WO2013169890A1 - Systems and methods for assessing disease risk, status, and prognosis - Google Patents

Abstract

Described herein are systems and methods for stratification of patients into cohorts having defined disease risks, disease statuses, or prognoses. In some embodiments, patients are divided into cohorts based on the quantitation of levels of galectin-3 and at least one additional biomarker in patient samples.

Description

SYSTEMS AND METHODS FOR ASSESSING DISEASE RISK. STATUS. AND

PROGNOSIS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent

Application No. 61/644,293, filed May 8, 2012, which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] Heart failure (HF) is a major public health problem. In the United States, approximately 5.7 million people suffer from HF, including roughly 670,000 new cases each year. Of these patients, 277,000 die each year from the disease, and the aggregate mortality and morbidity attributed to HF exceeds that of all major cancers combined. (See Roger VL, Go AS, Lloyd- Jones DM, et al. Heart Disease and Stroke Statistics 2011 Update: A Report From the American Heart Association. Circulation. 2011 Feb I ;123(4):el8-e209; Stewart S, Ekman I, Ekman I, Oden A, Rosengren A; Population Impact of Heart Failure and the Most Common Forms of Cancer: A Study of 1 162 309 Hospital Cases in Sweden (1988 to 2004) Qual Outcomes 2010;3;573-580; originally published online October 5; and Lloyd-Jones D, Adams RJ, Brown TM, et al. Heart Disease and Stroke Statistics 2010 Update: A Report From the American Heart Association. Circulation. 2010 Feb 23; 121(7):e46-e215.) [0003] In HF, the heart fails to pump enough blood to meet the body's needs, leading to the congestion of blood and other fluid in the liver, abdomen, lower extremities, and lungs. Thus, HF has also been called congestive heart failure (CHF), although the term HF is preferred because not all patients exhibit fluid congestion. HF results in a gradual deterioration of the patient often leading to cardiovascular mortality. Thus, a large number of patients die within one to five years after diagnosis. However, others may remain stable for prolonged periods. [0004] Symptoms of HF include fatigue, weakness, rapid or irregular heartbeat, shortness of breath, persistent cough or wheezing, swelling of lower extremities or abdomen, sudden weight gain from fluid retention, lack of appetite or nausea, and chest pain. Currently, an important diagnostic test for HF is the comprehensive 2-dimensional echocardiogram together with Doppler flow studies to determine whether structural abnormalities exist. This test can determine the fraction of blood being pumped out of the ventricle (the ejection fraction, EF), which is an important measurement of heart function. The ejection fraction for a normal heart is approximately 60%. One or more of the following tests may also be used: radionuclide ventriculography, magnetic resonance imaging (MRI), a complete blood count, urinalysis, serum electrolytes, glycohemoglobin and blood lipids, tests of renal and hepatic function, tests of thyroid function, a chest radiograph, and a 12-lead electrocardiogram. Additionally, blood tests for biomarkers, such as B-type natriuretic peptide (BNP), can be performed. BNP is upregulated in response to the stretching of cardiomyocytes. Thus, a high level of BNP is indicative of a heart under stress, and is a good indicator of heart failure. However, other mechanisms that play a role in heart failure, such as inflammation, may not be reflected by an increase in BNP.

[0005] Galectins are a family of proteins characterized by their galactose-specific binding. All share common amino acid sequence in regions of their structure known as the carbohydrate recognition domain or CRD. Currently, 15 mammalian galectins have been identified. One subgroup contains galectins 1, 2, 5, 7, 10, 13, 14, and 15, each of which comprises a single CRD. A second subgroup contains a single species, galectin-3, which comprises a single CRD linked to an N-terminal domain comprising repeats of short amino acid sequences such as PGA. A third galectin subgroup contains galectins 4, 6, 8, 9, and 12, each of which comprises two CRDs joined by a linker of variable length. All of the galectins have significant amino acid sequence homology, and many appear in the human circulatory system.

[0006] Among galectins, galectin-3 is implicated in a variety of cellular processes, including cell-cell adhesion, cell-matrix interactions, phagocytosis, cell cycle, apoptosis, angiogenesis and mRNA splicing, and to act through both intracellular and extracellular mechanisms. The inventors have recently developed and received FDA clearance for a clinical assay for galectin-3 (BGM Galectin-3™, BG Medicine, Waltham MA) that is helpful in assessing the prognosis of patients with chronic HF. While this clinical assay addresses a long- standing need in the field for reliable clinical detection of galectin-3, an equally long-standing need remains for systems and methods useful in assessing patient prognoses, predicting patient outcomes and/or selecting treatments for patients with HF or those at risk of incident HF based on molecular and cellular markers for HF disease and/or progression.

SUMMARY [0007] The technology disclosed herein relates, in general, to medical assays and, more specifically, to immunoassays for galectin-3 and systems and methods using the same. In some embodiments, systems and methods described herein may be used for assessing disease status and risk in patients with diseases in which galectin-3 levels are elevated, including HF and other diseases in which galectin-3 mediated fibrosis is implicated. [0008] In one aspect, kits for detecting galectin-3 in a sample are provided.

Contemplated kits contain a first binding moiety that includes all or part of a monoclonal antibody produced by the 87B5 cell line, and a second binding moiety including all or part of a monoclonal antibody produced by the M3/38 cell line, and provide results that indicate detection of galectin-3 above a 100 pg/mL threshold. In various embodiments, contemplated kits can be provided in a form suitable for use in a sandwich assay, the first and/or second binding moieties may be operably bound to a solid support. The second binding moiety can be provided in a solution including lOmM phosphate buffered saline with a pH of 7.4 and 1% bovine serum albumin. Contemplated kits may also include a recombinant galectin-3 polypeptide. In various embodiments, samples used with the kit can include whole or fractional blood, serum, plasma, or urine, and may be taken from a human subject with a known risk or symptom of heart failure or from a patient whose risk or symptom of heart failure is sought to be determined, and in some embodiments the kit may include third and fourth binding moieties specific for one of cardiac troponin I (cTNI), cardiac troponin T (cTNT), tropomyosin, myosin light chain- 1, and heart-type fatty acid binding protein. [0009] In another aspect, methods of patient stratification are provided. The methods include quantitating a level of galectin-3 and at least one additional biomarker in a sample of fluid or tissue from a patient, comparing the quantitated levels to multiple thresholds for galectin-3 and any other quantitated markers, and assigning the patient to a cohort that is defined by one or more of the thresholds, which cohort has a specified risk of incident heart failure or a specified heart failure status. In various embodiments, the additional biomarker(s) can be selected from various subsets of the following: markers of fibrosis, markers of myocyte injury or necrosis, markers of myocyte stretching, markers of extracellular matrix remodeling, inflammatory markers and cell adhesion markers, collagen type I, collagen type III, type I collagen telopeptidase, ΡΓΝΡ, PIIINP, MMPl, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP 10, MMP1 1, MMP12, MMP13, MMPl 4, MMPl 5, MMPl 6, MMPl 7, MMP 19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, TIMP1, TIMP2, TIMP3, TIMP4, elastin, fibronectin, LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB 1, LAMB2, LAMB 3, LAMB4, LAMC1, LAMC2, and LAMC3, cTNI, cTNT, tropomyosin, myosin light chain- 1, heart-type fatty acid binding protein, CRP, adiponectin, MCP-1, CD40 ligand, Lp-PLA2, BNP and NT-proBNP. The method may also include selecting a treatment for the patient that has been shown to be effective for the patient's cohort or determining an efficacy of a treatment for the patient's cohort. [0010] In still another aspect, a system for stratifying patients is provided. The system includes means for determining levels of galectin-3 and at least one additional biomarker, one or more memories for storing at least one patient data record and multiple thresholds for galectin-3 and the additional biomarker(s), and a processor that stratifies patients by executing instructions to compare the levels of galectin-3 and any additional biomarker' s in the patient record to the thresholds and associating that patient record with a patient cohort defined by at least one of the thresholds, which patient cohort is characterized by a specified risk of incident heart failure and/or a specified heart failure status. The additional biomarker(s), in various embodiments, can be selected from various subsets of the following: markers of fibrosis, markers of myocyte injury or necrosis, markers of myocyte stretching, markers of extracellular matrix remodeling, inflammatory markers and cell adhesion markers, collagen type I, collagen type III, type I collagen telopeptidase, ΡΓΝΡ, PIIINP, MMPl, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP 12, MMP13, MMPl 4, MMP 15, MMPl 6, MMP 17, MMP 19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, TIMP1, TIMP2, TIMP3, TIMP4, elastin, fibronectin, LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMB 3, LAMB4, LAMC1,

LAMC2, and LAMC3, cTNI, cTNT, tropomyosin, myosin light chain-1, heart-type fatty acid binding protein, CRP, adiponectin, MCP-1, CD40 ligand, Lp-PLA2, BNP and NT-proBNP. [0011] In yet another aspect, a method of determining the efficacy of a drug is provided. The method includes administering the drug to multiple patients, quantitating levels of galectin-3 and one or more additional biomarker(s) in samples of fluid or tissue from the patients, comparing the quantitated levels of galectin-3 and the additional biomarker(s) to multiple thresholds for galectin-3 and the additional biomarker(s) and assigning each patient to one of multiple cohorts that are defined by at least one of the thresholds, and evaluating the efficacy of the drug in that cohort.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 includes a schematic illustration of exemplary patient subpopulations that can be resolved using contemplated systems and methods. [0013] FIG. 2 includes a schematic illustration of a system according to certain embodiments.

