U.S. patent application number 16/221673 was filed with the patent office on 2019-04-25 for diagnosis of cardiovascular disease.
This patent application is currently assigned to Critical Care Diagnostics, Inc.. The applicant listed for this patent is Critical Care Diagnostics, Inc.. Invention is credited to Sven Jacobson, James V. Snider.
Application Number | 20190120852 16/221673 |
Document ID | / |
Family ID | 38668487 |
Filed Date | 2019-04-25 |
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United States Patent
Application |
20190120852 |
Kind Code |
A1 |
Snider; James V. ; et
al. |
April 25, 2019 |
DIAGNOSIS OF CARDIOVASCULAR DISEASE
Abstract
This invention relates to methods for the detection of
cardiovascular disease, e.g., acute coronary syndrome, heart
failure and/or pulmonary embolism, in high body mass index (BMI)
individuals, e.g., with a BMI of 25-29, or 30 or above, and those
with impaired renal function.
Inventors: |
Snider; James V.; (San
Diego, CA) ; Jacobson; Sven; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Critical Care Diagnostics, Inc. |
San Diego |
CA |
US |
|
|
Assignee: |
Critical Care Diagnostics,
Inc.
San Diego
CA
|
Family ID: |
38668487 |
Appl. No.: |
16/221673 |
Filed: |
December 17, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12299177 |
Sep 2, 2009 |
10203339 |
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PCT/US2007/067914 |
May 1, 2007 |
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16221673 |
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60796912 |
May 1, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/7155 20130101;
G01N 2800/52 20130101; G01N 2800/32 20130101; G01N 2800/226
20130101; G01N 33/6893 20130101; G01N 33/6869 20130101; G01N
2800/325 20130101 |
International
Class: |
C12Q 1/6883 20180101
C12Q001/6883; G01N 33/68 20060101 G01N033/68 |
Claims
1. A kit for diagnosing cardiovascular disease (CVD), the kit
comprising antibodies that specifically bind to ST2, BNP, and
D-dimer, or nucleic acid probes that specifically bind to nucleic
acids encoding ST2, BNP, and D-dimer, and instructions for use in a
method of diagnosing heart failure (HF) or a pulmonary embolism
(PE) in a subject who has one or both of (i) a body mass index
(BMI) of greater than or equal to 25, or (ii) impaired renal
function.
2. A method of diagnosing cardiovascular disease (CVD) in a subject
who has one or both of (i) a body mass index (BMI) of greater than
or equal to 25, or (ii) impaired renal function, the method
comprising: one or both of: (A) determining the subject's BMI, and
if the subject's BMI is equal to or greater than 25, selecting the
subject; or (B) evaluating the subject's renal function, and if the
subject has impaired renal function, selecting the subject; and
determining levels of BNP, D-dimers, and ST2 in a biological sample
from the subject; wherein the subject's BNP level, D-dimer level,
and ST2 level indicates whether the subject has CVD.
3. The kit of claim 1 or the method of claim 2, wherein the CVD is
heart failure (HF) or pulmonary embolism (PE).
4. The kit of claim 1 or the method of claim 2, wherein the BNP
level, D-dimer level, and ST2 level are detected in a biological
sample comprising blood, plasma, or serum.
5. The method of claim 2, wherein if the subject's BNP level is
less than 500 pg/mL, and the D-dimer level is less than 500
.mu.g/L, then the relationship of the ST2 level to a reference
level of ST2 indicates whether the subject has HF.
6. The method of claim 2, wherein if the subject's BNP level is
less than 100 pg/mL, and the D-dimer level is 500-4000 .mu.g/L,
then the relationship of the ST2 level to a reference level of ST2
indicates whether the subject has PE.
7. The method of claim 5, wherein the reference level of ST2
represents a level in a subject who does not have HF.
8. The method of claim 5, wherein the reference level of ST2 is
about 0.2 to 0.3 ng/ml of serum, and values above that level
indicate the presence of HF.
9. The method of claim 6, wherein the reference level of ST2
represents a level in a subject who does not have PE.
10. The method of claim 6, wherein the reference level of ST2 is
about 0.2 to 0.3 ng/ml of serum, and values above that level
indicate the presence of PE.
11. The method of claim 2, wherein the subject's BNP level is
100-500 pg/mL.
12. The method of claim 2, further comprising determining a level
in the subject of one or more other biomarkers.
13. The method of claim 12, wherein the other biomarkers are
selected from the group consisting of NT-proANP, ANP, troponin,
CRP, creatinine, Blood Urea Nitrogen (BUN), liver function enzymes,
albumin, and bacterial endotoxin.
14. The method of claim 2, wherein determining whether the subject
has impaired renal function comprises determining glomerular
filtration rate (GFR) and/or serum creatinine level, and the
subject has impaired renal function if they have a GFR or serum
creatinine level shown in the following table: TABLE-US-00015 Grade
GFR (ml/minute) Serum Creatinine (.mu.mol/litre) mild 20-50 150-300
moderate 10-20 300-700 severe <10 >700
15. The kit of claim 1, wherein a subject has impaired renal
function if they have a GFR or less than 50 ml/minute.
16. The kit of claim 1 or the method of claim 2, wherein the
subject has a BMI of greater than or equal to 30.
17. The kit of claim 1 or method of claim 2, wherein the subject
has a BMI of 25 to 29.
18. A method of diagnosing cardiovascular disease (CVD) in a
subject who has impaired renal function, the method comprising:
evaluating the subject's renal function, and if the subject has
impaired renal function, selecting the subject; and determining an
ST2 level in a biological sample from the subject; wherein the
relationship of the ST2 level to a reference level of ST2 indicates
whether the subject has CVD.
19. The method of claim 18, wherein the reference level of ST2
represents a level in a subject who does not have CVD.
20. The method of claim 18, wherein the reference level of ST2 is
about 0.2 to 03 ng/ml of serum, and values above that level
indicate the presence of CVD.
21. The method of claim 18, wherein the reference level of ST2
represents a level in a subject who does not have CVD.
22. The method of claim 18, wherein the CVD is heart failure (HF)
or pulmonary embolism (PE).
23. The method of claim 18, wherein the biological sample comprises
blood, plasma, or serum.
24. The method of claim 18, further comprising determining a level
in the subject of one or more other biomarkers.
25. The method of claim 24, wherein the other biomarkers are
selected from the group consisting of BNP, D-Dimer, NT-proANP, and
ANP troponin, CRP, creatinine, Blood Urea Nitrogen (BUN), liver
function enzymes, albumin, and bacterial endotoxin.
26. The method of claim 18, wherein determining whether the subject
has impaired renal function comprises determining glomerular
filtration rate (GFR) and/or serum creatinine level, and the
subject has impaired renal function if they have a GFR or serum
creatinine level shown in the following table: TABLE-US-00016 Grade
GFR (ml/minute) Serum Creatinine (.mu.mol/litre) mild 20-50 150-300
moderate 10-20 300-700 severe <10 >700
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/299,177, filed on Oct. 31, 2008, which is
the U.S. national stage under 35 U.S.C. .sctn. 371 of International
Patent Application Serial No. PCT/US2007/067914, filed on May 1,
2007, which claims the benefit under 35 U.S.C. .sctn. 119(e) to
U.S. Provisional Patent Application Ser. No. 60/796,912 filed on
May 1, 2006, the entire contents of which are hereby incorporated
by reference.
