid the
physician to assess whether an individual is at risk of developing
heart failure, or aid the physician in his assessing of an HF
patient in one or several other areas of diagnostic relevance in
HF. Preferred areas of diagnostic relevance in assessing an
individual with HF are the staging of heart failure, differential
diagnosis of acute and chronic heart failure, judging the risk of
disease progression, guidance for selecting an appropriate drug,
monitoring of response to therapy, and the follow-up of HF
patients.
[0054] A "marker of heart failure" in the sense of the present
invention is a marker that if combined with the marker IGFBP-7 adds
relevant information in the assessment of HF to the diagnostic
question under investigation. The information is considered
relevant or of additive value if at a given specificity the
sensitivity, or if at a given sensitivity the specificity,
respectively, for the assessment of HF can be improved by including
said marker into a marker combination already comprising the marker
IGFBP-7. Preferably the improvement in sensitivity or specificity,
respectively, is statistically significant at a level of
significance of p=0.05, 0.02, 0.01 or lower. Preferably, the one or
more other marker of heart failure is selected from the group
consisting of a natriuretic peptide marker, a cardiac troponin
marker, and a marker of inflammation.
[0055] The term "sample" as used herein refers to a biological
sample obtained for the purpose of evaluation in vitro. In the
methods of the present invention, the sample or patient sample
preferably may comprise any body fluid. Preferred test samples
include blood, serum, plasma, urine, saliva, and synovial fluid.
Preferred samples are whole blood, serum, plasma or synovial fluid,
with plasma or serum representing the most convenient type of
sample. As the skilled artisan will appreciate, any such assessment
is made in vitro. The patient sample is discarded afterwards. The
patient sample is solely used for the in vitro method of the
invention and the material of the patient sample is not transferred
back into the patient's body. Typically, the sample is a liquid
sample, e.g., whole blood, serum, or plasma.
[0056] The expression "comparing the concentration . . . to the
concentration as established in a control sample" is merely used to
further illustrate what is obvious to the skilled artisan anyway.
The control sample may be an internal or an external control
sample. In one embodiment an internal control sample is used, i.e.,
the marker level(s) is(are) assessed in the test sample as well as
in one or more other sample(s) taken from the same subject to
determine if there are any changes in the level(s) of said
marker(s). In another embodiment an external control sample is
used. For an external control sample the presence or amount of a
marker in a sample derived from the individual is compared to its
presence or amount in an individual known to suffer from, or known
to be at risk of, a given condition; or an individual known to be
free of a given condition, i.e., "normal individual". For example,
a marker level in a patient sample can be compared to a level known
to be associated with a specific course of disease in HF. Usually
the sample's marker level is directly or indirectly correlated with
a diagnosis and the marker level is, e.g., used to determine
whether an individual is at risk for HF. Alternatively, the
sample's marker level can, e.g., be compared to a marker level
known to be associated with a response to therapy in HF patients,
the differential diagnosis of acute and chronic heart failure, the
guidance for selecting an appropriate drug to treat HF, in judging
the risk of disease progression, or in the follow-up of HF
patients. Depending on the intended diagnostic use an appropriate
control sample is chosen and a control or reference value for the
marker established therein. It will be appreciated by the skilled
artisan that such control sample in one embodiment is obtained from
a reference population that is age-matched and free of confounding
diseases. As also clear to the skilled artisan, the absolute marker
values established in a control sample will be dependent on the
assay used. Preferably samples from 100 well-characterized
individuals from the appropriate reference population are used to
establish a control (reference) value. Also preferred the reference
population may be chosen to consist of 20, 30, 50, 200, 500 or 1000
individuals. Healthy individuals represent a preferred reference
population for establishing a control value.
[0057] An increased value for IGFBP-7 as measured from a sample
derived from an individual is indicative for heart failure.
[0058] The values for IGFBP-7 as measured in a control group or a
control population are for example used to establish a cut-off
value or a reference range. A value above such cut-off value or
out-side the reference range and its higher end is considered as
elevated.
[0059] In a one embodiment a fixed cut-off value is established.
Such cut-off value is chosen to match the diagnostic question of
interest.
[0060] In one embodiment values for IGFBP-7 as measured in a
control group or a control population are used to establish a
reference range. In a preferred embodiment an IGFBP-7 concentration
is considered as elevated if the value measured is above the
90%-percentile of the reference range. In further preferred
embodiments an IGFBP-7 concentration is considered as elevated if
the value measured is above the 95%-percentile, the 96%-percentile,
the 97%-percentile or the 97.5%-percentile of the reference
range.
[0061] In one embodiment the control sample will be an internal
control sample. In this embodiment serial samples are obtained from
the individual under investigation and the marker levels are
compared. This may for example be useful in assessing the efficacy
of therapy.
[0062] The method according to the present invention is based on a
liquid sample which is obtained from an individual and on the
measurement of IGFBP-7 in such sample. An "individual" as used
herein refers to a single human or non-human organism. Thus, the
methods and compositions described herein are applicable to both
human and veterinary disease. Preferably the individual is a human
being.
[0063] IGFBP-7
[0064] Insulin-Like Growth Factors (IGFs) and Corresponding Binding
Proteins (BPs)
[0065] The Insulin like growth factor binding protein (IGFBP)
system plays an important role in cell growth and differentiation.
It comprises two ligands, IGF-I and IGF-II, two receptors, type 1
and type 2 IGF receptors, and as of 1995 six IGF-binding proteins
(IGFBPs), IGFBP-1 to -6 (Jones, J. I., et al., Endocr. Rev. 16
(1995) 3-34). Recently the IGFBP family has been expanded to
include the IGFBP-related proteins (IGFBP-rPs), which have
significant structural similarities with the IGFBPs (Hwa, V., et
al., Endocr. Rev 20 (1999) 761-787). Thus, the IGFBP superfamily
includes the six conventional IGFBPs, which have high affinity for
IGFs, and at least 10 IGFBP-rPs, which not only share the conserved
amino-terminal domain of the IGFBPs but also show some degree of
affinity for IGFs and insulin. The IGFBP-rPs are a group of
cysteine-rich proteins that control diverse cellular functions,
such as cellular growth, cell adhesion and migration, and synthesis
of the extracellular matrix. In addition, these proteins might be
involved in biological processes like tissue proliferation and
differentiation, reproduction, angiogenesis, wound repair,
inflammation, fibrosis, and tumorigenesis (Hwa, V., et al., Endocr.
Rev 20 (1999) 761-787).
[0066] IGF binding protein 7 (=IGFBP-7) (SEQ ID NO: 1) is a 30-kDa
modular glycoprotein known to be secreted by endothelial cells,
vascular smooth muscle cells, fibroblasts, and epithelial cells
(Ono, Y., et al., Biochem Biophys Res Comm 202 (1994) 1490-1496).
In the literature this molecule has also been denominated as FSTL2;
IBP 7; IGF binding protein related protein 1; IGFBP 7; IGFBP 7v;
IGFBP rP1; IGFBP7; IGFBPRP1; insulin like growth factor binding
protein 7; insulin like growth factor binding protein 7 precursor;
MAC25; MAC25 protein; PGI2 stimulating factor; and PSF or
Prostacyclin stimulating factor. Northern blot studies revealed a
wide expression of this gene in human tissues, including heart,
brain, placenta, liver, skeletal muscle, and pancreas (Oh, Y., et
al., J. Biol. Chem. 271 (1996) 30322-30325).
[0067] IGFBP-7 was initially identified as a gene differentially
expressed in normal leptomeningeal and mammary epithelial cells,
compared with their counterpart tumor cells, and named
meningioma-associated cDNA (MAC25) (Burger, A. M., et al., Oncogene
16 (1998) 2459-2467). The expressed protein was independently
purified as a tumor derived adhesion factor (later renamed
angiomodulin) (Sprenger, C. C., et al., Cancer Res 59 (1999)
2370-2375) and as a prostacyclin-stimulating factor (Akaogi, K., et
al., Proc Natl Acad Sci USA 93 (1996) 8384-8389). It has
additionally been reported as T1A12, a gene down-regulated in
breast carcinomas (StCroix, B., et al., Science 289 (2000)
1197-1202).
[0068] The biological roles of IGFBP-7 have not yet been clearly
established. Preliminary experimental data are somewhat
controversial and relate to diverse actions for IGFBP-7, such as
tumor suppression (Sprenger, C. C., et al., Cancer Res 59 (1999)
2370-2375), tumor growth promotion (Lopez-Bermejo, A., et al., J.
Clinical Endocrinology and Metabolism 88 (2003) 3401-3408,
stimulation of prostacyclin (Akaogi, K., et al., Proc. Natl. Acad.
Sci. USA 93 (1996) 8384-8389) and involvement in angiogenesis
(Yamauchi, T., et al., Biochem J. 303 (1994) 591-598) and
senescence (Lopez-Bermejo, A., et al., Endocrinology 141 (2000)
4072-4080).
[0069] Differential expression of IGFBP-7 mRNA was measured in
patients suffering from various diseases including cardiac disease,
kidney disease, inflammatory diseases (U.S. Pat. No. 6,709,855 to
Scios Inc.) and vascular graft disease (US 2006/0,003,338).
[0070] A number of different assays has been described and used to
test for the hormone binding properties of IGFBP-7. Low affinity
IGF binding was analyzed via competitive affinity cross-linking
assays. Recombinant human mac25 protein specifically binds IGF-I
and-II (Oh, Y., et al., J. Biol. Chem. 271 (1996) 30322-20325; Kim,
H. S., et al., Proc. Natl. Acad. Sci USA 94 (1997) 12981-12986.)
IGFBP activity can also be detected by measuring the ability of the
protein to bind radio-labeled IGF in Western ligand blotting.
[0071] Immunological determination of circulating IGFBP-7 was
performed recently. Low levels of this analyte were detected in
random human sera and increased serum levels have been seen in
association with insulin-resistance (Lopez-Bermejo, A., et al., J.
Clinical Endocrinology and Metabolism 88 (2003) 3401-3408,
Lopez-Bermejo, A., et al., Diabetes 55 (2006) 2333-2339).
[0072] Several patent applications deal with the potential utility
of IGFBPs as diagnostic agents, preventive agents and therapeutic
agents for a broad variety of diseases.
[0073] US 2003/0186308 discloses a human polypeptide called
Prostacyclin-Stimulating Factor-2 (PSF-2; =IGFBP-7) and its
optional diagnostic application to detect a pathological
condition.
[0074] WO 2003/54004 speculates that IGFBP-7 may be used for
diagnosis of a large variety of diseases. It is alleged that it may
be used to diagnose a cell proliferative disorder, an
autoimmune/inflammatory disorder, a cardiovascular disorder, a
neurological disorder, a developmental disorder, a metabolic
disorder, a reproductive disorder, an infection, a growth disorder
such as growth hormone deficiency, acromegaly, IUGR, macrosomia,
tumorigenesis and cancer (e.g., breast cancer), diabetes and its
complications (e.g., diabetic kidney disease), chronic renal
failure, vascular disease, asthma, atherosclerosis and restenosis
and other pathological conditions.
