WO2016033650A1 - Markers of disease susceptibility and onset and uses therefor - Google Patents

Abstract

The present invention discloses methods, compositions and kits for making clinical assessments, such as early diagnostic, diagnostic, disease stage, disease severity, disease subtype, disease susceptibility, response to therapy or prognostic assessments. More particularly, the present invention discloses methods, compositions and kits for identifying a subject with, or at risk of developing, Type 1 diabetes (T1D), or stratifying a subject with risk of development of T1D to a treatment regimen based on a biomarker profile including hematopoietic cell number or heparanase expression.

Description

TITLE OF THE INVENTION

"MARKERS OF DISEASE SUSCEPTIBILITY AND ONSET AND USES THEREFOR"

RELATED APPLICATIONS

[0001] This application claims priority to Australian Provisional Application No. 2014903514 entitled "Markers of Disease Susceptibility and Onset and Uses therefor", filed on 3 September 2014, the entire subject matter of which is hereby incorporated by reference herein .

FIELD OF THE INVENTION

[0002] The present invention relates to methods, compositions and kits for making clinical assessments, such as ea rly diagnostic, diagnostic, disease stage, disease severity, disease subtype, disease susceptibility, response to therapy or prognostic assessments. More particularly, the present invention relates to methods, compositions and kits for identifying a subject with, or at risk of developing, Type 1 diabetes (T1 D), or stratifying a subject with risk of development of T1D to a treatment regimen based on a biomarker profile.

BACKGROUND OF THE INVENTION

[0003] Heparanase is an endo-3-d-glucuronidase that degrades the

glycosaminoglycan heparan sulfate (HS) . Cloning studies have identified that catalytically active heparanase is encoded by a single mammalian gene ( 1 -3). Heparanase is initially produced as an inactive pre-proenzyme which undergoes post-translational processing to yield a 65 kDa proenzyme for secretion . Proteolytic cleavage of proheparanase by the cysteine protease Cathepsin L leads to the formation of catalytically active heparanase, a heterodimer consisting of a 50 kDa (human) or 48 kDa (mouse) polypeptide non- covalently bound to a 8 kDa peptide ( 1, 2, 4-8) . HS is a linear polysaccharide that consists of a repeating disaccharide composed of N -acetylated glucosamine (GlcNAc) and uronic acid [glucuronic acid (GIcA) or iduronic acid (IdoA)] . HS biosynthesis occurs in the Golgi compartment of cells, with the assembly of component sugar residues occurring directly onto the core proteins of heparan sulfate proteoglycans (HSPGs) (9-11) . During the polymerization of HS chains, selected sugar residues are chemically modified by a suite of enzymes (N -deacetylase-N -sulfotranferase, C5 epimerase, and 2, 3, and 6-0- sulfotransferases), resulting in HS chains with regions that are highly sulfated and other regions of lower or no sulfation ( 10, 11) . The sulfated regions of HS, in particular, bind to a vast array of bioactive ligands that include cytokines, chemokines, g rowth factors, adhesion molecules, lipases, and proteases ( 12, 13) .

[0004] Typically, HSPGs are localized at the cell surface (e.g. , syndecans 1-4, glypicans 1-6), in the extracellular matrix (ECM), in basement membra nes (BMs) (e.g., perlecan, collagen type XVIII, and agrin) and have been identified in the nucleus of certain cells (14, 15). Secreted proheparanase rapidly interacts with cell surface HSPGs and the proheparanase-HSPG complex subsequently undergoes endocytosis. Similarly, heparanase can be internalized after binding to cell surface lipoprotein receptor-related proteins (LPRs) and mannose-6-phosphate receptors (MPRs) (16). Internalized

proheparanase is cleaved by intracellular Cathepsin L at acidic pH in late endosomes or lysosomes, to form catalytically active heparanase which can either degrade co- endocytosed HS, thereby regulating the turnover of cell-associated HS, or undergo storage within the lysosomes for subsequent secretion (6, 17-21). Optimal heparanase- mediated cleavage of glycosidic bonds in HS occurs at pH 5.5-6.0 and typically at sites adjacent to N - or 6-O-sulfated glucosamine (16, 22), e.g., the linkage of glucuronic acid to 6- O-sulfated glucosamine (23). HS in BMs and ECM is degraded by heparanase secreted by platelets, endothelial cells, leukocytes, and metastasizing tumor cells (12). In these settings, heparanase activity can result from (i) activation of proheparanase bound to cell surface HSPG or to cation-independent MPRs (CIMPRs) by an extracellular source of Cathepsin L, e.g., produced by macrophages (24, 25); (ii) cytokine-, fatty acid-, or nucleotide-stimulated release of an intracellular pool of catalytically active heparanase (26-29) which may be subsequently captured by cell surface receptors such as CIMPRs (25); or (iii) platelet degranulation (30). This regulated release of heparanase in the local microenvironment limits the availability of heparanase activity, preserving the essential and diverse biological functions of HS.

[0005] Heparanase also exhibits non-enzymatic functions which impact on cell signaling, adhesion, and migration, as well as on gene expression. Such functions are generally expressed at neutral pH (31-33). Interaction of heparanase with cell surface receptors on endothelial cells activates intracellular Akt, PI3K, and p38 kinase signaling to stimulate cell migration and Src kinase-mediated upregulation of vascular endothelial growth factor (VEGF) for angiogenesis (6, 18, 34). Heparanase lacking catalytic enzyme activity has been shown to increase the expression of certain growth factors (35) and to facilitate cell binding to HS in the ECM and to endothelial cells in vitro (32).

[0006] Intra-nuclear heparanase modulates intra-nuclear HS/HSPGs and exerts direct effects on gene transcription. Transfer of heparanase to the nucleus occurs via

Hsp90 in endothelial cells following fatty acid stimulation (29). Intra-nuclear heparanase decreases the level of the HSPG syndecan-1 in the nucleus of myeloma cells (14) and cleaves nuclear HS which in turn inhibits histone acetyltransferases (36). Recently, active heparanase has been reported to directly mediate epigenetic effects by regulating histone methylation, a process that directly influences the transcription of certain immune response genes involved in T-cell migration and function, e.g. , IL-2 and IFN-γ (37). Heparanase was also found to bind to the promoters of micro-RNAs involved in T-cell differentiation (37) and to influence the transcription of genes encoding enzymes involved in glucose metabolism (29). Such nuclear roles for heparanase, either with or without HS-degrading activity, would be expected to impact on T cells in inflammatory responses.

Heparanase and Inflammation

[0007] Heparan sulfate has several important biological functions which are regulated by heparanase in inflammation . An inflammatory response is generated when leukocytes are rapidly recruited from the blood to sites of tissue inj ury. In the ea rly stages of infla mmation, cell surface HS on cytokine-activated or inflamed endothelial cells functions in presenting lymphocyte-attractant chemokines to leukocytes in the vascular lumen ( 12, 38) . The subsequent immobilization of the leukocytes (e.g. , T cells) at the endothelial cell surface is enhanced by the binding of chemokine-activated integrins on the leukocytes to adhesion molecules such as ICAM - 1 or VCAM- 1 expressed on endothelial cells. Such interactions could potentially be facilitated by the binding of T cell - bound inactive heparanase to HS expressed on the surface of endothelial cells ( 12, 32, 33) . The chemokine-binding role for endothelial cell surface HS may also function in establishing a chemokine gradient to direct leukocyte migration across the endothelium ( 12) . Having crossed the blood vessel wall, most probably by passing between

endothelial cells, inflammatory leukocytes employ degradative mechanisms to traverse the sub-endothelial BM . In fact BM HS, particularly associated with the HSPG perlecan, helps the BM to act as a barrier to leukocyte migration . This barrier property is attributed to the length of HS chains (up to 400 sugar residues) and to their intrinsic capacity to interact with other BM matrix proteins, forming a cell -impenetrable scaffold ( 12) . To overcome this obstacle, leukocytes including T cells (39, 40), nearby endothelial cells (26) and possibly platelets (40) produce hepara nase to degrade BM HS and proteases to destroy BM matrix proteins. The disassembly of the BM matrix components aids the passage of leukocytes across the BM and their entry into the surrounding tissue.

Similarly, heparanase is released by inflammatory leukocytes to solubilize HS in the ECM of underlying tissues and to aid their navigation to sites of inflammation ( 12) . During the course of the degradation of extracellular HS, HS-bound cytokines and chemokines can be liberated into the local microenvironment, potentially augmenting cell recruitment and exacerbating the inflammatory response ( 12) .

[0008] The role for heparanase as a "path-maker" required by migrating leukocytes is of particular significance for T cell-mediated autoimmune diseases. Indeed, heparanase activity represents a prime target for anti -inflammatory drug development. Experimental autoimmune encephalitis (EAE; an experimental model of multiple sclerosis) in rats was largely prevented by in vivo treatment with sulfated

polysaccharides. This effect was attributed to the inhibition of heparanase produced by activated T cells, which in turn blocked the solubilization of the sub-endothelial BM (41, 42) . In a delayed-type hypersensitivity (DTH) experimental model of inflammation, inhibition of endothelial cell-derived heparanase prevented the degradation of sub- endothelial BM HS and leukocyte migration (27). Heparanase, possibly produced by inflammatory cells in rheumatoid arthritis in humans, may release cytokines and/or chemokines from degraded HS in the ECM of rheumatoid joints, promoting joint destruction (43). In ulcerative colitis and Crohn's disease, which represent chronic inflammatory disorders, heparanase is preferentially produced by inflamed gut epithelial cells to drive a local circuit of inflammation (24, 44). A role for heparanase has therefore been established in a broad range of inflammatory conditions.

Heparanase and pathogenesis of Type 1 Diabetes

[0009] Type 1 diabetes is an autoimmune disease which has been extensively studied in non-obese diabetic (NOD) mice, a recognized preclinical model of T1D in humans. During T1D, the insulin-producing beta cells in the islets of Langerhans in the pancreas are selectively destroyed by a T cell-mediated autoimmune response (45). The priming of autoreactive T cells to their cognate beta cell-specific autoantigens most probably occurs in the draining pancreatic lymph nodes, possibly as a consequence of both the abnormal responsiveness of effector T cells and inadequate control by regulatory T cells (46). Histological studies of NOD female mice at an early age (~6-7 weeks) revealed that leukocytes initially accumulate around the periphery of the islets, forming foci of non-destructive inflammation (insulitis). In adult pre-diabetic mice, the insulitis advances to a destructive phenotype, with peri-islet inflammatory leukocytes invading the islet cell mass (45). However, this pathology does not occur as a

synchronized process throughout the pancreas, and the proportion of affected islets as well as the seventy of leukocyte invasion progressively increases with time. Clinical symptoms of T1D are observed in 60-80% of female NOD mice from ~100 days of age or older, and are characterized by blood glucose levels exceeding >20 mmol/L

(hyperglycemia).

[0010] In view of the established role for heparanase in leukocyte migration in other experimental models of inflammation (see above), the present inventors predicted that during T1D development, heparanase produced by islet beta cell-specific

autoreactive T cells, inflammatory leukocytes, and possibly endothelial cells in the pancreatic vasculature, would be required to degrade HS in the sub-vascular endothelial BM. Thereafter, heparanase-mediated degradation of HS in the underlying pancreatic ECM would allow the inflammatory cells to migrate to individual islets and destroy the islet beta cells. Since T1D development is a chronic disease process, it was further speculated that there would be an on-going need for this degradative activity. Their studies identified, however, that the requirement for heparanase extended far beyond the enzymatic activity necessary for leukocyte migration and the establishment of chronic inflammation. Indeed a critical role for heparanase was discovered at the level of the islets themselves. This local involvement of heparanase stemmed from the exceptionally high levels of HS normally associated with the islets in situ (8). Initially the presence of a BM was confirmed at the islet periphery (i.e., peri-islet BM), which revealed the HSPG perlecan as a previously unrecognized constituent (47). This HS +ve islet BM was predicted to act as a barrier to invading cells, a nalogous to the sub-endothelial BM. On further investigation of the distribution of HS in normal mouse islets in situ, it was found that HS was expressed not only in the islet BM but at extraordinarily high levels throughout the islet cell mass (8). Immunohistochemical studies demonstrated that insulitis mononuclear cells (MNCs) in NOD mice strongly expressed cell surface heparanase. Furthermore, Western blot analyses showed that the insulitis leukocytes expressed high levels of catalytically active heparanase at the time of diabetes onset in NOD mice, in contrast to the expression of inactive heparanase by peri-islet leukocytes in young pre-diabetic mice (8). Intra-islet infiltration by insulitis MNCs correlated with disruption of the islet BM, loss of the islet BM matrix proteins including the HSPG perlecan (47), progressive loss of intra-islet HS and beta cell death (8). These studies indicated that such processes are mediated by catalytically active heparanase.

[0011] In vitro studies of beta cells isolated from normal mouse islets revealed both the unique intracellular localization of HS and its function in maintaining the viability of beta cells (8). Loss of intracellular HS correlated with beta cell death and conversely, the restoration of intracellular HS after culture of the beta cells with HS mimetics, correlated with beta cell survival. HS replacement not only preserved beta cell viability but rendered the beta cells resistant to oxidative damage induced by treatment with hydrogen peroxide [a source of reactive oxygen species (ROS)] (8). Collectively these findings led to the proposal that the intrinsic role of intracellular beta cell HS in situ in the pancreas is to protect the beta cells from the physiological levels of ROS generated as a consequence of their high metabolic and biosynthetic activity (8). Furthermore, it was reasoned that such a mechanism could compensate for the low levels of free radical scavenger enzymes in beta cells (48).

[0012] Together, these in vivo and in vitro studies identified multiple roles for heparanase in T1D, namely promoting the migration of leukocytes from pancreatic blood vessels (i.e., across the sub-endothelial BM and through the pancreatic ECM), aiding the passage of leukocytes across the islet BM and depleting islet beta cells of the intracellular HS needed for their survival. In support of this new paradigm, in vivo treatment of pre-diabetic adult NOD female mice with the heparanase inhibitor/HS mimetic, PI-88, for 180 days significantly delayed T1D onset and reduced the incidence of diabetes by ~50% (8). Compared to saline-treated control NOD mice, PI-88 treatment significantly increased the proportion of pancreatic islets that were intact, markedly reduced the proportion of islets that showed destructive insulitis and better preserved the HS content of the islets (8). This hallmark study has therefore unveiled T1D disease to be largely heparanase-dependent. The extraordinarily high HS content of the beta cells, which is essential for their survival, renders them particu larly vulnerable to heparanase- mediated damage. The localization of HS in the islet BM, which by convention acts as a barrier to impede leukocyte infiltration, has also been confirmed in normal human islets (49). These studies suggest that intracellular HS maintains beta cell survival at least in part, by acting as a "free radical sink," protecting the beta cells against harmful chemical species generated endogenously.

SUMMARY OF THE INVENTION

[0013] The present invention is predicated in part on the determination that hematopoietic cells, particularly leukocytes including myeloid cells such as neutrophils, eosinophils and inflammatory macrophages, of prediabetic individuals have significantly increased heparanase expression and that heparanase expression correlates with progression from an asymptomatic prediabetic state to onset of TID. The present inventors have also determined that the number of such hematopoietic cells in the peripheral circulation significantly decreases in prediabetic individuals as compared to the number of these cells in the peripheral circulation of healthy subjects, and that this decrease in cell number also correlates with progression from an asymptomatic prediabetic state to onset of TID. Based on these determinations, it is proposed that hematopoietic cell heparanase expression, including heparanase expression on myeloid cells such as neutrophils, eosinophils and inflammatory macrophages, and/or peripheral hematopoietic cell number are biomarkers for the progression of TID in asymptomatic prediabetic subjects, with utility for tracking TID development in subjects at a risk of developing TID, for identifying individuals who can benefit from intervention therapy designed to prevent TID disease progression a nd for monitoring the therapeutic efficacy of TID therapies, as described hereafter.