[0014] FIG. 3 includes a schematic representation of an exemplary system for assigning patients to particular cohorts according to certain embodiments.

[0015] FIG. 4 depicts survival data for patients stratified by galectin-3 level. [0016] FIG. 5 depicts risk data for patients stratified by galectin-3 level.

[0017] FIG. 6 depicts risk data for patients stratified by galectin-3 level.

[0018] FIG. 7 depicts risk data for patients stratified by galectin-3 level.

[0019] FIG. 8 depicts survival data for patients stratified by galectin-3 level

[0020] FIG. 9 depicts certain data for galectin-3 assays according to certain

embodiments.

FIG. 10 depicts certain data for galectin-3 assays according to certain

[0022] FIG. 11 depicts certain data for galectin-3 assays according to certain embodiments. [0023] FIG. 12 depicts certain data for galectin-3 assays according to certain embodiments.

[0024] FIG. 13 depicts certain data for galectin-3 assays according to certain embodiments.

[0025] FIG. 14 depicts a process flow chart for an exemplary embodiment. DETAILED DESCRIPTION

Galectin-3

[0026] Galectin-3 (GenBank Accession Nos.: NC_000014.7 (gene) and NP_002297.2

(protein)) is one of 15 mammalian beta galactoside-binding lectins, or "galectins,"

characterized by their galactose-specific binding. Galectin-3 has variously been referred to in the literature as LGALS3, MAC-2 antigen, Carbohydrate binding protein (CBP)-35, laminin binding protein, galactose-specific lectin 3, mL-34, L-29, hL-31, epsilon BP, and IgE-binding protein. Galectin-3 is composed of a carboxyl-terminal carbohydrate recognition domain (CRD) and amino-terminal tandem repeats (Liu, F.-T. (2000) Role of galectin-3 in

inflammation. In Lectins and Pathology. M. Caron and D. Seve, eds. Harwood Academic Publishers, Amsterdam, p. 51 ; Liu, F.-T. et al. (1995) Am. J. Pathol. 147: 1016). Galectin-3 normally distributes in epithelia of many organs and various inflammatory cells, including macrophages as well as dendritic cells and Kupfer cells (Flotte, T. J. et al. (1983) Am. J. Pathol. 1 11 : 112).

[0027] Galectin-3 has been implicated in a variety of cellular process, including cell- cell adhesion, cell-matrix interactions, phagocytosis, cell cycle, apoptosis, angiogenesis and mRNA splicing. Galectin-3 has been shown to function through both intracellular and extracellular actions (Sano, H. et al. (2000) The Journal of Immunology, 165:2156-2164). It is a component of heterogeneous nuclear ribonuclear protein (hnRNP) (Laing, J. G. et al. (1998) Biochemistry 27:5329), a factor in pre-mRNA splicing (Dagher, S. F. et al. (1995) Proc. Natl. Acad. Sci. USA 92: 1213) and has been found to control the cell cycle (Kim, H.-R. C. et al. (1999) Cancer Res. 59:4148) and prevent T-cell apoptosis through interaction with the Bcl-2 family members (Yang, R.-Y. et al. (1996) Proc. Natl. Acad. Sci. USA 93:6737). On the other hand, galectin-3, which is secreted from monocytes/macrophages (Sato, S. et al. (1994) J. Biol. Chem. 269:4424) and epithelial cells (Lindstedt, R. G. et al. (1993) J. Biol. Chem. 268: 11750) has been demonstrated to function as an extracellular molecule in activating various types of cells such as monocytes/macrophages (Liu, F.-T. (1993) Immunol Today 14:486), mast cells, neutrophils and lymphocytes (Hsu, D. K., S. R. et al. (1996). Am. J. Pathol. 148: 1661).

Galectin-3 has been shown to act as a novel chemoattractant for monocytes and macrophages (Sano, H. et al. (2000) The Journal of Immunology, 2000, 165:2156-2164). Galectin-3 has been implicated in diseases and conditions such as cancer, inflammation, and heart failure.

[0028] Unlike the other mammalian galectins, galectin-3 comprises an atypical N- terminal domain, comprising the amino acid sequence: MADNFS LHDA LSGS GNPNPQ GWPGAWGNQP AGAGGYPGAS YPGAYPGQAP

PGAYPGQAPP GAYPGAPGAY PGAPAPGVYP GPPSGPGAYP S SGQPSATGA

YPATGPYGAP AGP (SEQ ID O: l) .

[0029] As can be appreciated from an inspection, the sequences of the N-terminus of galectin-3 are dissimilar to sequences of other mammalian galectins but comprise multiple repeats of the type PGAYPG(X)l-4 (SEQ ID NO:2), with intervening proline-, glycine-, and tyrosine-rich regions. The existence of repeated sequences in the N-terminal domain decreases the number of different potential epitopes specific to galectin-3, complicating the development of a detection assay. However, it has now been discovered that N-terminal epitopes can reliably distinguish galectin-3 from other mammalian galectins. Using the assays disclosed herein it is possible to correlate reliably and reproducibly galectin-3 clinical results with the presence, severity and stage of progression of HF in a subject. Exemplary N-terminal epitopes include, but are not limited to, MADNF SLHD ALS (amino acids 1-12 of SEQ ID NO: 1),

MADNFSLHDALSGS (amino acids 1-14 of SEQ ID NO: l), GNPNPQGWPGA (amino acids 15-25 of SEQ ID NO: 1), WGNQPAGAGG (amino acids 26-35 of SEQ ID NO: 1),

YPGQAPPGAYPGQAPPGA (amino acids 45-62 of SEQ ID NO: 1),

YPGAPGAYPGAPAPGV (amino acids 63-78 of SEQ ID NO: 1),

YPGAPAPGVYPGPPSGPGA (amino acids 70-88 of SEQ ID NO: l), YPSSGQPSATGA (amino acids 89-100 of SEQ ID NO: l), where the letters represent standard amino acid code. Other epitopes appear on the N-terminus comprising amino acids spaced apart on the primary structure but presented together in the tertiary structure, and these also can be addressed by the binders used in the assays described herein.

[0030] Galectin-3 may exist in a sample in a plurality of different forms characterized by detectably different masses. These forms can result from pre-translational modifications, post-translational modifications or both. Pre-translational modified forms include allelic variants, splice variants, and RNA-editing forms. Post-translationally modified forms include forms resulting from, among other things, proteolytic cleavage (e.g., fragments of a parent protein), complexation, glycosylation, phosphorylation, lipidation, oxidation, methylation, cystinylation, sulphonation and acetylation. Modified forms of galectin-3, as long as they retain the relevant N-terminal epitopes, may be detected according to the methods contemplated herein.

[0031] The expression of this galectin-3 is up-regulated during inflammation (Flotte et al. (1983) Am. J. Pathol. 1 1 1 : 1 12.), cell proliferation (Agrwal, et al. (1999) J. Biol. Chem. 264: 17236) and cell differentiation (Nangia-Makker et al. (1993). Cancer Res. 53 : 1) and through transactivation by viral proteins (Hsu, D. et al. (1996) Am. J. Pathol. 148: 1661). Its expression is also affected by neoplastic transformation. For example, galectin-3 upregulation is found in certain types of lymphomas (Hsu, D. et al. (1996) Am. J. Pathol. 148: 1661), and thyroid carcinoma (Fernadez, P. L. et al. (1997) J. Pathol. 181 :80), while galectin-3 is down- regulated in other types of malignancies such as colon (Lotz, M. M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3466), breast (Castronovo, V., F. A. et al. (1996) J. Pathol. 179:43.), ovarian (Van den Brule, F. A. et al. (1994) Eur. J. Cancer 30A: 1096) and uterine (Van den Brule, F. A. et al. (1996) Hum. Pathol. 27: 1 185) carcinomas. The expression of galectin-3 has a strong correlation with the grade and malignant potential of primary brain tumors (Bresalier, R. et al. (1997). Cancer 80:776). [0032] Galectin-3 plays a role in many other diseases, conditions and disorders, including autoimmune disorders and vascular complications in diabetes and hypertension. Galectin-3 has been detected in tissues affected by inflammatory diseases. For example, galectin-3 was detected in the tears of patients with inflammatory ocular diseases (Hrdlickova- Cela et al. (2001), Br J Opthalmol, 85: 1336-40). Increased galectin-3 levels have also been noted in human atherosclerotic lesions (Ohshima et al. (2003), Arthritis Rheum, 48:2788-95; Nachtigal et al. (1998), Am J Pathol, 152: 1199-208).