[0075] US 2004/0072238 relates to the detection of differentially
expressed or mutated Insulin like growth factor binding proteins
for diagnosis of: diseases accompanying abnormal cell growth,
diseases accompanying angiopathy, diseases accompanying abnormal
bone metabolism, diseases accompanying disorders of insulin-like
growth factors or growth hormone action, diseases accompanying
abnormal differentiation or growth of smooth muscle cells, diseases
accompanying abnormal differentiation or growth of skeletal muscle
cells, diseases accompanying abnormal gastric acid secretion, and
inflammatory diseases. Almost any medically relevant condition is
then mentioned in a further list said to be comprised in one of the
diseases accompanying one or the other disorder as mentioned
before.
[0076] WO 2004/042000 describes the therapeutic application of more
than 150 secreted proteins for various medical indications. One of
the sequences mentioned is the sequence of IGFBP-7.
[0077] WO 1994/029448 describes the use of a PGI2 production
promoting protein (PGI2 stimulating factor) for therapy of several
diseases like hemolytic uremic syndrome, thrombotic
thrombocytopenic purpura, peripheral embolism, cardiac ischemia,
cerebral ischemia, arteriosclerosis, cerebral infarction,
hyperlipemia, diabetes, cardiac failure, angina pectoris, ischemic
heart disease, congestive heart disease, choroidal circulatory
disturbance, bronchial disease, gastric ulcer and eclampsia of
pregnancy on the basis of the platelet aggregation inhibitor
activity, smooth muscle relaxant activity and gastric
secretion.
[0078] Preferably the marker IGFBP-7 is specifically measured from
a liquid sample by use of a specific binding agent.
[0079] A specific binding agent is, e.g., a receptor for IGFBP-7, a
lectin binding to IGFBP-7 or an antibody to IGFBP-7. A specific
binding agent has at least an affinity of 10.sup.7 l/mol for its
corresponding target molecule. The specific binding agent
preferably has an affinity of 10.sup.8 l/mol or even more preferred
of 10.sup.9 l/mol for its target molecule. As the skilled artisan
will appreciate the term specific is used to indicate that other
biomolecules present in the sample do not significantly bind to the
binding agent specific for IGFBP-7. Preferably, the level of
binding to a biomolecule other than the target molecule results in
a binding affinity which is only 10% or less, more preferably only
5% or less of the affinity to the target molecule, respectively. A
preferred specific binding agent will fulfill both the above
minimum criteria for affinity as well as for specificity.
[0080] A specific binding agent preferably is an antibody reactive
with IGFBP-7. The term antibody refers to a polyclonal antibody, a
monoclonal antibody, antigen binding fragments of such antibodies,
single chain antibodies as well as to genetic constructs comprising
the binding domain of an antibody.
[0081] Any antibody fragment retaining the above criteria of a
specific binding agent can be used. Antibodies are generated by
state of the art procedures, e.g., as described in Tijssen
(Tijssen, P., Practice and theory of enzyme immunoassays, Elsevier
Science Publishers B.V., Amsterdam (1990), the whole book,
especially pages 43-78). In addition, the skilled artisan is well
aware of methods based on immunosorbents that can be used for the
specific isolation of antibodies. By these means the quality of
polyclonal antibodies and hence their performance in immunoassays
can be enhanced (Tijssen, P., supra, pages 108-115).
[0082] For the achievements as disclosed in the present invention
polyclonal antibodies raised in goats may be used. However, clearly
also polyclonal antibodies from different species, e.g., rats,
rabbits or guinea pigs, as well as monoclonal antibodies can be
used. Since monoclonal antibodies can be produced in any amount
required with constant properties, they represent ideal tools in
development of an assay for clinical routine.
[0083] The generation and the use of monoclonal antibodies to
IGFBP-7 in a method according to the present invention,
respectively, represent yet other preferred embodiments.
[0084] It is not easy to purify IGFBP-7 from a natural source. The
recombinant production of IGFBP-7 is a method of choice to obtain
higher amounts of IGFBP-7. In a preferred embodiment IGFBP-7 is
produced by recombinant expression using an eukaryotic expression
system. Examples of eukaryotic expression systems are baculovirus
expression, expression in yeast and expression in a mammalian
expression system. In one preferred embodiment the expression of
IGFBP-7 will be performed in a mammalian expression system.
Examples of mammalian expression systems are CHO cells, HEK cells,
myeloma cells, etc. In a further preferred embodiment the
recombinantly produced IGFBP-7 is used as an antigen in the
production of poly- or monoclonal antibodies against IGFBP-7. It
may be also preferable to purify polyclonal antibodies by
immunoadsorption over an IGFBP-7 immunoadsorber make use of a
recombinantly produced IGFBP-7 as described herein above.
[0085] As the skilled artisan will appreciate now, that IGFBP-7 has
been identified as a marker which is useful in the assessment of
HF, alternative ways may be used to reach a result comparable to
the achievements of the present invention. For example, alternative
strategies to generate antibodies may be used. Such strategies
comprise amongst others the use of synthetic or recombinant
peptides, representing a clinically relevant epitope of IGFBP-7 for
immunization. Alternatively, DNA immunization also known as DNA
vaccination may be used.
[0086] For measurement the liquid sample obtained from an
individual is incubated with the specific binding agent for IGFBP-7
under conditions appropriate for formation of a binding agent
IGFBP-7-complex. Such conditions need not be specified, since the
skilled artisan without any inventive effort can easily identify
such appropriate incubation conditions. The amount of binding agent
IGFBP-7-complex is measured and used in the assessment of HF. As
the skilled artisan will appreciate there are numerous methods to
measure the amount of the specific binding agent IGFBP-7-complex
all described in detail in relevant textbooks (cf., e.g., Tijssen
P., supra, or Diamandis, E. P. and Christopoulos, T. K. (eds.),
Immunoassay, Academic Press, Boston (1996)).
[0087] Preferably IGFBP-7 is detected in a sandwich type assay
format. In such assay a first specific binding agent is used to
capture IGFBP-7 on the one side and a second specific binding
agent, which is labeled to be directly or indirectly detectable, is
used on the other side. Preferably, an antibody to IGFBP-7 is used
in a qualitative (IGFBP-7 present or absent) or quantitative
(amount of IGFBP-7 is determined) immunoassay.
[0088] As described in detail in the Examples section, two mouse
models have been used to identify polypeptides found in heart
tissue of experimental animals by advanced proteomics methods.
However these models did yield at least partially conflicting data,
and, of course tissue data for polypeptides are not representative
to the presence or absence of these polypeptides in the
circulation. A marker found to be differentially expressed in one
model may not be differentially expressed in a second model or even
show conflicting data in yet a further model. Even if a protein may
be differentially expressed in tissue this protein in most cases is
not of any diagnostic relevance if measured from a bodily fluid,
because it may not be released to the circulation, may become
fragmented or modified, e.g., upon release from a cell or tissue,
may not be stable in the circulation, may not be measurable in the
circulation, may not be specific for a given disease, etc.
[0089] The inventors of the present invention surprisingly are able
to detect protein IGFBP-7 in a bodily fluid sample. Even more
surprising they are able to demonstrate that the presence of
IGFBP-7 in such liquid sample obtained from an individual can be
correlated to HF. No tissue and no biopsy sample is required to
make use of the marker IGFBP-7 in the assessment of HF. Measuring
the level of protein IGFBP-7 is considered very advantageous in the
field of HF.
[0090] In a preferred embodiment the method according to the
present invention is practiced with serum as liquid sample
material. In a further preferred embodiment the method according to
the present invention is practiced with plasma as liquid sample
material. In a further preferred embodiment the method according to
the present invention is practiced with whole blood as liquid
sample material.
[0091] In a further preferred embodiment, the present invention
relates to use of protein IGFBP-7 as a marker molecule in the
assessment of heart failure from a liquid sample obtained from an
individual.
[0092] The ideal scenario for diagnosis would be a situation
wherein a single event or process would cause the respective
disease as, e.g., in infectious diseases. In all other cases
correct diagnosis can be very difficult, especially when the
etiology of the disease is not fully understood as is the case of
HF. As the skilled artisan will appreciate, no biochemical marker
in the field of HF is diagnostic with 100% specificity and at the
same time 100% sensitivity for a certain diagnostic question.
Rather, biochemical markers are used to assess with a certain
likelihood or predictive value an underlying diagnostic question.
The skilled artisan is fully familiar with the
mathematical/statistical methods that routinely are used to
calculate a relative risk or likelihood for the diagnostic question
to be assessed. In routine clinical practice various clinical
symptoms and biological markers are generally considered together
by a physician in the diagnosis, treatment, and management of the
underlying disease.
[0093] Preferably in a further preferred embodiment of the present
invention the method for assessment of HF is performed by measuring
the concentration of IGFBP-7 and of one or more other marker and by
using the concentration of IGFBP-7 and of the one or more other
marker in the assessment of HF.
[0094] In the assessment of HF the marker IGFBP-7 will aid the
physician in one or more of the following aspects: to assess an
individual's risk for heart failure or to assess a patient having
heart failure, e.g., with the intention to identify the stage of
heart failure, to differentiate between acute and chronic heart
failure, to judge the risk of disease progression, to provide
guidance in selecting an appropriate therapy, to monitor a
patient's response to therapy, and to monitor the course of
disease, i.e., in the follow-up of HF patients.
[0095] Screening (Assessment Whether Individuals are at Risk for
Developing Heart Failure):
[0096] In a preferred embodiment the present invention relates to
an in vitro method for assessing whether an individual is at risk
for developing heart failure comprising the steps of measuring in a
sample the concentration of the marker IGFBP-7, of optionally
measuring in the sample the concentration of one or more other
marker(s) of heart failure, and of assessing said individual's risk
for developing heart failure by comparing the concentration for
IGFBP-7 and optionally the concentration(s) determined for the
optionally one or more other marker(s) to the concentration of this
marker or these markers to its or their reference value(s).
[0097] Screening in the sense of the present invention relates to
the unbiased assessment of individuals regarding their risk for
developing heart failure. Whereas such screening may in theory be
performed on any sample, in clinical practice such screening option
will usually be given to individuals somehow at risk for
development of heart failure. As discussed above, such individuals
may clinically be asymptomatic, i.e., they have no signs or
symptoms of HF. In one preferred embodiment screening for HF will
be given to individuals at risk of developing heart failure, e g ,
falling into the stages A or B as defined by the ACC/AHA practice
guidelines.
[0098] As mentioned above, heart failure is one of the most
prevalent, costly and life-threatening diseases in developed
countries. Because of its high prevalence and its long asymptomatic
phase identification of individuals at risk for developing HF would
be of utmost importance to intervene in and if possible to
interrupt the course of disease. Without a very early risk
assessment, prevention of disease progression from the asymptomatic
state into a symptomatic phase of HF appears impossible.
[0099] The risk for heart failure is assessed by
mathematical/statistical methods fully known and understood by the
skilled artisan. Preferably an individual's risk for heart failure
is expressed in relative terms and given as the so-called relative
risk (=RR). In order to calculate such RR for heart failure an
individual's value for IGFBP-7 is compared to the values
established for IGFBP-7 in a reference population, preferably
healthy individuals not developing heart failure. Also preferred
the assessment of such RR for heart failure is based on a group of
individuals that have developed heart failure within the study
period, preferably within one or also preferred within two years,
and a group of individuals that did not develop heart failure in
the same study period.