[0014] Accordingly, in one aspect, the present invention provides methods for determining whether a subject is at risk of developing TID. These methods generally comprise, consist or consist essentially of: determining the presence of a TID

susceptibility biomarker profile in the subject, which indicates that the subject is at risk of developing TID, wherein the TID susceptibility biomarker profile comprises a TID susceptibility hematopoietic cell heparanase expression level, or a TID susceptibility hematopoietic cell number, or both a TID susceptibility hematopoietic cell heparanase expression level and a TID susceptibility hematopoietic cell number. The hematopoietic cells are suitably leukocytes. In some embodiments, the hematopoietic cells express the surface marker CD45 and in representative examples of this type, the hematopoietic cells comprise myeloid cells, which are suitably selected from neutrophils, eosinophils and inflammatory macrophages and combinations thereof. The hematopoietic cells may additionally express any one or more of the surface markers CD3e, CD9, CD10, CDl lb, CDl lc, CD13, CD14, CD15s, CD16a, CD17, CD20, CD23, CD25, CD26, CD40, CD40L, CD97 (Ly6G), CD170 (Siglec F), CD177, Ly6E and Ly6C. [0015] In specific embodiments, the hematopoietic cells are peripheral blood cells. In some embodiments, the hematopoietic cells are enriched for leukocytes (e.g., myeloid cells, which are suitably selected from neutrophils, eosinophils and inflammatory macrophages and combinations thereof) prior to determining heparanase expression.

[0016] The TI D susceptibility heparanase expression level is suitably

determined by comparing the level of heparanase expression of the subject's

hematopoietic cells to a control heparanase expression level . In illustrative examples of this type, the control heparanase expression level is selected from : (i) a heparanase expression level of healthy control hematopoietic cells, wherein the healthy control hematopoietic cells are selected from hematopoietic cells of normal subjects or of subjects in which prediabetes and TI D are absent; and (ii) a heparanase expression level of TI D control hematopoietic cells (e.g., from one or more TI D subjects) . Suitably, the TID susceptibility heparanase expression level in hematopoietic cells is higher than the heparanase expression level of healthy control hematopoietic cells. In specific

embodiments, the TID susceptibility heparanase expression level is at least about 105%, 106%, 107% 108%, 109%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% of the healthy control heparanase expression level . In certain embodiments, the TID susceptibility heparanase expression level is lower than the heparanase expression level of TID control hematopoietic cells. In non-limiting examples of this type, the TID susceptibility heparanase expression level is no more than about 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, of the TI D control heparanase expression level . Heparanase expression may be evaluated at the gene, protein or activity level .

[0017] The TI D susceptibility hematopoietic cell number is suitably determined by comparing the number of hematopoietic cells in the peripheral circulation of the subject to a control number of hematopoietic cells. In illustrative examples of this type, the control number of hematopoietic cells is selected from : (i) the number of

hematopoietic cells in a healthy control subject or in a sample obtained therefrom, wherein the healthy control subject is from a normal subject or a subject in which prediabetes and TI D are absent; and (ii) the number of hematopoietic cells in a TID control subject or in a sample obtained therefrom. Suitably, the TID susceptibility hematopoietic cell number is lower than the healthy control hematopoietic cell number. In specific embodiments, the TI D susceptibility hematopoietic cell number is no more than about 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, of the healthy control hematopoietic cell number. In certain embodiments, the TID susceptibility hematopoietic cell number is higher than the TI D control

hematopoietic cell number. In non-limiting examples of this type, the TI D susceptibility hematopoietic cell number is at least about 105%, 106%, 107% 108%, 109%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% of the T1D control hematopoietic cell number.

[0018] In some embodiments, the subject lacks autoantibody directed to islets of Langerhans cells (also referred to herein "islet autoantibody") and is thus an islet autoantibody negative subject. In other embodiments, the subject is an islet

autoantibody positive subject. In illustrative examples of this type, the autoantibody positive subject expresses a single islet autoantibody. In other illustrative examples, the autoantibody positive subject expresses a plurality of islet autoantibodies (e.g., 2, 3, 4, 5 or more islet autoantibodies) . In some embodiments, the subject has a HLA haplotype that is associated with the presence or risk of development of T1 D (e.g., DRB1*0301- DQB1*0201 and DRB1*04-DQB1*0302) . Suitably, a subject that is determined to be at risk of developing T1D has prediabetes or has a n increased likelihood of having prediabetes.

[0019] In a related aspect, the present invention provides compositions for determining the heparanase expression level of hematopoietic cells in a subject, suitably a subject that is determined to be at risk of developing T1D, as broadly described above and elsewhere herein . These compositions generally comprise, consist or consist essentially of hematopoietic cells, as broadly described above and elsewhere herein, obtained from the subject and a reagent that detects the level or activity of hepa ranase expressed by the hematopoietic cells. The hematopoietic cells are suitably obtained from a suitably sample taken from the subject, as broadly described above and elsewhere herein.

[0020] In another aspect, the present invention provides methods for determining the likelihood of the presence or a bsence of a condition selected from a healthy condition (e.g. , a normal condition or one in which prediabetes and T1D a re absent), prediabetes and T1 D. These methods generally comprise, consist or consist essentially of: ( 1) providing a correlation of a reference biomarker profile with the presence or absence of a condition selected from a healthy condition , prediabetes and T1D, wherein the reference biomarker profile evaluates at least one biomarker selected from hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic cell number; (2) obtaining a biomarker profile of a sample from a subject, wherein the sample biomarker profile evaluates for an individual biomarker in the reference biomarker profile a corresponding biomarker; and (3) determining a likelihood of the su bject having or not having the condition based on the sample biomarker profile and the reference biomarker profile. An individual biomarker profile suitably indicates the level of heparanase expressed by the hematopoietic cells and/or the number of hematopoietic cells, which correlate(s) with the presence or absence of a condition, as defined above. In some embodiments, the methods comprise compa ring the sample biomarker profile with the reference biomarker profile and determini ng a likelihood of the presence or absence of the condition based on that comparison . Suitably, the methods further comprise correlating the reference biomarker profile with the presence or absence of a respective condition .

[0021] The inventors' findings enable treatment regimens, which can be used to treat subjects with biomarker profiles that correlate with the presence of prediabetes or TID. In some embodiments, these treatment regimens can be adopted or prescribed, particularly at an earlier stage in the prog ression towards TID, with a view to treating TID or preventing or delaying the onset of TI D in a subject. Thus, in another aspect, the present invention provides methods for treating or preventing a condition selected from prediabetes or TI D, or a symptom thereof, in a subject. These methods generally comprise, consist or consist essentially: (a) determining whether prediabetes or TID is present in the subject according to the methods broadly described above and elsewhere herein; and (b) exposing the subject, on the basis that the subject has the condition, to a treatment regimen for treating or preventing the condition, or a symptom thereof.

[0022] In a related aspect of the present invention, methods are provided for preventing or delaying the onset of TI D or a symptom thereof in a subject. These methods generally comprise, consist or consist essentially: (a) determining whether a subject is at risk of developing TID according to the methods broadly described above and elsewhere herein; and (b) exposing the subject, on the basis that the subject has an increased risk or likelihood of developing TID, to a treatment regimen for preventing or delaying the onset of TI D or a symptom thereof.

[0023] The inventors' findings also enable methods of monitoring the efficacy of a treatment regimen for treating TID, or for preventing or delaying the onset of TI D, and determining a subject's response to such treatment (e.g., whether it is a positive or negative response to such treatment) . Thus, in another aspect, methods are provided for monitoring the efficacy of a treatment regimen in a subject with TI D or at risk of developing TID. These methods generally comprise : ( 1) providing a correlation of a reference biomarker profile with a likelihood of having a healthy condition, wherein the reference biomarker profile evaluates at least one biomarker selected from hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic cell number; (2) obtaining a

corresponding biomarker profile of a subject with TI D or at risk of developing TI D after commencement of the treatment regimen, wherein a similarity of the subject's biomarker profile after commencement of the treatment regimen to the reference biomarker profile indicates the likelihood that the treatment regimen is effective for changing (e.g., improving) the health status of the subject. [0024] In another aspect, the present invention provides methods of correlating a reference biomarker profile with an effective treatment regimen for treating TID or for preventing or delaying the onset of TID, or a symptom thereof, wherein the reference biomarker profile evaluates at least one biomarker selected from hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic cell number. These methods generally comprise: (1) determining a sample biomarker profile from a subject with TID or at risk of developing TID prior to commencement of the treatment regimen, wherein the sample biomarker profile evaluates, for an individual biomarker in the reference biomarker profile, a corresponding biomarker; and (2) correlating the sample biomarker profile with a treatment regimen that is effective for treating TID or for preventing or delaying the onset of TID, or a symptom thereof.

[0025] In yet another aspect, the present invention provides methods of determining whether a treatment regimen is effective for treating TID or for preventing or delaying the onset of TID, or a symptom thereof, in a subject with TID or at risk of developing TID. These methods generally comprise : (1) correlating a reference biomarker profile prior to treatment with an effective treatment regimen for treating TID or for preventing or delaying the onset of TID, or a symptom thereof, wherein the reference biomarker profile evaluates at least one biomarker selected from hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic cell number; and (2) obtaining a sample biomarker profile from the subject after commencement of the treatment regimen, wherein the sample biomarker profile evaluates, for an individual biomarker in the reference biomarker profile, a corresponding biomarker, and wherein the sample biomarker profile after commencement of treatment indicates whether the treatment regimen is effective for treating TID or for preventing or delaying the onset of TID, or a symptom thereof, in the subject.

[0026] In still another aspect, the present invention provides methods of correlating a biomarker profile with a positive or negative response to a treatment regimen for treating TID or for preventing or delaying the onset of TID, or a symptom thereof. These methods generally comprise: (1) obtaining a sample biomarker profile from a subject with TID or at risk of developing TID following commencement of the treatment regimen, wherein the sample biomarker profile evaluates at least one biomarker selected from hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic cell number; and (2) correlating the sample biomarker profile from the subject with a positive or negative response to the treatment regimen.

[0027] In a further aspect, the present invention provides methods of determining a positive or negative response to a treatment regimen by a subject with T1D or at risk of developing T1D. These methods generally comprise: (a) correlating a reference biomarker profile with a positive or negative response to a treatment regimen for treating T1D or for preventing or delaying the onset of T1D, or a symptom thereof, wherein the reference biomarker profile evaluates at least one biomarker selected from hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic cell number; (b) determining a sample biomarker profile from the subject following commencement of the treatment regimen, wherein the sample biomarker profile evaluates, for an individual biomarker in the reference biomarker profile, a corresponding biomarker; and (c) determining a positive or negative response to the treatment regimen based on a comparison of the sample biomarker profile with the reference biomarker profile.

[0028] Another aspect of the present invention provides kits comprising one or more reagents and/or devices for use in performing any one of the methods of the present invention as broadly described above and elsewhere herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is graphical representation showing (A) heparanase and (B) Cathepsin L expression of various cell types including (i) myeloid, lymphoid and conventional dendritic cells (CDC) and (ii)T cells, B cells, inflammatory macrophages, resident macrophages, eosinophils, neutrophils and CDC isolated from islets of prediabetic NOD mice and onset diabetic NOD mice, which both have active insulitis. (C) Sixty percent of CD45+ insulitis leukocytes were (i) lymphocytes made up of about 40% T cells, about 20% B cells, (ii) about 2-3% dendritic cells and about 0.1-0.2% myeloid cells represented by (iii) inflammatory macrophages, eosinophils and neutrophils and other non-characterized myeloid cells. The strongest heparanase expression was observed in the myeloid cells, including macrophages, eosinophils and neutrophils with much weaker expression in B cells. A similar trend was observed for Cathepsin L expression.

[0030] Figure 2 is a graphical representation showing (A) heparanase and (B) Cathepsin L expression as well as (C) cell number of various cell types in peripheral blood from normal B6.SJL , prediabetic and new-onset NOD mice. Peripheral blood obtained from prediabetic and new-onset NOD mice show some changes in the T cell and B cell numbers, but a dramatic decline is shown in the peripheral blood myeloid cells, neutrophil numbers, inflammatory macrophages and eosinoph ils, as well as an increase in dendritic cells. In contrast to the observed decline in the peripheral blood myeloid cell population and in specific myeloid populations, the eosinophil and neutrophil sub- populations express substantially elevated levels of heparanase. Elevated Cathepsin L in myeloid cell populations was also observed.

[0031] Figure 3 is a graphical representation showing flow cytometry of heparanase expressed in normal B6.SJL (Norm; blue bar; n = 14), pre-TID NOD (orange bar; n = 10) and recent onset T1D NOD (green bar; n=6) peripheral blood and insulitis (a) T1D or at risk of developing T1D. These methods generally comprise: (a) correlating a reference biomarker profile with a positive or negative response to a treatment regimen for treating T1D or for preventing or delaying the onset of T1D, or a symptom thereof, wherein the reference biomarker profile evaluates at least one biomarker selected from hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic cell number; (b) determining a sample biomarker profile from the subject following commencement of the treatment regimen, wherein the sample biomarker profile evaluates, for an individual biomarker in the reference biomarker profile, a corresponding biomarker; and (c) determining a positive or negative response to the treatment regimen based on a comparison of the sample biomarker profile with the reference biomarker profile.

[0028] Another aspect of the present invention provides kits comprising one or more reagents and/or devices for use in performing any one of the methods of the present invention as broadly described above and elsewhere herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is graphical representation showing heparanase and Cathepsin L expression of various cell types including T cells, B cells, inflammatory macrophages, resident macrophages, eosinophils, neutrophils and conventional dendritic cells isolated from islets of prediabetic NOD mice and onset diabetic NOD mice, which both have active insulitis. Sixty percent of CD45+ insulitis leukocytes were lymphocytes made up of about 40% T cells, about 20% B cells, about 2-3% dendritic cells and about 0.1-0.2% myeloid cells represented by inflammatory macrophages, eosinophils and neutrophils and other non-characterized myeloid cells. The strongest heparanase expression was observed in the myeloid cells, including macrophages, eosinophils and neutrophils with much weaker expression in B cells. A similar trend was observed for Cathepsin L expression.

[0030] Figure 2 is a graphical representation showing heparanase and

Cathepsin L expression as well as cell number of various cell types in peripheral blood from normal B6.SJL , prediabetic and new-onset NOD mice. Peripheral blood obtained from prediabetic and new-onset NOD mice show some changes in the T cell and B cell numbers, but a dramatic decline is shown in the peripheral blood myeloid cells, neutrophil numbers, inflammatory macrophages and eosinophils, as well as an increase in dendritic cells. In contrast to the observed decline in the peripheral blood myeloid cell population and in specific myeloid populations, the eosinophil and neutrophil sub- populations express substantially elevated levels of heparanase. Elevated Cathepsin L in myeloid cell populations was also observed.

[0031] Figure 3 is a graphical representation showing flow cytometry of heparanase expressed in normal B6.SJL (Norm; blue bar; n= 14), pre-TID NOD (orange bar; n = 10) and recent onset T1D NOD (green bar; n=6) peripheral blood and insulitis (a)

- 11 - myeloid cells (pooled) and myeloid sub-populations, (b) macrophages, (c) eosinophils and (d) neutrophils. Data show + SEM GMFI (geometric mean fluorescence intensity) of cell surface heparanase staining, with ANOVA statistical analysis.

[0032] Figure 4 is a graphical representation showing heparanase (Hpse) activity in cell extracts of NOD mouse lymph nodes (LN) and peripheral blood leukocytes (PB). Hpse activity was detected using Fondaparinux (as a HS substrate) and a modified colorimetric assay (Hammond et a/., 2010. Anal Biochem 396 (1) : 112-116). The samples were prepared to remove the high background levels associated with tissue lysates. Hpse showed stronger activity in NOD female LN than NOD male LN, and was detected in NOD male PB leukocytes. Samples from NOD. Hpse knockout (NOD.HpseKO) mice showed no Hpse activity. Recombinant human Hpse served as a positive control.