[0033] Various galectin-3 activities such as binding to AGE receptors, etc. may be potentiated or inhibited by galectin-3 binding to carbohydrates. Sandwich Assays for Galectin-3

[0034] In some embodiments, concentrations of galectin-3 may be quantitated in a sample such as a bodily fluid (e.g. urine, whole blood, blood serum, blood plasma,

cerebrospinal fluid, synovial fluid, cytoplasm, lymph, saliva, stool, bile, sweat, etc.) or biopsied tissue using one or more binding moieties that bind specifically to N-terminal portions of galectin-3. A "binding moiety" refers to a molecule that binds or interacts selectively or preferentially with a polypeptide or peptide. In preferred embodiments, the binding moieties are monoclonal or polyclonal antibodies, though other molecules may be used as binding moieties, as described in U.S. patent application 12/608,821, which is incorporated by reference herein.

[0035] In some embodiments, galectin-3 is detected and quantified using a "sandwich" assay. In certain embodiments, two molecules ("binding moieties") such as monoclonal antibodies that specifically bind to non-overlapping sites ("epitopes") on the N-terminus of galectin-3 are used. Typically, one binding moiety is immobilized (for example on a solid surface such as the surface of a microtiter well, the interior or exterior surface of a pipette tip, or the surface of a magnetic or non-magnetic bead) where it binds with and captures galectin-3. This first binding moiety is therefore also referred to herein as the capture binding moiety. A second binding moiety is detectably labeled with any suitable label, for example, with a fluorophore, chemiluminescent agent, enzyme, or colored particle, such that binding of the second binding moiety to the galectin-3 -complex indicates that galectin-3 has been captured. The intensity of the signal is proportional to the concentration of galectin-3 in the sample. The second binding moiety is therefore also referred to herein as the detection binding moiety or label binding moiety. A binding moiety can be any type of molecule, as long as it specifically binds to a portion of the N-terminus of galectin-3. In a preferred embodiment, the binding moieties used are monoclonal anti-galectin-3 antibodies, i.e., monoclonals raised against or otherwise selected to bind to separate portions of the N-terminal 1 13 amino acids of galectin-3. [0036] Capture binding moieties, such as capture antibodies, may be attached to any suitable substrate, for example the surface of a microtiter well, a non-magnetic bead, a magnetic bead, and an affinity reagent such as avidin, streptavidin, biotin, etc.

[0037] The assay procedures described herein can be referred to as two-site

immunometric assay methods, "sandwich" methods or (when antibodies are the binders)

"sandwich immunoassays." As is known in the art, the capture and detection antibodies can be contacted with the test sample simultaneously or sequentially. Sequential methods, sometimes referred to as the "forward" method, can be accomplished by incubating the capture antibody with the sample, and adding the labeled detection antibody at a predetermined time thereafter. Alternatively, the labeled detection antibody can be incubated with the sample first and then the sample can be exposed to the capture antibody (sometimes referred to as the "reverse" method). After any necessary incubation(s), which may be of short duration, the label is detected and may also be measured. Such assays may be implemented in many specific formats known to those of skill in the art, including through use of various high throughput clinical laboratory analyzers or with point of care or home testing devices.

[0038] In one embodiment, a lateral flow device may be used in the sandwich format, wherein the presence of galectin-3 above a baseline sensitivity level in a biological sample will permit formation of a sandwich interaction upstream of or at the capture zone in the lateral flow assay. See, for example, U.S. Pat. No. 6,485,982. The capture zone as used herein may contain capture binding moieties such as antibody molecules, suitable for capturing galectin-3, or immobilized avidin or the like for capture of a biotinylated complex. See, for example, U.S. Pat. No. 6,319,676. The device may also incorporate a luminescent label suitable for capture in the capture zone, the concentration of galectin 3 being proportional to the intensity of the signal at the capture site. Suitable labels include fluorescent labels immobilized on polystyrene microspheres. Colored particles also may be used.

[0039] The most common enzyme immunoassay is the "Enzyme-Linked

Immunosorbent Assay" (ELISA). ELISA is a technique for detecting and measuring the concentration of an antigen using a labeled (e.g., enzyme linked) form of the antibody. There are different forms of ELISA, which are well known to those skilled in the art. The standard techniques known in the art for ELISA are described in "Methods in Immunodiagnosis", 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; Campbell et al, "Methods and Immunology", W. A. Benjamin, Inc., 1964; and Oellerich, M. (1984), J. Clin. Chem. Clin. Biochem. 22:895-904.

[0040] In a "sandwich ELISA," an antibody (e.g., anti-galectin-3) is linked to a solid phase (i.e., a microtiter plate) and exposed to a biological sample containing antigen (e.g., galectin-3). The solid phase is then washed to remove unbound antigen. A labeled antibody (e.g., enzyme linked) is then bound to the bound antigen, forming an antibody-antigen-antibody sandwich. Examples of enzymes that can be linked to the antibody are alkaline phosphatase, horseradish peroxidase, luciferase, urease, and .beta.-galactosidase. The enzyme-linked antibody reacts with a substrate to generate a colored reaction product that can be measured. This measurement can be used to derive the concentration of galectin-3 present in a sample, for example, by comparing the measurement to a galectin-3 standard curve. Galectin-3

concentration in a sample from a subject may be determined to be above or below a threshold. For example, as is discussed in greater detail below, a threshold of approximately 17.8 ng/mL galectin-3 in plasma is predictive of significantly increased HF risk in chronic HF patients.

[0041] In some embodiments, the antibodies used to detect galectin-3 are monoclonal antibodies, for example, M3/38, 9H3.2, and 87B5. M3/38 detects a linear epitope

(YPGQAPPGAYPGQAPPGA (amino acids 45-62 of SEQ ID NO: l)) on the N-terminus of galectin-3. M3/38 was prepared from the supernatant of the rat hybridoma M3/38.1.2.8 HL.2, a clone of which can be found in the American Type Culture Collection with ATCC^® number TIB-166. 9H3.2 detects a linear epitope (MADNFS LHDALSGS (amino acids 1-14 of SEQ ID NO: l) at the extreme N-terminus of galectin-3. 9H3.2 is a mouse monoclonal IgG, affinity purified using protein A. 9H3.2 is available from Millipore (Millipore, 290 Concord Road, Billerica, MA 01821, USA), catalog no.: MAB4033. 87B5 detects a non-linear epitope comprising portions of GNPNPQGWPGA (amino acids 15-25 of SEQ ID O: l) and

YPGAPAPGVYPGPPSGPGAYPS SGQPSATGA (amino acids 70-100 of SEQ ID O: l). 87B5 was prepared from the mouse-mouse hybridoma (X63-Ag8.653 xBALB/c mouse spleen cells) clone 87B5, and is an IgG2a that was affinity purified using Protein A. 87B5 is available from Immuno-Biological Laboratories (IBL, 8201 Central Ave E, Suite P, Minneapolis, MN 55432 USA). [0042] In a currently preferred embodiment, the capture binding moiety is the anti- galectin-3 monoclonal antibody, M3/38 and the labeled detection binding moiety is a second anti-galectin-3 monoclonal antibody, 87B5. The given designations for these antibodies are not limiting. In another embodiment, the capture antibody is 9H3.2 and the labeled detection binding moiety is M3/38. Other antibodies which recognize the epitopes described above also may be used.

[0043] In some embodiments, the label used for the detection antibody can be selected from any of those known conventionally in the art. Preferred labels are those that permit more precise quantitation. Examples of labels include but are not limited to a fluorescent moiety (e.g. rhodamine, fluorescein, cyanine dye, etc.), an enzyme (e.g. malate dehydrogenase,

staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, acetylcholinesterase, etc.) an

electrochemically active species, a radioactive isotope (e.g. 3H, 23P, 35S, 14C, 13 II, etc.), a chemiluminescent molecule, a latex or gold particle, a detectable ligand (e.g., detectable by secondary binding of a labeled binding partner for the ligand), etc. In a preferred embodiment, the label is an enzyme or a fluorescent molecule. Methods for affixing the label to the binding moiety are well known in the art, and include covalent and non-covalent linkage. [0044] In certain embodiments, systems and methods described herein can detect concentrations down to 1.4 ng/mL in circulation, which is lower than the concentrations previously described. In some instances, assay buffers (into which detection antibodies may be diluted, for example) typically include 10 mM phosphate buffered saline (PBS) at pH 7.4 and 1% bovine serum albumin (BSA). In some embodiments, the pH of the assay buffer is selected to improve a binding specificity of at least one binding moiety diluted therewithin.

[0045] Those of skill in the art will appreciate that, while the foregoing disclosure has focused on quantitation of galectin-3, other peptide markers may be quantitated using similar sandwich assays, as is discussed in more detail below. Assays for Heart Failure Status or Risk Based on Fibrotic Disease Progression

[0046] While not wishing to be bound to any theory, it is believed that, in a large percentage of HF patients, the disease is driven by a diffuse progressive cardiac fibrosis.

Patients with underlying cardiac fibrosis may be asymptomatic, but may be at increased risk of Incident HF. Incident HF in these patients may be precipitated by an acute (e.g. trauma, infarction etc.) or chronic (e.g. hypertension, diabetic cardiomyopathy, etc.) injury to the myocardium. It is believed that the progression to HF in these patients is related to or mediated by aldosterone, and that their underlying fibrosis is mediated by galectin-3.