[0100] In another preferred embodiment the present invention
relates to the use of the marker IGFBP-7 in the screening for heart
failure. As the skilled artisan knows the term "use as a marker"
implies that the concentration of a marker molecule is quantified
by appropriate means and that value measured for such marker is
then used to indicate, i.e., to mark, the presence or absence of a
disease or clinical condition. Appropriate means for quantitation
for example are specific binding agents, like antibodies.
[0101] Preferably the screening for HF will be performed in
individuals suspected to be at risk of future heart failure.
Patients at risk of future heart failure in this sense are patients
diagnosed with hypertension, atherosclerotic disease, diabetes,
obesity and metabolic syndrome. Preferably the risk for future
heart failure is assessed with individuals suffering from
hypertension, atherosclerotic disease, diabetes, and/or metabolic
syndrome.
[0102] Also preferred is the use of the marker IGFBP-7 in assessing
the risk for future heart failure for an individual in stage B
according to the ACC/AHA practice guidelines, i.e., an individual
exhibiting a structural change at the heart but not showing
symptoms of heart failure.
[0103] In a further preferred embodiment the present invention
relates to the use of IGFBP-7 as one marker of a HF marker
combination for HF screening purposes.
[0104] In the screening setting an elevated level of IGFBP-7 is a
positive indicator for an individual's increased risk to develop
heart failure.
[0105] Staging of Patients
[0106] In a preferred embodiment the present invention relates to
an in vitro method aiding in the staging of heart failure patients,
comprising the steps of a) measuring in a sample the concentration
of the marker IGFBP-7, of b) optionally measuring in the sample the
concentration of one or more other marker(s) of heart failure, and
staging heart failure by comparing the concentration determined in
step (a) and optionally the concentration(s) determined in step (b)
to the concentration of this marker or these markers to its or
their reference value(s). Preferably the level of marker IGFBP-7 is
used as an aid in classifying the individuals investigated into the
groups of individuals that are clinically "normal" (i.e.,
individuals in stage A according to the ACA/ACC classification),
asymptomatic patients having structural heart disease (stage B
according to the ACA/ACC classification) and the group of patients
having heart failure (i.e., patients in stage C or stage D
according to the ACA/ACC classification).
[0107] Differentiation Between an Acute Cardiac Event and Chronic
Cardiac Disease
[0108] In a preferred embodiment the present invention relates to
an in vitro method aiding in the differential diagnosis between an
acute cardiac event and chronic cardiac disease, comprising the
steps of measuring in a sample the concentration of the marker
IGFBP-7, of optionally measuring in the sample the concentration of
one or more other marker(s) of heart failure, and establishing a
differential diagnosis between an acute cardiac event and chronic
cardiac disease by comparing the concentration determined in step
(a) and optionally the concentration(s) determined in step (b) to
the concentration of this marker or these markers to its or their
reference value(s).
[0109] The person skilled in the art is familiar with the meanings
of "acute cardiac event" and of "chronic cardiac disease".
[0110] Preferably, an "acute cardiac event" relates to an acute
condition, disease or malfunction of the heart, particularly to
acute heart failure, e.g., myocardial infarction (MI) or
arrhythmia. Depending on the extent of an MI, it may be followed by
LVD and CHF.
[0111] Preferably, a "chronic cardiac disease" is a weakening of
heart function, e.g., due to ischemia of the heart, coronary artery
disease, or previous, particularly small, myocardial infarction(s)
(possibly followed by progressing LVD). It may also be a weakening
due to inflammatory diseases, heart valve defects (e.g., mitral
valve defects), dilatative cardiomyopathy, hypertrophic
cardiomyopathy, heart rhythm defects (arrhythmias), and chronic
obstructive pulmonary disease. Thus, it is clear that a chronic
cardiac disease may also include patients who had suffered from an
acute coronary syndrome, e.g., MI, but who are presently not
suffering from an acute cardiac event.
[0112] It is important to differentiate between an acute cardiac
event and chronic cardiac disease, because an acute cardiac event
and chronic cardiac disease may require quite different treatment
regimens. For example, for a patient presenting with acute
myocardial infarction early treatment for reperfusion may be of
utmost importance. Whereas a treatment for reperfusion performed on
a patient with chronic heart failure at best is of no or only
little harm to this patient.
[0113] In a further preferred embodiment according to the present
invention the marker IGFBP-7 is used in the differential diagnosis
of acute and chronic heart failure.
[0114] Assessing the Risk of Disease Progression
[0115] In a preferred embodiment the present invention relates to
an in vitro method for assessing an HF-patient's risk for disease
progression, comprising the steps of measuring in a sample the
concentration of the marker IGFBP-7, of optionally measuring in the
sample the concentration of one or more other marker(s) of heart
failure, and of establishing said individual's risk for disease
progression by comparing the concentration for IGFBP-7 and
optionally the concentration(s) determined for the optionally one
or more other marker(s) to the concentration of this marker or
these markers to its or their reference value(s).
[0116] At present it is very difficult to assess or to even predict
with a reasonable likelihood whether a patient diagnosed with HF
has a more or less stable status or whether the disease will
progress and the patient's health status as result is likely to
worsen. Severity and progression of heart failure is clinically
usually established by assessing the clinical symptoms or by
identification of adverse changes by using imaging technologies
such as echocardiography. In one embodiment the worsening of heart
failure is established by monitoring the left ventricular ejection
fraction (LVEF). A deterioration in LVEF by 5% or more is
considered as disease progression.
[0117] In a further preferred embodiment the present invention
therefore relates to the use of the marker IGFBP-7 in assessing the
risk of disease progression for a patient suffering from HF. In the
assessment of disease progression for patients suffering from HF an
elevated level of IGFBP-7 is an indicator for an increased risk of
disease progression.
[0118] Guidance in Selecting an Appropriate HF Therapy
[0119] In a preferred embodiment the present invention relates to
an in vitro method, aiding in the selection of an appropriate
HF-therapy, comprising the steps of measuring in a sample the
concentration of the marker IGFBP-7, of optionally measuring in the
sample the concentration of one or more other marker(s) of heart
failure, and of selecting an appropriate therapy by comparing the
concentration for IGFBP-7 and optionally the concentration(s)
determined for the optionally one or more other marker(s) to the
concentration of this marker or these markers to its or their
reference value(s).
[0120] It is expected that the marker IGFBP-7 will be of help in
aiding the physician to select the most appropriate treatment
regimen from the various treatment regimens at hand in the area of
heart failure. In a further preferred embodiment therefore relates
to the use of the marker IGFBP-7 in selecting a treatment regimen
for a patient suffering from HF.
[0121] Monitor a Patient's Response to Therapy
[0122] In a preferred embodiment the present invention relates to
an in vitro method for monitoring a patient's response to
HF-therapy, comprising the steps of a) measuring in a sample the
concentration of the marker IGFBP-7, of b) optionally measuring in
the sample the concentration of one or more other marker(s) of
heart failure, and of monitoring a patient's response to HF-therapy
by comparing the concentration determined in step (a) and
optionally the concentration(s) determined in step (b) to the
concentration of this marker or these markers to its or their
reference value(s).
[0123] Alternatively the above method for motoring a patient's
response to therapy can be practiced by establishing the pre- and
post-therapeutic marker level for IGFBP-7 and for the optionally
one or more other marker and by comparing the pre- and the
post-therapeutic marker level(s).
[0124] The diagnosis of heart failure is clinically established.
According to the present invention HF is considered clinically
established if a patient meets the criteria of stages C or D as
defined by the ACC/AHA practice guidelines. According to these
guidelines stage C refers to patients with structural heart disease
and with prior or current symptoms of heart failure. Patients in
stage D are those patients with refractory heart failure that
require specialized interventions.
[0125] As indicated further above the values measured for NT-proBNP
are highly correlated to the severity of heart failure. However,
both BNP and NT-proBNP appear to be not ideal in monitoring a
patient's response to therapy, cf. e.g., Beck-da-Silva, L., et al.,
Congest. Heart Fail. 11 (2005) 248-253, quiz 254-255.
[0126] The marker IGFBP-7 appears to be appropriate to monitor a
patient's response to therapy. The present invention thus also
relates to the use of IGFBP-7 in monitoring a patient's response to
therapy. In that diagnostic area the marker IGFBP-7 can also be
used for establishing a baseline value before therapy and to
measure IGFBP-7 at one time-point or several time-points after
therapy. In the follow-up of HF patients an elevated level of
IGFBP-7 is a positive indicator for an effective treatment of
HF.
[0127] Marker Combination
[0128] Biochemical markers can either be determined individually
or, in a preferred embodiment of the invention, they can be
measured simultaneously using a chip- or a bead-based array
technology. The concentrations of the biomarkers are then
interpreted independently using an individual cut-off for each
marker or they are combined for interpretation, i.e., they form a
marker combination.
[0129] As the skilled artisan will appreciate the step of
correlating a marker level to a certain likelihood or risk can be
performed and achieved in different ways. Preferably the values
measured for the marker IGFBP-7 and the one or more other marker(s)
are mathematically combined and the combined value is correlated to
the underlying diagnostic question. Marker values may be combined
with the measurement of IGFBP-7 by any appropriate state of the art
mathematical method.
[0130] Preferably the mathematical algorithm applied in the
combination of markers is a logistic function. The result of
applying such mathematical algorithm or such logistical function
preferably is a single value. Dependent on the underlying
diagnostic question such value can easily be correlated to e.g.,
the risk of an individual for heart failure or to other intended
diagnostic uses helpful in the assessment of patients with HF. In a
preferred way such logistic function is obtained by a)
classification of individuals into the groups, e.g., into normals,
individuals at risk for heart failure, patients with acute or
chronic heart failure and so on, b) identification of markers which
differ significantly between these groups by univariate analysis,
c) logistic regression analysis to assess the independent
discriminative values of markers useful in assessing these
different groups and d) construction of the logistic function to
combine the independent discriminative values. In this type of
analysis the markers are no longer independent but represent a
marker combination.
[0131] In a preferred embodiment the logistic function used for
combining the values for IGFBP-7 and the value of at least one
further marker is obtained by a) classification of individuals into
the groups of normals and individuals at risk of heart failure,
respectively, b) establishing the values for IGFBP-7 and the value
of the at least one further marker c) performing logistic
regression analysis and d) construction of the logistic function to
combine the marker values for IGFBP-7 and the value of the at least
one further marker.
[0132] A logistic function for correlating a marker combination to
a disease preferably employs an algorithm developed and obtained by
applying statistical methods. Appropriate statistical methods,
e.g., are 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 statistical method to evaluate a marker combination of
the present invention and thereby to obtain an appropriate
mathematical algorithm. Preferably the statistical method employed
to obtain the mathematical algorithm used in the assessment of
heart failure 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., et al., J. of Computational and
Graphical Statistics 12 (2003) 475-511; Friedman, J. H., J. of the
American Statistical Association 84 (1989) 165-175; Hastie, T., et
al., The Elements of Statistical Learning, Springer Verlag (2001);
Breiman, L., et al. Classification and regression trees, Wadsworth
International Group, California (1984); Breiman, L., Machine
Learning 45 (2001) 5-32; Pepe, M. S., The Statistical Evaluation of
Medical Tests for Classification and Prediction, Oxford Statistical
Science Series, 28, Oxford University Press (2003); and Duda, R.
O., et al., Pattern Classification, John Wiley & Sons, Inc.,
2nd ed. (2001).