DETAILED DESCRIPTION OF THE INVENTION

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0034] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0035] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

- 12 - 1. Heparanase-expressing hematopoietic cell profiles

[0036] The present invention is predicated in part on the surprising finding that subjects at risk of developing T1D such as prediabetic individuals, have a hematopoietic cell profile that distinguishes them from individuals who are not at risk of developing T1D, including healthy individuals, and from individuals with T1D. Specifically, the present inventors have found that heparanase expression is significantly increased in a subset of hematopoietic cells of prediabetic individuals, as compared to the

corresponding subset of healthy subjects and that heparanase expression correlates with progression from an asymptomatic prediabetic state to onset of T1D. The subset of hematopoietic cells includes myeloid cells, such as but not limited to neutrophils, eosinophils and inflammatory macrophages. The present inventors have also found that there are fewer hematopoietic cells of this type in the peripheral circulation of prediabetic individuals as compared to the peripheral circulation of healthy subjects and that there are even fewer of these cells in the peripheral circulation of subjects with T1D. Based on these findings, a subject's hematopoietic cell profile can be used as a biomarker to determine the subject's T1D status, including the presence or absence of T1D or risk of developing T1D.

[0037] Thus, disclosed herein are methods for determining whether a subject has T1D, is at risk of developing T1D or is otherwise healthy or lacking T1D or is not at risk of developing T1D based on the subject's biomarker profile, including the subject's hematopoietic cell profile of heparanase expression and cell number. These profiles will usually be compared to a reference biomarker profile that correlates with the presence or absence of a healthy condition, prediabetes or T1D, to thereby determine whether the subject has T1D, is at risk of developing T1D or is otherwise healthy or lacking T1D or is not at risk of developing T1D.

[0038] In accordance with the present invention, a biomarker profile that includes the level of expression of heparanase in a subset of hematopoietic cells and/or the number of such cells is indicative of a healthy state or an increased risk that the subject has or will develop T1D, or a symptom thereof. Thus, a change in the biomarker profile, including a change in heparanase expression level of a hematopoietic cell subset and/or change in the number of such cells, may reflect the nature (e.g., severity) of the physiological or pathophysiological state, or symptom thereof, of the subject to be tracked over a period of time. This approach may be useful, for example, in monitoring a treatment regimen for the purpose of assessing its effectiveness (or otherwise) in a subject. As used herein the term "heparanase expression" refers to transcription and/or translation and/or activity of heparanase. Several methods can be utilized to determine the level of heparanase expression, as described for example below.

[0039] Hematopoietic cell heparanase expression may be determined in cells derived from the bone marrow, the lymph node, or peripheral blood. In specific embodiments, the hematopoietic cells are peripheral blood cells. Heparanase expression may be determined in hematopoietic cell populations, whereupon heparanase-expressing versus heparanase non-expressing cells are readily determined. The hematopoietic cells in which heparanase is differentially expressed between healthy and prediabetic subjects and between prediabetic subjects and T1D subjects are suitably myeloid cells expressing the surface marker CD45, for example neutrophils, eosinophils and inflammatory macrophages, and combinations thereof. These hematopoietic cells are also referred to herein as "TID-associated hematopoietic cells". The TID-associated hematopoietic cells may additionally express any one or more of the surface markers CD9, CD10, CDl lb, CDl lc, CD13, CD14, CD15s, CD16a, CD17, CD20, CD23, CD25, CD26, CD40, CD40L, CD97 (Ly6G), CD170 (Siglec F), CD177, Ly6E and Ly6C. While heparanase expression in hematopoietic cell populations may be determined individually, as a means of classifying expressing versus non-expressing cells, it is also to be envisioned that numerous other marker proteins may be concurrently evaluated, in order to further classify hematopoietic cell populations.

[0040] The term "biomarker" typically refers to a measurable characteristic that reflects the presence or nature (e.g., severity) of a physiological and/or

pathophysiological state, including an indicator of risk of developing a particular physiological or pathophysiological state. For example, a biomarker may be present in a sample obtained from a subject before the onset of a physiological or pathophysiological state, including a symptom, thereof. Thus, the presence of the biomarker in a sample obtained from the subject is likely to be indicative of an increased risk that the subject will develop the physiological or pathophysiological state or symptom thereof.

Alternatively, or in addition, the biomarker may be normally expressed in an individual, but its expression may change (i.e., it is increased (upregulated ; over-expressed) or decreased (downregulated ; under-expressed) before the onset of a physiological or pathophysiological state, including a symptom thereof. Thus, a change in the level of the biomarker is likely to be indicative of an increased risk that the subject will develop the physiological or pathophysiological state or symptom thereof. Alternatively, or in addition, a change in the level of a biomarker may reflect a change in a particular physiological or pathophysiological state, or symptom thereof, in a subject, thereby allowing the nature (e.g., severity) of the physiological or pathophysiological state, or symptom thereof, to be tracked over a period of time. This approach may be useful in, for example, monitoring a treatment regimen for the purpose of assessing its

effectiveness (or otherwise) in a subject. As herein described, reference to the level of a biomarker includes cell number or cell activity when the marker is a cell, the

concentration of a biomarker, or the level of expression of a biomarker, or the activity of the biomarker, as will be described in more detail below.

- 14 - [0041] The term "reference biomarker" is used herein to denote a biomarker that has been identified as being associated with the presence or risk of development of TID, including an increased risk of developing TID. For example, a reference biomarker can be differentially expressed for a sample population of reference individuals at risk of developing TID as compared to healthy controls or TID affected subjects. Reference individuals include, but are not limited to, healthy subjects, prediabetic subjects, subjects with TID and first degree relatives (i.e., siblings) of individuals who have TID.

[0042] As used herein, the terms "profile" and "biomarker profile" are used interchangeably herein to denote any set of data that represents the distinctive features or characteristics associated with a condition of interest, such as with a particular prediction, diagnosis and/or prognosis of a specified condition as taught herein. The term generally encompasses quantification of one or more biomarkers, inter alia, nucleic acid profiles, such as, for example, gene expression profiles (e.g. , sets of gene expression data that represents mRNA levels of one or more genes associated with a condition of interest), as well as protein, polypeptide or peptide profiles, such as, for example, protein expression profiles (e.g., sets of protein expression data that represents the levels of one or more proteins associated with a condition of interest), cell number profiles including the number of cell types associated with the condition of interest (e.g., hematopoietic cells or subsets thereof), and any combinations thereof.

[0043] The term "reference biomarker profile" is used herein to denote a pattern of hematopoietic cell heparanase expression and/or hematopoietic cell number that has been identified as being associated with healthy or normal state, with TID or with a risk of developing TID, particularly an increased risk of developing TID. Reference individuals include, but are not limited to, normal or healthy subjects, prediabetic subjects and TID subjects. A reference biomarker profile provides a compositional analysis (e.g., concentration, number ratio or mole percentage (%) of the biomarker) in which one or more, two or more, three or more, four or more or a greater number of biomarkers are evaluated. A suitable biomarker is typically a biological characteristic that can be detected and measured in a subject in situ or in a biological sample obtained from an subject (e.g., ex vivo or in vitro). Examples of suitable biomarkers include specific cells (e.g., hematopoietic cells as described herein), molecules, or genes, gene products, enzymes, or hormones. Complex organ functions or general characteristic changes in biological structures can also serve as biomarkers. For example, body temperature is a well-known biomarker for fever and blood pressure can be used to determine the risk of stroke. Exemplary biomarkers in accordance with the present invention include hematopoietic cell heparanase expression level and hematopoietic cell number.

[0044] The terms "subject," "individual" and "patient" are used interchangeably herein to refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum

Chordata including primates, rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g. , pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars, etc.), marine mammals (e.g. , dolphins, whales), reptiles (snakes, frogs, lizards, etc.), and fish. A preferred subject is a primate (e.g. , a human, ape, monkey, chimpanzee).

[0045] As used herein, the term "prediabetes" refers to a condition

characterized by one or more of the following factors: the presence of islets

autoantibodies, reduced numbers of islets of Langerhans cells, suppression of the early peak of insulin secretion, glucose intolerance, an impairment in fasting glycaemia and/or any diabetic risk factor. Non-limiting examples of islet autoantibodies include those described by Buysschaert et al., Louvain Med. 119, S251-S258, 2000, representative examples of which include anti-islet (ICA), anti-glutamic acid decarboxylase (GAD), anti- tyrosine phosphatase (IA-2) and anti-(pro)insulin (AIA) auto-antibodies, or the anti- carboxypeptidase H, anti-64 kDa and anti-heat shock protein antibodies. As used herein, "reduced numbers of islets of Langerhans cells" refers to a decrease of at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85% in the number of islets of Langerhans cells relative to a healthy subject. The expression "impairment in fasting glycaemia and/or glucose intolerance", as used herein, refers to a fasting glycaemia of between 1.10 g/L and 1.26 g/L and a glycaemia after meals of between 1.40 g/L and 2 g/L. Diabetic risk factors include and encompass familial history, gestational diabetes, excess weight, obesity, insufficient physical exercise, high blood pressure, a high level of triglycerides, inflammation, hyperlipidaemia, etc.

[0046] The terms "normal subject" and "healthy subject", are used

interchangeably herein to refer to a healthy subject without disabilities ("Koujien", the sixth edition, by Iwanami Shoten, Publishers) or one lacking prediabetes or TID or a recognizable risk of developing prediabetes or TID.

[0047] As used herein, the term "TID subject" refers to individuals with insulin- dependent diabetes mellitus ("Type I" diabetics, IDD, or IDM). The hyperglycemia present in individuals with TID is associated with deficient, reduced, or nonexistent levels of insulin which are insufficient to maintain blood glucose levels within the physiological range. TID, often called juvenile or insulin-dependent diabetes results from altered metabolism of carbohydrates (including sugars such as glucose), proteins, and fats. In TID, the beta cells of the pancreas produce little or no insulin, the hormone that allows glucose to enter body cells. Once glucose enters a cell, it is used as fuel. Without adequate insulin, glucose builds up in the bloodstream instead of going into the cells. The body is unable to use this glucose for energy despite high levels in the bloodstream, leading to increased hunger. In addition, the high levels of glucose in the blood cause the patient to urinate more, which in turn causes excessive thirst. Other symptoms include increased weight loss and episodic ketoacidosis. Within 5 to 10 years after diagnosis, the insulin-producing beta cells of the pancreas are completely destroyed, a nd no more insulin is produced.

1.1 Evaluation of heparanase expression

[0048] Heparanase expression may be evaluated at the level of protein expression, either by demonstration of the presence of the protein, or by its catalytic endoglycosidase activity. Anti heparanase antibodies for use in heparanase-specific protein detection are described for example in U.S. Pat. No. 6,177,545; U.S. patent application Ser. Nos. 09/704,772; 09/322,977; 09/186,200; 09/944,602; 09/759,207; and PCT Application Nos. US99/25451 and US99/09255, which are incorporated by reference herein in their entirety. The antibodies bind both native and denatured heparanase protein and may be detected by several well-known assays in the art, including ELISA, RIA, light emission immunoassays, Western blot analysis,

immunofluorescence assays, immunohistochemistry and FACS analysis.

[0049] Enzyme linked immunosorbent (ELISA) assays and radioimmunoassays (RIA) follow similar principles for detection of specific antigens, in this case, heparanase. In RIA a heparanase-specific antibody is radioactively labeled, typically with ¹²⁵I. In ELISA assays a heparanase-specific antibody is chemically linked to an enzyme.

Heparanase specific capturing antibody is immobilized onto a solid support. Unlabeled specimens, e.g., protein extracts from biological samples are then incubated with the immobilized antibody under conditions where non-specific binding is blocked, and unbound antibody and/or protein removed by washing. Bound heparanase is detected by a second heparanase specific labeled antibody. Antibody binding is measured directly in RIA by measuring radioactivity, while in ELISA binding is detected by a reaction converting a colorless substrate into a colored reaction product, as a function of linked - enzyme activity. Changes can thus readily be detected by spectrophotometry (Janeway C. A. et a\. (1997). "Immunobiology" 3.sup. rd Edition, Current Biology Ltd., Garland Publishing Inc. ; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed.

(1994); "Current Protocols in Immunology" Volumes I-III Coligan 1 E., ed. (1994); Stites et al. (eds)). Both assays therefore provide a means of quantification of heparanase protein content in a biological sample.

[0050] The term "antibody" and its grammatical equivalents refer to a protein which is capable of specifically binding to a target antigen such as heparanase or a surface molecule on a hematopoietic cell and includes any substance, or group of substances, which has a specific binding affinity for an antigen, suitably to the exclusion of other substances. This term encompasses an immunoglobulin molecule capable of specifically binding to a target antigen by virtue of an antigen binding site contained within at least one variable region. This term includes four chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, primatized antibodies, de-immunized antibodies, half antibodies, bispecific antibodies) and single domain antibodies such as domain antibodies and heavy chain only antibodies (e.g. , camelid antibodies or cartilaginous fish

immunoglobulin new antigen receptors (IgNARs)) . An antibody generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallizable (Fc) . In specific embodiments, the antibodies comprise a four- chain structure as their basic unit. Full-length antibodies comprise two heavy chains («50-70 kDa) covalently linked and two light chains («23 kDa each) . A light chain generally comprises a variable region and a constant domain and in mammals is either a K light chain or a λ light chain . A heavy chain generally comprises a va riable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s) . Heavy chains of mammals are of one of the following types α, δ, ε, γ, or μ . Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by inter-chain disulfide bonds and by non-covalent interactions. The number of inter-chain disulfide bonds can vary among different types of antibodies. Each chain has an N -terminal variable region (V_H or V_L wherein each a re «110 amino acids in length) and one or more constant domains at the C-terminus. The constant domain of the light chain (C_L which is «110 amino acids in length) is aligned with and disulfide bonded to the first constant domain of the heavy chain (C_H which is «330-440 amino acids in length) . The light chain variable region is aligned with the variable region of the heavy chain. The antibody heavy chain can comprise 2 or more additional C_H domains (such as, C_H2, C_H3 and the like) and can comprise a hinge region can be identified between the C_H_ and Cm constant domains. Antibodies can be of any type (e.g., IgG, Ig E, IgM, IgD, IgA, and IgY), class (e.g ., IgGi, IgG₂, IgG₃, IgG₄, IgA_x and IgA₂) or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (suitably human) antibody. The term "antibody" encompasses not only intact polyclonal or monoclonal antibodies, but also variants, fusion proteins comprising an antibody portion with an antigen binding site, humanized antibodies, human antibodies, chimeric antibodies, primatized antibodies, de-immunized antibodies or veneered antibodies. Also within the scope of the term "a ntibody" are antigen binding fragments that retain specific binding affinity for an antigen, suitably to the exclusion of other substances. This term includes a Fab fragment, a Fab' fragment, a F(ab') fragment, a single chain antibody (SCA or SCAB) amongst others. An "Fab fragment" consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain . An "Fab' fragment" of an a ntibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact

- 18 - light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner. An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A (Fab')₂ fragment. An "Fv fragment" is a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains. A "single chain antibody" (SCA) is a genetically engineered single chain molecule containing the variable region of a light chain and the variable region of a heavy chain, linked by a suitable, flexible polypeptide linker.

[0051] Heparanase protein expression may also be detected via light emission immunoassays. Much like ELISA and RIA, in light emission immunoassays the biological sample/protein extract to be tested is immobilized on a solid support, and probed with a specific label, labeled anti-heparanase antibody. The label, in turn, is luminescent, and emits light upon binding, as an indication of specific recognition. Luminescent labels include substances that emit light upon activation by electromagnetic radiation, electro chemical excitation, or chemical activation and may include fluorescent and

phosphorescent substances, scintillators, and chemiluminescent substances. The label can be a part of a catalytic reaction system such as enzymes, enzyme fragments, enzyme substrates, enzyme inhibitors, coenzymes, or catalysts; part of a chromogen system such as fluorophores, dyes, chemiluminescers, luminescers, or sensitizers; a dispersible particle that can be non-magnetic or magnetic, a solid support, a liposome, a ligand, a receptor, a hapten radioactive isotope, and so forth (U.S. Pat. Nos. 6,410,696, U.S. Pat. No. 4,652,533 and European Patent Application No. 0,345,776), and provide an additional, highly sensitive method for detection of heparanase protein expression.