[0047] In some embodiments, patients may be stratified into cohorts characterized by specific disease status, prognosis, or risk of developing a disease such as incident heart failure using a method including quantitating of a galectin-3 level of the patient, as well as one or more levels of an additional marker or markers of HF or a process related to HF. Suitable markers that may be quantitated alongside galectin-3 using methods described herein include, without limitation, markers of fibrotic processes, markers of myocyte injury or necrosis, markers of myocyte stretching, markers of extracellular matrix remodeling, inflammatory markers, cell adhesion molecules and the like.

[0048] Suitable markers of fibrotic processes include collagens, such as collagen type I, collagen type III, type I collagen telopeptidase, aminoterminal propeptide of type I procollagen (ΡΓΝΡ), aminoterminal propeptide of type III procollagen (ΡΙΙΓΝΡ), matrix metalloproteinases (MMPs) such as MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9,

MMP10, MMP 11, MMP 12, MMP13, MMP 14, MMP15, MMP16, MMP 17, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, etc., and tissue inhibitors of metalloproteinases (TIMPs) such as TIMP1, TIMP2, TIMP3, TIMP4, etc, and other molecules of the extracellular matrix, including without limitation elastin, fibronectin, laminins (LAMs) such as LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMB3, LAMB4, LAMC1, LAMC2, LAMC3, etc., proteoglycans and other extracellular matrix glycans and glycoconjugates, etc.

[0049] Suitable markers of myocyte stretching include, without limitation, brain natriuretic peptide (BNP) and N-terminal pro-brain natriuretic peptide (NT-proBNP). [0050] Suitable markers of myocyte injury or necrosis include, without limitation, cardiac troponin I (cTNI), cardiac troponin T (cTNT), tropomyosin, myosin light chain- 1 , heart-type fatty acid binding protein (hFABP), etc.

[0051] Suitable markers of inflammation include, without limitation, C-reactive protein (CRP), adiponectin, monocyte chemoattractant protein 1 (MCP-1), CD40 ligand, lipoprotein- associated phospholipase A2 (Lp-PLA2), etc.

[0052] Quantitated levels of galectin-3 and other markers may be used in methods described herein to define patient cohorts characterized a particular risk profile, disease profile or prognostic profile and/or to assign patients to one or more of such cohorts. Specific profiles can be established based on one or more reference or standard profiles characterized by a specific disease state, disease risk, or prognosis, which are associated with particular levels of galectin-3 and/or other markers listed above. An exemplary system 200 for profiling patients based on levels of galectin-3 and one other marker is depicted in FIGs. 2-3. System 200 (FIG. 2) makes use of a schema 205, represented graphically in FIG. 3, that is defined by varying levels of a first biomarker 210, preferably galectin-3, and at least one additional biomarker 215. The schema 205 includes four subspaces 206, 207, 208, 209, which are defined, for example, by a first threshold value 225 of galectin-3 and a second threshold value 230 of the second marker 215. The threshold values 225, 230 may be linear or non-linear, and may be constant, as shown in FIG. 3A, or they may vary, as shown in FIG. 3B. In some embodiments, a threshold may be a function of a level of one or more markers 210, 215, and may depend as well on another measured variable such as ejection fraction, age, etc. Where the first marker 210 is galectin-3, a suitable constant value for the first threshold 225 is a galectin-3

concentration of 17.8 ng/mL in plasma or serum, and a second threshold galectin-3

concentration of 25.9 ng/mL may also be used in some embodiments. In the example of FIG. 3 A, because elevated galectin-3 levels are positively correlated with elevated risk of incident HF and with poorer prognosis in patients with chronic HF, subspaces 206 and 207 may represent higher risk profiles than subspaces 208 and 209, respectively. The second marker may also be positively correlated with elevated HF risk, in which case subspaces 207 and 209 may also represent higher risk profiles than subspaces 206 and 208, respectively; alternatively, the second marker may be inversely correlated with elevated HF risk, in which case subspaces 207 and 209 may represent lower risk cohorts than subspaces 206 and 208, respectively. Other relationships may be possible depending upon the nature of the relationship between a particular marker and various disease states, risk states, or prognoses.

[0053] While the schema 205 depicted in FIG. 3A utilizes only two thresholds and only four subspaces corresponding to up to four cohorts, any number of thresholds may be used to create any number of subspaces. Additionally, multiple subspaces may correspond to the same patient cohort. For example, in the system 200 depicted in FIG. 3A, the risk profiles associated with subspaces 206 and 209 may be substantially identical, such that subspace 207 represents the highest risk cohort, subspace 208 represents the lowest risk cohort, and subspaces 206 and 209 represent an intermediate risk cohort. [0054] While FIGs. 2-3 depict a system utilizing only two markers 210 215, any number of markers may be measured. The number of markers and identity of the markers measured may be selected by any appropriate means, including by reference to epidemiological data or models, or to test a particular hypothesis. In some embodiments, the number and identity of the markers selected of is selected to minimize certain false negative results whereby a patient who is subsequently found to have a relatively high risk, or a relatively more advanced disease state or prognosis is erroneously assigned a profile associated with a relatively low risk or relatively less advanced disease state or prognosis by the system 200. Alternatively, the number of markers measured may selected to account for a desired extent of the variance in a particular patient population or subpopulation. For example, a plurality of high-variance principal components may be defined for a patient population of interest and an output of interest by multivariate analysis methods such as principal component analysis, and the number of markers used in a contemplated system may then be selected so that one, two, three or more high-variance principal components can be determined.

[0055] Small numbers of patients can be assigned to cohorts manually, however the initial definition of cohorts, and the assignment of more than a handful of patients to individual cohorts, will entail working with datasets that necessitate processing with a computer. In certain embodiments, the assignment process is performed by comparing the marker levels to thresholds presented in tabulated or graphical form. Threshold levels corresponding to various cohorts may be provided to users in tangible, hard-copy form, or in machine-readable format on tangible media, which may then be processed by a computer and displayed on a screen or otherwise provided as output to a user. Alternatively, in some embodiments, the threshold levels corresponding to various cohorts may be provided in machine-readable form on fixed media along with machine-readable instructions for performing the comparisons and cohort assignments described above, and the marker levels in one or more patient samples may be provided as input to a computer, which may then assign one or more patients to cohorts based on the marker levels in their samples.

[0056] Patient cohorts may be developed using the methods described herein and, once developed, may be used to segment or stratify patient data, for example drug-safety or efficacy data. Without wishing to be bound to any theory, it is believed that a subpopulation of individuals at risk of HF, or who have developed HF, have underlying cardiac fibrosis implicating galectin-3, as is depicted in FIG. 1. It is further believed that, while certain treatment regimens for HF such as specific drugs or other medical or surgical interventions may not show efficacy when evaluated over the undifferentiated HF patient population, these regimens may be effective for treating HF when evaluated across narrower patient cohorts defined by levels of galectin-3 above certain thresholds, such as 17.8 ng/niL or 25.9 ng/niL in serum or plasma.

[0057] According to certain embodiments, a method of selecting a suitable treatment for a patient at risk of, or having, HF will include the steps of obtaining a sample from the patient and quantitating levels for one or more of the markers described above, including galectin-3; assigning the patient to a cohort based on the quantitation of these markers either manually or by computer, as described above; identifying one or more treatments demonstrated to be effective for the applicable patient cohort, and selecting such one or more treatments for the patient. In some embodiments, the treatment selected for a patient cohort characterized by high galectin-3 levels may include an aldosterone antagonist or another drug that blocks or inhibits, directly or indirectly, an action of aldosterone.

[0058] According to certain embodiments, a method of determining the efficacy of a medical treatment proceeds along similar lines: patients are divided into cohorts as described above, then the treatment efficacy is assessed using ordinary methods known in the art for one or more of the patient cohorts. Patients may be divided into cohorts for purposes of assessing treatment efficacy at any suitable time, including before treatment is administered (e.g. at the time of enrollment in a clinical trial), during a course of treatment, or after a treatment or series of treatments (e.g. patients may be divided into cohorts for post-hoc efficacy evaluations).

Detection of Biomarkers in Various Sample Types

[0059] In some embodiments, the systems and methods described herein, particularly in Example 1, may be used to quantitate galectin-3 levels in urine as follows: dilute the sample 1 to 10-fold in the assay buffer; add 100 uL to each well; run the assay according to the package insert. Note: adjust the dilution factor if needed.

[0060] In some embodiments, the systems and methods described herein, particularly in

Example 1, may be used to quantitate galectin-3 levels in Bronchoalveolar lavage fluid as follows: dilute the sample 1 to 10-fold in the assay buffer; add 100 uL to each well; run the assay according to the package insert. Note: adjust the dilution factor if needed.

[0061] In some embodiments, the systems and methods described herein, particularly in

Example 1, may be used to quantitate galectin-3 (Gal3) levels in tissue (e.g. liver, heart, lung, kidney, cancer tissues) homogenates as follows: suspend tissue slices in 0.5 mL pre-chilled homogenizing buffer (e.g. 10 mM PBS, pH7,4 with protease inhibitor cocktail); homogenize the slices with a homogenizer such as Covaris E100; centrifuge samples forl5 min at 3200 g; collect the supernatant and measure Gal3. Adjust the amount of tissue, the volume of homogenizing buffer, and /or the dilution factor if needed.