[0133] It is a preferred embodiment of the invention to use an
optimized multivariate cut-off for the underlying combination of
biological markers and to discriminate state A from state B, e.g.,
normals and individuals at risk for heart failure, HF patients
responsive to therapy and therapy failures, patients having an
acute heart failure and HF patients having chronic heart failure,
HF patients showing disease progression and HF patients not showing
disease progression, respectively.
[0134] The area and the receiver operator curve (=AUC) is an
indicator of the performance or accuracy of a diagnostic procedure.
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.
[0135] 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 disease progression versus no
disease progression.
[0136] 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 distributions of results for the two
groups) is a 45.degree. 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.degree.
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.
[0137] 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
(AUC). By convention, this area is always .gtoreq.0.5 (if it is
not, one can reverse the decision rule to make it so). 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).
[0138] The overall assay sensitivity will depend on the specificity
required for practicing the method disclosed here. In certain
preferred settings a specificity of 75% may be sufficient and
statistical methods and resulting algorithms can be based on this
specificity requirement. In one preferred embodiment the method
used to assess individuals at risk for heart failure is based on a
specificity of 80%, of 85%, or also preferred of 90% or of 95%.
[0139] As discussed above, the marker IGFBP-7 aids in assessing an
individuals risk of developing heart failure as well as in the
further in vitro diagnostic assessment of a patient having heart
failure. A preferred embodiment accordingly is the use of IGFBP-7
as a marker molecule in the assessment of heart failure.
[0140] The use of a marker combination comprising IGFBP-7 and one
or more other marker(s) of HF in the assessment of HF patients or
in the assessment of individuals at risk for HF represents a
further preferred embodiment of the present invention. In such
marker combination the one or more other marker(s) preferably is
selected from the group consisting of a natriuretic peptide marker,
a cardiac troponin marker, and a marker of inflammation.
[0141] The one or more preferred selected other HF marker(s) with
which the measurement of IGFBP-7 may be combined preferably is or
are selected from the group consisting of a natriuretic peptide
marker, a cardiac troponin marker, and a marker of inflammation.
These preferred other markers whose measurement(s) preferably are
combined with the measurement of IGFBP-7 or which form part of the
HF marker combination comprising IGFBP-7, respectively, are
discussed in more detail below.
[0142] Natriuretic Peptide Marker
[0143] A natriuretic peptide marker in the sense of the present
invention is either a marker selected from the atrial natriuretic
peptide (ANP) family or a marker selected from the brain
natriuretic peptide (BNP) family.
[0144] The polypeptide markers in either the atrial natriuretic
peptide family or in the brain natriuretic peptide family are
derived from the preproforms of the corresponding active
hormones.
[0145] Preferred natriuretic peptide markers according to the
present invention are NT-proANP, ANP, NT-proBNP, BNP, and
immunologically detectable physiological fragments thereof. As the
skilled artisan readily appreciates, the immunologically detectable
fragment has to comprise at least one epitope allowing for the
specific detection of such physiological fragment. A physiological
fragment is a fragment as naturally present in an individual's
circulation.
[0146] The markers in both the natriuretic peptide families
represent fragments of the corresponding pro-hormones, i.e., proANP
and proBNP, respectively. Since similar considerations apply for
both families, only the BNP marker family shall be described in
some detail. The pro-hormone of the BNP family, i.e., proBNP
consists of 108 amino acids. proBNP is cleaved into the 32
C-terminal amino acids (77-108) representing the biologically
active hormone BNP and the N-terminal amino acids 1-76 called
N-terminal proBNP (or NT-proBNP). BNP, N-terminal proBNP (1-76) as
well as further breakdown products (Hunt, P. J., et al., Biochem.
Biophys. Res. Com. 214 (1995) 1175-1183) circulate in blood.
Whether the complete precursor molecule (proBNP 1-108) also occurs
in the plasma is not completely resolved. It is however described
(Hunt, P. J., et al., Peptides 18 (1997) 1475-1481) that a low
release of proBNP (1-108) in plasma is detectable but that due to
the very quick partial breakdown at the N-terminal end some amino
acids are absent. Today it is generally accepted that e.g., for
NT-proBNP the central portion of the molecule, residing in between
the amino acids 10 to 50 represents a physiologically rather stable
part. NT-proBNP molecules comprising this central part of NT-proBNP
can be reliably measured from bodily fluids. Detailed disclosure
relating to methods based on the immunological detection of this
central part of the NT-proBNP molecule is given in WO 00/45176 and
the reader is referred thereto for details. It may be of further
advantage to measure only a certain subfraction of NT-proBNP for
which the term native NT-proBNP has been proposed. Detailed
disclosure relating to this subfraction of NT-proBNP is found in WO
2004/099253. The artisan will find all necessary instructions
there. Preferably the NT-proBNP measured is or corresponds to the
NT-proBNP as measured with the ELECSYS (Roche Diagnostics GmbH)
NT-proBNP assay from Roche Diagnostics, Germany.
[0147] Preanalytics are robust with NT-proBNP, which allows easy
transportation of the sample to a central laboratory (Mueller, T.,
et al., Clin. Chem. Lab. Med. 42 (2004) 942-944). Blood samples can
be stored at room temperature for several days or may be mailed or
shipped without recovery loss. In contrast, storage of BNP for 48
hours at room temperature or at 4.degree. Celsius leads to a
concentration loss of at least 20% (Mueller, T., et al., supra; Wu,
A. H., et al., Clin. Chem. 50 (2004) 867-873).
[0148] The brain-derived natriuretic peptide family (especially BNP
and NT-proBNP) has been thoroughly investigated in the screening of
certain populations for HF. The findings with these markers,
especially with NT-proBNP are quite encouraging. Elevated values of
NT-proBNP even in asymptomatic "patients" are clearly indicative
for "heart problems" (Gremmler, B., et al., Exp. Clin. Cardiol. 8
(2003) 91-94). These authors showed that an elevated NT-proBNP
indicates the presence of `cardio-renal distress` and should prompt
referral for further investigation. In line with several other
groups of investigators Gremmler, et al., also find that an
abnormal NT-proBNP concentration is an accurate diagnostic test
both for the exclusion of HF in the population and in ruling out
left ventricular dysfunction (=LVD) in breathless subjects. The
role of negative BNP or NT-proBNP values in ruling out HF or LVD is
corroborated by other groups of investigators, cf., e.g., McDonagh,
T. A., et al., Eur. J. Heart Fail. 6 (2004) 269-273; and
Gustafsson, F., et al., J. Card. Fail. 11, Suppl. 5 (2005)
S15-20.
[0149] BNP is produced predominantly (albeit not exclusively) in
the ventricle and is released upon increase of wall tension. Thus,
an increase of released BNP reflects predominantly dysfunctions of
the ventricle or dysfunctions which originate in the atria but
affect the ventricle, e.g., through impaired inflow or blood volume
overload. In contrast to BNP, ANP is produced and released
predominantly from the atrium. The level of ANP may therefore
predominantly reflect atrial function.
[0150] ANP and BNP are the active hormones and have a shorter
half-life than their respective inactive counterparts, NT-proANP
and NT-proBNP. BNP is metabolised in the blood, whereas NT-proBNP
circulates in the blood as an intact molecule and as such is
eliminated renally. The in-vivo half-life of NT-proBNP is 120 min
longer than that of BNP, which is 20 min (Smith, M. W., et al., J.
Endocrinol. 167 (2000) 239-246).
[0151] Therefore, depending on the time-course or properties of
interest, either measurement of the active or the inactive forms of
the natriuretic peptide can be advantageous.
[0152] In the assessment of an individual at risk for heart failure
the value measured for IGFBP-7 is preferably combined with the
value for NT-proANP and/or NT-proBNP. Preferably the value for
NT-proBNP is combined with the value for IGFBP-7. Similar
considerations apply for selecting an appropriate therapy, judging
the risk of disease progression, and to monitoring the course of
disease.
[0153] In case IGFBP-7 is used in assessing a patient's response to
therapy its measurement is preferably combined with the measurement
of ANP or BNP.
[0154] In case IGFBP-7 is used to differentiate between acute and
chronic heart failure the preferred marker combination comprises
IGFBP-7, ANP or proANP and BNP or proBNP.
[0155] Cardiac Troponin Marker
[0156] The term cardiac troponin relates to the cardiac isoforms of
troponin I and troponin T. As already indicated above the term
marker also relates to physiological variants of the marker
molecule, like physiological fragments or complexes. For the
cardiac troponin markers their physiologically occurring complexes
are known to be of diagnostic relevance and are herewith expressly
included.
[0157] 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) 1333-1341; Christenson, R. H., et
al., Clin. Chem. 44 (1998) 494-501; and EP 0 394 819.
[0158] 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 Ca.sup.2+, 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). cTnI is released into the bloodstream
following cardiac cell death; thus, the plasma cTnI concentration
is elevated in patients with acute myocardial infarction (Benamer,
H., et al., Am. J. Cardiol. 82 (1998) 845-850).
[0159] The unique cardiac isoforms of troponin I and T allow them
to be distinguished immunologically from the other troponins of
skeletal muscle. Therefore, the release into the blood of troponin
I and T from damaged heart muscle can be specifically related to
damage of cardiac tissue. It is nowadays also appreciated by the
skilled artisan that the cardiac troponins may be detected from the
circulation either in their free form or as a part of a complex
(cf. e.g., U.S. Pat. No. 6,333,397, U.S. Pat. No. 6,376,206 and
U.S. Pat. No. 6,174,686).
[0160] In the assessment of an individual at risk for heart failure
as well as in the assessment of a patient suffering from heart
failure, the value measured for IGFBP-7 is preferably combined with
the value for cardiac isoform of troponin T and/or troponin I. A
preferred cardiac troponin used in combination with the marker
IGFBP-7 is cardiac troponin T.
[0161] Marker of Inflammation
[0162] The skilled artisan is familiar with the term marker of
inflammation. Preferred markers of inflammation are interleukin-6,
C-reactive protein, serum amyloid A and a S100 protein.
[0163] Interleukin-6 (IL-6) is a 21 kDa secreted protein that has
numerous biological activities that can be divided into those
involved in hematopoiesis and into those involved in the activation
of the innate immune response. IL-6 is an acute-phase reactant and
stimulates the synthesis of a variety of proteins, including
adhesion molecules. Its major function is to mediate the acute
phase production of hepatic proteins, and its synthesis is induced
by the cytokines IL-1 and TNF-.alpha.. IL-6 is normally produced by
macrophages and T lymphocytes. The normal serum concentration of
IL-6 is <5 pg/ml.
[0164] C-reactive protein (CRP) is a homopentameric
Ca.sup.2+-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 .mu.g/ml (30 nM) in 90% of the
healthy population, and <10 .mu.g/ml (100 nM) in 99% of healthy
individuals. Plasma CRP concentrations can, e.g., be measured by
Serum amyloid A (=SAA) is an acute phase protein of low molecular
weight of 11.7 kDa. It is predominantly synthesized by the liver in
response to IL-1, IL-6 or TNF-.alpha. stimulation and is involved
in the regulation of the T-cell dependent immune response. Upon
acute events the concentration of SAA increases up to 1000-fold
reaching one milligram per milliliter. It is used to monitor
inflammation in diseases as divers as cystic fibrosis, renal graft
refection, trauma or infections. In rheumatoid arthritis is has in
certain cases been used as a substitute for CRP, but, SAA is not
yet as widely accepted.