[0052] Western blot analysis is another means of assessing heparanase protein content in a biological sample. Protein extracts from biological samples of hematopoietic cells, particularly TID-associated hematopoietic cells, are solubilized in a denaturing ionizing environment, and aliquots are applied to polyacrylamide gel matrixes. Proteins separate based on molecular size properties as they migrate toward the anode. Antigens are then transferred to nitrocellulose, PVDF or nylon membranes, followed by membrane blocking to minimize non-specific binding. Membranes are probed with antibodies directly coupled to a detectable moiety, or are subsequently probed with a secondary antibody containing the detectable moiety. Typically the enzymes horseradish peroxidase or alkaline phosphatase are coupled to the antibodies, and chromogenic or luminescent substrates are used to visualize activity (Harlow E. et al (1998) Immunoblotting. In

Antibodies: A Laboratory Manual, pp. 471-510 CSH Laboratory, cold Spring Harbor, N.Y. and Bronstein I. Et al. ( 1992) Biotechniques 12: 748-753).

[0053] Unlike RIA, ELISA, light emission immunoassays and immunoblotting, which quantify heparanase protein content in whole samples,

- 19 - immunofluorescence/immunocytochemistry may be used to detect proteins in a cell- specific manner, though quantification is compromised.

[0054] TlD-associated hematopoietic cells may be isolated or enriched by methods known in the art. Isolation or enrichment of the hematopoietic cells refers to a process wherein the percentage of hematopoietic cells is increased (relative to the percentage in the sample before the enrichment procedure). Purification is one example of enrichment. In certain embodiments, the increase in the number of TlD-associated hematopoietic cells of the invention as a percentage of cells in the enriched sample, relative to the sample prior to the enrichment procedure, is at least 25-, 50-, 75-, 100-, 150-, 200-, 250-, 300-, 350-fold, and suitably is 100-200 fold. In specific embodiments, antibodies to surface markers on TlD-associated hematopoietic cells may be attached to a solid support to allow for separation. Procedures for separation may include magnetic separation, using antibody magnetic beads (e.g., Miltenyi™ beads), affinity

chromatography, "panning" with antibody attached to a solid matrix or any other convenient technique such as Laser Capture Microdissection. In specific embodiments, the TlD-associated hematopoietic cells are enriched using an antibody to CD45, which antibody is conjugated to a magnetic bead, and a magnetic cell separation device to separate out the CD45⁺ cells. Alternatively, or in addition, the TlD-associated

hematopoietic cells are enriched using at least one other antibody to a surface marker selected from CD9, CD10, CDl lb, CDl lc, CD13, CD14, CD15s, CD16a, CD17, CD20, CD23, CD25, CD26, CD40, CD40L, CD97 (Ly6G), CD170 (Siglec F), CD177, Ly6E and Ly6C. Other techniques providing particularly accurate separation include fluorescence activated cell sorting (FACS). Alternatively, blood smears may be prepared via standard hematological processes. Once cells are deposited on slides, they may be fixed, and probed with labeled antibody for detection of heparanase in a cell specific fashion.

[0055] Anti-heparanase antibodies may be directly conjugated to fluorescent markers, including fluorescein, FITC, rhodamine, Texas Red, Cy3, Cy5, Cy7, and other fluorescent markers, and viewed in a fluorescent microscope, equipped with the appropriate filters. Antibodies may also be conjugated to enzymes, which upon addition of an appropriate substrate commence a reaction providing a colored precipitate over cells with detected heparanase protein. Slides may then be viewed by standard light microscopy. Alternatively, primary antibodies specific for heparanase may be further bound to secondary antibodies conjugated to the detectable moieties. Cell surface expression can be thus assessed, and the addition of cell permeabilization solutions, such as Triton-X and saponin may be applied to facilitate reagent penetration within cell cytoplasms ("Cell Biology: A Laboratory Handbook", Volumes 1-111 Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan 1 E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk,

- 20 - Conn. (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980)).

[0056] Immunohistochemistry is quite similar to immunofluorescence or immunocytochemistry, in principle, however tissue specimens are probed with

heparanase antibody, as opposed to cell suspensions. Biopsy specimens are fixed and processed and optionally embedded in paraffin, sectioned if needed, providing cell or tissue slides subsequently probed with heparanase specific antibodies. Alternatively, frozen tissue may be sectioned on a cryostat, with subsequent antibody probing, obviating fixation-induced antigen masking. Antibodies, as in immunofluorescence or immunocytochemistry, are coupled to a detectable moiety, either fluorescent, or enzyme-linked, and are used to probe tissue sections by methods described for immunofluorescence, and are subsequently visualized by fluorescent or confocal microscopy, depending upon the detection method employed. Visualization of a reaction product precipitate may be viewed by standard light microscopy, if an enzymatic detectable moiety was utilized, following development of the reaction product ("Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980)).

[0057] In specific embodiments, FACS analysis is used to assess heparanase expression. A general description of FACS apparatus and methods in provided in U.S. Pat. Nos. 4,172,227; 4,347,935; 4,661,913; 4,667,830; 5,093,234; 5,094,940; and 5,144,224. Cells are introduced into the FACS machine and are delivered via tubing into the FACS cell, which they pass through as single cells. A laser beam is directed at the FACS cell, and forward laser scatter is collected by a photodiode, side laser scatter is directed to a PMT tube via a lens, directed to PMT1. Specific filters direct fluorescence from the side scatter to other PMT tubes for multivariate analysis. Side laser scatter is a reflection of cell size and granularity, and may be used to identify cell populations in mixed samples. Cells labeled with fluorescent anti-heparanase antibody may be detected by laser excitation and collection via PMT tubes, which can be identified for cell type via size and granularity, or via incorporation of additional cell surface markers for

identification, as for example disclosed above. Typically, FACS analysis is used for determination of cell surface expression of a particular protein, and hence heparanase specific antibodies may be utilized for probing detection of cell surface heparanase expression in TID-associated hematopoietic cell populations. Specific hematopoietic cell subtypes expressing surface heparanase protein may be ascertained by size and granularity characteristics, or alternatively by co-staining with additional cell surface marker proteins.

- 21 - [0058] Demonstration of the absence or presence of heparanase activity within a sample is an additional means of distinguishing heparanase expressing versus non- expressing populations. Heparanase catalytic activity assays are described in U.S. Pat. No. 6,190,875; U.S. patent application Ser. No. 09/753,692 and PCT Application No. US99/15643, which are incorporated by reference herein in their entirety. In principle, heparanase activity is measured in the presence of a heparanase substrate, wherein cleavage of the substrate into specific products serves as an indicator for enzyme activity.

[0059] The heparanase substrates may be selected from soluble or immobilized heparan sulfate proteoglycans, heparan sulfate or heparin. A detectable moiety is suitably incorporated within the assay, to effect ease of identification of heparanase activity. Detectable moieties used as such may be selected from the group consisting of chromogenic moieties, fluorogenic moieties, radioactive moieties and light-emitting moieties, enabling quantitative evaluation of heparanase activity via a suitable detecting equipment, e.g., a spectrophotometer, fluorimeter or luminometer, β-emission counter, a densitometer, and others. An exemplary quantitative calorimetric assay is the tetrazolium blue (an oxidative reagent) assay in which the reagents are reduced to a soluble colored formazan salt by the degraded substrate.

[0060] In other embodiments, heparanase expression is monitored by determining heparanase transcript levels. In order to isolate heparanase RNA, biological samples must be obtained for processing. For example, heparinized peripheral blood may be drawn and RNA extracted from the sample, or alternatively, if desired, leukocytes, including myeloid cells, may be isolated by differential gradient separation, using, for example, Ficoll-hypaque or sucrose gradient solutions for cell separations, followed by ammonium chloride or hypotonic lysis of rema ining contaminating erythrocytes ("Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994) ; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds)). Bone marrow and lymph node biopsies may be processed by collagenase/dispase treatment of the biopsy material, or by homogenization in order to obtain single cell suspensions ("Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed.

(1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds)) whereupon RNA is subsequently isolated.

[0061] RNA may be extracted from biological samples via a number of standard techniques (see Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Md . (1989)). Guanidium-based methods for cell lysis enabling RNA isolation, with subsequent cesium chloride step gradients for separation of the RNA from other cellular macromolecules, followed by RNA precipitation and resuspension, is an older, less commonly employed method of RNA isolation (Glisin, Ve. Et al (1973) Biochemistry 13 :

- 22 - 2633). Alternatively, RNA may be isolated in a single step procedure (U.S. Pat. No.

4,843,155, and Puissant, C. And Houdebine L. M. (1990) Biotechniques 8: 148-149). Single step procedures include the use of Guanidium isothiocyanate for RNA extraction, and subsequent phenol/chloroform/isoamyl alcohol extractions facilitating the separation of total RNA from other cellular proteins and DNA. Commercially available single-step formulations based on the above-cited principles may be employed, including, for example, the use of the TRIZOL reagent (Life Technologies, Gaithersburg, Md .).

[0062] Heparanase RNA/gene expression can be monitored via a number of other standard techniques, illustrative examples of which include Northern blot and dot blot analysis, primer extension, RNase protection, RT-PCR, in-situ hybridization and chip hybridization.

[0063] Specific heparanase RNA sequences can be readily detected by hybridization of labeled probes to blotted RNA preparations extracted as above. In Northern blot analysis, fractionated RNA is subjected to denaturing agarose gel electrophoresis, which prevents RNA from assuming secondary structures that might inhibit size based separation. RNA is then transferred by capillary transfer to a nylon or nitrocellulose membrane support and may be probed with a labeled oligonucleotide probe complementary to the heparanase sequence (Alwine, et al. (1977). Proc. Natl. Acad. Sci. USA 74: 5350-5354 and Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989)).

[0064] Alternatively, unfractionated RNA may be immobilized on a nylon or nitrocellulose membrane, and similarly probed for heparanase-specific expression, by Slot/Dot blot analysis. RNA slot/dot blots can be prepared by hand, or alternatively constructed using a manifold apparatus, which facilitates comparing hybridization signals by densitometry scanning (Chomczynski P. (1992) Anal. Biochem. 201 : 134-139).

[0065] Primer extension is another means whereby quantification of the RNA may be accomplished. Primer extension provides an additional benefit in mapping the 5' terminus of a particular RNA, by extending a primer using the enzyme reverse

transcriptase. In this case, the primer is an oligonucleotide (or restriction fragment) complementary to a portion of the heparanase mRNA. The primer is end-labeled, and is allowed to hybridize to template heparanase mRNA. Once hybridized, the primer is extended by addition of reverse transcriptase, and incorporation of unlabeled

deoxynucleotides to for a single-stranded DNA complementary to template heparanase mRNA. DNA is then analyzed on a sequencing gel, with the length of extended primer serving to map the 5' position of the mRNA, and the yield of extended product reflecting the abundance of RNA in the sample (Jones et al (1985) Cell 42: 559-572 and Mierendorf R. C. And Pfeffer, D. (1987). Methods Enzymol. 152: 563-566).

- 23 - [0066] RNase protection assays provide a highly sensitive means of quantifying heparanase RNA, even in low abundance. In protection assays, sequence-specific hybridization of ribonucleotide probes complementary to heparanase RNA, with high specific activity are generated, and hybridized to sample RNA. Hybridization reactions are then treated with ribonuclease to remove free probe, leaving intact fragments of annealed probe hybridized to homologous heparanase sequences in sample RNA.

Fragments are then analyzed by electrophoresis on a sequencing gel, when

appropriately-sized probe fragments are visualized (Zinn K. et al (1983) Cell 34: 865-879 and Melton S. A., et al (1984). Nucl. Acids Res. 12: 7035-7056).

[0067] RT-PCR is another means by which heparanase expression may be analyzed . RT-PCR employs the use of reverse transcriptase to prepare cDNA from RNA samples, using deoxynucleotide primers complementary to the hepara nase mRNA. Once the cDNA is generated, it is amplified through the polymerase chain reaction, by the addition of deoxynucleotides and a DNA polymerase that functions at high temperatures. Through repetitive cycles of primer annealing, incorporation of deoxynucleotides facilitating cDNA extension, followed by strand denaturation, amplification of the desired sequence occurs, yielding an appropriately sized fragment that may be detected by agarose gel electrophoresis. Optimal reverse transcription, hybridization, and

amplification conditions will vary depending upon the sequence composition and length(s) of the primers and target(s) employed, and the experimental method selected by the practitioner. Various guidelines may be used to select appropriate primer sequences and hybridization conditions (see, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, (Volumes 1-3) Cold Spring Harbor Press, N.Y. ; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.).

[0068] In-situ hybridization provides may be used for detecting and localizing cell/tissue specific heparanase RNA expression. Labeled anti-sense RNA probes are hybridized to mRNAs in cells singly, or in processed tissue slices, which are immobilized on microscope glass slides (In Situ Hybridization : Medical Applications (eds. G. R.

Coulton and J. de Belleroche), Kluwer Academic Publishers, Boston (1992); In Situ Hybridization : In Neurobiology; Advances in Methodology (eds. J. H. Eberwine, K. L. Valentino, and J. D. Barchas), Oxford University Press Inc., England (1994); and In Situ Hybridization : A Practical Approach (ed. D. G. Wilkinson), Oxford University Press Inc., England (1992)). Numerous non-isotopic systems have been developed to visualize labeled DNA probes including; a) fluorescence-based direct detection methods, b) the use of digoxigenin- and biotin-labeled DNA probes coupled with fluorescence detection methods, and c) the use of digoxigenin-and biotin-labeled DNA probes coupled with antibody-enzyme detection methods. When fluorescence-labeled anti-sense RNA probes are hybridized to cellular RNA, the hybridized probes can be viewed directly using a

- 24 - fluorescence microscope. Direct fluorochrome-labeling of the nucleic acid probes eliminate the need for multi-layer detection procedures (e.g., antibody-based-systems), which allows fast processing and also reduces non-specific background signals, hence providing a versatile and highly sensitive means of identifying heparanase gene expression.

[0069] Chip hybridization utilizes heparanase specific oligonucleotides attached to a solid substrate, which may consist of a particulate solid phase such as nylon filters, glass slides or silicon chips [Schena et al . ( 1995) Science 270 : 467-470] designed as a microarray. Microarrays are known in the art and consist of a surface to which probes that correspond in sequence to gene products (such as cDNAs) can be specifically hybridized or bound at a known position for the detection of heparanase gene

expression.

[0070] Quantification of the hybridization complexes is well known in the art and may be achieved by any one of several approaches. These approaches are generally based on the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. A label can be applied to either the oligonucleotide probes or the RNA derived from the biological sample.

[0071] In general, mRNA quantification is suitably effected alongside a calibration curve so as to enable accurate mRNA determination . Furthermore, quantifying transcript(s) originating from a biological sample is preferably effected by comparison to a normal sample, which sample is characterized by normal expression pattern of the examined transcript(s) .

[0072] In certain embodiments, hepa ranase expression is monitored by determining the expression of Cathepsin L. As known in the art, proteolytic cleavage of proheparanase by the Cathepsin L leads to the formation of catalytically active heparanase and thus, Cathepsin L can be used as a surrogate marker of hepa ranase expression.