[0062] In some embodiments, the systems and methods described herein, particularly in Example 1 , may be used to quantitate galectin-3 levels in portal venous serum and hepatic serum as follows: dilute the sample 1 to 10-fold in the assay buffer; add 100 uL to each well; run the assay according to the package insert. Note: adjust the dilution factor if needed.

Assessing Disease Risk, Status and Prognosis in Patients With Conditions Involving Fibrosis

[0063] While the systems and methods described herein have been illustrated using embodiments specific to HF and cardiac disease, those of skill in the art will appreciate that fibrosis-mediated HF may share certain mechanisms with other diseases implicating fibrosis. Accordingly, the systems and methods described above may be adapted to assess disease risk, disease status, or prognosis for any disease or indication having a fibrotic endpoint. Examples of suitable indications for which the systems and methods described herein may be used include pulmonary diseases such as pulmonary fibrosis, cystic fibrosis, progressive massive fibrosis mediastinitis, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), etc., liver diseases such as cirrhosis, hepatitis, nonalcoholic steatohepatitis, etc., myeloproliferative diseases such as myelofibrosis, etc. digestive diseases such as Crohn's disease, etc., kidney diseases such as renal fibrosis, etc, diseases associated with organ and tissue transplantation such as organ rejection and graft versus host disease, and diseases of the skin and connective tissue such as scleroderma, arthrofibrosis, capsulitis, systemic fibrosis, etc. Systems and methods described herein may also be used to evaluate certain cancers with fibrotic endpoints, including thyroid cancers, etc.

[0064] The following documents are hereby incorporated by reference for all purposes herein: U.S. Patent Application No. 12/608,821, filed October 29, 2009; and U.S. Provisional Patent Application No. 61/109,366, filed Oct. 29, 2008.

EXAMPLES [0065] Certain aspects of the invention are illustrated by the following examples:

Example 1 :

[0066] BGM Galectin-3™ (BG Medicine, Waltham, MA) is an in vitro diagnostic device that quantitatively measures galectin-3 in serum or plasma by enzyme linked immunosorbent assay (ELISA) on a microtiter plate platform, to be used in conjunction with clinical evaluation as an aid in assessing the prognosis of patients diagnosed with chronic heart failure (HF).

[0067] BGM Galectin-3 is a microtiter plate-based ELISA for the quantitative determination of galectin-3 levels in human serum and plasma.

[0068] BGM Galectin-3 utilizes two monoclonal antibodies against galectin-3. One rat monoclonal anti-mouse galectin-3 antibody is coated onto the surface of the wells in a microtiter plate and serves as the capture antibody to bind galectin-3 molecules in samples, while the other mouse monoclonal anti-human galectin-3 antibody is provided in solution and functions as the tracer antibody for detecting galectin-3 molecules bound to the capture antibody. The microtiter plate is ready to use.

[0069] Standards, quality control materials, and patient specimens are introduced in duplicate into the wells and incubated for 60 minutes. During this incubation, the galectin-3 present in the standards and specimens binds to the capture antibody coated onto the well surface. Subsequent washing removes all unbound material introduced with the samples, including unbound galectin-3.

[0070] The tracer antibody, a horseradish peroxidase (HRP)-labeled anti-galectin-3 antibody, is then introduced into the well and incubated for 60 minutes. During this time, an antibody-antigen-antibody complex is formed.

[0071] After a wash step to remove any unbound tracer antibody, the

tetramethylbenzidine (TMB) substrate is added, yielding a blue color in the presence of HRP. The color development is stopped after 20 minutes by the addition of sulfuric acid, changing the color to yellow, which is read at an absorbance of 450 nm. [0072] The absorbance is proportional to the galectin-3 levels in the specimens. The test results of the specimens are read from the standard curve.

[0073] A detailed protocol includes the following steps, shown schematically in FIG.

14:

1. Reconstitute Galectin-3 Standard (S I)

BGM Galectin-3 is calibrated with a set of seven standards that are prepared by serial dilution of the standard (SI) that is supplied with each kit. The calibration range is 0.156 ng/mL to 10.0 ng/mL.

Open vacuum sealed vial slowly to avoid loss of material due to aerosol formation. Note: To avoid potential cross-contamination, write "SI" on the stopper with permanent ink. Before use, reconstitute one vial of the galectin-3 standard (SI) with 300 of deionized water, immediately followed by addition of 900 μϊ_^ of Assay Buffer. Allow the vial to stand for a minimum of 15 minutes at 20-25°C with periodic vortexing and gentle inversion ensuring that the reconstitution water wets the entire surface area inside the vial. Complete dissolution of the standard is critical. Verify dissolution is complete prior to use. After use, remaining reconstituted standard may be stored for a maximum of 10 days at 2-8°C, if reuse is desired. 2. Reconstitute Galectin-3 Controls (CI and C2)

The controls are comprised of a protein matrix spiked with recombinant human galectin 3. The BGM Galectin-3 Controls are supplied with assigned QC ranges that are printed on the vials and in the accompanying Value Assignment Sheet. Ranges are lot-specific and the user must confirm the appropriate range with each new lot of a BGM Galectin-3 kit.

Open vacuum sealed vials slowly to avoid loss of material due to aerosol formation. Note: To avoid potential cross-contamination, write "CI or "C2", as applicable on the appropriate stopper of each vial with permanent ink. Reconstitute one vial of CI and C2 with 250 μϊ_^ deionized water. Allow the vials to stand for a minimum of 15 minutes at 20-25°C with periodic vortexing and gentle inversion ensuring that the reconstitution water wets the entire surface area inside the vial. Verify complete dissolution prior to use. After use, remaining reconstituted controls may be stored for a maximum of 10 days at 2-8°C, if reuse is desired. 3. Define a Plate Map

Designate microtiter plate wells for each of the controls, test specimens, diluted standards and blank. All samples are preferably be tested in duplicate (i.e. blank, diluted standards, controls and test specimens).

4. Prepare Diluted Specimens and Blank

While the standard and controls are reconstituting, dilute each test specimen 10-fold (1 : 10) using the Assay Buffer (AB). Final dilution volume is preferably sufficient for duplicate measurement. Therefore, it is recommended that a minimum of 30 μϊ_^ of the specimen be used for the dilution (i.e. 30 μΐ., specimen + 270 μΐ., AB). An assay blank is preferably prepared using just Assay Buffer (AB).

Dilutions must be performed externally in a separate transfer vessel (i.e. off-line and not in the BGM Galectin-3 antibody-coated plate). Recommended transfer vessels are a non-binding 96- well U-bottom microtiter plate ("transfer plate") or disposable test tubes composed of borosilicate glass, polypropylene or other low protein-binding plastic. If a transfer plate is used, ensure plate is clean by inspecting for dust particles prior to use and make sample dilutions in the corresponding wells per the plate map defined in step 3 above. Mix each dilution by performing multiple aspiration and dispense cycles with the pipette (if transfer plate is used), or by vortexing or inversion (if test tubes are used). Note: BGM Galectin-3 is designed to analyze samples (patient specimens and controls) that are diluted 10-fold (1 : 10) in Assay Buffer prior to analysis. This provides the proper sample to reagent ratio that yields optimal results within the measurement range up to 94.8 ng/mL.

Patient specimens that yield galectin-3 results greater than 94.8 ng/mL are preferably not be further diluted.

5. Prepare Diluted Controls

Dilute each reconstituted Control (CI and C2) 10-fold (1 : 10) using the Assay Buffer (AB) in transfer vessels (i.e., in designated wells of transfer plate, or disposable test tubes composed of borosilicate glass, polypropylene or other low protein-binding plastic). Mix each dilution by pipette aspiration, vortexing or inversion. Final dilution volume is preferably sufficient for duplicate measurement. It is recommended that a minimum of 30 μϊ_^ of the reconstituted control be used for the dilution (i.e. 30 μΐ, CI or C2 + 270 μΐ, AB). Dilutions must be performed externally (i.e. off-line and not in the BGM Galectin-3 antibody-coated plate). After use, remaining reconstituted or diluted (1 : 10) control material may be stored for a maximum of 10 days at 2-8°C, if reuse is desired.

6. Prepare Set of Diluted Standards

Dilute the standards immediately before use. Label 6 disposable test tubes with numbers S2 to S7 according to the dilution scheme illustrated in FIG. 1. Pipette 300 Assay Buffer (AB) into each labeled tube. Next, pipette 300 μΐ_^ Galectin-3 Standard (SI) to tube S2 and mix gently by pipette aspiration, vortexing or inversion. Then, transfer 300 μΐ_^ from tube S2 to tube S3 and mix gently, transfer 300 μΐ_^ from tube S3 to S4, etc. If using a transfer plate, pipette 300uL of SI through S7 into the corresponding wells of the transfer plate per the plate map defined in step 3 above. After use, remaining reconstituted or diluted standard material may be stored for a maximum of 10 days at 2 8°C, if reuse is desired.

7. Prepare Samples for Transfer

Samples are transferred from the transfer vessel (i.e. transfer plate or test tubes) using a multichannel pipette. If a transfer plate was used as the transfer vessel, the samples are already prepared for transfer to the BGM Galectin-3 antibody-coated plate using adjustable or multichannel pipette. If test tubes or vials were used as the transfer vessel, arrange all test tubes in a suitable rack corresponding to the sample order per the plate map defined in step 3 above so samples can be readily transferred to the BGM Galectin-3 antibody-coated plate using a multichannel pipette. 8. Transfer Samples

Transfer is preferably completed within 5 minutes, regardless of method.