TECHNICAL FIELD
[0002] This invention relates to methods for the detection of heart
failure and pulmonary embolism in high body mass index (BMI)
individuals and those with impaired renal function.
BACKGROUND
[0003] Levels of natriuretic peptides such as B-type natriuretic
peptide (BNP) and N terminal-pro BNP (NT-proBNP) have been shown to
be diagnostic of cardiovascular disease (Clerico and Emdin, Clin.
Chem. 50:33-50 (2004)). However, it is known and accepted in the
field that certain subjects have levels of natriuretic peptide that
are lower than expected relative to a "normal" subject for the same
level of disease. The exact mechanism for this phenomenon is not
known. These subjects include people with impaired renal function
(Anwaruddin et al., J. Am. Coll. Cardiol. 47(1):91-7 (2006);
McCullough et al., Am. J. Kidney Dis. 41(3):571-9 (2003)), and
those who are overweight (Body Mass Index (BMI) of 25-29) or obese
(BMI.gtoreq.30) (Krauser et al., Am. Heart J. 149(4):744-50 (2005);
McCord et al., Arch. Intern. Med. 164(20):2247-52 (2004)).
SUMMARY
[0004] The present invention is based, at least in part, on the
surprising discovery that, unlike the natriuretic peptides (NPs),
the biomarker ST2 (also known as Interleukin 1 Receptor Like-1
(IL1RL1)) is not affected by high body mass index (BMI) or by
impaired renal function, and therefore provides better prognostic
and diagnostic information than NPs in subjects with high (BMI) or
impaired renal function. Thus, the methods described herein include
determining whether a subject has a high BMI and/or has renal
failure, and if the subject has one or both conditions, selecting
the subject, and determining levels of IL1LR1, and, optionally, BNP
and/or D-dimer in the subject. These methods can be used to
diagnose cardiovascular disease (CVD), e.g., acute coronary
syndrome (ACS), heart failure (HF), and pulmonary embolism (PE) in
the subject, e.g., in subjects with dyspnea.
[0005] In some embodiments, the methods include determining levels
of IL-33 in addition to or as an alternative to determining levels
of ST2.
[0006] In one aspect, the invention provides methods for diagnosing
cardiovascular disease (CVD), e.g., acute coronary syndrome (ACS),
heart failure (HF), or pulmonary embolism (PE) in a subject who has
a body mass index (BMI) of greater than or equal to 25. The methods
include determining the subject's BMI, and if the subject's BMI is
equal to or greater than 25, selecting the subject; and determining
levels of ST2, and optionally one or both of BNP level and D-dimer
level, in the subject's blood, plasma, or serum. The relationship
of the ST2 level to a reference level of ST2, e.g., a reference
level that represents a level of ST2 in a subject who does not have
CVD, indicates whether the subject has CVD. In some embodiments, if
the subject's BNP level is less than 500 pg/mL, e.g., 100-500
pg/mL, and the D-dimer level is less than 500 .mu.g/L, then the
relationship of the ST2 level to a reference level of ST2, e.g., a
reference level that represents a level of ST2 in a subject who
does not have HF, indicates whether the subject has HF. In some
embodiments, if the subject's BNP level is less than 100 pg/mL, and
the D-dimer level is 500-4000 .mu.g/L, then the relationship of the
ST2 level to a reference level of ST2, e.g., a reference level that
represents a level of ST2 in a subject who does not have PE,
indicates whether the subject has PE.
[0007] In another aspect, the invention provides methods for
diagnosing cardiovascular disease (CVD), e.g., acute coronary
syndrome (ACS), heart failure (HF), or pulmonary embolism (PE) in a
subject who has impaired renal function. The methods include
evaluating the subject's renal function, and if the subject has
impaired renal function, selecting the subject; and determining an
ST2 level, and optionally BNP level and/or D-dimer level, in the
subject's blood, plasma or serum. The relationship of the ST2 level
to a reference level of ST2, e.g., a reference level that
represents a level of ST2 in a subject who does not have CVD,
indicates whether the subject has CVD. In some embodiments, if the
subject's BNP level is less than 500 pg/mL, e.g., 100-500 pg/mL,
and the D-dimer level is less than 500 .mu.g/L, then the
relationship of the ST2 level to a reference level of ST2, e.g., a
reference level that represents a level of ST2 in a subject who
does not have HF, indicates whether the subject has HF. In some
embodiments, if the subject's BNP level is less than 100 pg/mL, and
the D-dimer level is 500-4000 .mu.g/L, then the relationship of the
ST2 level to a reference level of ST2, e.g., a reference level that
represents a level of ST2 in a subject who does not have PE,
indicates whether the subject has PE.
[0008] In some embodiments, the reference level represents a level
in a subject who does not have CVD, e.g., does not have ACS, HF,
and/or PE. In some embodiments, e.g., wherein the biomarker level
of ST2 is measured using an immunoassay, e.g., an enzyme-linked
immunosorbent assay (ELISA), e.g., as described in Example 1, the
reference level is about 0.2 to 0.3 ng/ml, e.g., the level can be
0.20, 0.23, 0.25, 0.27, or 0.29 ng/ml of serum, and values above
that level indicate the presence of CVD, e.g., ACS, HF and/or PE.
If an analytical technique other than the ELISA described in
Example 1 is employed, the reference ST2 level may be different
than described herein. However, the specific numbers recited herein
should be construed to be equivalent to corresponding numbers
generated using other analytical techniques.
[0009] In general, determining a level of ST2, BNP, and/or D-dimer
in a subject includes obtaining a biological sample from the
subject, contacting binding compositions to the sample, wherein the
binding compositions specifically bind to ST2, BNP and D-dimer, and
measuring or determining the specific binding of the binding
composition to the sample. The binding compositions can be, e.g.,
antibodies that bind specifically to ST2, BNP, and D-dimer
polypeptides (e.g., an anti-ST2 Ab, an anti-BNP Ab, and an
anti-D-dimer Ab), or oligonucleotide probes that bind specifically
to ST2, BNP and D-dimer polynucleotides (e.g., an ST2-specific
probe, a BNP-specific probe, and a D-dimer-specific probe).
[0010] The methods can also include determining levels of one or
more additional biomarkers, e.g., NT-proANP, proANP, ANP, troponin,
CRP, creatinine, Blood Urea Nitrogen (BUN), liver function enzymes,
albumin, and bacterial endotoxin.
[0011] In some embodiments, determining whether a subject has
impaired renal function includes determining a glomerular
filtration rate (GFR) and/or serum creatinine level. The subject
has mildly, moderately, or severely impaired renal function if they
have a GFR or serum creatinine level shown in Table 1:
TABLE-US-00001 TABLE 1 Grade GFR (ml/minute) Serum Creatinine
(.mu.mol/litre) mild 20-50 150-300 moderate 10-20 300-700 severe
<10 >700
[0012] Also provided herein are kits for diagnosing cardiovascular
disease (CVD), that include three different antibodies that
specifically bind to ST2, BNP, and D-dimer polypeptides,
respectively, or three different nucleic acid probes that
specifically bind to nucleic acids encoding ST2, BNP, and D-dimer,
respectively, and instructions for use in a method described
herein.