1.2 Evaluation of TI D-associated hematopoietic cell number

[0073] Hematopoietic cell numbers need not be evaluated as absolute values. In some embodiments, cell numbers may be expressed as a percentage or ratio of the total number of cells in the blood (e.g., cells per ml. blood) or of the total number of a subset of cells, such as peripheral blood mononuclear cells (PBMC) . Methods by which the number of hematopoietic cells can be determined will be known to persons skilled in the art. As an illustrative example, cell numbers may be measured by flow cytometry, including fluorescence-activated cell sorting (FACS), using detectable binding agents (e.g., fluorescein labeled antibodies) that selectively bind to markers on the surface of the cells, illustrative exa mples of which are listed above. Flow cytometry can also be

- 25 - used to determine whether the hepa ranase expression status of the cells by measuring the presence and intensity of staining of an anti-heparanase antibody.

1.3 TlD-associated biomarker profiles

[0074] A reference biomarker profile may be identified based on reference data measured for individuals in the sample population (e.g., healthy subjects, prediabetic subjects, and TID subjects). Reference data typically include the measurement of at least one biomarker, including heparanase expression of TI D-associated hematopoietic cells and/or hematopoietic cell number. The measurement may include information rega rding cell activity, level or abundance of an expression product or measurable molecule, as will be described in more detail herein. The reference data may also include other additional relevant information, such as clinical data, including, but not limited to, information regarding age-adj usted body-mass index (BMI) percentile, BMI standard deviation score (BMI-SDS), waist circumference, fasting lipid profile and homeostatic model assessment of insulin resistance (HOMA-IR), the presence, absence, degree, severity or progression of a symptom associated with TI D, phenotypic information, such as details of phenotypic traits, genetic or genetically regulated information associated with TID, amino acid or nucleotide related genomics information associated with TID and the like and this is not intended to be limiting, as will be apparent from the description below.

[0075] The reference data may be acquired in any appropriate manner, such as obtaining TI D-associated hematopoietic cell data (e.g., heparanase expression and or hematopoietic cell number) from a plurality of subjects, selected to include healthy subjects, prediabetic subjects, and TID subjects. Quantified values indicative of the relative activity can then be stored as part of the reference data . Distinct reference profiles may represent the deg ree of risk (e.g., an abnormally elevated risk) of having or developing prediabetes or TID, as compared no or normal risk of having or developing the prediabetes or TI D. In another example, distinct reference profiles may represent predictions of differing degrees of risk of having or developing prediabetes or TID.

[0076] A reference biomarker profile can be quantitative, semi-quantitative and/or qualitative. For example, the biomarker profile can evaluate the presence of heparanase expression above or below a particular threshold, and/or can evaluate the relative or absolute amount of heparanase expression and/or the relative or absolute numbers of TID-associated hematopoietic cells.

[0077] In some embodiments, the subject's risk of having prediabetes or developing TID is determined by comparing a biomarker cell profile in a sample obtained from the subject (e.g. , a biomarker profile including data relating to hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic cell number) with a corresponding

- 26 - reference biomarker profile in a healthy control population. An illustrative example is provided in Figure 2, which shows a comparison of the level of hematopoietic cell heparanase expression between prediabetic and healthy controls. Alternatively, the subject's risk of having TID is determined by comparing the hematopoietic cell profile of heparanase expression in a sample obtained from the subject (i.e. , the sample biomarker profile) with a corresponding reference biomarker profile from a diabetic population. For example, a subject's risk of having TID is determined by comparing the level of expression of a hematopoietic cell heparanase expression in the sample obtained from the subject with a level that is representative of a mean or median level of the

hematopoietic cell heparanase expression in the diabetic population, as for example illustrated in Figure 1.

[0078] In some embodiments, the subject's risk of having prediabetes or developing TID is determined by comparing a biomarker profile that includes data relating to the number of TID-associated hematopoietic cells in a sample obtained from the subject (i.e. , the sample biomarker profile) with a corresponding reference biomarker profile in a healthy control population. An illustrative example is provided in Figure 2, which shows a comparison of the hematopoietic cell number between prediabetic and healthy controls. Alternatively, the subject's risk of having TID is determined by comparing a biomarker profile that includes data relating to the number of hematopoietic cells in a sample obtained from the subject (i.e., the sample biomarker profile) with a corresponding reference biomarker profile from a diabetic population. For example, a subject's risk of having TID is determined by comparing the hematopoietic cell number in the sample obtained from the subject with a cell number that is representative of a mean or median hematopoietic cell number in the diabetic population, as for example illustrated in Figure 2.

[0079] By "obtained" is meant to come into possession. The terms "sample" and "biological sample" are used interchangeably herein to refer a variety of sample types obtained from an organism and can be used in a diagnostic or monitoring assay. The term encompasses blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The terms encompass samples that have been manipulated in any way after their procurement, such as by treatment with reagents, sol ubilization, or

enrichment for certain components. The terms encompass clinical samples, and also include cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. Also encompassed are samples that stored for subsequent analysis. If storage of a sample is desired or required, it would be understood by persons skilled in the art that it should ideally be stored under conditions that preserve the integrity of the biomarker of interest within the sample (e.g., at -80°C).

- 27 - [0080] In some embodiments, a biomarker profile (e.g. , one that includes data relating to hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic cell number) in a sample population of reference individuals, as broadly defined herein, is used to generate a biomarker profile; namely, of subjects at risk of developing T1D or having T1D (the reference g roup) and healthy controls (the control group) . These data may be

represented as an overall signature score or the profile may be represented as a barcode, heat-map, z-score, receiver-operator characteristics (ROC) curve or other graphical representation known to persons skilled in the art to facilitate the

determination of a test subject's risk of developing T1D or having T1 D. The

corresponding biomarker profile data in a test subject may be represented in the same way, thereby providing a sample biomarker profile, such that a comparison of the sample profile with the reference profile may be undertaken to determine the test subject's risk of developing T1D or having T1D.

[0081] Additionally, the reference data may include details of one or more phenotypic traits of the individuals and/or their relatives. Phenotypic traits can include information such as the gender, ethnicity, age, and the like. Additionally, in the case of the technology being applied to individuals other than humans, this can also include information such as designation of a species, breed or the like. Accordingly, in one example, the reference data can include for each of the reference individuals an indication of the reference biomarkers (e.g., hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic cell number), a presence, absence degree or prog ression of a condition, phenotypic information such as phenotypic traits, genetic information and a physiological score such as a SOFA score.

[0082] It will be appreciated that once collected, the reference data can be stored in a database allowing them to be subsequently retrieved, for example, by a processing system for subsequent use in accordance with the present invention. The processing system may also store an indication of the identity of an individual reference hematopoietic cell profile as a reference profile of heparanase expression and/or hematopoietic cell number collection or panel .

2. Risk or likelihood of developing T1D or having T1D

[0083] The term "risk" is used to denote a subject's likelihood, based on the sample biomarker profile as determined for that subject, of developing T1D (or not) or having T1 D (or not) on the basis of the reference biomarker profile, as herein described . Accordingly, the terms "risk" and "likelihood" are used interchangeably herein, unless otherwise stated .

- 28 - [0084] It would be apparent to persons skilled in the art that the risk that a subject will develop or have T1D will vary, for example, from being at low or decreased risk of developing or having T1 D to being at high or increased risk of developing or having T1D. By "low or decreased risk" is meant that the subject is less likely to develop or have T1D as compa red to a subject determined to be a "high or increased risk" subject. Conversely, a "high or increased risk" subject is one who is more likely to develop or have T1 D as compared to a subject who is not at risk or a "low risk" subject. For example, a healthy subject may be regarded as being at low risk of developing or having T1D.

[0085] Likelihood is suitably based on mathematical modeling . An increased likelihood, for example, may be relative or absolute and may be expressed qualitatively or quantitatively. For instance, an increased risk may be expressed as simply

determining the subject's level of a given biomarker (e.g., hematopoietic cell heparanase expression level, hematopoietic cell number, or both) and placing the test subject in an "increased risk" category, based upon the corresponding reference hematopoietic cell profile as determined, for example, from previous population studies. Alternatively, a numerical expression of the test subject's increased risk may be determined simply based upon an analysis of the subjects biomarker level (e.g., hematopoietic cell heparanase expression level, hematopoietic cell number, or both) .

[0086] In some embodiments, likelihood is assessed by comparing the level or abundance of at least one biomarker (e.g. , hematopoietic cell heparanase expression level, hematopoietic cell number, or both) to one or more preselected level, also referred to herein as a threshold or reference levels. Thresholds may be selected that provide an acceptable ability to predict risk, treatment success, etc. In illustrative examples, receiver operating characteristic (ROC) curves are calculated by plotting the value of a variable versus its relative frequency in two populations in which a first population is considered at risk of developing T1 D (e.g., prediabetes, T1D first degree relatives) and a second population that is not considered to be at risk, or have a low risk, of developing T1D (called arbitrarily, for example, "healthy controls") .

[0087] In some embodiments, the subject is considered at risk of developing

T1D where the hematopoietic cell heparanase expression in the sample hematopoietic cell profile for the subject is increased as compared to the corresponding hematopoietic cell profile in a healthy subject.

[0088] In other embodiments, the subject is considered at risk of having T1D where the hematopoietic cell heparanase expression in the sample hematopoietic cell profile for the subject is increased as compared to the corresponding hematopoietic cell profile in a prediabetic subject.

- 29 - [0089] In some embodiments, the subject is considered at risk of developing T1D where the hematopoietic cell number in the sample hematopoietic cell profile for the subject is reduced relative to the corresponding hematopoietic cell profile in a healthy subject.

[0090] In other embodiments, the subject is considered at risk of having T1D where the hematopoietic cell number in the sample hematopoietic cell profile for the subject is reduced relative to the corresponding hematopoietic cell profile in a prediabetic subject.

[0091] Occasionally, a distribution of biomarkers, including hematopoietic cell heparanase expression levels and/or hematopoietic cell numbers, for subjects who are at risk or not at risk of developing T1D or having T1D may overlap. Under such conditions, a test may not absolutely distinguish a subject who is at risk of developing T1D or having T1D from a subject who is not at risk of developing T1D or having T1D with absolute (i.e., 100%) accuracy, and the area of overlap indicates where the test cannot distinguish the two subjects. A threshold can be selected, above which (or below which, depending on how the biomarker, e.g., hematopoietic cell heparanase expression level or hematopoietic cell number, or both changes with risk) the test is considered to be "positive" and below which the test is considered to be "negative." The area under the ROC curve (AUC) provides the C-statistic, which is a measure of the probability that the perceived measurement will allow correct identification of a condition (see, e.g., Hanley et al., Radiology 143 : 29-36 (1982)).

[0092] As used herein, the term "probability" refers to the probability of class membership for a sample as determined by a given mathematical model and is construed to be equivalent likelihood in this context.

[0093] Alternatively, or in addition, thresholds may be established by obtaini ng a biomarker profile from the same patient, to which later results may be compared. In these embodiments, the individual in effect acts as their own "control group." In biomarkers (e.g. , hematopoietic cell heparanase expression level) that increase with, for example, prognostic risk, an increase over time in the same patient can indicate a failure of a treatment regimen, while a decrease over time can indicate success of a treatment regimen.

[0094] In some embodiments, a positive likelihood ratio, negative likelihood ratio, odds ratio, and/or AUC or receiver operating characteristic (ROC) values are used as a measure of a method's ability to predict risk of developing T1D or having T1D. As used herein, the term "likelihood ratio" is the probability that a given test result would be observed in a subject with a likelihood of such risk, divided by the probability that that same result would be observed in a subject without a likelihood of such risk. Thus, a positive likelihood ratio is the probability of a positive result observed in subjects with the

- 30 - specified risk divided by the probability of a positive results in subjects without the specified risk. A negative likelihood ratio is the probability of a negative result in subjects without the specified risk divided by the probability of a negative result in subjects with specified risk. The term "odds ratio," as used herein, refers to the ratio of the odds of an event occurring in one g roup (e.g., a healthy control group) to the odds of it occurring in another group (e.g. , a prediabetic group or T1D group), or to a data-based estimate of that ratio. The term "area under the curve" or "AUC" refers to the a rea under the curve of a receiver operating characteristic (ROC) curve, both of which are well known in the art. AUC measures a re useful for comparing the accuracy of a classifier across the complete data range. Classifiers with a greater AUC have a greater capacity to classify unknowns correctly between two groups of interest (e.g., a healthy control group and a T1D risk group, or a T1 D risk group and a T1D group) . ROC curves are useful for plotting the performance of a particular feature (e.g. , any of the biomarkers described herein and/or any item of additional biomedical information) in distinguishing or discriminating between two populations (e.g., cases having a condition and controls without the condition) . Typically, the feature data across the entire population (e.g. , the cases and controls) are sorted in ascending order based on the value of a single feature. Then, for each value for that feature, the true positive and false positive rates for the data a re calculated . The sensitivity is determined by counting the number of cases above the value for that feature and then dividing by the total number of cases. The specificity is determined by counting the number of controls below the value for that feature and then dividing by the total number of controls. Although this definition refers to scenarios in which a feature is elevated in cases compared to controls, th is definition also applies to scena rios in which a feature is lower in cases compared to the controls (in such a scena rio, samples below the value for that feature would be counted) . ROC curves can be generated for a single feature as well as for other single outputs, for example, a combination of two or more features can be mathematically combined (e.g., added, subtracted, multiplied, etc.) to produce a single value, and this single value can be plotted in a ROC curve. Additionally, any combination of multiple features, in which the combination derives a single output value, can be plotted in a ROC curve. These combinations of features may comprise a test. The ROC curve is the plot of the sensitivity of a test against the specificity of the test, where sensitivity is traditionally presented on the vertical axis and specificity is traditionally presented on the horizontal axis. Thus, "AUC ROC values" are equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one. An AUC ROC value may be thought of as equivalent to the Mann-Whitney U test, which tests for the median difference between scores obtained in the two g roups considered if the groups are of continuous data, or to the Wilcoxon test of ranks.

- 31 - [0095] In some embodiments, at least one biomarker (e.g. , hematopoietic cell heparanase expression level and/or hematopoietic cell number) is selected to

discriminate between subjects with or without risk of developing T1D or having T1D with at least about 50%, 55% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% accuracy or having a C-statistic of at least about 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95.

[0096] In the case of a positive likelihood ratio, a value of 1 , in embodiments for example comparing T1 D risk subjects and healthy controls, indicates that a positive result is equally likely among subjects in both the "T1 D risk" and "healthy control" groups; a value greater than 1 indicates that a positive result is more likely in the T1D risk group; and a value less than 1 indicates that a positive result is more likely in the healthy control group. In this context, "T1D risk group" is meant to refer to a population of reference individuals considered to be at risk of developing T1D (e.g., prediabetes subjects) and a "control group" is meant to refer to a group of subjects considered not to be at risk of developing T1 D (e.g., healthy controls) . In the case of a negative likelihood ratio, a value of 1 indicates that a negative result is equally likely among subjects in both the "T1D risk" and "control" groups; a value greater than 1 indicates that a negative result is more likely in the "T1D risk" group; and a value less than 1 indicates that a negative result is more likely in the "control" group. In the case of an odds ratio, a value of 1 indicates that a positive result is equally likely among subjects in both the "T1D risk" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the "T1 D risk" group; and a value less than 1 indicates that a positive result is more likely in the "control" group. In the case of an AUC ROC value, this is computed by numerical integration of the ROC curve. The range of this value can be 0.5 to 1.0. A value of 0.5 indicates that a classifier (e.g., a hematopoietic cell profile) is no better than a 50% chance to classify unknowns correctly between two groups of interest, while 1.0 indicates the relatively best diagnostic accuracy. In certain embodiments, biomarkers (e.g., hematopoietic cell heparanase expression level, hematopoietic cell number, or both) are selected to exhibit a positive or negative likelihood ratio of at least about 1.5 or more or about 0.67 or less, at least about 2 or more or about 0.5 or less, at least about 5 or more or about 0.2 or less, at least about 10 or more or about 0.1 or less, or at least about 20 or more or about 0.05 or less.