[0165] S100-proteins form a constantly increasing family of
Ca.sup.2+-binding proteins that today includes more than 20
members. The physiologically relevant structure of S100-proteins is
a homodimer but some can also form heterodimers with each other,
e.g., S100A8 and S100A9. The intracellular functions range from
regulation of protein phosphorylation, of enzyme activities, or of
the dynamics of the cytoskeleton to involvement in cell
proliferation and differentiation. As some S100-proteins are also
released from cells, extracellular functions have been described as
well, e.g., neuronal survival, astrocyte proliferation, induction
of apoptosis and regulation of inflammatory processes. S100A8,
S100A9, the heterodimer S100A8/A9 and S100A12 have been found in
inflammation with S100A8 responding to chronic inflammation, while
S100A9, S100A8/A9 and S100A12 are increased in acute inflammation.
S100A8, S100A9, S100A8/A9 and S100A12 have been linked to different
diseases with inflammatory components including some cancers, renal
allocraft rejection, colitis and most importantly to RA
(Burmeister, G., and Gallacchi, G., Inflammopharmacology 3 (1995)
221-230; Foell, D., et al., Rheumathology 42 (2003) 1383-1389). The
most preferred S100 markers for assessing an individual at risk for
HF or a patient having HF e.g., for use in a marker combination
according to the present invention are S100A8, S100A9, S100A8/A9
heterodimer and S100A12.
[0166] sE-selectin (soluble endothelial leukocyte adhesion
molecule-1, ELAM-1) is a 115 kDa, type-I transmembrane glycoprotein
expressed only on endothelial cells and only after activation by
inflammatory cytokines (IL-1.beta., TNF-.alpha.) or endotoxin.
Cell-surface E-selectin is a mediator of the rolling attachment of
leucocytes to the endothelium, an essential step in extravasion of
leucocytes at the site of inflammation, thereby playing an
important role in localized inflammatory response. Soluble
E-selectin is found in the blood of healthy individuals, probably
arising from proteolytic cleavage of the surface-expressed
molecule. Elevated levels of sE-selectin in serum have been
reported in a variety of pathological conditions (Gearing, A. J.
and Hemingway, I., Ann. N.Y. Acad. Sci. 667 (1992) 324-331).
[0167] In a preferred embodiment the present invention relates to
the use of IGFBP-7 as a marker molecule for HF in combination with
one or more marker molecule(s) for HF in the assessment of HF from
a liquid sample obtained from an individual.
[0168] As indicated above, in a preferred method according to the
present invention the value measured for IGFBP-7 is at least
combined with the value of at least one further marker selected
from the group consisting of a natriuretic peptide marker, a
cardiac troponin marker, and a marker of inflammation.
[0169] In a preferred embodiment the present invention relates to
the use of the marker combination IGFBP-7 and NT-proBNP in the
assessment of heart failure.
[0170] In a preferred embodiment the present invention relates to
the use of the marker combination IGFBP-7 and troponin T in the
assessment of heart failure.
[0171] In a preferred embodiment the present invention relates to
the use of the marker combination IGFBP-7 and CRP in the assessment
of heart failure.
[0172] In a further preferred embodiment the present invention
relates to a marker combination comprising the markers IGFBP-7,
troponin T, NT-proBNP and CRP.
[0173] In yet a further preferred embodiment the present invention
relates to a marker panel used in a method for assessing HF in
vitro by biochemical markers, comprising measuring in a sample the
concentration of IGFBP-7 and of one or more other marker of HF and
using the concentrations determined in the assessment of HF.
[0174] A marker panel according to the present invention is
preferably measured using a protein array technique. An array is a
collection of addressable individual markers. Such markers can be
spacially addressable, such as arrays contained within microtiter
plates or printed on planar surfaces where each marker is present
at distinct X and Y coordinates. Alternatively, markers can be
addressable based on tags, beads, nanoparticles, or physical
properties. The microarrays can be prepared according to the
methods known to the ordinarily skilled artisan (see for example,
U.S. Pat. No. 5,807,522; Robinson, W. H., et al., Nat. Med. 8
(2002) 295-301; Robinson, W. H., et al., Arthritis Rheum. 46 (2002)
885-893). Array as used herein refers to any immunological assay
with multiple addressable markers. In one embodiment the
addressable markers are antigens. In another embodiment the
addressable elements are autoantibodies. A microarray is a
miniaturized form of an array. Antigen as used herein refers to any
molecule that can bind specifically to an antibody. The term
autoantibody is well-defined in the art.
[0175] In a preferred embodiment the present invention relates to a
protein array comprising the marker IGFBP-7 and optionally one or
more other marker of HF.
[0176] In a preferred embodiment the present invention relates to a
protein array comprising the markers IGFBP-7 and NT-proBNP.
[0177] In a preferred embodiment the present invention relates to a
protein array comprising the markers IGFBP-7 and troponin T.
[0178] In a preferred embodiment the present invention relates to a
protein array comprising the markers IGFBP-7 and CRP.
[0179] In a further preferred embodiment the present invention
relates to a protein array comprising the markers IGFBP-7, troponin
T, NT-proBNP and CRP.
[0180] In yet a further preferred embodiment the present invention
relates to a kit comprising the reagents required to specifically
measure IGFBP-7. Also preferred is a kit comprising the reagents
required to specifically measure IGFBP-7 and the reagents required
to measure the one or more other marker of heart failure that are
used together in an HF marker combination.
[0181] The following examples, sequence listing and figures 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
Mouse Models of Heart Failure 1.1 The R9C Mouse Model
[0182] It has been reported that an inherited human dilated
cardiomyopathy resulted from the conversion of Arg9 to Cys in the
human phospholamban (PLN) gene (PLN-R9C) (Schmitt, J. P., et al.,
Science 299 (2003) 1410-1413). The onset of dilated cardiomyopathy
in affected patients typically commenced during adolescence,
followed by progressive deterioration in cardiac function leading
to crisis and mortality. A transgenic mouse model of this mutation
showed similar cardiac phenotype as the affected patients and
presented with dilated cardiomyopathy, decreased cardiac
contractility, and premature death (Schmitt et al., 2003,
supra).
[0183] We established a survival curve for the transgenic mice. The
PLN-R9C mice had a median survival of only .about.20 weeks with
fewer than 15% persisting past 24 weeks (FIG. 1A). The first
recorded deaths in the PLN-R9C line are observed at 12 weeks of
age, while only one wild-type control mouse died over the 24 week
period. Eight weeks is selected as representative time point of
`early` stage disease prior to the first recorded mortality, while
16 weeks is chosen as it is the midpoint between 8 and 24 weeks
(classic DCM). A detailed analysis of the pathology of isolated
hearts shows evidence of ventricle and atria enlargement even at 8
weeks of age in the PLN-R9C mice. Cross-sections of isolated
cardiac muscle (obtained from wild-type and PLN-R9C mice followed
by hematoxylin and eosin staining also shows evidence of left
ventricular dilatation, or thinning of the ventricular wall, in the
transgenic animals beginning at 8 weeks, with continued progression
of dilatation with age.
[0184] Functional cardiac measurements are performed by
echocardiography on the 8, 16 and 24 week old male mice (summarized
in Table 1). Echocardiography measurements of the thickness of the
anterior and posterior wall show that the R9C mice have significant
dilatation at 8 weeks, which continues to deteriorate throughout
the lifespan of the mice. Contractility, as assessed by cardiac
shortening (FIG. 1B), is also slightly, but significantly, reduced
at 8 weeks, while a more pronounced decrease is clearly evident by
16 weeks. Female mice analyzed show identical findings as the males
(data not shown).
TABLE-US-00001 TABLE 1 Echocardiographic and hemodynamic parameters
in wildtype and R9C mice at 8, 24 weeks in male mice. WT R9C WT R9C
WT R9C Age 8 wks 8 wks 16 wks 16 wks 24 wks 24 wks Gender M M M M M
M HR (bpm) 560 .+-. 6 567 .+-. 5 569 .+-. 5 552 .+-. 15 565 .+-. 9
502 .+-. 15* AW (mm) 0.66 .+-. 0.01 0.60 .+-. 0.01* 0.70 .+-. 0.01
0.58 .+-. 0.01* 0.71 .+-. 0.01 0.57 .+-. 0.01* PW (mm) 0.66 .+-.
0.01 0.61 .+-. 0.01* 0.70 .+-. 0.01 0.59 .+-. 0.01* 0.71 .+-. 0.01
0.57 .+-. 0.01* LVEDD (mm) 3.82 .+-. 0.05 4.01 .+-. 0.03* 3.92 .+-.
0.07 5.01 .+-. 0.06* 3.99 .+-. 0.05 5.48 .+-. 0.08* LVESD (mm) 1.82
.+-. 0.05 2.13 .+-. 0.04* 1.84 .+-. 0.06 3.36 .+-. 0.09* 1.89 .+-.
0.03 4.23 .+-. 0.09* FS (%) 52.7 .+-. 0.9 47.6 .+-. 1.2* 53.1 .+-.
0.7 32.9 .+-. 1.9* 52.9 .+-. 1.5 22.6 .+-. 2.1* VCFc (circ/s) 10.5
.+-. 0.2 9.1 .+-. 0.2* 10.5 .+-. 0.1 7.0 .+-. 0.5* 10.9 .+-. 0.3
5.1 .+-. 0.5* PAVc (cm/s) 102.4 .+-. 2.4 97.8 .+-. 2.6 110.1 .+-.
3.7 85.3 .+-. 3.2* 111.3 .+-. 2.9 73.6 .+-. 3.1* AVA (m/s2) 65.7
.+-. 1.3 60.6 .+-. 1.6 66 .+-. 3.2 47.9 .+-. 2.5* 67.1 .+-. 3.1 40
.+-. 2.2* Samples (n) 6 9 6 9 5 5
[0185] Values in Table 1 are mean.+-.SEM. Symbols used in Table 1:
HR=Heart Rate; AW, PW=Anterior and Posterior Wall Thickness (Left
Ventricle); LVEDD, LVESD=Left Ventricular End Diastolic and
Systolic Dimension, respectively; FS=Fractional
Shortening=(LVEDD-LVESD)/LVEDD.times.100%; ETC=Ejection Time
corrected for HR; VCFC=Velocity of Circumferential Shortening
corrected for HR=FS/ETC; PAVC=Peak Aortic Velocity corrected for
HR; E-wave=Early-filling transmitral diastolic wave; LVESP,
LVEDP=Left Ventricular End Systolic and Diastolic Pressure;
+dP/dtmax=Maximum positive 1st derivative of the left ventricular
pressure; -dP/dtmax=Maximum negative 1st derivative of the left
ventricular pressure; AVA=aortic velocity acceleration
(PAVc/Acceleration Time); *P<0.05 compared with WT.
[0186] 1.2 The Aortic Banding (AB) Mouse Model
[0187] In this mouse model pressure-overload caused by aortic
banding (AB) induces cardiac-hypertrophy.