[0013] "Upregulated," as used herein, refers to increased
expression of a gene and/or its encoded polypeptide. "Increased
expression" refers to increasing (i.e., to a detectable extent)
replication, transcription, and/or translation of IL-33, since
upregulation of any of these processes results in an increase in
concentration/amount of the polypeptide encoded by the gene.
Conversely, "downregulation," or "decreased expression" as used
herein, refers to reduced replication, transcription, and/or
translation of the IL-33 gene and/or its encoded polypeptide. The
upregulation or downregulation of gene expression can be directly
determined by detecting an increase or decrease, respectively, in
the level of mRNA for the gene, or the level of protein expression
of the gene-encoded polypeptide, using any suitable means known to
the art, such as nucleic acid hybridization or antibody detection
methods, respectively, and in comparison to controls. "Expression,"
as used herein, refers to nucleic acid and/or polypeptide
expression.
[0014] As used herein, a "subject" is a mammal, e.g., a human. In
all embodiments, human nucleic acids, polypeptides, and human
subjects can be used.
[0015] As used herein, a "biological sample" includes one or more
of blood, serum, plasma, urine, and body tissue. In some
embodiments, a sample is a serum or blood sample.
[0016] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control.
[0017] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
DESCRIPTION OF DRAWINGS
[0018] FIG. 1 is a Receiver Operating Characteristics (ROC) curve
of the Second Prospective Randomized Amlodipine Survival Evaluation
(PRAISE-2) study, illustrating the characteristics of the study
population for age, weight, height, BMI, left ventricular ejection
fraction (LVEF), creatinine, ST2t1, norepinephrine (NEM,
epinephrine (Et1), dopamine (DAt1), angiotensin (ANGt1),
malondialdehyde (MDAt1), adrenolutin (ADRt1), ANPt1, and BNPt1.
"t1" refers to a level taken at a first time.
[0019] FIG. 2 is a ROC curve for BNP and ST2 Ratio in the PRAISE-2
study; the two measures have similar AUC, with BNP somewhat
higher.
[0020] FIG. 3 is a ROC curve for prognostic utility of BNP and ST2
ratio in high BMI individuals; in this case, ST2 ratio has a
greater AUC.
[0021] FIG. 4 is a box graph of ST2 levels in subjects with various
BMIs (<25, 25-29, and >30), showing no significant difference
in ST2 levels between the BMIs.
[0022] FIGS. 5A-B are box graphs illustrating mean Glomerular
Filtration Rate (GFR, 5A) and ST2 levels (5B) in a population of
133 subjects with moderate to severe renal insufficiency.
[0023] FIG. 6 is a bar graph illustrating the distribution of ST2
levels in the population described in Example 3, showing that the
vast majority of subjects in the population have levels of ST2 that
are below 0.2 ng/ml.
DETAILED DESCRIPTION
[0024] Clinical evaluation of cardiovascular disease (CVD) using
natriuretic peptides (NPs) in subjects with high body mass index
(BMI) or impaired renal function is complicated by the fact that
these subjects have levels of natriuretic peptide that are lower
than expected relative to a "normal" subject for the same level of
disease. The exact mechanism for this phenomenon is not known.
However, one theory, not meant to be limiting, is that lower NP
levels in obese and overweight subjects and those with impaired
renal function may be related to the clearance mechanisms for NPs,
which may have both a renal and epithelial component. ST2, although
possibly produced in a similar manner as NPs, does not suffer from
these limitations. Therefore, the methods described herein include
the use of ST2 (and/or IL-33, the ligand for ST2) in these special
subjects, for whom NPs may provide misleading information.
[0025] General Methodology
[0026] General methods for using levels of ST2 for diagnosis are
described in, e.g., U.S. Pat. App. No. 2004/0048286 to Lee et al.,
the entire contents of which are incorporated herein by reference.
The methods described herein are particularly useful in populations
of subjects for whom NPs are less useful in the diagnosis and
prognosis of CVD. These subjects include those with high BMI, e.g.,
overweight subjects (BMI of 25-29) or obese subjects
(BMI.gtoreq.30). Thus, in some embodiments, the methods include
determining a subject's BMI, and if the subject is overweight or
obese, selecting the patient (e.g., selecting the subjects on the
basis of their BMI). These subjects also include those with renal
impairment. Thus, in some embodiments, the methods include
determining whether a subject has impaired renal function, and if
the subject has impaired renal function, selecting the patient.
[0027] In general, the methods described herein include evaluating
levels of ST2 in a biological sample (e.g., a blood, serum, plasma,
urine, or body tissue sample), and optionally BNP and/or D-dimer in
a subject, e.g., a mammal, e.g., a human. These levels provide
information regarding the presence of CVD, e.g., HF and/or PE in a
subject. For example, a diagnosis of CVD, e.g., HF in a subject
with an ambiguous level of BNP can be confirmed by the presence of
elevated ST2 and low D-dimer levels. A diagnosis of CVD, e.g., PE
in a subject with ambiguous levels of D-dimer can be confirmed by
the presence of high ST2 and low BNP.
[0028] Evaluating circulating levels of ST2, BNP, or D-dimer in a
subject typically includes obtaining a biological sample, e.g.,
serum or blood, from the subject. Levels of ST2, BNP, and D-dimer
in the sample can be determined by measuring levels of polypeptide
in the sample, using methods known in the art and/or described
herein, e.g., immunoassays such as enzyme-linked immunosorbent
assays (ELISA). Alternatively, levels of ST2, BNP, and D-dimer mRNA
can be measured, again using methods known in the art and/or
described herein, e.g., by quantitative PCR or Northern blotting
analysis.
[0029] An antibody that "binds specifically to" an antigen, binds
preferentially to the antigen in a sample containing other
proteins. The term "antibody" as used herein refers to an
immunoglobulin molecule or immunologically active portion thereof,
i.e., an antigen-binding portion. Examples of immunologically
active portions of immunoglobulin molecules include F(ab) and
F(ab').sub.2 fragments which can be generated by treating the
antibody with an enzyme such as pepsin. The antibody can be
polyclonal, monoclonal, recombinant, e.g., a chimeric or humanized,
fully human, non-human, e.g., murine, monospecific, or single chain
antibody. In some embodiments it has effector function and can fix
complement.
[0030] A "probe" is a nucleic acid that is at least 10, and less
than 200 (typically less than about 100 or 50) base pairs in
length. A probe that "binds specifically to" a target nucleic acid
hybridizes to the target under high stringency conditions. As used
herein, the term "hybridizes under high stringency conditions"
describes conditions for hybridization and washing. As used herein,
high stringency conditions are 0.5M sodium phosphate, 7% SDS at
65.degree. C., followed by one or more washes at 0.2.times.SSC, 1%
SDS at 65.degree. C. Methods for performing nucleic acid
hybridization assays are known to those skilled in the art and can
be found in Ausubel et al., Eds., Current Protocols in Molecular
Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
[0031] Detection can be facilitated by coupling (e.g., physically
linking) the antibody or probe to a detectable substance (e.g.,
antibody labeling). Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride, quantum dots,
or phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
materials include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0032] Diagnostic assays can be used with biological matrices such
as live cells, cell extracts, cell lysates, fixed cells, cell
cultures, bodily fluids, or forensic samples. Conjugated antibodies
useful for diagnostic or kit purposes, include antibodies coupled
to dyes, isotopes, enzymes, and metals, see, e.g., Le Doussal et
al., New Engl. J. Med. 146:169-175 (1991); Gibellini et al., J.