[0097] In some cases, multiple thresholds may be determined in so-called "tertile," "quartile," or "quintile" analyses. In these methods, the "T1D risk" and "healthy control" groups or the "T1 D" and "T1D risk" groups are considered together as a single population, and are divided into 3, 4, or 5 (or more) "bins" having equal numbers of individuals. The boundary between two of these "bins" may be considered "thresholds. " The degree of risk can then be assigned based on which "bin" a test subject falls into.

- 32 - [0098] In other embodiments, particular thresholds for the reference biomarker(s) (e.g., hematopoietic cell heparanase expression level, hematopoietic cell number, or both) measured are not relied upon to determine if the biomarker level(s) obtained from a subject are correlated to risk of developing TID or having TID. For example, a temporal change in the biomarker(s) can be used to rule in or out such risk. In the case of biomarker profiles, the present invention may utilize an evaluation of the profile of hematopoietic cell heparanase expression levels and hematopoietic cell numbers to provide a single result value (e.g., a "panel response" value expressed either as a numeric score or as a percentage risk) .

[0099] In certain embodiments, a panel of biomarkers (e.g., one that includes hematopoietic cell heparanase expression level and hematopoietic cell number) is selected to assist in distinguishing between "TI D risk" and "healthy control" groups or between "with at least about 70%, 80%, 85%, 90% or 95% sensitivity, suitably in combination with at least about 70% 80%, 85%, 90% or 95% specificity. In some embodiments, both the sensitivity and specificity are at least about 75%, 80%, 85%, 90% or 95%.

[OIOO] The phrases "assessing the likelihood" and "determining the likelihood," as used herein, refer to methods by which the skilled artisan can predict a subject's risk of developing TID or of having TID. The skilled artisan will understand that this phrase includes within its scope a n increased probability that the subject has TID or will develop TID; that is, such risk is more likely to be present or absent in a subject. For example, the probability that an individual identified as being at risk of developing TI D or have TID may be expressed as a "positive predictive value" or "PPV." Positive predictive value can be calculated as the number of true positives divided by the sum of the true positives and false positives. PPV is determined by the characteristics of the predictive methods of the present invention as well as the prevalence of the condition in the population analyzed . The statistical algorithms can be selected such that the positive predictive value in a population considered to be at risk of developing TID or having TID is in the range of 70% to 99% a nd can be, for example, at least 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0101] In other examples, the probability that a subject is identified as not being at risk of developing TI D or having TI D may be expressed as a "negative predictive value" or "NPV. " Negative predictive value can be calculated as the number of true negatives divided by the sum of the true negatives and false negatives. Negative predictive value is determined by the characteristics of the diagnostic or prognostic method, system, or code as well as the prevalence of risk in the population analyzed . The statistical methods and models can be selected such that the negative predictive value in a population considered at risk of developing TID is in the range of about 70% to about

- 33 - 99% and can be, for example, at least about 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0102] In some embodiments, a subject is determined as being at significant risk of developing T1D or having T1D. By "significant risk" is meant that the subject has a reasonable probability (e.g., 0.6, 0.7, 0.8, 0.9 or more) of developing T1D or having T1D.

[0103] The methods of the present invention, as broad ly described herein, also permit the generation of high-density data sets that can be evaluated using informatics approaches. High data density informatics analytical methods are known and software is available to those in the art, e.g., cluster analysis (Pirouette, Informetrix), class prediction (SIMCA-P, Umetrics), principal components analysis of a computationally modeled dataset (SIMCA-P, Umetrics), 2D cluster analysis (GeneLinker Platinum, Improved Outcomes Software), and metabolic pathway analysis

(biotech. icmb. utexas.edu) . The choice of softwa re packages offers specific tools for questions of interest (Kennedy et al ., Solving Data Mining Problems Through Pattern Recognition. Indianapolis : Prentice Hall PTR, 1997; Golub et al ., (2999) Science 286 : 531 - 7; Eriksson et al ., Multi and Megavariate Analysis Principles and Applications: Umetrics, Umea, 2001) . In genera l, any suitable mathematic analyses can be used to evaluate at least one biomarker (e.g., hematopoietic cell heparanase expression level, hematopoietic cell number, or both) in a hematopoietic cell profile with respect to determining the likelihood that the subject is at risk of developing T1D or having T1 D. For exa mple, methods such as multivariate analysis of variance, multivariate regression, and/or multiple regression can be used to determine relationships between dependent variables (e.g., clinical measures) and independent variables (e.g., levels of biomarkers) .

Clustering, including both hierarchical and non-hierarchical methods, as well as nonmetric Dimensional Scaling can be used to determine associations or relationships among variables and a mong changes in those variables.

[0104] In some embodiments, a biomarker profile, which includes heparanase expression level and/or hematopoietic cell number, is used to assign a risk score which describes a mathematical equation for evaluation or prediction of risk. The evaluation of risk may also take into account genotype (including descri bed HLA genes, e.g.,

DRB1*0301-DQB1*0201 and DRB1*04-DQB1*0302), islet autoantibodies species (e.g., the number of autoantibody target antigens) and other clinical features into account, including age-adjusted BMI, fasting and 2h glucose measurements on an oral glucose tolerance test, age and first-phase insulin response to a glucose load .

[0105] In addition, principal component analysis is a common way of reducing the dimension of studies, and can be used to interpret the va riance-covariance structure

- 34 - of a data set. Principal components may be used in such applications as multiple reg ression and cluster a nalysis. Factor analysis is used to describe the covariance by constructing "hidden" variables from the observed variables. Factor analysis may be considered an extension of principal component analysis, where principal component analysis is used as pa ra meter estimation along with the maximum likelihood method . Furthermore, simple hypothesis such as equality of two vectors of means can be tested using Hotelling's T squa red statistic.

[0106] In some embodiments, the data sets corresponding to biomarker profiles (which include for exa mple data on hematopoietic cell heparanase expression level and/or on hematopoietic cell number) are used to create a diagnostic or predictive rule or model based on the application of a statistical and machine learning algorithm. Such an algorithm uses relationships between a biomarker profile and risk of developing TID or having TI D observed in control subjects or typically cohorts of control subjects (sometimes referred to as training data), which provides combined control or reference biomarker profiles for comparison with biomarker profiles of a subject. The data are used to infer relationships that are then used to predict the status of a su bject and the presence or absence of risk of developing TI D.

[0107] Persons skilled in the art of data analysis will recognize that many different forms of inferring relationships in the training data may be used without materially changing the present invention . The data presented in the examples herein has been used to generate illustrative minimal combinations of biomarkers (models) that differentiate between TI D risk subjects and control subjects or between TID risk subjects and TID subjects using feature selection based on AUC maximization in combination with support vector machine classification .

3. Monitoring a response to treatment

[0108] The methods of the present invention, as broad ly described herein, can also be used to monitor the efficacy of treatment regimen for treating TI D or for preventing or delaying the onset of TI D. Therefore, the present invention further contemplates methods for (i) determining whether a treatment regimen is effective for treating TI D or for preventing or delaying the onset of TID, or a symptom thereof in a subject, (ii) monitoring the efficacy of a treatment regimen in a subject with TID or at risk of developing TID; (iii) correlating a reference biomarker profile (e.g., a biomarker profile comprising hematopoietic cell heparanase expression level and/or on

hematopoietic cell number) with an effective treatment regimen for treating TI D or for preventing or delaying the onset of TI D, or a symptom thereof, (iv) determining whether a treatment regimen is effective for treating TID or for preventing or delaying the onset of TI D, or a symptom thereof, in a subject with TID or at risk of developing TID, (v) correlating a biomarker profile (e.g., a biomarker profile comprising hematopoietic cell heparanase expression level and/or on hematopoietic cell number) with a positive or

- 35 - negative response to a treatment regimen for treating TI D or for preventing or delaying the onset of TI D, or a symptom thereof, and (vi) determining a positive or negative response to a treatment regimen by a subject with TID or at risk of developing TID.

[0109] For example, in a method of monitoring the efficacy of a treatment regimen in a subject with TID or at risk of developing TID, the method may comprise: ( 1) providing a correlation of a biomarker profile (e.g., a biomarker profile comprising hematopoietic cell heparanase expression level and/or on hematopoietic cell number) with a likelihood of having a healthy condition ; (2) obtaining a corresponding biomarker profile of a subject with TI D or at risk of developing TI D after commencement of the treatment regimen, wherein a similarity of the subject's biomarker profile after commencement of the treatment regimen to the reference biomarker profile indicates the likelihood that the treatment regimen is effective for changi ng (e.g. , improving) the health status of the subject.

[0110] With respect to correlating a reference biomarker profile (e.g., a biomarker profile comprising hematopoietic cell heparanase expression level and/or on hematopoietic cell number) with an effective treatment regimen for preventing or delaying the onset of TI D, or a symptom thereof, the method may comprise : ( 1) determining a sample biomarker profile (e.g., a biomarker profile comprising

hematopoietic cell heparanase expression level and/or on hematopoietic cell number) from a subject with TI D or at risk of developing TI D prior to commencement of the treatment regimen ; and (2) correlating the sample biomarker profile with a treatment regimen that is effective for treating TI D or for preventing or delaying the onset of TI D, or a symptom thereof.

[0111] For determining whether a treatment regimen is effective for treating TID or for preventing or delaying the onset of TID, or a symptom thereof, in a subject with TID or at risk of developing TID, the method may comprise : ( 1) correlating a reference biomarker profile (e.g., a biomarker profile comprising hematopoietic cell heparanase expression level and/or on hematopoietic cell number) prior to treatment with an effective treatment regimen for treating TID or for preventing or delaying the onset of TID, or a symptom thereof; and (2) obtaining a corresponding sample biomarker profile from the subject after commencement of the treatment regimen, wherein the sample biomarker profile after commencement of treatment, when compared to the reference biomarker profile, indicates whether the treatment regimen is effective for treating TID or for preventing or delaying the onset of TID, or a symptom thereof, in the subject.

[0112] For correlating a biomarker profile (e.g., a biomarker profile comprising hematopoietic cell heparanase expression level and/or on hematopoietic cell number) with a positive or negative response to a treatment regimen for treating TID or for

- 36 - preventing or delaying the onset of T1D, or a symptom thereof, the method may comprise: (1) obtaining a sample biomarker profile (e.g., a biomarker profile comprising hematopoietic cell heparanase expression level and/or on hematopoietic cell number) from a subject at risk of developing T1D following commencement of the treatment regimen; and (2) correlating the sample biomarker profile from the subject with a positive or negative response to the treatment regimen. This enables an evaluation as to whether a T1D subject or an at-risk subject is responding (i.e., a positive response) or not responding (i.e., a negative response) to a treatment regimen.

[0113] The invention also provides methods of determining a positive and/or negative response to a treatment regimen by a subject. This aspect of the invention can be practiced to identify responders or non-responders relatively early in the treatment process, i.e., before clinical manifestations of efficacy. In this way, the treatment regimen can optionally be discontinued, a different treatment protocol can be

implemented and/or supplemental therapy can be administered. The method may comprise: (a) correlating a reference biomarker profile (e.g. , a biomarker profile comprising hematopoietic cell heparanase expression level and/or on hematopoietic cell number) with a positive or negative response to the treatment regimen for treating T1D or for preventing or delaying the onset of T1D, or a symptom thereof; (b) determining a corresponding sample biomarker profile from the subject following commencement of the treatment regimen; and (c) determining a positive or negative response to the treatment regimen based on a comparison of the sample biomarker profile and the reference biomarker profile.

[0114] In some embodiments, the reference biomarker profile further evaluates at least one other biomarker selected from genotype (including described HLA genes, e.g., DRB1*0301-DQB1*0201 and DRB1*04-DQB1*0302), islet autoantibodies species (e.g., the number of autoantibody target antigens) and other clinical features into account, including age-adjusted BMI, fasting and 2h glucose measurements on an oral glucose tolerance test, age and first-phase insulin response to a glucose load .

[0115] In some embodiments, the methods comprise the analysis of a series of samples obtained over a period of time from the subject during treatment. Without being bound by theory or a particular mode of practice, it is expected that a change in a sample biomarker profile over the period of time will be indicative of treatment efficacy and a change in the subject's risk of developing T1D or having T1D. Conversely, it would be understood that no change in the sample biomarker profile over the period of time is indicative of lack of an effective treatment regimen, where that treatment regimen was prescribed for reducing the subjects risk of developing T1D or having T1D.

[0116] Where there has been no change or an increase in the likelihood that a subject will develop T1D or have T1D, based on the sample biomarker profile and

- 37 - reference biomarker profile in accordance with the present invention, the method may further comprise exposing the subject to a treatment regimen for treating TI D or for preventing or delaying the onset of TI D. This may comprise administering to the subject additional doses of the same agent with which they a re being treated or changing the dose and/or type of medication. Illustrative exa mples of suitable treatment regimens will be discussed in more detail herein below.

[0117] The diagnostic methods of the present invention, as d isclosed herein, further enable determination of endpoints in pharmacotranslational studies. For example, clinical trials can take many months or even years to establish the pharmacological parameters for a medicament to be used in treating TI D or in preventing or delaying the onset of TID, particularly in subjects at risk of developing TID or having TI D. However, these parameters may be associated with the biomarker profiles as herein described . Hence, the clinical trial can be expedited by selecting a treatment regimen (e.g., medicament and pharmaceutical parameters), which results in a biomarker profile associated with low or lower risk of developing TI D, including a healthy state (e.g. , healthy condition) . This may be determined for example by ( 1) providing a correlation of a reference biomarker profile with the likelihood of having the healthy condition; (2) obtaining a sample biomarker profile from a subject suspected of having TI D or being at risk of developing TID, wherein a similarity of the subject's biomarker profile after treatment to the reference biomarker profile indicates the likelihood that the treatment regimen is effective for changing the health status of the subject to the desired health state (e.g., healthy condition) . This aspect of the present invention advantageously provides methods of monitoring the efficacy of a particular treatment regimen in a subject (for example, in the context of a clinical trial) already diagnosed as having TID or being at risk of developing TI D. Thus, in another aspect, the present invention provides a method of correlating a reference biomarker profile (e.g., a biomarker profile comprising hematopoietic cell heparanase expression level and/or on hematopoietic cell number) with an effective treatment regimen for TID, wherein the method comprises : ( 1) determining a sample biomarker profile (e.g., a biomarker profile comprising hematopoietic cell heparanase expression level and/or on hematopoietic cell number) from a subject prior to commencement of the treatment regimen ; and (2) correlating the sample biomarker profile with a treatment regimen that is effective for treating TID or for preventing or delaying the onset of TID, or a symptom thereof.

[0118] The term "correlating" generally refers to determining a relationship between one type of data with another or with a state (physiological and/or

pathophysiological). In various embodiments, correlating a biomarker profile with the presence or absence of TI D or of risk of development of TI D comprises determining the presence, absence or level of at least one biomarker (e.g. , a biomarker profile comprising hematopoietic cell heparanase expression level and/or on hematopoietic cell number) in

- 38 - a subject that suffers from that condition; or in persons known to be free of that condition. In specific embodiments, a profile of biomarker levels, absences or presences is correlated to a global probability or a particular outcome, using receiver operating characteristic (ROC) curves.

[0119] Thus, in some embodiments, evaluation of biomarkers includes determining the levels of individual biomarkers, which correlate with the presence, absence or degree of having T1D or risk of developing T1D, as herein described. In certain embodiments, the techniques used for detection of biomarkers will include internal or external standards to permit quantitative or semi-quantitative determination of those biomarkers, to thereby enable a valid comparison of the level of the biomarkers in a sample with the corresponding biomarkers in a reference sample or samples. Such standards can be determined by the skilled practitioner using standard protocols, illustrative examples of which are disclosed herein.