Transfer 100 μΐ., of each sample (blank, diluted standards, diluted controls, and diluted test specimens) to duplicate wells of the BGM Galectin-3 antibody-coated plate using a multi- channel pipette and according to the plate map defined in step 3 above.

9. Seal and Incubate

Cover the wells with a clean plate seal and incubate for 1 hour ± 5 minutes at 20-25°C without shaking. The incubation time at this step is critical. The plate should incubate for 1 hour ± 5 minutes. Use of a timer is strongly recommended.

10. Remove Seal and Wash

Carefully remove the plate seal and wash wells with diluted wash buffer.

Mechanical washer: 400 μΐ., per well, 4 cycles. Dispensed wash should remain in wells a minimum of 15 seconds before aspiration step. After the fourth wash, empty wells by tapping on an absorbent paper towel. Inspect wells for any remaining wash and repeat tapping on absorbent paper towel if necessary.

Note: Prior to mechanical washing, ensure wash/aspirator tips have been adjusted to be close to the bottom of the wells but not touching or scratching the surface. If the mechanical washer model does not have the ability to adjust the washer wash/aspirator tip height, an additional wash cycle may be added if blank wells are inconsistent or the absorbance reading is too high. Manual wash: Empty wells, add 300 μΐ., wash buffer per well with a wash bottle and soak for 15 seconds; empty wells by tapping on an absorbent paper towel. Repeat 3 more times for a total of 4 wash cycles.

1 1. Prepare Diluted Tracer

Dilute the Tracer Concentrate 1 :30 with the Assay Buffer according to the dilution scheme shown in Table 1. After use, remaining diluted Tracer material may be stored for a maximum of 10 days at 2-8°C, if reuse is desired.

Note: This step may be performed while the plate is being washed. Table 1: Recommended Dilution Scheme for Tracer Concentrate (30x)*.

12. Add Diluted Tracer

Pipette 100 μΐ., diluted tracer solution to each well.

13. Seal and Incubate

Cover the wells with a clean plate seal and incubate 1 hour at 20-25°C without shaking.

14. Remove Seal and Wash

Carefully remove plate seal and wash wells with diluted wash buffer.

Mechanical washer: 400 μΐ., per well, 4 cycles. Dispensed wash should remain in wells a minimum of 15 seconds before aspiration step. After the fourth wash, empty wells by tapping on an absorbent paper towel. Inspect wells for any remaining wash and repeat tapping on absorbent paper towel if necessary.

Manual wash: Empty wells, add 300 μΐ., wash buffer per well with a wash bottle and soak for 15 seconds; empty wells by tapping on an absorbent paper towel. -^■Repeat 3 more times for a total of 4 wash cycles.

15. Add TMB Substrate and Incubate in the Dark

Pipette 100 TMB-substrate (TS) to each well and incubate the plate for 20 minutes at 20- 25°C in the dark. Note: Avoid pipetting directly from the TS bottle. Pour volume needed into intermediate 15 mL conical tube to measure volume needed, then transfer to the reservoir.

16. Add Stop Solution

Pipette 50 μΐ., stop solution (ST) to each well. Mix well by drawing up and down using a clean pipette tip, or by gently tapping the side of the plate. The contents of the well will turn from blue to yellow. Note: Other steps have required 100 μΐ,; this step requires only 50 μΐ,.

17. Remove Bubbles

Check for and remove any bubbles from the liquid surface of each well. Remove any dirt or liquid from the well exterior. Note: Bubbles may be removed by using a clean pipette tip to gently touch and burst the air bubble; be certain to use a clean tip for each well. 18. Measure Absorbance

Measure the absorbance of each well in a microtiter plate reader at 450 nm within 30 minutes of adding the stop solution.

[0074] Calculation of Results: BGM Galectin-3 is based on traditional

spectrophotometry and a multi-point standard (calibration) curve. After completing the assay steps, the absorbance of each specimen is read at 450 nm using the microplate reader. The absorbance is proportional to the concentration of galectin-3 in the specimens. Galectin-3 concentrations in the specimens and controls are based on the relationship of the absorbance of the specimens compared to that of the standards, which have a known concentration of galectin-3 and is preferably assigned using the following procedure:

• Verify that the average absorbance of the blank is less than the average absorbance of the lowest standard. If the absorbance of the blank exceeds the absorbance of the lowest standard (e.g. the low standard absorbance is negative when the blank absorbance is subtracted), the entire plate is preferably repeated.

· Subtract the average absorbance of the blank from each individual replicate absorbance of the standards, controls and test specimens.

• Calculate the average absorbance for each of the blank-corrected standards. Use the average of the blank-corrected absorbance for each standard to generate the standard curve using either 4-parameter logistic (4PL), 5-parameter logistic (5PL) or third order polynomial (cubic) curve fitting with least squares optimization (note: avoid use of

"Cubic-Spline" curve fit methods). See 'Materials Required but Not Provided' section for more details.

• Calculate concentrations for each of the duplicate measurements of unknown test specimens and controls based upon the selected curve fit equation using the corresponding blank-corrected absorbances. Multiply the measured concentration of specimens and controls by 10 (dilution factor of specimens and controls).

• Calculate the average, standard deviation, and coefficient of variation (CV) of the assigned concentration for each set of duplicate controls and test specimens.

• The coefficient of variation (CV) of the duplicate measurements of controls and test specimens is preferably within 20%. Specimens with duplicate CVs greater than 20% are preferably re-analyzed. If either of the controls has a duplicate CV greater than 20%, the entire plate is rejected and all specimens is preferably re-analyzed.

• Verify that the average concentration of the duplicate measurement for each control is within the corresponding acceptable range. If the average concentration of either control is out of the acceptable range, the assay is preferably repeated.

• Report the average concentration of the duplicate measurement of each test specimen as the galectin-3 concentration.

[0075] To validate the clinical effectiveness of the BGM Galectin-3 assay, galectin-3 levels were measured in a set of 895 banked EDTA plasma samples from participants in the United States and Canada in a controlled multi center clinical study, the Heart Failure: A

Controlled Trial Investigating Outcomes of Exercise Training (HF -ACTION) study. The HF- ACTION study involved 2,331 chronic HF patients with left ventricular dysfunction and with NYHA class II, III or IV symptoms. The average age of the 895 participants whose galectin-3 levels were assessed in the clinical validation study was 58 years, 29% were female, and 36% were non-white. Sensitivity analysis was performed comparing the set of 895 HF -ACTION subjects having evaluable galectin-3 values with all other HF ACTION participants, and it was found that the clinical validation results based on the evaluable set of subjects were robust and representative of the larger study population. The median follow-up time was approximately 30 months. Participants were categorized at baseline based on the following risk categories: · galectin-3 greater than 25.9 ng/mL

• galectin-3 greater than 17.8 ng/mL and less than or equal to 25.9 ng/mL

• galectin-3 less than or equal to 17.8 ng/mL

For the clinical validation study, Cox regression models were used to evaluate the association of baseline galectin-3 levels in HF patients with the endpoints of: (i) composite of all-cause mortality and all-cause hospitalization, (ii) cardiovascular mortality, (iii) composite of cardiovascular mortality and heart failure-related hospitalization, and (iv) all-cause mortality. Galectin-3 levels were found to be significantly associated with increased risk of each of these endpoints in Cox regression models (Table 2, Table 4, Table 6 and Table 9). Galectin-3 remained significantly associated with increased risk upon adjustment for baseline risk factors of age, gender, NYHA functional classification, left ventricular ejection fraction, diabetes status, and smoking status. FIG. 4 displays Kaplan Meier curves for the composite endpoint of all cause mortality and all-cause hospitalization, for HF Subjects in the Clinical Validation Study, by baseline galectin-3 category. FIG. 5, FIG. 6, and FIG. 7 display cumulative probabilities for events for the endpoints of the composite of all-cause mortality and all-cause hospitalization, cardiovascular mortality, and the composite of cardiovascular mortality and heart failure-related hospitalization, respectively, for HF subjects in the clinical validation study, by baseline galectin-3 category, and at timepoints of 6, 12, 24 and 36 months after baseline. FIG. 8 displays Kaplan-Meier curves for the endpoint of all-cause mortality, for HF Subjects in the Clinical Validation Study, by baseline galectin-3 level.

Table 2: Hazard Ratios for All-Cause Mortality and All-Cause Hospitalization Events for HF Subjects in the Clinical Validation Study.

The reference category is the < 17.8 ng/mL galectin-3 category.

Table 3: Cumulative Probability (with 95% Confidence Intervals) of Event for the Composite Endpoint of All-Cause Mortality and All-Cause Hospitalization, at Various Time Points and By Baseline Galectin-3 Level for HF Subjects in the Clinical Validation Study.

17.8 and < 25.9 ng mL 39.5 (32.5-47.3) 58.1 (50.8-65.7) 74.7 (67.7-81.1) 81.7 (74.9-87.6)

^*■ 25.9 na niL 44.2 (33.9-55.9) 55.8 (45.2-67.1) 79.8 (69.9-88.2) 82.1 (72.0-90.0)

Table 4: Hazard Ratios for Cardiovascular Mortality Events for HF Subjects in the Clinical Validation Study.