Immunol. 160:3891-3898 (1998); Hsing and Bishop, New Engl. J. Med.
162:2804-2811 (1999); Everts et al., New Engl. J. Med. 168:883-889
(2002). Various assay formats exist, such as radioimmunoassays
(RIA), ELISA, and lab on a chip (U.S. Pat. Nos. 6,176,962 and
6,517,234).
[0033] Known techniques in biochemistry and molecular biology can
be used in the methods described herein (see, e.g., Maniatis et
al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1982); Sambrook and
Russell, Molecular Cloning, 3.sup.rd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (2001); Wu, Recombinant
DNA, Vol. 217, Academic Press, San Diego, Calif (1993); and Ausbel
et al., Current Protocols in Molecular Biology, Vols. 1-4, John
Wiley and Sons, Inc. New York, N.Y. (2001)).
[0034] Once a level of ST2 has been determined, the level can be
compared to a reference level, or directly correlated with a value
known to correspond to the presence or absence of CVD. In some
embodiments, the reference level will represent a threshold level,
above which the subject has CVD, e.g., ACS, PE, or HF, and/or has a
given severity of CVD, e.g., ACS, HF, or PE, e.g., severe disease.
The reference level chosen may depend on the methodology used to
measure the levels of ST2. For example, in some embodiments, where
circulating levels of soluble ST2 are determined using an
immunoassay, e.g., as described herein, the reference level is
about 0.2 to 0.3 ng/ml, e.g., 0.20, 0.23, or 0.29 ng/ml of serum,
and a level of ST2 above that reference level indicates that the
subject has CVD, e.g., ACS, PE, or HF, and/or has severe CVD, e.g.,
severe ACS, PE, or HF; these reference levels apply when the levels
are determined using the method describe in Example 1 herein. In
some embodiments, the reference level is a range of levels.
[0035] In some embodiments, the methods described herein include
determining levels of IL-33 in addition to, or as an alternative
to, ST2. In some embodiments, both levels of ST2 and IL-33 are
determined, and the information from the comparison of both
biomarkers with their respective reference levels provides
cumulative information regarding the presence of CVD, and/or
presence of severe CVD in the subject. In some embodiments, the
ratio of ST2 to IL-33 may be determined, and the ratio compared to
a reference ratio that represents a threshold ratio above which the
subject has CVD, and/or has severe CVD. Alternatively or in
addition, the presence and/or levels of IL-33/ST2 complexes can be
determined and compared with a reference level to provide
information regarding the presence of CVD, e.g., ACS, PE, or HF, in
a subject; for example, levels of the complex above a selected
threshold would indicate that the subject has CVD, e.g., ACS, PE,
or HF.
[0036] In some embodiments, the methods include the use of
additional diagnostic methods. Any diagnostic methods known in the
art can be used, and one of skill in the art will be able to select
diagnostic methods that are appropriate for the subject's symptoms.
In some embodiments, the methods described herein include other
diagnostic methods in addition to or as an alternative to the
measurement of other biomarkers, e.g., physical measurements of
lung function or cardiac function as are known in the art.
[0037] Thus, the methods described herein can also include
measuring levels of ST2, optionally BNP and/or D-dimer, and one or
more additional biomarkers, e.g., biomarkers that aid in the
subject's diagnosis. As one example, for a subject who has chest
pain or dyspnea, biomarkers indicative of cardiac or cardiovascular
disease can be measured, e.g., cardiac troponin (cTn), e.g., cTnl
or cTnT, NT-proBNP, proBNP, NT-proANP, proANP, and/or ANP;
alternatively or in addition, additional biomarkers of pulmonary
disease can be measured. Thus, in subjects presenting with symptoms
that include MI in their differential diagnoses, the methods can
include measuring levels of cTnl, to determine whether the subject
is having an MI. One of skill in the art will appreciate that there
are a number of additional diagnostic methods that can be used,
depending on the situation and the subject's condition.
[0038] Also included herein are kits that include a reagent for the
detection of ST2, BNP, and D-dimer polypeptide or nucleic acid,
e.g., antibodies (i.e., antibodies that bind specifically to one of
ST2, BNP, and D-dimer polypeptides), or nucleic acid probes (i.e.,
probes that are complementary to all or part of one of ST2, BNP,
and D-dimer nucleic acids) and instructions for use in a method
described herein.
[0039] The methods described herein are useful in the diagnosis of
subjects with CVD, e.g., ACS, PE, or HF. In the methods described
herein, if an overweight or obese subject (e.g., a subject with a
BMI of 25-29, or 30 or above) has ambiguous, e.g., low or moderate,
BNP (i.e., <500 pg/ml of serum), D-dimer levels of less than 500
.mu.g/L of plasma, and elevated ST2 (e.g., levels above a
reference, e.g., 0.2 ng/ml of serum), then the subject can be
diagnosed with CVD, e.g., HF and treated accordingly, e.g., with
surgical or pharmaceutical intervention, and/or lifestyle change,
in spite of the low or moderate BNP levels.
[0040] In the methods described herein, if a subject (e.g., a
subject with a BMI of 25-29, or 30 or above) has low BNP (i.e.,
<100 pg/ml of serum), ambiguous D-dimer levels, e.g., 500-4000
.mu.g/L of plasma, and elevated ST2 (e.g., levels above a
reference, e.g., 0.2 ng/ml of serum), then the subject can be
diagnosed with CVD, e.g., PE, and treated accordingly, e.g., with
anticoagulant therapy, in spite of their ambiguous D-dimer
levels.
[0041] ST2/Interleukin 1 Receptor-Like 1 (IL1RL1)
[0042] The ST2 gene is a member of the interleukin-1 receptor
family, whose protein product exists both as a trans-membrane form,
as well as a soluble receptor that is detectable in serum (Kieser
et al., FEBS Lett. 372(2-3):189-93 (1995); Kumar et al., J. Biol.
Chem. 270(46):27905-13 (1995); Yanagisawa et al., FEBS Lett.
302(1):51-3 (1992); Kuroiwa et al., Hybridoma 19(2):151-9 (2000)).
ST2 was recently described to be markedly up-regulated in an
experimental model of heart failure (Weinberg et al., Circulation
106(23):2961-6 (2002)), and preliminary results suggest that ST2
concentrations may be elevated in those with chronic severe HF
(Weinberg et al., Circulation 107(5):721-6 (2003)) as well as in
those with acute myocardial infarction (MI) (Shimpo et al.,
Circulation 109(18):2186-90 (2004)).