[0120] In some embodiments, the methods comprise comparing the level of at least one biomarker (e.g., hematopoietic cell heparanase expression level and/or on hematopoietic cell number) in the subject's sample biomarker profile to the expression of a corresponding biomarker in a reference biomarker profile from at least one control subject or population of subjects selected from a healthy control subject or group (i.e., "reference biomarker profile"), wherein a similarity between the level of the at least one biomarker in the sample biomarker profile and the level of the corresponding biomarker in the reference biomarker profile identifies that the subject has a biomarker profile that correlates with the presence of a healthy condition, or alternatively the absence of risk (or low risk) of having T1D or developing T1D and/or wherein a similarity between the level of the at least one biomarker in the sample biomarker profile and the level of the corresponding biomarker in the reference biomarker profile identifies that the subject has a biomarker profile that correlates with an increased risk of having T1D or developing T1D or, alternatively, the absence of a healthy condition.

4. Treatment regimen

[0121] The present invention also extends to the management of T1D or risk of developing T1D in a subject. The management of can include identification and amelioration of the underlying cause and use of therapeutic agents or treatment regimens for preventing or delaying the onset of T1D, or a symptom thereof. Treatment regimens may include dietary restrictions (e.g., limiting caloric intake) and exercise. In some embodiments, a treatment regimen will be administered in pharmaceutical (or veterinary) compositions together with a pharmaceutically acceptable carrier and in an effective amount to achieve their intended purpose. The dose of active compounds administered to a subject should be sufficient to achieve a beneficial response in the subject. The quantity of the pharmaceutically active compounds(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general

- 39 - health condition thereof. In this regard, precise amounts of the active compound(s) for administration will depend on the judgment of the practitioner. In determining the effective amount of the active compound(s) to be administered for treating TID or for preventing or delaying the onset of TID, the medical practitioner or veterinarian may evaluate severity of any symptom associated with the presence of TID including abnormal blood pressure and vascular disease (e.g., atherosclerosis). In any event, those of skill in the art may readily determine suitable dosages of the therapeutic agents and suitable treatment regimens without undue experimentation.

[0122] Thus, also disclosed herein is a method for treating TID or for preventing or delaying the onset of TID or a symptom thereof in a subject, wherein the method comprises: (a) determining whether a subject has TID or is at risk of developing TID in accordance with the method of the present invention, as broadly described above and elsewhere herein; and (b) exposing the subject, on the basis that the subject has an increased likelihood of having or developing TID, to a treatment regimen for treating TID or for preventing or delaying the onset of TID or a symptom thereof.

[0123] As used herein, the term "treatment regimen" includes reference to a prophylactic regimen (i.e., before the onset of TID), or to a therapeutic regimen (i.e., after the onset of TID) . The term "treatment regimen" encompasses natural substances and pharmaceutical agents (i.e., "drugs") as well as any other treatment regimen including but not limited to dietary treatments, physical therapy or exercise regimens, surgical interventions, and combinations thereof.

[0124] The term "treating" as used herein, unless otherwise indicated, means treating TID, or alleviating, inhibiting the progress of, or preventing, either partially or completely, the onset of TID, or a symptom thereof. The term "treatment" as used herein, unless otherwise indicated, refers to the act of treating.

[0125] Following diagnosis, the treatment regimen to be adopted or prescribed may depend on several factors, including the age, weight and general health of the subject. Another determinative factor may be the degree of risk of having or developing TID determined by the sample biomarker profile in accordance with the present invention, as herein described. For instance, where the subject is determined to be at high risk of having or developing TID, a more aggressive treatment regimen may be prescribed as compared to a subject who is determined to be at low risk of having or developing TID. The treatment regimen may also depend on existing clinical parameters relevant to TID, including body mass index, weight, glucose intolerance and homeostatic insulin resistance.

[0126] Thus, the present invention contemplates exposing the subject to a treatment regimen if the subject is determined to be at risk of having or developing TID in accordance with the methods of the present invention. Non-limiting examples of such

- 40 - treatment regimens include exposing the at-risk subject to metformin, glucagon-like peptide (GLP)-l, diet (e.g., caloric intake restrictions), exercise, anti-CD3 monoclonal antibodies (mAb), rituximab, abatacept, IL-l-receptor antagonist, TNF-inhibitors, other anti-cytokine mAb or soluble receptors, strategies to induce antigen-specific tolerance (including curcusomes encapsulating islet antigenic peptides, DNA vaccines encoding islet antigenic peptides, islet antigenic peptide immunotherapy, dendritic cell targeting strategies using monoclonal antibodies fused to islet antigens).

5. Kits

[0127] Also contemplated are kit for use in the methods of the present invention. These kits may contain reagents for obtaining a sample biomarker profile in accordance with the methods as herein described. Kits for carrying out the methods of the present invention typically include, in suitable container means, (i) a reagent for detecting the at least one biomarker (e.g. , at least a portion of heparanase or of a transcript encoding heparanase), (ii) a probe that comprises an antibody or nucleic acid sequence that specifically binds to the at least one biomarker (e.g. , at least a portion of heparanase or of a transcript encoding heparanase), (iii) a label for detecting the presence of the probe and (iv) instructions for how to measure the level of expression of the at least one biomarker (e.g., at least a portion of heparanase or of a transcript encoding heparanase) . The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe and/or other container into which a first antibody specific for the at least one biomarker or a first nucleic acid specific for the at least one biomarker may be placed and/or suitably aliquoted. Where a second and/or third and/or additional component is provided, the kit will also generally contain a second, third and/or other additional container into which this component may be placed. Alternatively, a container may contain a mixture of more than one reagent, each reagent specifically binding a different biomarker in accordance with the present invention, when required. The kits of the present invention will also typically include means for containing the reagents (e.g., polypeptides, nucleic acids, etc.) in close confinement for commercial sale. Such containers may include injection and/or blow-molded plastic containers into which the desired vials are retained.

[0128] The kits may further comprise positive and negative controls, including a reference biomarker profile, as well as instructions for the use of kit components contained therein, in accordance with the methods of the present invention.

[0129] In some embodiments, the kit comprises a set of antibodies for identifying the heparanase-expressing hematopoietic cells described above and elsewhere herein.

[0130] All the essential materials and reagents required for detecting and quantifying biomarker expression products may be assembled together in a kit, which is

- 41 - encompassed by the present invention. The kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, blotting membranes, microtiter plates dilution buffers and the like. For example, a nucleic acid-based detection kit may include (i) a biomarker polynucleotide, which encodes heparanase or portion thereof (which may be used as a positive control), (ii) a primer or probe that specifically hybridizes to a biomarker polynucleotide. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (Reverse Transcriptase, Taq, Sequenase™D DNA ligase etc. depending on the nucleic acid amplification technique employed), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Such kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe. Alternatively, a protein-based detection kit may include (i) a biomarker polypeptide (e.g., heparanase) (which may be used as a positive control), (ii) an antibody that binds specifically to a biomarker polypeptide. The kit can also feature various devices (e.g. , one or more) and reagents (e.g. , one or more) for performing one of the assays described herein; and/or printed instructions for using the kit to quantify the expression of a biomarker gene.

[0131] It will be appreciated that the above described terms and associated definitions are used for the purpose of explanation only and are not intended to be limiting.

[0132] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting example.

EXAMPLE EXAMPLE 1

FLOW CYTOMETRY METHOD FOR PHENOTYPING BLOOD (OR ISLET-DERIVED INSULITIS) LEUKOCYTE SUB-POPULATIONS EXPRESSING HEPARANASE AND CATHEPSIN L IN PREDIABETIC

AND TI D-ONSET NOD MICE

[0133] Blood (200 μΙ_) is collected (from prediabetic NOD, TID-onset NOD, or B6.SJL mice) via a retro-orbital bleed into a tube containing 400 μΙ_ BSCG buffer to prevent coagulation Note: blood from B6.SJL is used as a CD45.1 positive control for neutrophils.

[0134] After 1 hr. red blood cells are removed by treatment with red cell lysis buffer (RCLB) three times.

[0135] PBL are stained with a mix of anti-heparanase mAb (HP 3/17) and anti-

Cathepsin L mAb, followed by goat anti-mouse Ig R-PE (to detect cell surface

heparanase) and then mouse anti-rat IgG FITC (to detect cell surface Cathepsin L).

- 42 - [0136] The cells are stained with a mix of directly conjugated antibodies for detection of leukocyte sub-populations using a gating strategy based on cell surface expression of CDl lc (+ve/-ve), CDl lb (+ve/-ve), Ly6C (hi or med), Ly6G (+ve for neutrophils) and Siglec F (+ve for eosinophils). Anti-CD3 is used for T cells and B220mAb is used for B lymphocytes. This gating strategy is a modification of Rose et al.

(Cytometry Part A; 81A, 343-350, 2012) :

[0137] Antibodies used :

[0138] PE CF594 Hamster anti-mouse CDl lc

[0139] Per CP Cy5.5 Rat anti-mouse CD45R/B220

[0140] PE Cy7 Rat anti-mouse Ly6G

[0141] BV421 Rat anti-mouse Siglec F

[0142] APC anti-mouse CD45.1, clone A2

[0143] AF700 Rat anti-mouse CDl lb, clone Ml/70

[0144] APC-Cy7 Hamster anti-mouse CD3e, clone 145-2C11

[0145] BV605 Rat anti-mouse Ly6C, clone AL-21

[0146] The same staining protocol is used for characterizing the insulitis mononuclear cells isolated from islets with associated insulitis from pancreas of prediabetic or TlD-onset NOD mice.

Results

[0147] Using an eight-color staining and gating strategy for flow cytometry analyses, the present inventors routinely detect inflammatory macrophages, eosinophils and neutrophils (specific myeloid cell sub-populations), conventional dendritic cells, T lymphocytes and B lymphocytes in the peripheral blood mononuclear cells (PBMC) of normal (B6.SJL) and NOD (prediabetic (n=2) or onset diabetic (n=4)) mice.

Representative results are presented in Figures 1 and 2.

[0148] In six experiments, it was found that compared to normal PBMC (B6.SJL mice), there was a consistent and substantial decline in the pooled myeloid cells in the NOD PBMC (3-5 fold reduction), specifically the neutrophil, eosinophil and inflammatory macrophage sub-populations. In general, there was also a decline in the B lymphocytes in the NOD PBMC and sometimes an increase in T lymphocytes.

[0149] Within the myeloid cells in prediabetic or onset NOD PBMC, in 4 of 5 experiments, the neutrophils showed a 5-10 fold increase in the cell surface expression of heparanase, as compared to normal B6.SJL mice (in the 5^th experiment, the increase was 2.5-fold). In five experiments, the eosinophils showed a 2.5-7-fold increase in heparanase expression and in four of five experiments, the inflammatory macrophages

- 43 - showed a 4-10-fold increase in heparanase expression. By contrast, T cells showed little if any cell surface heparanase expression. In general, B lymphocytes showed cell surface heparanase expression similar to control B6.SJL mice. Usually, heparanase expression was not increased in conventional NOD dendritic cells.

[0150] In three of four experiments, cell surface expression of Cathepsin L, a cysteine protease that cleaves proheparanase to catalytically active heparanase, was increased 1.2-4 fold on NOD neutrophils and a 1.5 to over 10-fold increase was seen in NOD eosinophils.

[0151] Results from another experiment investigating the expression of heparanase by peripheral blood in normal female (B6.SJL) mice, female prediabetic and recent onset NOD mice and in insulitis leukocytes from the same NOD donors, are presented in Figure 3. Although normal and recent onset TID lymphoid (T and B) cells showed no differences in heparanase staining, heparanase expression by circulating recent onset TID myeloid cells was 4-fold higher than in normal blood (Figure 3(a);

P<0.05). Furthermore, the level of heparanase on insulitis recent onset TID myeloid cells was ~6-fold higher than circulating recent onset TID myeloid cells and 24-fold higher than myeloid cells in normal blood. Heparanase expressed on myeloid cells in prediabetic NOD peripheral blood and insulitis were elevated to a similar extent (Figure 3(a)).

Myeloid cell sub-populations in recent onset TID NOD PBMC, i.e., macrophages, eosinophils and neutrophils revealed a 9-fold, 8-fold (P<0.05) and 3.7-fold (P<0.01) increase in heparanase expression, respectively, compared to corresponding normal controls (Figures 3 (b-d)).

[0152] Based on these studies, the cell surface expression of heparanase in PBMC myeloid cells, and particularly neutrophils, eosinophils and inflammatory macrophages, correlates with TID disease progression in prediabetic NOD mice as well as TID onset in NOD female mice. These findings indicate that cell surface heparanase expression on myeloid cells, including neutrophils, eosinophils and inflammatory macrophages, in peripheral blood represents a biomarker for heparanase expressed and utilized by insulitis myeloid cells to aid intra-islet leukocyte invasion, degradation of beta cell heparan sulfate and TID pathogenesis. In essence, the present inventors have identified a novel biomarker for beta cell damage in recent onset TID peripheral blood, and for the progression of autoimmune TID disease in asymptomatic prediabetic subjects and onset TID subjects.

[0153] Thus, it is proposed that heparanase expression by myeloid cell populations in peripheral blood will be a useful biomarker for tracking TID development in humans at a high risk of developing TID, for identifying individuals who can benefit from intervention therapy designed to prevent TID disease progression and for monitoring the therapeutic efficacy of treatment in prediabetic and TID patients.

- 44 - EXAMPLE 2

DETECTION OF HEPARANASE ACTIVITY IN BLOOD LEUKOCYTES DURING THE DEVELOPMENT OF

TID IN NOD MICE

[0154] Heparanase (Hpse) activity was measured using a colorimetric assay (Hammond et a/., 2010. Anal Biochem 396 (1) : 112-116). The reducing disaccharides formed by cleavage of Fondaparinux (a synthetic heparin pentasaccharide; Arixtra, GlaxoSmithKline, Boronia, VIC, Australia) by recombinant active human heparanase (R&D Systems, Minneapolis, MN) were detected using the tetrazolium salt WST-1. The spin columns used in the modified assay allow background levels of cell or tissue lysates to be minimized. The recombinant Hpse activity was determined by measuring the optical density at 584 nm using a plate reader (Tecan, Infinite M200 Pro, Maennedorf,

Switzerland). The protocol used was as follows:

[0155] Blood (200 μΙ_) was collected (from male or female prediabetic NOD, TID-onset NOD, or Heparanase knockout NOD mice) via a retro-orbital bleed into a tube containing 400 μΙ_ BSCG buffer to prevent coagulation . Lymph nodes (inguinal, brachial, axillary, superficial cervicals and mesenteric) were collected from the same donors into 20mM HPES/Hanks (pH 8) on ice.

[0156] After 1 hr. red blood cells were removed from the blood sample by treatment with red cell lysis buffer (RCLB) three times and washed. The blood leukocytes were resuspended in 40 μί 1% CHAPS (pH 8) containing 50 mM EDTA, 50 μΜ E64, 1 mg/ml Pefabloc SC and Complete Protease Inhibitor Cocktail Tablets (~100-mouse). The cells/buffer were mixed and frozen immediately on dry ice. Lymph nodes were

transferred to another tube containing 1% CHAPS (pH 8) containing 50 mM EDTA, 50 μΜ E64, 1 mg/ml Pefabloc SC and Complete Protease Inhibitor Cocktail Tablets (~100-200 μΙ/mouse) . The sample was then frozen immediately on dry ice.

[0157] Frozen samples were stored at -80 °C until cell lysis was performed.

[0158] Tissue and leukocyte samples were lysed by three rounds of freezing on dry ice and thawing on normal ice. Cell debris was pelleted by centrifugation at 4° C. Supernatant was collected into a fresh microfuge tube and the protein concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies,

Wilmington, DE). The protein concentration of each sample was adjusted to ensure equal loading of protein/ sample for the assay.

[0159] For the heparanase activity assay, two 30 kDa spin columns (Cat No. : UFC 503024, Amicon Ultra 0.5 mL centrifugal filter unit with Ultracel -30 membrane, Millipore, Billerica, MA, USA) are prepared for each sample (one is for sample-only control and the other with sample and Fondaparinux).