[0188] By surgical intervention pressure-overload is performed in
C57BL mice. The coarction of the ascending aorta (known as aortic
banding) induces cardiac hypertrophy and growth of the myocardial
muscle, especially in the left ventricle as a primary response to
coarction of the aorta. In the later stages of this mouse model the
heart becomes hypertrophic and finally dilated. This model is well
characterized and has proven to be highly reproducible with a low
mortality rate of 10-15% or less based on experience. After
coarction this animal model allows for evaluating the progress of
development of left ventricular hypertrophy and heart failure in
response to hemodynamic stress.
[0189] Briefly C57BL mice are anesthetized with mixed Ketamine (90
mg/kg) and Rompun (10 mg/Kg) and the aorta is ligated using
25-gauge needle. Sham operated mice undergo the same surgical
procedure, except that the ligation is not tightened against the
needle.
[0190] Experimental Time Points
[0191] To examine the hypertrophic response, banded animals and
sham-operated controls are sacrificed at one, two, four, and eight
weeks post intervention. Cardiac function and the development of
hypertrophy are assessed by echocardiographic analysis and
confirmed post mortem by examining the histology. Table 2 shows an
overview over the cardiac function evaluated at the various time
points by echocardiography. Details on the echocardiographic
parameters given in Table 2 are known to the artisan and can e.g.,
be found in Asahi, M., et al., Proc. Natl. Acad. Sci. USA 101
(2004) 9199-9204, and Oudit, G. Y., et al., Nat. Med. 9 (2003)
1187-1194.
TABLE-US-00002 TABLE 2 Parameter 2 wk sham 2 wk AB 4 wk sham 4 wk
AB 8 wk sham 8 wk AB Heart rate (bpm) 271.6 .+-. 31.2 286.3 .+-.
39.1 275.3 .+-. 25.8 276.5 .+-. 28.1 255.5 .+-. 23.9 310.8 .+-.
18.0 Maximum Volume (uL) 32.2 .+-. 2.3 36.4 .+-. 3.4 36.9 .+-. 1.1
40.8 .+-. 1.6 38.1 .+-. 1.5 48.9 + 4.4 Minimum Volume (uL) 13.7
.+-. 2.4 15.8 .+-. 3.3 14.7 .+-. 1.9 25.7 .+-. 0.9 18.4 .+-. 0.5
36.5 .+-. 3.7 End-systolic Volume 14.7 .+-. 2.8 16.9 .+-. 3.3 15.5
.+-. 2.1 28.0 .+-. 0.7 19.3 .+-. 0.5 40.2 .+-. 4.3 (uL)
End-diastolic Volume 30.6 .+-. 2.4 34.5 .+-. 3.2 35.2 .+-. 1.1 39.8
.+-. 1.6 36.8 .+-. 1.4 47.2 .+-. 4.1 (uL) Maximum Pressure 93.1
.+-. 3.5 149.2 .+-. 4.8 92.6 .+-. 0.8 153.5 .+-. 6.1 93.6 .+-. 5.0
169.8 .+-. 10.2 (mmHg) Minimum Pressure 4.9 .+-. 1.3 3.2 .+-. 0.4
3.6 .+-. 0.1 7.3 .+-. 3.6 4.1 .+-. 0.5 6.2 .+-. 1.9 (mmHg)
End-systolic Pressure 87.3 .+-. 4.3 139.4 .+-. 2.8 89.2 .+-. 1.0
149.6 .+-. 5.0 90.5 .+-. 4.9 168.3 .+-. 9.8 (mmHg) End-diastolic
Pressure 14.0 .+-. 3.2 10.6 .+-. 2.7 13.0 .+-. 0.7 16.8 .+-. 4.8
16.5 .+-. 1.4 16.9 .+-. 3.1 (mmHg) Stroke Volume (uL) 18.6 .+-. 1.0
20.6 .+-. 0.7 22.2 .+-. 2.3 15.1 .+-. 1.2 19.7 .+-. 1.4 12.4 .+-.
1.0 Ejection Fraction (%) 58.7 .+-. 5.1 57.9 .+-. 4.5 60.0 .+-. 5.3
36.8 .+-. 1.9 51.5 .+-. 1.6 25.8 .+-. 2.0 Cardiac Output 5113.5
.+-. 819.2 5879.1 .+-. 714.0 6114.8 .+-. 897.0 4108.6 .+-. 310.3
5066.0 .+-. 653.3 3893.8 .+-. 466.1 (uL/min) Stroke Work 1339.6
.+-. 134.0 2196.3 .+-. 94.6 1577.8 .+-. 134.4 1477.8 .+-. 99.6
1451.8 .+-. 130.4 1179.2 + 104.1 (mmHg*uL) Arterial Elastance (Ea)
4.8 .+-. 0.4 6.8 .+-. 0.3 4.1 .+-. 0.4 10.1 .+-. 0.7 4.7 .+-. 0.4
14.1 .+-. 1.7 (mmHg/uL) dPdt max (mmHg/sec) 5481.6 .+-. 491.1
6785.3 .+-. 434.2 6036.0 .+-. 352.9 5133.2 .+-. 621.4 5755.8 .+-.
652.9 6454.4 .+-. 712.0 dPdt min (mmHg/sec) -5049.6 .+-. 426.9
-7427.5 .+-. 685.3 -4743.3 .+-. 287.7 -5484.75 .+-. 412.2 -4564.5
.+-. 525.8 -7625 .+-. 586.5 dVdt max (uL/sec) 883.0 .+-. 61.2 758.0
.+-. 29.8 856.5 .+-. 27.4 1152.8 .+-. 206.3 1188.0 .+-. 114.1
1041.2 .+-. 109.6 dVdt min (uL/sec) -679.6 .+-. 71.4 -696.3 .+-.
30.6 -703.5 .+-. 52.2 -921.0 .+-. 158.0 -1000.5 .+-. 76.8 -938.4
.+-. 126.2 P@dVdt max (mmHg) 9.0 .+-. 2.5 7.4 .+-. 2.6 4.6 .+-. 0.4
10.3 .+-. 3.4 6.2 .+-. 1.0 13.3 .+-. 4.5 P@dPdt max (mmHg) 44.1
.+-. 2.1 46.3 .+-. 3.5 49.0 .+-. 2.6 47.1 .+-. 2.8 49.6 .+-. 5.6
52.8 .+-. 3.6 V@dPdt max (uL) 31.2 .+-. 2.4 35.5 .+-. 3.5 35.0 .+-.
1.1 39.7 .+-. 1.6 37.0 .+-. 1.5 47.3 .+-. 4.4 V@dPdt min (uL) 14.7
.+-. 2.6 17.1 .+-. 3.2 15.6 .+-. 1.9 27.0 .+-. 0.7 19.2 .+-. 0.4
39.0 .+-. 4.3 Tau_w (msec) 11.4 .+-. 1.2 8.6 .+-. 0.7 10.7 10.9
11.2 .+-. 1.3 11.3 .+-. 0.5 8.8 .+-. 0.4 Tau_g (msec) 15.8 .+-. 1.5
12.1 .+-. 1.2 17.5 .+-. 0.7 17.4 .+-. 1.0 17.5 .+-. 1.0 15.6 .+-.
1.0 Maximal Power 6.4 .+-. 0.6 9.5 .+-. 0.4 6.8 .+-. 0.5 8.8 .+-.
0.5 7.3 .+-. 0.7 9.0 .+-. 0.5 (mWatts) Preload adjusted 74.8 .+-.
16.5 85.0 .+-. 12.9 55.5 .+-. 2.4 57.3 .+-. 7.4 53.6 .+-. 3.0 46.1
.+-. 11.5 maximal power (mWatts/.rarw. N5L{circumflex over (
)}2)
[0192] In addition to functional parameters histology by
Hematoxylin/Eosin (HE) staining is performed on cardiac tissue from
AB mice and control mice at 2, 4, and 8 weeks. Histology confirms
the expected necrotic and remodeling processes for the AB mice,
whereas heart tissue in sham operated mice does not show any
significant changes. At two weeks after surgery the ventricle of a
ligated mouse shows significant left ventricular hypertrophy which
after four weeks has further progressed and at eight weeks post
surgery closely resembles end stage dilated cardiomyopathy.
EXAMPLE 2
Sample Preparation and Mass Spectroscopy
[0193] Heart Homogenization and Organelle Isolation
[0194] Hearts are isolated, atria removed, the ventricles carefully
minced with a razor blade and rinsed extensively with ice-cold PBS
(phosphate buffered saline) to remove excess blood. Tissue is
homogenized for 30 s using a loose fitting hand-held glass
homogenizer in 10 ml lysis buffer (250 mM sucrose, 50 mM Tris-HCl
pH 7.6, 1 mM MgCl2, 1 mM DDT (dithiothreitol), and 1 mM PMSF
(phenylmethylsulphonyl fluoride). All subsequent steps are
performed at 4.degree. C. The lysate is centrifuged in a benchtop
centrifuge at 800.times.g for 15 min; the supernatant serves as a
source for cytosol, mitochondria, and microsomal fractions. The
pellet containing nuclei is diluted in 8 ml of lysis buffer and
layered onto 4 ml of 0.9 M sucrose buffer (0.9 M sucrose, 50 mM
Tris-HCl pH 7.6, 1 mM MgCl2, 1 mM DDT, and 1 mM PMSF) and
centrifuged at 1000.times.g for 20 min at 4.degree. C. The
resulting pellet is resuspended in 8 ml of a 2 M sucrose buffer (2
M sucrose, 50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM DTT, and 1 mM
PMSF), layered onto 4 ml of 2 M sucrose buffer and pelleted by
ultracentrifugation at 150,000.times.g for 1 h (Beckman SW40.1
rotor). The nuclei are recovered as a pellet. The mitochondria are
isolated from the supernatant by re-centrifugation at 7500.times.g
for 20 min at 4.degree. C.; the resulting pellet is washed twice in
lysis buffer. Microsomes are pelleted by ultracentrifugation of the
post-mitochondrial cytoplasm at 100,000.times.g for 1 h in a
Beckman SW41 rotor. The supernatant served as the cytosolic
fraction (=cyto).
[0195] Organelle Extraction
[0196] Soluble mitochondrial proteins are extracted by incubating
the mitochondria in hypotonic lysis buffer (10 mM HEPES, pH 7.9, 1
mM DTT, 1 mM PMSF), for 30 min on ice. The suspension is sonicated
briefly and debris removed by centrifugation at 13,000.times.g for
30 min. The supernatant serves as the "mito 1" fraction. The
resulting insoluble pellet is resuspended in membrane detergent
extraction buffer (20 mM Tris-HCl, pH 7.8, 0.4 M NaCl, 15%
glycerol, 1 mM DTT, 1 mM PMSF, 1.5% Triton-X-100) and shaken gently
for 30 min followed by centrifugation at 13,000.times.g for 30 min;
the supernatant served as "mito 2" fraction.
[0197] Membrane-associated proteins are extracted by resuspending
the microsomes in membrane detergent extraction buffer. The
suspension is incubated with gentle shaking for 1 h and insoluble
debris removed by centrifugation at 13,000.times.g for 30 min. The
supernatant serves as the "micro" fraction.