[0043] The transmembrane form of ST2 is thought to play a role in
modulating responses of T helper type 2 cells (Lohning et al.,
Proc. Natl. Acad. Sci. U.S.A. 95(12):6930-5 (1998); Schmitz et al.,
Immunity 23(5):479-90 (2005)), and may play a role in development
of tolerance in states of severe or chronic inflammation (Brint et
al., Nat. Immunol. 5(4):373-9 (2004)), while the soluble form of
ST2 is up-regulated in growth stimulated fibroblasts (Yanagisawa et
al., 1992, supra). Experimental data suggest that the ST2 gene is
markedly up-regulated in states of myocyte stretch (Weinberg et
al., 2002, supra) in a manner analogous to the induction of the BNP
gene (Bruneau et al., Cardiovasc. Res. 28(10):1519-25 (1994)).
[0044] Tominaga, FEBS Lett. 258:301-304 (1989), isolated murine
genes that were specifically expressed by growth stimulation in
BALB/c-3T3 cells; they termed one of these genes St2 (for Growth
Stimulation-Expressed Gene 2). The St2 gene encodes two protein
products: ST2 (IL1RL1), which is a soluble secreted form; and ST2L,
a transmembrane receptor form that is very similar to the
interleukin-1 receptors. The HUGO Nomenclature Committee designated
the human homolog of ST2, the cloning of which was described in
Tominaga et al., Biochim. Biophys. Acta. 1171:215-218 (1992), as
Interleukin 1 Receptor-Like 1 (IL1RL1). The two terms are used
interchangeably herein.
[0045] The mRNA sequence of the shorter, soluble isoform of human
ST2 can be found at GenBank Acc. No. NM_003856.2, and the
polypeptide sequence is at GenBank Acc. No. NP_003847.2; the mRNA
sequence for the longer form of human ST2 is at GenBank Acc. No.
NM_016232.4; the polypeptide sequence is at GenBank Acc. No.
NP_057316.3. Additional information is available in the public
databases at GeneID: 9173, MIM ID #601203, and UniGene No. Hs.66.
In general, in the methods described herein, the soluble form of
ST2 polypeptide is measured.
[0046] Methods for detecting and measuring ST2 are known in the
art, e.g., as described in U.S. Pat. Pub. Nos. 2003/0124624,
2004/0048286 and 2005/0130136, the entire contents of which are
incorporated herein by reference. Kits for measuring ST2
polypeptide are also commercially available, e.g., the ST2 ELISA
Kit manufactured by Medical & Biological Laboratories Co., Ltd.
(MBL International Corp., Woburn, Mass.), no. 7638. In addition,
devices for measuring ST2 and other biomarkers are described in
U.S. Pat. Pub. No. 2005/0250156.
[0047] In some embodiments, the level of ST2 is determined once,
e.g., at presentation. In some embodiments, the level of ST2 is
determined at one or more of 2, 4, 6, 8, 12, 18, and/or 24 hours,
and/or 1-7 days after the onset of symptoms.
[0048] In some embodiments, the level of ST2 is determined more
than once; in that case, the higher measurement can be used. In
embodiments where the level of ST2 is determined more that once,
the highest level can be used, or the change in levels can be
determined and used. Levels of ST2 can also be determined multiple
times to evaluate a subject's response to a treatment. For example,
a level of ST2 taken after administration of a treatment, e.g., one
or more doses or rounds of a treatment, can be compared to levels
of ST2 before the treatment was initiated, e.g., a baseline level.
The change in ST2 levels would indicate whether the treatment was
effective; e.g., a reduction in ST2 levels would indicate that the
treatment was effective.
[0049] In some embodiments, the methods include determining the
identity of the nucleotide sequence at RefSNP ID: rs1041973.
[0050] Interleukin-33 (IL-33)
[0051] In the methods described herein, IL-33 can be measured
instead of or in addition to ST2.
[0052] IL-33 was recently identified as the ligand for ST2, and the
presence of increased levels of IL-33 in various inflammatory
disorders has been described (see Schmitz et al., Immunity
23(5):479-90 (2005); U.S. Pat. Pub. No. 2005/0203046). The ratio of
ST2 to IL-33 can also be determined.
[0053] IL-33 protein is expressed as an inactive molecule,
pre-IL-33, that is activated after cleavage by Caspase I resulting
in the active IL-33 peptide as well as the cleavage peptide
product, pro-IL-33. Therefore, the methods described herein can
include measuring one, two, or all three of mature IL-33,
pre-IL-33, and/or pro-IL-33, all of which are included in the term
"IL-33."
[0054] The nucleic acid sequence of IL-33 can be found at GenBank
Acc. No. NM_033439.2, and the polypeptide sequence is at GenBank
Acc. No. NP_254274.1. Additional information is available in the
public databases at GenelD: 90865, MIM ID #*608678, and UniGene No.
Hs.348390. IL-33 is also known as Chromosome 9 Open Reading Frame
26 (C9ORF26); Nuclear Factor from High Endothelial Venules (NFHEV);
and Interleukin 33. See also Baekkevold et al., Am. J. Path. 163:
69-79 (2003).
[0055] Methods for measuring levels of IL-33 polypeptide and
nucleic acid are known in the art, see, e.g., Schmitz et al.,
Immunity 23(5):479-90 (2005); U.S. Pat. Pub. No. 2005/0203046.
[0056] Body Mass Index (BMI)
[0057] Obesity influences the expression of BNP in chronic HF. It
is known that there is a significant inverse relationship between
body mass index (BMI) and BNP levels.
[0058] BMI is determined by weight relative to height, and equals a
person's weight in kilograms divided by height in meters squared
(BMI=kg/m.sup.2). Accepted interpretations are given in Table
2.
TABLE-US-00002 TABLE 2 Category BMI Underweight .ltoreq.18.5 Normal
weight 18.5-24.9 Overweight 25-29.9 Obese .gtoreq.30
[0059] Thus, the methods described herein can include determining a
subject's height, determining a subject's weight, and calculating
BMI from the values determined thereby. Alternatively, the methods
described herein can include reviewing a subject's medical history
to determine their BMI.
[0060] In some embodiments, the methods described herein include
selecting subjects who have a BMI of 30 or above (i.e., obese
subjects).
[0061] Renal Function
[0062] Measures of renal function can include serum creatinine
results as well as estimated glomerular filtration rate (GFR) (see,
e.g., Levey et al., Ann. Intern. Med. 130(6):461-70 (1999)). Renal
impairment is usually divided into three grades, shown in Table
3.
TABLE-US-00003 TABLE 3 Grade GFR (ml/minute) Serum Creatinine
(.mu.mol/litre) mild 20-50 150-300 moderate 10-20 300-700 severe
<10 >700
[0063] Thus, the methods described herein can include determining a
subject's serum creatinine levels and/or GFR. Alternatively, the
methods described herein can include reviewing a subject's medical
history to determine their serum creatinine levels and/or GFR.