Table 5: Cumulative Probability (with 95% Confidence Intervals) of Event for the Cardiovascular Mortality.

at Various Time Points and By Baseline Galectin-3 Level, for HF Subjects in the Clinical Validation Study.

Table 6: Hazard Ratios for Cardiovascular Mortality and Heart Failure-Related Hospitalization Events for HF Subjects in the Clinical Validation Study.

Abbreviations: LVEF = left ventricular ejection fraction; NYHA = New York Heart Association. The reference category is the < 17.8 ng/mL galectin-3 category.

Table 7: Cumulative Probability (with 95% Confidence Intervals) of Event for Cardiovascular Mortality and Heart Failure-Related Hospitalization, at Various Time Points and By Baseline Galectin-3 Level, for HF Subjects in the Clinical Validation Study.

Table 8: Cumulative Probability (with 95% Confidence Intervals) of Event for the Endpoint of All-Cause Mortality, at Various Time Points and By Baseline Galectin-3 Level, for HF Subjects in the Clinical Validation Study.

< 1 7.X ng ^'niL 1.2% (0.6%-2.5%) 3.3 (2.1 -5.0) 8.7 (6.7-1 1.3) 15.3 (12.4-18.8) I 7.S and < 25.9 ng/mL 5.3 (2.8-10.0) 8.9 (5.5-14.4) 22.0 (16.3-29.4) 30.5 (23.4-39.1) 25. ng mL 2.6 (0.6-9.9) 9.1 (4.4-18.1) 26.6 (17.5-39.1) 35.5 (24.5-49.5)

Table 9: Hazard Ratios for All-Cause Mortality Events for HF Sub ects in the Clinical Validation Study.

A rev at ons: LVEF = e t vent cu ar e ect on ract on; NYHA = New Yor Heart Assoc at on. T e reference category is the < 17.8 ng/mL galectin-3 category.

[0076] Interpretation: The BGM Galectin-3 assay results are preferably interpreted in conjunction with clinical evaluation as an aid in assessing the prognosis of patients diagnosed with chronic heart failure. [0077] Patients with chronic heart failure with galectin-3 levels over 17.8 ng/mL were found to have a higher risk of adverse outcomes including mortality or hospitalization compared to patients with levels below 17.8 ng/mL. Galectin-3 levels between 17.8 ng/mL and 25.9 ng/mL are preferably interpreted with caution because these values lie within the reference range. [0078] Interpretation Relative to Natriuretic Peptides: Galectin-3 and natriuretic peptides are measures of separate and distinct biological processes. Each marker provides independent and complementary information on the prognosis of patients with chronic heart failure.

[0079] Table 10 illustrates this for N-terminal pro B-type natriuretic peptide (NT- proBNP) in the clinical validation study by evaluating primary endpoint event rates by categories of galectin-3 and NT-proBNP.

Table 10: Event Rates at 6, 12, 24 and 36 Months for the Composite Endpoint of All-Cause Mortality and All-Cause Hospitalization, by Galectin-3 Category and NT-proBNP level, for HF Subjects in the Clinical Validation Study. The median value for NT-proBNP in the Clinical Validation Study was 848 pg/mL.

[0080] Precision: Precision of BGM Galectin-3 was assessed in an evaluation according to the CLSI EP5-A2 guideline. Six (6) EDTA-plasma pools spanning a range of galectin-3 concentrations were analyzed in duplicate with two (2) runs per day over twenty (20) days using one (1) reagent lot, two (2) operators and one (1) microtiter plate reader. Estimates of within-run, run-to-run, day-to-day and total precision were calculated and met acceptance criteria. Results are summarized in Table 1 1.

Table 11: Precision of BGM Galectin-3.

5 46.2 ! 1.1 2.4 1.6 3.6 ! 0.5 1.1 2.0 4.4

6 1 72.2 ! 2.4 3.3 4.3 6.0 2.9 4.0 5.7 8.0

[0081] Table 1 1 shows the results of the precision evaluation with EDTA-plasma pools.

An additional experiment was performed using serum pools which yielded similar results. Additional test pools at multiple galectin-3 concentrations were also tested and yielded similar results.

[0082] Clinical Laboratory Precision: Precision was also evaluated at three (3) CLIA- certified clinical laboratories according the CLSI EP5-A2 guideline. The study included testing of EDTA-plasma pools spanning three (3) galectin-3 concentrations, across two (2) reagent lots, using three (3) different models of microtiter plate readers, and a total of four (4) different operators. Total testing days were 17, 18 and 20 days across the three sites, yielding 1 10 unique analytical runs. Results from each of the CLIA laboratories were within acceptable limits. Within run and total imprecision estimates are summarized in Table 12.

Table 12: Clinical Laboratory Precision - Within Run and Total Imprecision

[0083] Detection Limit: The limit of detection and limit of quantitation of BGM

Galectin-3 were established according to the recommendation of the CLSI EP 17-A guideline. The limit of blank (LoB) was determined as the 95th percentile value of forty-eight (48) replicate measurements of the BGM Galectin-3 Assay Buffer. The limit of detection (LoD) was LoD = LoB + c(beta) SDs, where SDs is the pooled standard deviations from four (4) serum samples with different levels of galectin-3, each of which was measured in sixteen (16) replicates (for a total of sixty-four (64) measurements) and c(beta) is the 95th percentile of the standard Gaussian distribution corrected for the degree of freedom. The Limit of quantitation (LoQ) was specified as the lowest galectin-3 concentration of the serum samples closest to while above the LoD, which is 1.32 ng/mL. For this sample, the coefficient of variation (CV) for the galectin-3 measurement was 10.4%. Limit of Blank (LoB): LoB = 0.86 ng/mL

Limit of Detection (LoD): LoD = 1.13 ng/mL

Limit of Quantitation (LoQ): LoQ = 1.32 ng/mL

[0084] Note: The LoQ does not represent the lower end of the measuring range and is preferably not be used for that purpose. The measuring range is 1.4 to 94.8 ng/mL as reported in the Measuring Range and Linearity sections of this package insert.

[0085] Cross Reactivity: BGM Galectin-3 displayed no significant cross-reactivity when tested in the presence of the following compounds: galectin-1, galectin-2, galectin-4, galectin-7, galectin-8, galectin-9, galectin-12, collagen I and collagen III, all at a concentration of 500 ng/mL. The mean % cross-reactivity of the above potential cross-reactants is at or below 0.3%.

[0086] Linearity: The linearity of BGM Galectin-3 was established according to the recommendations of the Clinical and Laboratory Standards Institute Evaluation Protocol 6 (CLSI EP6-A guideline). Samples were prepared to span a clinically-meaningful measurement range of galectin-3 concentrations. Linearity of BGM Galectin-3 was demonstrated between 1.4 and 94.8 ng/mL. These linearity data are shown FIG. 9.

[0087] High Dose Hook Effect: There is no high dose hook effect at galectin-3 levels up to 500 ng/mL.

[0088] Dilution Parallelism: Dilution parallelism was evaluated by analyzing ten (10) clinical specimens with endogenous native galectin-3 concentrations from 21.6 ng/mL to 88.5 ng/mL at 1 :20, 1 :40, 1 :80 and 1 : 160 dilutions. The grand mean recovery was 97.6%. For patient samples, dilute ten- fold (1 : 10) prior to measurement according to the instructions provided in the Procedure Section. Dilutions other than ten-fold are not recommended. [0089] Sample Matrices: The BGM Galectin-3 assay has been validated for use with plasma and serum. The equivalence of serum (no anticoagulant, no gel barrier) and EDTA plasma sample matrices were demonstrated in a study of forty-nine (49) matched serum and EDTA plasma samples with values spanning the measurement range. The regression equation is shown in the x/y scatter plot in FIG. 10 (BGM Galectin-3 Serum and EDTA-Plasma

Regression).

[0090] A study in forty one (41) subjects with matched samples (with values spanning the measuring range), demonstrated the equivalence of serum (no anticoagulant, no gel barrier), and each of the following matrices: · lithium heparin plasma

• serum (gel barrier tube)

• RST serum (gel barrier)

[0091] The regression equation demonstrating the equivalence of serum (no

anticoagulant, no gel barrier) and lithium heparin plasma is shown in the x/y scatter plot in FIG. 1 1 (BGM Galectin-3 Serum and Lithium Heparin Plasma Regression). FIG. 12 is an x/y scatter plot including the regression equation demonstrating the equivalence of serum (no

anticoagulant, no gel barrier) and serum (gel barrier tube). FIG. 13 is an x/y scatter plot including the regression equation demonstrating the equivalence of serum (no anticoagulant, no gel barrier) and RST serum (gel barrier). [0092] Reference Range Study: A reference distribution for galectin-3 was determined through an observational study. Galectin-3 levels were measured in 1,099 banked plasma samples from a population of apparently healthy subjects without known heart disease but that otherwise resemble, by age and gender distribution, the HF patient population. Specimens were from women between the ages of 60 and 80 years (n=575) and men between the ages of 55 and 80 (n=524). This reference population comprised individuals of different ethnic background, as follows: Black or African-American (n=307, 27.9%), Caucasian (n=691, 62.9%), Hispanic (n=42, 3.8%), Asian or Pacific Islander (n=30, 2.7%), and not specified (n=29, 2.6%). All subjects had detectable galectin-3 levels (min-max, 3.2 - 94.6 ng/niL) within the measuring range of BGM Galectin-3. Blood plasma samples were collected from study participant into tubes containing EDTA. The blood was processed and blood plasma was subsequently frozen at 20 C or colder. Table 13 summarizes the galectin-3 distribution results. The 97.5th percentile of the galectin-3 distribution from this reference population is 26.2 ng/niL.