[0198] Digestion of Organelle Extracts and MudPITAnalysis
[0199] An aliquot of about 100 .mu.g total protein (as determined
by Bradford assay) from each fraction is precipitated overnight
with 5 vol of ice-cold acetone at about 20.degree. C., followed by
centrifugation at 13,000.times.g for 15 min. The protein pellet is
solubilized in a small volume of 8 M urea, 50 mM Tris-HCl, pH 8.5,
1 mM DTT, for 1 h at 37.degree. C., followed by
carboxyamidomethylation with 5 mM iodoacetamide for 1 h at
37.degree. C. in the dark. The samples are then diluted to 4 M urea
with an equal vol of 100 mM ammonium bicarbonate, pH 8.5, and
digested with a 1:150-fold ratio of endoproteinase Lys-C (Roche
Diagnostics, Laval, Quebec, Canada) at 37.degree. C. overnight. The
next day, the samples are diluted to 2 M urea with an equal vol of
50 mM ammonium bicarbonate pH 8.5, supplemented with CaCl2 to a
final concentration of 1 mM, and incubated overnight with Poroszyme
trypsin beads (Applied Biosystems, Streetsville, Ontario, Canada)
at 30.degree. C. with rotation. The resulting peptide mixtures are
solid phase-extracted with SPEC-Plus PT C18 cartridges (Ansys
Diagnostics, Lake Forest, Calif.) according to the instructions of
the manufacturer and stored at -80.degree. C. until further use. A
fully-automated 20 h long 12-step multi-cycle MudPIT procedure is
set up as described (Kislinger, T., et al., Mol. Cell Proteom. 2
(2003) 96-106). Briefly, an HPLC quaternary pump is interfaced with
an LCQ DECA XP ion trap mass spectrometer (Thermo Finnigan, San
Jose, Calif.). A 100-.mu.m i.d. fused silica capillary microcolumn
(Polymicro Technologies, Phoenix, Ariz.) is pulled to a fine tip
using a P-2000 laser puller (Sutter Instruments, Novato, Calif.)
and packed with 8 cm of 5 .mu.m Zorbax Eclipse XDB-C18 resin
(Agilent Technologies, Mississauga, Ontario, Canada), followed by 6
cm of 5 .mu.m Partisphere strong cation exchange resin (Whatman,
Clifton, N.J.). Individual samples are loaded manually onto
separate columns using a pressure vessel. Chromatography solvent
conditions are exactly as described in Kislinger, T., et al., Mol.
Cell Proteom. 2 (2003) 96-106.
[0200] Protein Identification and Validation
[0201] The SEQUEST database search algorithm is used to match
peptide tandem mass spectra to peptide sequences in a
locally-maintained minimally redundant FASTA formatted database
populated with mouse and human protein sequences obtained from the
Swiss-Prot/TrEMBL and IPI databases. To statistically assess the
empirical False-Discovery Rate to control for, and hence, minimize
false positive identifications, all of the spectra are searched
against protein sequences in both the normal (Forward) and inverted
(Reverse) amino acid orientations (Kislinger, T., et al., Mol. Cell
Proteom. 2 (2003) 96-106). The STATQUEST filtering algorithm is
then applied to all putative search results to obtain a measure of
the statistical reliability (confidence score) for each candidate
identification (cutoff p-value.ltoreq.0.15, corresponding to an 85%
or greater likelihood of being a correct match). High-confidence
matches are parsed into an in-house SQL-type database using a
Perl-based script. The database is designed to accommodate database
search results and spectral information (scan headers) for multiple
peptides matching to a given protein, together with information
regarding the sample name, experiment number, MudPIT step,
organelle source, amino acid sequence, molecular mass, isoelectric
point, charge, and confidence level. Only those proteins with a
predicted confidence p value of 95% or more, and for which at least
two spectra are collectively detected, are retained for further
analysis.
EXAMPLE 3
Statistical Evaluation of the Data Obtained in the Model
Systems
[0202] 3.1 Statistical Methods Used to Generate p-Values of
Differential Expression for the R9C Mouse Model
[0203] The raw data obtained with the methods as described in
Example 2 consists of 6190 proteins each with spectral counts, the
sum of all spectra associated with the protein, for each of the 137
different experimental runs. The raw data, 6190 subset of proteins,
is subjected to global normalization which first separates the data
within each run into an equal number of groups, set at 100 for our
analysis, based on their spectral counts. LOESS (Cleveland, W. S.
and Devlin, S. J., Journal of the American Statistical Association
83 (1988) 596-610) is then performed on each group (1-100)
adjusting for differences in spectral counts across a set of genes
with similar spectral counts.
[0204] Based on our raw data we constructed two linear models, the
first model uses control/disease, time (8W, 16W, end) and location
(cyto, micro, mitol, mitoll) as factors and is described using:
run count=.beta.0+.beta.1time+.beta.2time.sup.2+.beta.3
location+.beta.4control (1)
[0205] The second model uses only time (8W, 16W, end) and location
(cyto, micro, mitol, mitoll) as factors and is described using:
run count=.beta.0+.beta.1time+.beta.2 time.sup.2+.beta.3 location
(2)
[0206] where .beta.0 is the intercept term and .beta.1, .beta.2,
.beta.3, and .beta.4 are the slope estimates for the variables
time, time squared, location, and control/disease.
[0207] The two models are compared using Anova, with the null
hypothesis being that there is no difference between the two
models. A low p-value then indicates that there is not enough proof
to say the two models are the same. The extra information indicates
the state (i.e., control/disease) appears to be a significant
component of the model. In order to extract proteins that have a
significant change in relative protein abundance between our
control and disease models our list of 6190 proteins is ranked
based on their computed p-values. This generates a set of 593
proteins with p-values<0.05.
[0208] In order to account for multiple hypothesis testing from the
above model the p-values are then corrected using false discovery
rate (FDR) correction, specifically Benjamini-Hochberg FDR
correction (Benjamini, Y., and Hochberg, Y., Journal of the Royal
Statistical Society B. 57 (1995) 289-300). This generates a set of
40 proteins with corrected p-values<0.05 for the R9C mouse
model.
[0209] 3.2 Statistical Methods Used to Generate p-Values of
Differential Expression for the Aortic Banding Mouse Model
[0210] From 68 experimental runs in the aortic banding mouse model
3152 proteins with spectral counts are identified. The same data
analysis is applied to the datasets for the aortic banding mouse
model as described above for the R9C mouse model.
EXAMPLE 4
Detection of the Marker IGFBP-7 by Western Blot Assay
[0211] Crude tissue lysates are obtained from R9C mouse heart
tissue samples. In brief, heart tissue is minced, ground up in a
dounce homogenizer and subjected to a centrifuge spin of 8,000 g
for 30 min to remove nuclei and cell debris. The supernatant is
used for Western Blotting.
[0212] SDS-PAGE and Western-Blotting are carried out using reagents
and equipment of Invitrogen, Karlsruhe, Germany. For each tissue
sample tested, 10 .mu.g of the cytosolic fraction are diluted in
reducing NuPAGE (Invitrogen) SDS sample buffer and heated for 10
min at 95.degree. C. Samples are run on 4-12% NuPAGE gels
(Tris-Glycine) in the MES running buffer system. The gel-separated
protein mixture is blotted onto nitrocellulose membranes using the
Invitrogen XCell II Blot Module (Invitrogen) and the NuPAGE
transfer buffer system. The membranes are washed 3 times in
PBS/0.05% TWEEN-20 (ICI Americas Inc.) and blocked with Roti-Block
blocking buffer (A151.1; Carl Roth GmbH, Karlsruhe, Germany) for 2
h. The primary antibody, rabbit polyclonal to calreticulin
(PA1-903, Affinity Bioreagents (ABR), Golden, Colo.) is diluted
1:500 in Roti-Block blocking buffer and incubated with the membrane
for 1 h. The membranes are washed 6 times in PBS/0.05% TWEEN-20.
The specifically bound primary IGFBP-7 antibody is labeled with a
POD-conjugated polyclonal anti-rat IgG antibody, diluted to 10
mU/ml in 0.5.times. Roti-Block blocking buffer. After incubation
for 1 h, the membranes are washed 6 times in PBS/0.05% TWEEN-20.
For detection of the bound POD-conjugated anti-rabbit antibody, the
membrane is incubated with the Lumi-Light.sup.PLUS Western Blotting
Substrate (Order-No. 2015196, Roche Diagnostics GmbH, Mannheim,
Germany) and exposed to an autoradiographic film.
[0213] Results of a typical experiment are shown in FIG. 3. A
strong overexpression of IGFBP-7 is observed in tissue samples
derived R9C experimental animals suffering from heart failure
versus tissue samples derived from a healthy mouse at matching time
points.
EXAMPLE 5
[0214] 5.1. ELISA for the Measurement of IGFBP-7 in Human Serum and
Plasma Samples
[0215] For detection of IGFBP-7 in human serum or plasma, a
sandwich ELISA is developed. For capture and detection of the
antigen, aliquots of an anti-IGFBP-7 polyclonal antibody from
R&D Systems (Catalogue number: AF 1334) is conjugated with
biotin and digoxygenin, respectively.
[0216] Streptavidin-coated 96-well microtiter plates are incubated
with 100 .mu.l biotinylated anti-IGFBP-7 polyclonal antibody for 60
min at 1 .mu.g/ml in 1.times. PBS solution. After incubation,
plates are washed three times with 1.times. PBS+0.02% TWEEN-20,
blocked with PBS+1% BSA (bovine serum albumen) and then washed
again three times with 1.times. PBS+0.02% TWEEN-20. Wells are then
incubated for 1.5 h with either a serial dilution of the
recombinant IGFBP-7 as standard antigen or with diluted serum or
plasma samples (1:50) from patients or control individuals,
respectively. After binding of IGFBP-7, plates are washed three
times with 1.times. PBS+0.02% TWEEN-20. For specific detection of
bound IGFBP-7, wells are incubated with 100 .mu.l of
digoxigenylated anti-IGFBP-7 polyclonal antibody for 60 min at 1
.mu.g/ml in 1.times. PBS+1% BSA. Thereafter, plates are washed
three times to remove unbound antibody. In a next step, wells are
incubated with 75 mU/ml anti-digoxigenin-POD conjugates (Roche
Diagnostics GmbH, Mannheim, Germany, Catalog No. 1633716) for 60
min in 1.times. PBS+1% BSA. Plates are subsequently washed six
times with the same buffer. For detection of antigen-antibody
complexes, wells are incubated with 100 .mu.l ABTS solution (Roche
Diagnostics GmbH, Mannheim, Germany, Catalog No. 11685767) and the
optical density (OD) is measured after 15 min at 405 and 492 nm
with an ELISA reader.