[0064] BNP
[0065] B-type natriuretic peptide (BNP), is a marker of heart
failure. Levels of BNP can be determined, e.g., in whole blood or
serum, using standard methodology. For example, a number of assay
kits are commercially available, e.g., the Triage BNP Test
(Biosite, Inc., San Diego, Calif.), a point-of-care assay that
whole blood or plasma and produces results in about 15 minutes; a
chemiluminescent sandwich immunoassay (Bayer HealthCare
Diagnostics, Tarrytown, N.Y.) for BNP that is run on the ADVIA
Centaur and ACS:180 platforms; a microparticle-based immunoassay
(Abbott Laboratories, Abbott Park, Ill.) for BNP that is run on the
AxSYM platform; and a chemiluminescent immuno-enzymatic assay
(Biosite, Inc., San Diego, Calif.) for BNP that is run on the
following Beckman Coulter platforms: Access, Access 2, Synchron LXI
and the UniCel DXI. An electrochemiluminescent assay (Roche
Diagnostics, Indianapolis, Ind.) available for measuring
NT-proBNP.
[0066] The reference ranges for BNP and NTproBNP vary depending on
a number of factors. The following ranges are for use where BNP
levels are measured using an ELISA-type method, and one of skill in
the art will be able to determine what levels obtained using other
methods are equivalent. If the BNP level is >500 pg/mL, then HF
is highly likely. Levels of BNP of 100-500 pg/mL are often
described as a "grey zone," in which diagnosis is less certain. In
lean subjects, if the BNP is <100 pg/mL, then HF is unlikely,
however, obesity influences the expression of BNP in chronic HF
(Mehra et al., J Am Coll Cardiol. 43(9):1590-1595 (2004)), so
levels of <100 pg/mL do not rule out heart failure in obese
subjects (Silver et al., Cong. Heart Fail. 10(5 suppl. 3):1-30
(2004)).
[0067] D-Dimers
[0068] A D-dimer is a stable end-product of fibrin degradation.
Increased levels of D-dimers in the blood are associated with
enhanced fibrin formation and fibrinolysis, and thus are diagnostic
of conditions associated with these processes.
[0069] Methods for assaying D-dimer levels in the blood are known
in the art. Commercially available assay kits include the VIDAS
D-Dimer Exclusion (bioMerieux, Durham, N.C.) a rapid, automated
ELISA; Minutex.RTM. D-dimer, Biopool Auto-Dimer.TM. (an automated,
immunoturbidimetric assay for analysers reading at wavelengths of
540-880 nm), MiniQuant.TM., AMAX Auto D-Dimer.TM. (Automated
D-dimer assay for AMAX instruments), and Accuclot D-Dimer.TM.
assays (a semi-quantitative assay) (Trinity Biotech, Bray, Co.
Wicklow, Ireland); and the HemosIL.TM. D-Dimer assay
(Instrumentation Laboratory, distributed by Beckman Coulter), a
fully automated immunoturbidimetric assay.
[0070] Plasma D-Dimer levels above 4000 .mu.g/L are highly
correlated with the presence of acute PE, and levels below 500 can
be used to rule out PE (see, e.g., Perrier et al., Am. J. Respir.
Crit. Care Med., 156(2):492-496 (1997)). Plasma D-dimer level of
500-4000 .mu.g/L are more ambiguous, due to the number of
conditions that activate the coagulation and fibrinolytic
processes.
[0071] Other Biomarkers
[0072] The methods described herein can also include measuring
levels of other biomarkers in addition to ST2 and/or IL-33.
Suitable biomarkers include NT-proBNP, proBNP, BNP, NT-proANP,
proANP, ANP, troponin, CRP, creatinine, D-dimers (degradation
products of cross-linked fibrin, whose level becomes elevated
following clot formation), BUN (blood-urea-nitrogen), liver
function enzymes, albumin, IL-6 and/or bacterial endotoxin. Methods
for measuring these biomarkers are known in the art, see, e.g.,
U.S. Pat. Pub. Nos. 2004/0048286 and 2005/0130136 to Lee et al.;
Dhalla et al., Mol. Cell Biochem. 87:85-92 (1989); Moe et al., Am.
Heart J. 139:587-95 (2000), the entire contents of which are
incorporated herein by reference. Liver function enzymes include
Alanine transaminase (ALT); Aspartate transaminase (AST) Alkaline
phosphatase (ALP) and Total bilirubin (TBIL).
[0073] In these embodiments, levels of ST2 and one or more
additional biomarkers are determined, and the information from the
comparison of the biomarkers with their respective reference levels
provides additional information regarding the presence of CVD in
the subject, and/or the level of severity of CVD in the
subject.
EXAMPLES
[0074] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Example 1
Sandwich ELISA Assay
[0075] This example uses the ST2 ELISA Kit manufactured by Medical
& Biological Laboratories Co., Ltd. (MBL International Corp.,
Woburn, Mass.), no. 7638. This kit is a sandwich ELISA assay
utilizing monoclonal antibodies for both capture and detection.
This procedure is intended to analyze a full plate of samples
assayed in replicates at a 1:3 dilution factor and closely follows
the manufacturers' protocol. Kits should be stored at 4.degree. C.
until use. The procedure described in this example is optimized for
human serum or plasma collected in citrate or EDTA anticoagulant
tubes. Plasma collected in heparin anticoagulant tubes should not
be used in this assay as heparin binds ST2 and inhibits measurement
by this ELISA protocol. Plasma or serum samples may be used fresh
or stored frozen. This assay is not adversely affected by up to 3
freeze and thaw cycles of plasma samples.
[0076] Reagents should be prepared fresh from a new kit immediately
before performing the assays. Allow the kit to equilibrate to room
temperature prior to use. Reagents not explicitly discussed below
are provided by the manufacturer ready to use. [0077] 1. Wash
solution--wash solution is provided by the manufacturer as a
10.times. concentrate solution. To make 1 liter of wash solution
dilute 100 ml of the 10.times. concentrate provided with 900 ml of
distilled water. [0078] 2. Detector solution--the detector solution
is prepared by diluting the detector concentrate 1:101 with the
detector diluent. For a full 96 well plate of samples 10 ml of
detector solution is required. To prepare 10 ml of detector
solution use a pipette to transfer 10 ml of the blue colored
detector diluent to a 15 ml orange top polypropylene tube. Ad 100
.mu.l of the detector concentrate to this volume of detector
diluent. [0079] a. NOTE: this reagent should be prepared during the
first assay incubation step. [0080] 3. Calibrator
stock--reconstitute the calibrator protein by dissolving the
lyophilized protein in the amount of distilled water defined by the
manufacturer for this manufacturing lot to yield a stock solution
of 8 ng/ml. This volume specification is included in the product
insert.
[0081] Preparation of standards and samples: [0082] All of the
following should be prepared in labeled 1.5 ml polypropylene tubes
to be transferred to the assay plate with the P200 pipetter.
[0083] Standards:
[0084] The standard curve is prepared by making 2 fold serial
dilutions of the 8 ng/ml stock solution. [0085] 1. Using a P1000
pipette transfer 250 .mu.l of Assay Diluent to 8 1.5 ml
polypropylene tubes labeled S1-S8 [0086] 2. Using the same P1000
pipette transfer 250 .mu.l of the 8 ng/ml Calibrator stock solution
to tube S1. This tube is now 4 ng/ml calibrator protein. [0087] a.