- 45 - [0160] Lysed sample (20 μΙ_) was added to a 30 kDa spin column (Cat No. : 7570-GH-005, R&D Systems, Minneapolis, MN, USA) . Recombinant Heparanase (at optimal concentration 0.8-1.1 nM) and Fondaparinux (Arixtra, GlaxoSmithKline, Boronia, VIC, Australia) are used as separate controls. (Note: recombinant Hpse is diluted in the same lysis buffer).

[0161] Bovine serum albumin (BSA; 100 μΙ_ of 0.1 mg/mL in PBS; Cat No. : A3294 or A7030, Sigma-Aldrich, St. Louis, MO, USA) was added and the spin column was centrifuged at 14,000 rpm for 5 min at 4° C using a microcentrifuge (Model 5417R, Eppendorf, Hamburg, Germany).

[0162] The columns were washed 3x with 200 μΙ_ ice-cold PBS at 14 000 rpm for 5 min at 4° C (per wash). Note: After the third wash, the lymph node lysate samples were centrifuged for an additional 5 min to reduce the concentrate to the same volume (~15-20 μΙ_) as the control (i.e. , recombinant Hpse and Fondaparinux-only sample).

[0163] The filtrates were discarded.

[0164] 70 μΙ_ of 57 mM sodium acetate (pH 5.0) was added to the spin columns followed by 10 μΙ_ 1 mM Fondaparinux (diluted with H₂0) and the columns were incubated at 37° C in 5% C0₂, in air for 20 hr.

[0165] For each sample, the tube was replaced with a fresh microfuge tube and the columns were centrifuged at 14,000 rpm for 5 min at room temperature. 70-100 μΙ_ of the filtrate was transferred to a 96 flat bottom well plate (Cat No. : 456537, MaxiSorp, Thermo Scientific Nunc, Waltham, MA, USA).

[0166] 70-100 μΙ_ of WST-1 solution 1 (4-[3-(4-iodophenol)-2-(4-nitrophenyl)- 2H-5-tetrazolio]-l,3-benzene disulfonate; Cat No. : W201, Dojindo Molecular

Technologies, Kumamoto, Japan) ; equal volume as filtrate) was added to individual wells and gently mixed. The plate was sealed with an adhesive cover and incubated at 60° C for an hour.

[0167] The absorbance was read at 584 nm using a microplate reader (Tecan, Infinite M200 Pro, Maennedorf, Switzerland).

[0168] Heparanase enzymatic activity was measured by subtracting the sample-only absorbance (AO) from the absorbance for sample + heparanase +

Fondaparinux (Al). Thereafter, the Fondaparinux-only absorbance (Af) was subtracted from the calculated absorbance, i.e., heparanase enzymatic activity per sample = (Al- A0)-Af.

Results

[0169] Results of the above experiments are presented in Figure 4. These results show that, using the modified heparanase activity assay, heparanase activity was detected in lymph node samples from female and male NOD mice, with females

- 46 - expressing >2-fold higher heparanase activity than males. This is consistent with the higher incidence of Type 1 diabetes in females (~60-70%) as compared to males (~10- 30%). Heparanase activity could not be detected in lymph nodes from TlD-onset male or female heparanase knockout mice.

[0170] In addition, heparanase activity was detected in peripheral blood leukocytes from prediabetic male NOD mice but not TlD-onset male heparanase knockout mice.

[0171] The results also show that heparanase activity in the blood leukocytes correlated with heparanase activity in lymph nodes, albeit at a slightly lower level.

[0172] These findings confirm that heparanase activity in blood leukocytes correlates with the development of T1D in the NOD mouse animal model.

[0173] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0174] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0175] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

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Claims

WHAT IS CLAIMED IS:

1. A method for determining whether a subject is at risk of developing TID, the method comprising, consisting or consisting essentially of: determining the presence of a TID susceptibility biomarker profile in the subject, which indicates that the subject is at risk of developing TID, wherein the TID

susceptibility biomarker profile comprises a TID susceptibility hematopoietic cell heparanase expression level, or a TID susceptibility hematopoietic cell number, or both a TI D susceptibility hematopoietic cell heparanase expression level and a TID susceptibility hematopoietic cell number.

2. A method according to claim 1, wherein the hematopoietic cells are leukocytes.

3. A method according to claim 1 or claim 2, wherein the hematopoietic cells express the surface ma rker CD45.

4. A method according to any one of claims 1 to 3, wherein the hematopoietic cells comprise myeloid cells.

5. A method according to claim 4, wherein the myeloid cells are selected from neutrophils, eosinophils and inflammatory macrophages and combinations thereof.

6. A method according to any one of claims 1 to 5, wherein the hematopoietic cells are peripheral blood cells.

7. A method according to any one of claims 1 to 6, wherein the TI D susceptibility heparanase expression level is determined by comparing the level of heparanase expression of the subject's hematopoietic cells to a control heparanase expression level .

8. A method according to claim 7, wherein the control heparanase expression level is selected from : (i) a heparanase expression level of healthy control hematopoietic cells, wherein the healthy control hematopoietic cells are selected from hematopoietic cells of normal subjects or of subjects in which prediabetes and TI D are absent; and (ii) a heparanase expression level of TID control hematopoietic cells (e.g., from one or more TID subjects) .

9. A method according to claim 8, wherein the TID susceptibility heparanase expression level is higher than the heparanase expression level of healthy control hematopoietic cells.

10. A method according to claim 9, wherein the TID susceptibility heparanase expression level is at least about 105%, 106%, 107% 108%, 109%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%,

- 53 - 400%, 500%, 600%, 700%, 800%, 900% or 1000% of the healthy control heparanase expression level.

11. A method according to any one of claims 8 to 10, wherein the T1D susceptibility heparanase expression level is lower than the heparanase expression level of T1D control hematopoietic cells.

12. A method according to claim 11, wherein the T1D susceptibility heparanase expression level is no more than about 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, of the T1D control heparanase expression level.

13. A method according to any one of claims 1 to 12, wherein the T1D susceptibility hematopoietic cell number is determined by comparing the number of hematopoietic cells in the peripheral circulation of the subject to a control number of hematopoietic cells.

14. A method according to claim 13, wherein the control number of hematopoietic cells is selected from: (i) the number of hematopoietic cells in a healthy control subject or in a sample obtained therefrom, wherein the healthy control subject is selected from a normal subject or a subject in which prediabetes and T1D are absent; and (ii) the number of hematopoietic cells in a T1D control subject or in a sample obtained therefrom.

15. A method according to claim 14, wherein the T1D susceptibility hematopoietic cell number is lower than the healthy control hematopoietic cell number.

16. A method according to claim 15, wherein the T1D susceptibility hematopoietic cell number is no more than about 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, of the healthy control hematopoietic cell number.

17. A method according to any one of claim 14 to 16, wherein the T1D susceptibility hematopoietic cell number is higher than the T1D control

hematopoietic cell number.

18. A method according to claim 17, wherein the T1D susceptibility hematopoietic cell number is at least about 105%, 106%, 107% 108%, 109%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% of the T1D control hematopoietic cell number.

19. A method according to any one of claim 1 to 18, wherein the subject is an islet autoantibody negative subject.

20. A method according to any one of claim 1 to 18, wherein the subject is an islet autoantibody positive subject.

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21. A method according to claim 20, wherein the autoantibody positive subject expresses a single islet autoantibody.

22. A method according to claim 20, wherein the autoantibody positive subject expresses a plurality of islet autoantibodies (e.g., 2, 3, 4, 5 or more islet autoantibodies).

23. A method according to any one of claim 1 to 22, wherein the subject has a HLA haplotype that is associated with the presence or risk of development of TID (e.g., DRB1*0301-DQB1*0201 and DRB1*04-DQB1*0302).

24. A method according to any one of claims 1 to 23, wherein the heparanase expression level is determined by determining heparanase RNA levels.

25. A method according to claim 24, wherein the heparanase RNA level is determined by an assay selected from the group consisting of RT-PCR, chip hybridization, RNase protection, in-situ hybridization, primer extension, Northern blot and dot blot analysis.

26. A method according to any one of claims 1 to 23, wherein the heparanase expression level is determined by determining heparanase protein level.

27. A method according to claim 26, wherein the heparanase protein level is determined by an assay selected from the group consisting of

immunohistochemistry, ELISA, RIA, Western blot analysis, FACS analysis, an immunofluorescence assay, and a light emission immunoassay.

28. A method according to any one of claims 1 to 23, wherein the heparanase expression level is determined by determining heparanase activity level.

29. A method according to claim 28, wherein the heparanase activity level is determined by using a heparanase substrate.

30. A method according to claim 29, wherein the heparanase substrate is selected from the group consisting of soluble or immobilized heparan sulfate proteoglycans, heparan sulfate, or heparin.

31. A method according to claim 29 or claim 30, wherein the heparanase substrate includes a detectable moiety selected from the group consisting of a chromogenic moiety, a fluorogenic moiety, a radioactive moiety and a light-emitting moiety.

32. A method according to any one of claims 1 to 31, wherein the hematopoietic cells are enriched for leukocytes prior to determining heparanase expression.

33. A method for determining the likelihood of the presence or absence of a condition selected from a healthy condition (e.g., a normal condition or one in

- 55 - which prediabetes and TID are absent), prediabetes and TID, the method comprising, consisting or consisting essentially of: (1) providing a correlation of a reference biomarker profile with the presence or absence of a condition selected from a healthy condition, prediabetes and TID, wherein the reference biomarker profile evaluates at least one biomarker selected from hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic cell number; (2) obtaining a biomarker profile of a sample from a subject, wherein the sample biomarker profile evaluates for an individual biomarker in the reference biomarker profile a corresponding biomarker; and (3) determining a likelihood of the subject having or not having the condition based on the sample biomarker profile and the reference biomarker profile.

34. A method according to claim 33, wherein an individual biomarker profile indicates the level of heparanase expressed by the hematopoietic cells and/or the number of hematopoietic cells, which correlates with the presence or absence of a respective condition.

35. A method according to claim 33 or claim 34, comprising comparing the sample biomarker profile with the reference biomarker profile and determining a likelihood of the presence or absence of the condition based on that comparison.

36. A method according to any one of claims 33 to 35, further comprising correlating the reference biomarker profile with the presence or absence of a respective condition.

37. A method for treating TID or for preventing or delaying the onset of TID or a symptom thereof in a subject, the method comprising, consisting or consisting essentially of: (a) determining whether a subject has TID or is at risk of developing TID according to the method of any one of claims 1 to 36; and (b) exposing the subject, on the basis that the subject has TID or an increased risk or likelihood of developing TID, to a treatment regimen for treating TID or for preventing or delaying the onset of TID, or a symptom thereof.

38. A method for monitoring the efficacy of a treatment regimen in a subject with TID or at risk of developing TID, the method comprising, consisting or consisting essentially of: (1) providing a correlation of a reference biomarker profile with a likelihood of having a healthy condition, wherein the reference biomarker profile evaluates at least one biomarker selected from hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic cell number; (2) obtaining a corresponding biomarker profile of a subject with TID or at risk of developing TID after commencement of a treatment regimen, wherein a similarity of the subject's

- 56 - biomarker profile after commencement of the treatment regimen to the reference biomarker profile indicates the likelihood that the treatment regimen is effective for changing (e.g., improving) the health status of the subject.

39. A method of correlating a reference biomarker profile with an effective treatment regimen for treating TI D or for preventing or delaying the onset of TID, or a symptom thereof, the method comprising, consisting or consisting essentially of: ( 1) determining a sample biomarker profile from a subject with TI D or at risk of developing TID prior to commencement of the treatment regimen, wherein the sample biomarker profile evaluates at least one biomarker selected from

hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic cell number; and (2) correlating the sample biomarker profile with a treatment regimen that is effective for treating TI D or for preventing or delaying the onset of TI D, or a symptom thereof.

40. A method of determining whether a treatment regimen is effective for treating TI D or for preventing or delaying the onset of TI D, or a symptom thereof, in a subject with TID at risk of developing TI D, the method comprising, consisting or consisting essentially of: ( 1) correlating a reference biomarker profile prior to treatment with an effective treatment regimen for treating TI D or for preventing or delaying the onset of TI D, or a symptom thereof, wherein the reference biomarker profile evaluates at least one biomarker selected from hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic cell number; and (2) obtaining a sample biomarker profile from the subject after commencement of the treatment regimen wherein the sample biomarker profile evaluates, for an individual biomarker in the reference biomarker profile, a corresponding biomarker, and wherein the sample biomarker profile after commencement of treatment indicates whether the treatment regimen is effective for treating TID or for preventing or delaying the onset of TI D, or a symptom thereof, in the subject.

41. A method of correlating a biomarker profile with a positive or negative response to a treatment regimen for treating TI D or for preventing or delaying the onset of TID, or a symptom thereof, the method comprising, consisting or consisting essentially of: ( 1) obtaining a sample biomarker profile from a subject with TID or at risk of developing TI D following commencement of the treatment regimen, wherein the sample biomarker profile evaluates at least one biomarker selected from hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic

- 57 - cell number; and (2) correlating the sample biomarker profile from the subject with a positive or negative response to the treatment regimen .

42. A method of determining a positive or negative response to a treatment regimen by a subject with T1D or at risk of developing T1 D, the method

comprising, consisting or consisting essentially of: (a) correlating a reference biomarker profile with a positive or negative response to the treatment regimen for treating T1 D or for preventing or delaying the onset of T1 D, or a symptom thereof, wherein the reference biomarker profile evaluates at least one biomarker selected from hematopoietic cell heparanase expression level, hematopoietic cell number, or both hematopoietic cell heparanase expression level and hematopoietic cell number; (b) determining a sample biomarker profile from the subject following commencement of the treatment regimen, wherein the sample biomarker profile evaluates, for an individual biomarker in the reference biomarker profile, a corresponding biomarker; and (c) determining a positive or negative response to the treatment regimen based on a comparison of the sample biomarker profile with the reference biomarker profile.

43. A kit for use in performing any one of the methods according to any one of claims 1 to 42, the kit comprising at least one container including at least one reagent for determining at least one biomarker as defined in those methods, and packaging material identifying the at least one reagent for use in determining a biomarker profile that correlates with the presence or absence of a condition selected from a healthy condition (e.g., a normal condition or one in which prediabetes and T1 D are absent), prediabetes and T1D .

44. A kit according to claim 43, wherein a reagent is provided for determining the presence of a T1 D susceptibility heparanase expression level of hematopoietic cells in a hematopoietic cell sample.

45. A kit according to claim 43 or claim 44, wherein a reagent is provided for determining the presence of a T1 D susceptibility hematopoietic cell number in a hematopoietic cell sample.

46. A composition for determining a heparanase expression level of hematopoietic cells in a subject that is at risk of developing T1D, the composition comprising, consisting or consisting essentially of hematopoietic cells obtained from the subject and at least one reagent that detects the level or activity of heparanase expressed by the hematopoietic cells.

47. A composition according to claim 46, wherein the hematopoietic cells are obtained from a blood sample taken from the subject.

48. A composition according to claim 46, wherein the blood sample is a peripheral blood sample.

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49. A composition according to claim 46 or claim 47, wherein the blood sample comprises leukocytes (e.g., myeloid cells, which are suitably selected from neutrophils, eosinophils and inflammatory macrophages and combinations thereof) .

50. A composition according to any one of claims 46 to 49, wherein the at least one reagent comprises an antibody that binds to heparanase.

51. A composition according to any one of claims 46 to 50, wherein the at least one reagent comprises an antibody that binds to one or more of the hematopoietic cells.

52. A composition according to claim 51, wherein the antibody binds to a leukocyte (e.g., a myeloid cell, which is suitably selected from neutrophils, eosinophils and inflammatory macrophages) .

53. A composition according to any one of claims 46 to 52, further comprising a labeled reagent for detecting the heparanase.

54. A composition according to any one of claims 46 to 53, further comprising a labeled reagent for detecting a hematopoietic cell .

55. A composition according to claim 53 or claim 54, wherein the labeled reagent is a labeled antibody.

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