[0217] 5.2. Test of IGFBP7 ELISA in Samples Derived from Patients
with Heart Failure and Apparently Healthy Donors, Respectively
[0218] For the establishment and optimization of the IGFBP-7 ELISA
10 serum samples obtained from different patients with heart
failure (HF samples) and 10 sera of normal healthy donors (NHS) are
tested. After performing the assay procedure as described in 5.1
the following results (see Table 3) are obtained with these
samples:
TABLE-US-00003 TABLE 3 IGFBP7 ELISA results (assay development
samples) ng/mL Delta OD Conc [ng/mL * 50 Delta OD Conc Conc
calculated HF 5078 0.351 0.108 5.4 Median 0.421 0.15 7.33 5084
0.582 0.231 11.55 Min 0.229 0.03 1.55 5085 0.435 0.154 7.7 Max
0.957 0.42 21.00 5100 0.304 0.080 4 MW 0.4645 0.17 8.30 5101 0.537
0.208 10.4 5104 0.556 0.218 10.9 5107 0.407 0.139 6.95 5112 0.287
0.070 3.5 5113 0.957 0.420 21 5144 0.229 0.031 1.55 NHS 34 0.372
0.120 6 Median 0.256 0.05 2.48 35 0.254 0.048 2.4 Min 0.204 0.01
0.55 36 0.254 0.048 2.4 Max 0.377 0.12 6.10 37 0.288 0.070 3.5 MW
0.2718 0.06 2.90 38 0.258 0.051 2.55 39 0.234 0.035 1.75 40 0.263
0.055 2.75 41 0.214 0.019 0.95 42 0.204 0.011 0.55 43 0.377 0.122
6.1
[0219] As obvious from FIG. 4 the IGFBP-7 levels are in average
much higher in the sera obtained from patients with HF as compared
to the levels found in the samples obtained from control
individuals.
[0220] 5.3. IGFBP7 ELISA with Sera of Patients have HF and Obtained
Out of the Clinical Routine and Apparently Healthy Donors,
Respectively
[0221] In order to further evaluate the utility of the IGFBP-7
assays under routine clinical conditions a panel of sera from HF
patients (n=45) and of 35 sera from apparently healthy control
patients is investigated. As mentioned before, sera are diluted
1:50 in 1.times. PBS+1% BSA. Table 4 shows the result for these
extended panels:
TABLE-US-00004 TABLE 4 IGFBP7 ELISA results (extended panel with HF
samples from clinical routine) Heart Failure Sera (HF-Panel) Normal
Controls n = 45 Conc [ng/mL] n = 35 Conc [ng/mL] Sample No. Delta
OD measured calculated (*50) Sample No. Delta OD measured
calculated (*50) 4143 0.236 0.07 3.40 31 0.172 0.02 0.95 4144 0.236
0.07 3.35 32 0.334 0.13 6.65 4145 0.223 0.06 2.90 33 0.21 0.05 2.45
4146 0.244 0.07 3.65 44 0.257 0.08 4.10 4150 0.35 0.14 7.20 45
0.489 0.23 11.55 4151 0.347 0.14 7.10 46 0.224 0.06 2.95 4152 0.437
0.20 9.95 47 0.226 0.06 3.00 4153 0.235 0.07 3.35 48 0.183 0.03
1.40 4154 0.253 0.08 4.00 49 0.171 0.02 0.90 4155 0.271 0.09 4.60
50 0.29 0.11 5.25 4157 0.615 0.31 15.45 51 0.314 0.12 6.00 4158
0.419 0.19 9.40 52 0.301 0.11 5.60 4159 0.36 0.15 7.55 53 0.232
0.07 3.25 4161 0.232 0.07 3.25 54 0.235 0.07 3.35 4162 0.345 0.14
7.05 55 0.222 0.06 2.90 4163 0.27 0.09 4.55 56 0.343 0.14 7.00 4164
0.402 0.18 8.85 57 0.214 0.05 2.60 4170 0.833 0.44 22.20 58 0.27
0.08 3.95 4171 0.625 0.32 15.75 59 0.389 0.15 7.65 4172 0.56 0.28
13.75 60 0.214 0.04 2.05 4173 0.396 0.17 8.70 61 0.252 0.07 3.35
4174 0.52 0.25 12.55 62 0.183 0.02 0.90 4175 0.251 0.08 3.90 63
0.204 0.03 1.70 4176 0.352 0.13 6.55 64 0.208 0.04 1.85 4178 0.224
0.05 2.40 65 0.228 0.05 2.55 4181 0.491 0.21 10.70 66 0.337 0.12
6.05 4182 0.352 0.13 6.55 67 0.239 0.06 2.95 4187 0.766 0.38 18.80
68 0.235 0.06 2.80 4189 0.578 0.27 13.25 69 0.163 0.01 0.55 4190
0.28 0.09 4.30 70 0.224 0.05 2.40 4191 0.297 0.10 4.85 71 0.214
0.04 2.05 4192 0.317 0.11 5.45 72 0.265 0.08 3.80 4193 0.476 0.21
10.25 73 0.231 0.05 2.65 4194 0.428 0.18 8.85 74 0.25 0.07 3.30
4196 0.236 0.06 2.80 75 0.281 0.09 4.35 4198 0.333 0.12 5.95 Min
0.163 0.01 0.55 4199 0.342 0.13 6.25 Max 0.489 0.23 11.55 4200
0.298 0.10 4.90 MW 0.252 0.07 3.57 4202 0.556 0.25 12.60 Median
0.232 0.06 2.95 4203 0.895 0.45 22.65 4204 0.52 0.23 11.60 4205
0.463 0.20 9.85 4206 0.333 0.12 5.95 4212 0.267 0.08 3.85 4213
0.754 0.37 18.45 Min 0.223 0.05 2.40 Max 0.895 0.45 22.65 MW 0.405
0.17 8.43 Median 0.350 0.14 7.05
[0222] The data summarized in Table 4 have been used to calculate
the box-blots shown in FIG. 5. FIG. 5 demonstartes that there is
quite a difference in the average IGFBP-7 values as measured in
sera derived from patients with heart failure as compared to
IGFBP-7 values as measured in sera derived from apparently healthy
control individuals.
EXAMPLE 6
Marker Combinations Comprising the Marker IGFBP-7 in the Assessment
of Heart Failure
EXAMPLE 6.1
The Marker Combination NT-proBNP and IGFBP-7
[0223] The marker combination NT-proBNP and IGFBP-7 is evaluated
for the differentiation of patients in stage B and stages C plus D,
respectively. Diagnostic accuracy is assessed by analyzing
individual liquid samples obtained from well-characterized groups
of individuals, i.e., 50 individuals in stage B according to the
ACA/ACC criteria for classification of HF and 50 patients suffering
from HF and having stage C according to the ACA/ACC criteria for
classification of HF. NT-proBNP as measured by a commercially
available assay (Roche Diagnostics, NT-proBNP-assay (Cat. No. 03
121 640 160 for ELECSYS Systems immunoassay analyzer) and IGFBP-7
measured as described above are quantified in a serum sample
obtained from each of these individuals. ROC-analysis is performed
according to Zweig, M. H., and Campbell, G., supra. Discriminatory
power for differentiating patients in stage C from individuals in
stage B for the combination of IGFBP-7 with the established marker
NT-proBNP is calculated by regularized discriminant analysis
(Friedman, J. H., Regularized Discriminant Analysis, Journal of the
American Statistical Association 84 (1989) 165-175).
EXAMPLE 6.2
The Marker Combination Troponin T and IGFBP-7
[0224] The marker combination troponin T and IGFBP-7 is evaluated
for the differentiation of patients suffering from an acute cardiac
event from patients suffering from chronic heart disease,
respectively. Diagnostic accuracy is assessed by analyzing
individual liquid samples obtained from well-characterized groups
of individuals, i.e., 50 individuals diagnosed as having an acute
cardiac event and 50 individuals diagnosed as having chronic
cardiac disease. Troponin T as measured by a commercially available
assay (Roche Diagnostics, troponin T-assay (Cat. No. 201 76 44 for
ELECSYS Systems immunoassay analyzer) and IGFBP-7 measured as
described above are quantified in a serum sample obtained from each
of these individuals. ROC-analysis is performed according to Zweig,
M. H., and Campbell, G., supra. Discriminatory power for
differentiating patients in stage C from individuals in stage B for
the combination of IGFBP-7 with the established marker NT-proBNP is
calculated by regularized discriminant analysis (Friedman, J. H.,
J. of the American Statistical Association 84 (1989) 165-175).
EXAMPLE 6.3
The Marker Combination NT-proBNP and CRP
[0225] The marker combination C-reactive protein and IGFBP-7 is
evaluated for the differentiation of patients diagnosed as having a
cardiomyopathy versus controls not suffering from any confounding
heart disease, respectively. Diagnostic accuracy is assessed by
analyzing individual liquid samples obtained from
well-characterized groups of 50 individuals with cardiomyopathy and
of 50 healthy control individuals. CRP as measured by a
commercially available assay (Roche Diagnostics, CRP-assay
(Tina-quant C-reactive protein (latex) high sensitive assay--Roche
Cat. No. 11972855 216) and IGFBP-7 measured as described above are
quantified in a serum sample obtained from each of these
individuals. ROC-analysis is performed according to Zweig, M. H.,
and Campbell, G., supra. Discriminatory power for differentiating
patients in stage C from individuals in stage B for the combination
of IGFBP-7 with the established marker NT-proBNP is calculated by
regularized discriminant analysis (Friedman, J. H., J. of the
American Statistical Association 84 (1989) 165-175).
[0226] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be
appreciated by one skilled in the art, from a reading of the
disclosure that various changes in form and detail can be made
without departing from the true scope of the invention in the
appended claims.
Sequence CWU 1
1
11282PRTHomo sapiens 1Met Glu Arg Pro Ser Leu Arg Ala Leu Leu Leu
Gly Ala Ala Gly Leu 1 5 10 15 Leu Leu Leu Leu Leu Pro Leu Ser Ser
Ser Ser Ser Ser Asp Thr Cys 20 25 30 Gly Pro Cys Glu Pro Ala Ser
Cys Pro Pro Leu Pro Pro Leu Gly Cys 35 40 45 Leu Leu Gly Glu Thr
Arg Asp Ala Cys Gly Cys Cys Pro Met Cys Ala 50 55 60 Arg Gly Glu
Gly Glu Pro Cys Gly Gly Gly Gly Ala Gly Arg Gly Tyr 65 70 75 80 Cys
Ala Pro Gly Met Glu Cys Val Lys Ser Arg Lys Arg Arg Lys Gly 85 90
95 Lys Ala Gly Ala Ala Ala Gly Gly Pro Gly Val Ser Gly Val Cys Val
100 105 110 Cys Lys Ser Arg Tyr Pro Val Cys Gly Ser Asp Gly Thr Thr
Tyr Pro 115 120 125 Ser Gly Cys Gln Leu Arg Ala Ala Ser Gln Arg Ala
Glu Ser Arg Gly 130 135 140 Glu Lys Ala Ile Thr Gln Val Ser Lys Gly
Thr Cys Glu Gln Gly Pro 145 150 155 160 Ser Ile Val Thr Pro Pro Lys
Asp Ile Trp Asn Val Thr Gly Ala Gln 165 170 175 Val Tyr Leu Ser Cys
Glu Val Ile Gly Ile Pro Thr Pro Val Leu Ile 180 185 190 Trp Asn Lys
Val Lys Arg Gly His Tyr Gly Val Gln Arg Thr Glu Leu 195 200 205 Leu
Pro Gly Asp Arg Asp Asn Leu Ala Ile Gln Thr Arg Gly Gly Pro 210 215
220 Glu Lys His Glu Val Thr Gly Trp Val Leu Val Ser Pro Leu Ser Lys
225 230 235 240 Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser Asn Ser
Gln Gly Gln 245 250 255 Ala Ser Ala Ser Ala Lys Ile Thr Val Val Asp
Ala Leu His Glu Ile 260 265 270 Pro Val Lys Lys Gly Glu Gly Ala Glu
Leu 275 280
* * * * *