Mix thoroughly by gently pipetting 3 times being careful not to
create bubbles. [0088] 3. Using the same P1000 pipette and a fresh
tip for each of the following transfer 250 .mu.l of the reagent in
tube S1 to tube S2, repeat the mixing. [0089] 4. Repeat step 3 for
S2 to S3, S3 to S4, S4 to S5, S5 to S6 and S6 to S7. S8 will be the
reagent blank so do not transfer the calibrant protein to this
well. [0090] a. Tubes S1-S6 and S8 will now have 250 .mu.l of
reagent and tube S7 will have 450 .mu.l.
[0091] Samples:
[0092] The plate is set up so that each sample is analyzed as a 1:3
dilution in duplicate. An exemplary set up is shown below in Table
4. [0093] 1. Label a 1.5 ml polypropylene tube for each sample.
[0094] 2. Using the P200 pipette transfer 160 .mu.l of Assay
Diluent to each tube. [0095] 3. Using a P200 pipette transfer 80
.mu.l of serum or plasma from sample 1 to tube [0096] 1. Mix
carefully by pipetting 3 times without making bubbles. [0097] 4.
Continue transferring samples to the sample tubes by repeating step
2 for each sample.
[0098] Procedure: [0099] 1. Use the P200 pipette transfer the
standards and diluted serum samples quickly to the 96 well assay
plate. [0100] a. Set the P200 pipette for 100 .mu.l [0101] b.
Transfer 100 .mu.l of the standard curve dilutions to each of
columns 1 & 2 in the assay plate [0102] c. Transfer 100 .mu.l
of each of the serum samples to the assay plate in exactly the same
positions as shown in the plate map below. [0103] 2. Cover the
assay plate with the provided shield and incubate at room
temperature for 60 minutes. [0104] 3. Using the plate autowasher
wash the plate 4 times. [0105] 4. Detector: using the 8 channel
multichannel pipette transfer 100 .mu.l of the detector solution to
each well and incubate at room temperature for 60 minutes. [0106]
a. NOTE: this reagent was to be prepared during the first
incubation step. [0107] b. NOTE: use a disposable reagent vessel
for this reagent addition.
[0108] ALWAYS use a fresh disposable reagent vessel for each
reagent. It is not necessary to change pipette tips during this
step. [0109] 5. Wash the plate as in step 3 [0110] 6. Substrate:
using the 8 channel multichannel pipette transfer 100 .mu.l of the
Substrate to each well and incubate at room temperature for 30
minutes. [0111] a. The Substrate reagent is provided ready to use
by the manufacturer. [0112] 7. Stop: at the completion of the
Substrate incubation using the 8 channel multichannel pipette
transfer 100 .mu.l of the Stop solution to each well. [0113] a. The
Stop Solution reagent is provided ready to use by the manufacturer.
[0114] 8. Read the plate at 450 nm with background correction at
620 nm. [0115] a. The plate should be read within 30 minutes after
stopping the reaction. [0116] 9. Enter the absorbance readings in
the provided spreadsheet for analysis.
TABLE-US-00004 [0116] TABLE 4 Map of Exemplary 96 Well Assay Plate
1 2 3 4 5 6 7 8 9 10 11 12 A 4.0 1 1 9 9 17 17 25 25 33 33 B 2.0 2
2 10 10 18 18 26 26 34 34 C 1.0 3 3 11 11 19 19 27 27 35 35 D 0.5 4
4 12 12 20 20 28 28 36 36 E 0.25 5 5 13 13 21 21 29 29 37 37 F
0.125 6 6 14 14 22 22 30 30 38 38 G 0.0625 7 7 15 15 23 23 31 31 39
39 H 0.0 8 8 16 16 24 24 32 32 40 40
Example 2
PRAISE-2
[0117] The Second Prospective Randomized Amlodipine Survival
Evaluation (PRAISE-2) study was a double-blind, randomized trial
prospectively designed to identify echocardiographic predictors of
survival among patients with non-ischemic cardiomyopathy and heart
failure and to determine if components of the echocardiographic
examination add prognostic information to baseline demographic and
clinical information (Cabell et al., Am. Heart J. 147(1):151-7
(2004)). One hundred patients participated in the PRAISE-2
echocardiographic study; of these, 93 had full and interpretable
echocardiographic examinations. Serum samples were drawn at
baseline and 2 weeks, and IL1LR1 levels were determined as
described in Example 1.
[0118] Receiver operating characteristic (ROC) curve analysis using
Analyse-It software (Analyse-It, Ltd, Leeds, UK). The ROC curve is
shown in FIG. 1, and AUC (area under the curve) information for the
same parameters shown in FIG. 1 is given below in Table 5. The ROC
analysis provides a summary of all of the markers that were
evaluated for prognostic value at baseline (t1). An AUC would
indicate a neutral result; any result above 0.5 indicates an
increase in accuracy of prediction based on that measurement,
whereas a result below 0.5 indicates a loss of accuracy (i.e., the
variability is high for that marker), and no correlation with the
measured parameter.
TABLE-US-00005 TABLE 5 PRAISE ROC Results Variable AUC p Age 0.620
0.027 Height 0.562 0.250 Weight 0.425 0.168 BMI 0.391 0.043 LVEF
0.421 0.146 Creatinine 0.599 0.066 ST2t1 0.611 0.040 NEt1 0.632
0.015 Et1 0.496 0.941 DAt1 0.637 0.012 ANGt1 0.471 0.587 MDAt1
0.541 0.451 ADRt1 0.504 0.934 ANPt1 0.811 0.000 BNPt1 0.779
0.000
[0119] The value of ST2 for endpoint prediction was compared to
other markers in three BMI groups. The results, shown in Table 6,
below, indicate that for patients with a high BMI, ST2 (e.g., the
ratio of ST2) is a stronger predictor than BNP. The negative
numbers in the middle weight group for ST2 may be due to the
presence of anomalous levels in some subjects.
TABLE-US-00006 TABLE 6 PRAISE Endpoint Prediction in 3 BMI Groups
BMI Group Predictor R S.E. Sig. Under 25 Log BNP 2.472 1.301 0.058
time 0 Age 0.032 0.032 0.319 Sex 0.274 0.986 0.781 ST2 Ratio 3.094
1.997 0.121 25 to 30 Log BNP 4.031 1.467 0.006 time 0 Age 0.042
0.037 0.258 Sex -0.516 0.960 0.591 ST2 Ratio -0.764 1.643 0.642 30
and Log BNP 1.283 0.966 0.184 over time 0 Age 0.008 0.039 0.844 Sex
-2.128 1.056 0.044 ST2 Ratio 6.581 2.539 0.010
[0120] The PRAISE ROC for BNP and ST2 Ratio was also calculated.
The results, shown in FIG. 2 and Table 7, indicate that ST2 ratio
is comparable to BNP across the entire PRAISE population, which
included both non-overweight, non-obese subjects, as well as
subjects in whom HF was stabilized; ST2 levels tend to return to
baseline when HF is stabilized.
TABLE-US-00007 TABLE 7 ROC for BNP and ST2 Ratio Predictor AUC SE p
Lower Upper BNPt1 0.783 0.043 0.000 0.698 0.868 ST2-Ratio 0.660
0.054 0.004 0.555 0.766
[0121] Prognostic utility of BNP and ST2 Ratio was calculated for
those individuals with high BMI; the results, shown in Table 8 and
FIG. 3, demonstrate that ST2 ratio is a better predictor than BNP
in the high BMI group, as it has a higher AUC and a better
correlation.