[0093] Each laboratory should establish a reference range that is representative of the patient population to be evaluated. Additionally, each laboratory should consider their current practice in the evaluation of heart failure patients at each institution.

Table 13: Distribution of Galectin-3 Levels in Subjects without Known Heart Disease

[0094] As used in this specification, the term "substantially" or "approximately" means plus or minus 10% (e.g., by weight or by volume), and in some embodiments, plus or minus 5%. Reference throughout this specification to "one example," "an example," "one embodiment," or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the example is included in at least one example of the present technology. Thus, the occurrences of the phrases "in one example," "in an example," "one embodiment," or "an embodiment" in various places throughout this specification are not necessarily all referring to the same example. Furthermore, the particular features, structures, routines, steps, or characteristics may be combined in any suitable manner in one or more examples of the technology. The headings provided herein are for convenience only and are not intended to limit or interpret the scope or meaning of the claimed technology.

[0095] The phrase "and/or," as used herein should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and/or B," when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0096] The term "bioactive material" as used herein should be understood to mean any substance that has a biological effect, including without limitation proteins and peptides, nucleic acids, lipids, carbohydrates, drugs, etc. The terms "quantify," "quantitate," and the like, as used herein, refer to the process of measuring a signal associated with binding of one material (as a non-limiting example, an antibody) to another material, (as a non-limiting example, an amount of galectin-3) in a sample and comparing the magnitude of the signal to the magnitude of the signal associated with a reference standard or another sample to determine a relative or absolute quantity associated with one of the materials (e.g., a concentration, mass, moles, volume in a sample, etc.).

[0097] As used herein, "fibrosis" refers to the formation of fibrous connective tissues within the organs or tissues of a patient. Fibrosis may be reactive, or may accompany tissue repair or regeneration, and may be diffuse or confluent, mild or severe.

[0098] It should also be noted that embodiments may be provided as one or more computer-readable programs embodied on or in one or more articles of manufacture. The article of manufacture may be any suitable hardware apparatus, such as, for example, a floppy disk, a hard disk, a CD ROM, a CD-RW, a CD-R, a DVD ROM, a DVD-RW, a DVD-R, a flash memory card, a PROM, a RAM, a ROM, or a magnetic tape. In general, the computer- readable programs may be implemented in any programming language. Some examples of languages that may be used include C, C++, or JAVA. The software programs may be further translated into machine language or virtual machine instructions and stored in a program file in that form. The program file may then be stored on or in one or more of the articles of manufacture.

[0099] The terms and expressions employed herein are used as terms and expressions of description and not of limitation, and there is no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof. In addition, having described certain embodiments, it will be apparent to those of ordinary skill in the art that other embodiments incorporating the concepts disclosed herein may be used without departing from the spirit and scope of the invention. Accordingly, the described embodiments are to be considered in all respects as only illustrative and not restrictive.

INCORPORATION BY REFERENCE

[00100] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

[00101] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. [00102] What is claimed is:

Claims

CLAIMS 1. A kit for detecting galectin-3 in a sample, the kit comprising a first binding moiety comprising a monoclonal antibody or functional fragment thereof produced by the cell line 87B5 and a second binding moiety comprising a monoclonal antibody or functional fragment thereof produced by the cell line M3/38, wherein the kit provides a result indicative of the detection of galectin-3 in the sample above a threshold of 100 picograms per milliliter.

2. The kit of claim 1, provided in a form suitable for use in a sandwich assay.

3. The kit of claim 1 or 2, wherein the first binding moiety is operably bound to a solid support.

4. The kit of any one of claims 1-3, wherein the second binding moiety is operably bound to a solid support.

5. The kit of any one of claims 1-4, wherein the second binding moiety is provided in a solution comprising 10 mM phosphate buffered saline (PBS) having a pH 7.4 and 1% bovine serum albumin (BSA).

6. The kit of any one of claims 1-5, further comprising a recombinant galectin-3 polypeptide.

7. The kit of any one of claims 1-6, wherein the sample comprises blood, serum, plasma, urine or a fraction thereof.

8. The kit of claim 7, wherein the sample is obtained from a human subject with a risk for heart failure or a symptom thereof or a human subject whose risk of heart failure or a symptom thereof is desired to be determined.

9. The kit of any one of claims 1-8, further comprising third and fourth binding moieties, each of the third and fourth binding moieties comprising at least one antibody or functional fragment of an antibody that immunospecifically binds to a biomarker selected from the group consisting of cardiac troponin I (cTNI), cardiac troponin T (cTNT), tropomyosin, myosin light chain- 1, and heart-type fatty acid binding protein.

10. A method for patient stratification, comprising the steps of:

quantitating, in at least one sample of fluid or tissue from a patient, levels of galectin-3 and at least one additional biomarker;

comparing the levels of galectin-3 and the at least one additional biomarker to a plurality of thresholds for galectin-3 and the at least one additional biomarker; and

assigning the patient to a cohort defined by one or more of the plurality of thresholds, wherein the cohort is characterized by at least one of a specified risk of incident heart failure and a specified heart failure status.

1 1. The method of claim 10, wherein at least one additional biomarker is selected from the group consisting of markers of fibrosis, markers of myocyte injury or necrosis, markers of myocyte stretching, markers of extracellular matrix remodeling, inflammatory markers and cell adhesion markers.

12. The method of claim 10 or 1 1, wherein at least one additional biomarker is selected from the group consisting of: collagen type I, collagen type III, type I collagen telopeptidase, ΡΓΝΡ, PIIINP, matrix metalloproteinase 1 (MMPl), MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP1 1, MMPl 2, MMP13, MMPl 4, MMP 15, MMPl 6, MMPl 7, MMP 19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, TIMP1, TIMP2, TIMP3, TIMP4, elastin, fibronectin, Laminin Al

(LAMA1), LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMB 3, LAMB4, LAMC 1 , LAMC2, and LAMC3.

13. The method of claim 10 or 1 1, wherein at least one additional biomarker is selected from the group comprising: cTNI, cTNT, tropomyosin, myosin light chain- 1 , and heart-type fatty acid binding protein.

14. The method of claim 10 or 1 1, wherein at least one additional biomarker is selected from the group comprising: CRP, adiponectin, MCP-1, CD40 ligand, and Lp-PLA2.

15. The method of claim 10 or 1 1, wherein at least one additional biomarker is selected from the group comprising BNP and NT-proBNP.

16. The method of any one of claims 10-15, further comprising the step of selecting a treatment for the patient based that has been shown to be effective for the patient's assigned cohort.

17. The method of any one of claims 10-16, further comprising the step of determining an efficacy of a treatment for the patient's assigned cohort.

18. A system for stratifying patients, comprising:

means for determining a level of galactin-3 and at least one level of an additional biomarker in a patient sample;

at least one memory for storing:

at least one patient data record including levels of galectin-3 and the at least one additional biomarker in a patient sample; and

a plurality of thresholds for galectin-3 and at least one additional biomarker; and a processor for stratifying patients by executing instructions for: comparing the levels of galectin-3 and the at least one additional biomarker to the plurality of thresholds, and associating the patient record with a patient cohort defined by one or more of the plurality of thresholds, wherein the patient cohort is characterized by at least one of a specified risk of incident heart failure and a specified heart failure status.

19. The system of claim 18, wherein at least one additional biomarker is selected from the group comprising markers of fibrosis, markers of myocyte injury or necrosis, markers of myocyte stretching, markers of extracellular matrix remodeling, inflammatory markers and cell adhesion markers.

20. The system of claim 18 or 19, wherein at least one additional biomarker is selected from the group comprising: cTNI, cTNT, tropomyosin, myosin light chain- 1, and heart-type fatty acid binding protein.

21. The system of claim 18 or 19, wherein at least one additional biomarker is selected from the group comprising: CRP, adiponectin, MCP-1, CD40 ligand, and Lp-PLA2.

22. The system of claim 18 or 19, wherein at least one additional biomarker is selected from the group comprising BNP and NT-proBNP.

23. A method of determining the efficacy of a drug, comprising the steps of:

administering the drug to a plurality of patients;

quantitating, in each of a plurality of samples of fluid or tissue from a plurality of patients, a level of galectin-3 and at least one additional biomarker;

comparing, for each patient, the levels of galectin-3 and at least one additional biomarker to a plurality of thresholds and assigning each patient associated with a patient sample to one of a plurality of cohorts, each cohort defined by at least one of the plurality of thresholds; and

evaluating the efficacy of the drug in at least one patient cohort.