TABLE-US-00008 TABLE 8 Prognostic Utility for BNP and ST2 Ratio in
High BMI BMI Group Predictor AUC SE p Lower Upper Under 25 BNP
0.788 0.077 0.002 0.637 0.939 Baseline ST2 Ratio 0.717 0.082 0.022
0.555 0.878 25-29 BNP 0.864 0.055 0.000 0.756 0.972 Baseline ST2
Ratio 0.521 0.097 0.829 0.330 0.711 30 and BNP 0.669 0.100 0.102
0.473 0.865 Above Baseline ST2 Ratio 0.772 0.083 0.009 0.609
0.934
[0122] These results indicate that ST2 is predictive of outcome in
the compensated heart failure patient when used as a change over
time, and provides additional prognostic resolution in high BMI
patients.
Example 2
ST2 is not Affected by BMI
[0123] 600 breathless subjects were enrolled in the PRIDE study to
analyze the utility of NT-proBNP for diagnosis and prognosis of
acute heart failure (HF). At enrollment, a blinded sample of blood
was obtained, processed and frozen at -80.degree. C. For the
purposes of ST2 analysis, an aliquot of citrated blood was thawed
(second freeze-thaw cycle) and analyzed for concentration of ST2
protein. The effect of BMI on ST2 levels was analyzed.
[0124] The results are shown in FIG. 4 and Table 9. ST2 median
values were the same across all three BMI groups, and the IQR was
nearly identical as well.
TABLE-US-00009 TABLE 9 BMI and ST2 levels BMI ST2 (median, ng/ml)
Interquartile range (ng/ml) <25 (n = 77) 0.56 0.31-1.39 25-29.9
(n = 65) 0.49 0.23-1.13 .gtoreq.30 (n = 66) 0.48 0.23-1.04
[0125] These results demonstrate that, unlike BNP, ST2 levels are
not affected by BMI.
Example 3
ST2 Concentrations are not Affected by Renal Insufficiency
[0126] The effect of renal impairment on ST2 concentrations was
evaluated in a population of 135 patients with moderate to severe
renal insufficiency. None of the patients were on dialysis, and
none were previously diagnosed with CVD. All of the patients were
evaluated using glomerular filtration rate (GFR in mls/min) as
determined by the Modification of Diet in Renal Disease (MDRD)
method as a measure of renal function. Echocardiography and
coronary artery calcium (CAC) measurements were also performed on
each subject to detect latent CVD. Multiple biomarkers were also
evaluated.
[0127] The descriptive statistics for this cohort are shown in
Table 10; the mean GFR and ST2 are illustrated graphically in FIGS.
5A-B.
TABLE-US-00010 TABLE 10 Glomerular Filtration Rate (GFR) and ST2
Levels GFR ST2 levels (ng/ml) Mean 34.5 0.122 Median 34 0.107 Std
Error 0.989 0.005 Std Dev. 11.4 0.059 Coeff. Var. 33.3 48.346 Lower
95% CL 32.5 0.112 Upper 95% CL 36.4 0.132 25th Percentile 27 0.090
75th Percentile 43 0.127 Minimum 9 0.068 Maximum 59 0.476 Count 135
135
[0128] In this cohort of patients with stable, chronic disease,
only ten (8%) had ST2 levels above 0.2, the highest of which was
0.476 ng/ml. This distribution of ST2 values is shown in FIG. 6.
This was as expected in this population of subjects with chronic,
managed renal insufficiency; one would not expect to see very high
ST2 levels.
[0129] Pearson Correlation analysis was performed in this
population to determine whether there was a correlation between ST2
levels and renal function, as measured by either GFR or creatinine
clearance. The results are shown in Tables 11 and 12.
TABLE-US-00011 TABLE 11 Pearson Correlation Results - GFR and ST2
Descriptive Statistics Variable Mean Std Dev. Std Err N GFR 34.5
11.5 0.989 135 ST2 (ng/mL) 0.122 0.059 0.005 135 GFR ST2 (ng/mL)
Correlation Matrix (R) GFR 1.000 0.028 ST2 (ng/mL) 0.028 1.000
Correlation Significance (P) GFR -- 0.748 ST2 (ng/mL) 0.748 --
TABLE-US-00012 TABLE 12 Pearson Correlation Results - Creatinine
Clearance and ST2 Descriptive Statistics Variable Mean Std Dev. Std
Err N Screening Cr 2.175 0.859 0.081 113 ST2 (ng/mL) 0.122 0.058
0.006 113 Screening Cr ST2 (ng/mL) Correlation Matrix (R) Screening
Cr 1.000 -0.018 ST2 (ng/mL) -0.018 1.000 Correlation Significance
(P) Screening Cr -- 0.851 ST2 (ng/mL) 0.851 --
[0130] These results demonstrate that, as was expected in this
population of subjects with chronic, managed renal insufficiency,
there is no correlation between ST2 levels and either GFR (p=0.75)
or creatinine clearance (p=0.851) in this population. This
indicates that renal insufficiency, by itself, does not cause an
elevation of ST2 levels.
[0131] The same analyses were carried out in a population of 139
subjects at the San Diego Veteran's Administration Hospital. All of
the subjects had previously been diagnosed with acute decompensated
heart failure (ADHF), and the mean ST2 level was about twice that
seen in the population of patients with chronic renal insufficiency
but no HF (see Tables 11-12). There is an almost ubiquitous
correlation between renal insufficiency and heart failure, with an
almost 80% confluence of patients with stage III/IV HF also having
impaired renal function (Fonarow and Heywood, Am. J. Med. (2006)
119(12A):S17-S25. Thus, because ADHF is correlated with ST2 levels,
one would expect to see a correlation between renal insufficiency
(as measured by GFR) and ST2 levels. This was exactly what was
seen, as shown in Tables 13 and 14.
TABLE-US-00013 TABLE 13 Pearson Correlation Results - GFR and ST2
in ADHF Descriptive Statistics Variable Mean Std Dev. Std Err N GFR
59.1 25.3 2.143 139 ST2 (ng/mL) 0.283 0.332 0.028 139 GFR ST2
(ng/mL) Correlation Matrix (R) GFR 1.000 -0.062 ST2 (ng/mL) -0.062
1.000 Correlation Significance (P) GFR -- 0.470 ST2 (ng/mL) 0.470
--
TABLE-US-00014 TABLE 14 Pearson Correlation Results - GFR and ST2
Ratios in ADHF Descriptive Statistics Variable Mean Std Dev. Std
Err N GFR 59.1 25.3 2.143 139 ST2 ratio 1.038 3.038 0.258 139 GFR
ST2 ratio Correlation Matrix (R) GFR 1.000 -0.161 ST2 ratio -0.161
1.000 Correlation Significance (P) GFR -- 0.058 ST2 ratio 0.058
--
[0132] These results demonstrate that, in subjects with ADHF, ST2
values, whether represented as a single level or a ratio, are
correlated with measures of renal insufficiency, but are
independent of the renal insufficiency; thus, there is no causative
1 o relationship between the two. Rather, both variables are
related to and independently interact with a third parameter (in
this case, heart failure).
Other Embodiments
[0133] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *