WO2012088105A2 - Methods and compositions for predicting disease status in cancer - Google Patents

Abstract

Methods for predicting disease statuses of cancers in subjects are provided. In some embodiments, the methods relate to determining a level of biological activity of a member of the MMP and/or the ADAM family of polypeptides in a biological sample isolated from or in the subject, wherein the determining provides a prediction of the disease status of the cancer in the subject. Also provided are methods for detecting enzyme activity of a member of the MMP and/or the ADAM family of polypeptides, peptides that include particular amino acid sequences, and methods for predicting disease status of breast cancer.

Description

DESCRIPTION

METHODS AND COMPOSITIONS FOR PREDICTING DISEASE STATUS IN CANCER CROSS REFERENCE TO RELATED APPLICATION

The subject matter disclosed herein claims the benefit of and priorityo U.S. Provisional Patent Application Serial. No. 61/424,895, filed December 20, 2010, the disclosure of which is incorporated herein by reference in its entirety.

GOVERNMENT INTEREST

This invention was made with government support under Grant No. PO1 CA45548 awarded by the National Institutes of Health of the United States of America. The United States Government has certain rights in the invention.

TECHNICAL FIELD

The presently disclosed subject matter related to methods and compositions for predicting disease status in cancer. In some embodiments, methods for using fluorescent substrates to predict disease statuses of cancers in subjects are provided. In some embodiments, the methods comprise determining a level of biological activity of a member of the MMP and/or the ADAM family of polypeptides in a biological sample isolated from or in the subject, wherein the determining provides a prediction of the disease status of the cancer in the subject.

BACKGROUND

Breast cancer is the second most common cancer among women, accounting for a third of the cancers diagnosed in the United States. One in nine women will develop breast cancer in her lifetime and about 192,000 new cases of breast cancer are diagnosed annually with about 42,000 deaths. Although men can get breast cancer, this is extremely rare. In the United States it is estimated there will be 217,440 new cases of breast cancer and 40,580 deaths due to breast cancer in 2004. With the exception of those cases with associated genetic factors, precise causes of breast cancer are not known.

In the treatment of breast cancer, there is considerable emphasis on detection and risk assessment because early and accurate staging of breast cancer has a significant impact on survival. For example, breast cancer detected at an early stage (stage TO, discussed below) has a five-year survival rate of 92%. Conversely, if the cancer is not detected until a late stage (e.g., stage T4 (IV)), the five-year survival rate plummets to 13%.

Current methods for predicting or detecting breast cancer risk are not optimal. One method for predicting the relative risk of breast cancer is by examining a patient's risk factors and pursuing aggressive diagnostic and treatment regiments for high risk patients. A patient's risk of breast cancer has been positively associated with increasing age, nulliparity, family history of breast cancer, personal history of breast cancer, early menarche, late menopause, late age of first full term pregnancy, prior proliferative breast disease, irradiation of the breast at an early age and a personal history of malignancy. Lifestyle factors such as fat consumption, alcohol consumption, education, and socioeconomic status have also been associated with an increased incidence of breast cancer although a direct cause and effect relationship has not been established. While these risk factors are statistically significant, their weak association with breast cancer limits their overall usefulness. For example, many women who develop breast cancer have none of the risk factors listed above.

Current screening methods for detecting cancer, such as breast self- exam, ultrasound, and mammography, also have drawbacks that reduce their effectiveness and/or prevent their widespread adoption. Breast self- exams, while useful, are unreliable for the detection of breast cancer in the critical initial stages where the tumor is small and difficult to detect by palpation. Ultrasound measurements require skilled operators at an increased expense. Mammography, while sensitive, is subject to over diagnosis in the detection of lesions that have questionable malignant potential. There is also the fear of the radiation used in mammography because prior exposure to chest radiation is a factor that is associated with an increase incidence of breast cancer.

For purpose of determining prognosis and treatment, these four breast cancer types have been staged according to the size of the primary tumor (T), the involvement of lymph nodes (N), and the presence of metastasis (M). Although DCIS by definition represents localized stage I disease, the other forms of breast cancer may range from stage II to stage IV. There are additional prognostic factors that further serve to guide surgical and medical intervention. The most common ones are total number of lymph nodes involved, ER (estrogen receptor) status, Her2/neu receptor status and histologic grades.

Stage determination has potential prognostic value and provides criteria for designing optimal therapy. Generally, pathological staging of breast cancer is preferable to clinical staging because the former gives a more accurate prognosis. However, clinical staging would be preferred if it were as accurate as pathological staging because it does not depend on an invasive procedure to obtain tissue for pathological evaluation. Staging of breast cancer would be improved by detecting new markers in cells, tissues, or bodily fluids which could differentiate between different stages of invasion. Progress in this field will allow more rapid and reliable method for treating breast cancer patients.

SUMMARY

This Summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.

The presently disclosed subject matter provides in some embodiments methods for predicting disease status of a cancer in a subject. In some embodiments, the method for predicting disease status of a cancer in a subject, the methods comprise (a) determining the level of enzyme activity of a member of the matrix metalloprotease (MMP) and/or the a disintegrin and metallopeptidase domain (ADAM) family of polypeptides in a biological sample from a source isolated from or in the subject; and (b) comparing the level of enzyme activity to a standard, wherein the comparing provides a prediction of the disease status of the cancer in the subject. In some embodiments, the biological sample is selected from the group consisting of serum, plasma, urine, cerebral spinal fluid, blood, saliva, synovial fluid, broncheolar lavage, and a biopsy sample of a tumor and/or a tissue. In some embodiments, the determining comprises employing a fluorescence resonance energy transfer (FRET) assay. In some embodiments, the FRET assay employs a fluorescent substrate selected from the group consisting of Fl₂i (Dabcyl- LAQA(homophenylalanine)RSK(5FAM)-NH₂; SEQ ID NO: 2), Fl₁₃ (Dabcyl- HGDQMAQKSK(5FAM)-NH₂; SEQ ID NO: 3), Flu (Dabcyl- GPLGMRGK(5FAM)-NH₂; SEQ ID NO: 4), Fl₁₀ (Dabcyl- SPLAQAVRSSK(5FAM)-NH₂; SEQ ID NO: 5), Fl₈ (Dabcyl- P(cyclohexylalanine)G(methylcysteine)HAK(5FAM)-NH₂; SEQ ID NO: 6), Fl₆₃ (Dabcyl-SNLAYYTAK(5FAM)-K-NH₂; SEQ ID NO: 7), FI_59A (Dabcyl- APRWLLTAC(FLU)-NH₂; SEQ ID NO: 8), Fl₁₅ (BTC sub; Dabcyl- VDLFYLQQPK(5FAM)-NH₂; SEQ ID NO: 9), and Fl₁₄ (TGFa sub; Dabcyl- EHADLLAWAK(5FAM)-NH₂; SEQ ID NO: 10), Dabcyl-APRWIQDK(5FAM)- NH₂ (SEQ ID NO: 1 1); Dabcyl-APFEMSAK(5FAM)-NH₂ (SEQ ID NO: 12); Dabcyl-APFEFSAK(5FAM)-NH₂ (SEQ ID NO: 13), Dabcyl- EHADLLAWAK(5FAM)-K-NH₂ (SEQ ID NO: 14), or a combination thereof. In some embodiments, the fluorescent substrate comprises Fl₁₃ and/or Fl₂₁ and the biological sample comprises urine. In some embodiments, the standard comprises a panel of average levels of biological activity of the member of the matrix metalloprotease (MMP) and/or the a disintegrin and metallopeptidase domain (ADAM) family of polypeptides in the same source or in normal subjects (i.e., subjects who do not have the cancer) and from subjects with various stages of the cancer. In some embodiments, the cancer is breast cancer and the standard comprises a panel of average levels of biological activity of the member of the matrix metalloprotease (MMP) and/or the a disintegrin and metallopeptidase domain (ADAM) family of polypeptides in the same source or in normal subjects, subjects with invasive breast cancer, subjects with advanced metastatic breast cancer. In some embodiments, the comparing comprises multivariable logistic regression analysis, optionally wherein the multivariable logistic regression is performed by a suitably programmed computer.

The presently disclosed subject matter also provides methods for detecting enzyme activity of a member of the MMP and/or the ADAM family of polypeptides. In some embodiments, the methods comprise contacting a sample suspected of comprising a member of the MMP and/or the ADAM family of polypeptides with a substrate comprising one or more of SEQ ID NOs: 2-14, and detecting the cleavage of the substrate, wherein the cleavage of the substrate is indicative of the enzyme activity of the member of the MMP and/or the ADAM family of polypeptides. In some embodiments, the substrate is detectably labeled with two fluorescent molecules and the cleavage results in the two fluorescent molecules being present on different fragments of the substrate.

The presently disclosed subject matter also provides peptides for use in the disclosed methods. In some embodiments, the peptides comprise, consist essentially of, and/or consist of any of the following amino acid sequences: SNLAYYTAKK (SEQ ID NO: 7); APRWLLTAC (SEQ ID NO: 8); APRWIQDK (SEQ ID NO: 11); APFEMSAK (SEQ ID NO: 12); APFEFSAK (SEQ ID NO: 13); and EHADLLAWAK (SEQ ID NO: 14), wherein the C- terminal lysine (K) of any of SEQ ID NOs: 7 and 11-14 is present or absent. In some embodiments, the peptides further comprise one or more detectable labels conjugated thereto. In some embodiments, the one or more detectable labels are selected from the group comprising of a fluorescent label, a radioactive label, a chemiluminescent label, a colorimetric label, and a tag labels, or a combination thereof. In some embodiments, the peptide comprises two fluorescent labels conjugated thereto such that the two fluorescent labels are on opposite sides of a cleavage site for an enzyme selected from the group consisting of MMP family members and ADAM family members present in the peptide. In some embodiments, a first of the two fluorescent labels comprises a dabcyl moiety or a dabsyl moiety conjugated at or near a first terminus of the peptide and a second of the two fluorescent labels comprises a fluorescein or a 5-carboxyflourescein moiety conjugated at or near the other terminus of the peptide. In some embodiments, the peptides are adapted for use in a fluorescence resonance energy transfer (FRET) assay to test for a biological activity of a member of the MMP and/or the ADAM family of polypeptides. In some embodiments, one or both of the two fluorescent labels comprises a moiety selected from the group consisting of FAM, HEX, TET, VIC, YAKIMAYELLOW™, JOE, NED, a CY® dyes, Texas Red, Rhod, Rhod6G, ROX, TAMRA, Alexa dyes (e.g., ALEXA FLUOR® dyes), LC Red dyes, CASCADE BLUE®, MARINA BLUE®, PACIFIC BLUE®, and BODIPY® FL.

In some embodiments, the peptides further comprise one or more tags conjugated thereto, where the tags are adapted for binding the peptide to a surface selected from the group consisting of glass, a membrane, a resin, a dip stick, plastic, and paper.

The presently disclosed subject matter also provides methods for predicting disease status of breast cancer in a subject. In some embodiments, the methods comprise determining an amount of an enzyme activity of an enzyme selected from the group consisting of a member of the disintegrin and metallopeptidase domain (ADAM) family of polypeptides, optionally, an ADAM8 polypeptide and/or an ADAM 2 polypeptide, in a biological sample from a source isolated from or in the subject; and comparing the amount of the enzyme activity determined to a standard, wherein the comparing provides a prediction of the disease status of the cancer in the subject. In some embodiments, the biological sample is selected from the group consisting of serum, plasma, urine, cerebral spinal fluid, blood, saliva, synovial fluid, broncheolar lavage, and a biopsy sample of a tumor and/or a tissue. In some embodiments, the determining step employs a substrate comprising two detectable moieties present on the substrate such that the enzyme activity, if present, cleaves the substrate resulting in the two detectable molecules being present on different fragments of the cleaved substrate. In some embodiments, the determining comprises employing a fluorescence resonance energy transfer (FRET) assay and the substrate comprises two fluorescent moieties present on the substrate such that the enzyme activity, if present in the biological sample, cleaves the substrate resulting in the two fluorescent moieties being present on different fragments of the cleaved substrate. In some embodiments, the determining step employs a fluorescent substrate comprising Fli₃ and/or Fl₂i and the biological sample comprises urine. In some embodiments, the determining step employs a peptide substrate that is cleavable by the enzyme, if present, and further wherein the peptide substrate comprises one or more tags conjugated thereto, at least one of which is adapted for binding the peptide substrate to a surface selected from the group consisting of glass, a membrane, a resin, a dip stick, plastic, and paper. In some embodiments, the standard is selected from the group consisting of an average level of enzymatic activity of ADAM8 and/or ADAM 12 in the same source isolated from normal subjects {i.e., subjects that do not have cancer) and an average level of enzymatic activity of ADAM8 and/or ADAM12 in the same source isolated from subjects with various stages of the cancer. In some embodiments, an average level of enzymatic activity of ADAM8 and/or ADAM 12 from a subject suspected of having a cancer or a pre-cancerous condition is compared to the average level of enzymatic activity of ADAM8 and/or ADAM12 present in the same source or in normal subjects, subjects with ductal carcinoma in situ, subjects with invasive breast cancer, and subjects with advanced metastatic breast cancer. In some embodiments, the comparing comprises multivariable logistic regression analysis, optionally performed by a suitably programmed computer.

It is thus an object of the presently disclosed subject matter to provide methods and compositions for predicting cancer status.

An object of the presently disclosed subject matter having been stated hereinabove, and which is achieved in whole or in part by the presently disclosed subject matter, other objects will become evident as the description proceeds when taken in connection with the accompanying Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A and 1 B are scatter plots showing that Fl₂i and Fl₁₃ cleavage activities are significantly higher in urine of patients with invasive and metastatic breast cancer compared to controls. Scatter plots representing urinary FI21 (Figure 1A) and FI-13 (Figure 1 B) cleavage activities from breast cancer groups; DCIS (triangle), IBC (inverted triangle), metastatic disease (diamonds) and normal controls (circles). Each assay was conducted in triplicate and the results are expressed as the mean ± standard error of the mean (SEM). Using Fl₂i , specific activity levels were significantly higher in urine samples from invasive (P < 0.001) and metastatic breast cancer (P < 0.001) compared to controls. Similarly for Fl¹³, significantly higher specific activities were observed for urines from patients with IBC (P < 0.001) and metastatic breast cancer (P < 0.001) compared to controls.

Figure 2A is a series of ROC curves depicting the diagnostic accuracy of urinary substrates FI21 (area under the curve (AUC) = 0.745, P = 0.001) and FI13 (AUC = 0.724, P = 0.002) based on actual numeric data (U/mg) in differentiating breast cancer patients from controls. AUC value for each biomarker was moderate and indicated significant classification of breast cancer patients and controls with a reasonably good tradeoff between true- positive fraction and false-positive fraction.

Figure 2B is a series of ROC curves depicting the diagnostic accuracy of urinary substrates Fl₂i (AUC = 0.842, P = 0.001 ) and Fl₁₃ (AUC = 0.811 , P = 0.001) based on actual numeric data (U/mg) in differentiating IBC/metastatic breast cancer patients from controls. AUC value for each biomarker was moderately high and indicated significant classification of IBC/metastatic breast cancer patients and controls with a very good tradeoff between true-positive fraction and false-positive fraction. BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 is the amino acid sequence of a matrix metalloprotease (MMP) conserved Zn²⁺-binding domain cores sequence: HEXGHXXGXXHS T (SEQ ID NO: 1 ).

SEQ ID NOs: 2-14 (Table 1 ) are the sequences of various substrates that were employed in the methods and compositions disclosed herein.

Table 1

Sequences of Exemplary MMP/ADAM Substrates

FI21 : LAQA(homophe)RSK SEQ ID NO: 2;

FI13: HGDQMAQKSK SEQ ID NO: 3;

FI11 : GPLGMRGK SEQ ID NO: 4;

Fho: SPLAQAVRSSK SEQ ID NO: 5;

Fl₈: P(Cha)G(C^Me)HAK SEQ ID NO: 6;

Fl₆₃: SNLAYYTAKK SEQ ID NO: 7;

FI59A^' APRWLLTAC SEQ ID NO: 8;

F ₅: VDLFYLQQPK SEQ ID NO: 9;

Flu: EHADLLAWAK SEQ ID NO: 10;

APRWIQDK SEQ ID NO: 1 1 ;

APFEMSAK SEQ ID NO: 12;

APFEFSAK SEQ ID NO: 13;

EHADLLAWAK SEQ ID NO: 14. wherein for any of SEQ ID NOs: 2-7 and 9-14, the C-terminal lysine (K) residue can be present or absent. In some embodiments, the C-terminal lysine residue (K) is present and a fluorescent moiety is conjugated thereto. In some embodiments, functional groups including, but not limited to amino acids, can be added to one or both termini of a peptide for coupling of detection moieties, fixation to solid supports, and/or for solubility purposes. In some embodiments, linker and/or spacer groups can be added to the peptides to attach detectable groups such as colorimetric labels, radioactive labels, chemiluminescent labels, fluorescent labels (e.g., FAM, HEX, TET, VIC, YAKIMAYELLOW™, JOE, NED, a CY® dyes, Texas Red, Rhod, Rhod6G, ROX, TAMRA, Alexa dyes (e.g., ALEXA FLUOR® dyes), LC Red dyes, CASCADE BLUE®, MARINA BLUE®, PACIFIC BLUE®, and BODIPY® FL), and/or tags that can be detected with specific antibodies (e.g., the hemagglutinin, Myc, HIS, VSV-G, HSV, V5, or FLAG® epitope tags, for which antibodies are available from Sigma-Aldrich Corp., St. Louis, Missouri, United States of America). In some embodiments, the function group can be used for attaching the peptide to a solid support such as a resin, glass (e.g., glass slides, beads, and/or membranes), or to any other desired surface, gel, and/or resin useful for a purpose including but not limited to facilitating detection of the peptide.

DETAILED DESCRIPTION

The present subject matter will be now be described more fully hereinafter with reference to the accompanying Examples, in which representative embodiments of the presently disclosed subject matter are shown. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the presently disclosed subject matter to those skilled in the art.

L General Considerations

Matrix metalloproteases (MMPs) comprise a multigene family of zinc- dependent endopeptidases that have been implicated in tumor growth, invasion, and metastasis in experimental cancer models and in human tumors (Chambers ef a/., 997; Benbow et al., 1999; Curran & Murray, 1999; Kleiner & Stetler-Stevenson, 1999; Bergers et al., 2000; Fang ef a/. , 2000; Egeblad & Werb, 2002; Roy et al., 2006). The characteristic domain structure of MMPs includes: (1) the signal peptide domain, which guides the enzyme into the rough endoplasmic reticulum during synthesis; (2) the propeptide domain, which sustains the latency of these enzymes until it is removed or disrupted; (3) the catalytic domain, which houses the highly conserved Zn²⁺ binding region (HEXGHXXGXXHS/T; SEQ ID NO: 1) and is responsible for enzyme activity; (4) the hemopexin domain, which determines the substrate specificity of MMPs; and (5) a small hinge region, which enables the hemopexin region to present substrate to the active core of the catalytic domain.

A subfamily of membrane-type MMPs (MT-MMPs) possesses an additional transmembrane domain. Two members of this family in particular, MMP-2 and MMP-9, degrade type IV collagen, fibronectin, and laminin, major components of the basement membrane and are commonly used as markers of the malignant phenotype. MMP activity is regulated by a group of four distinct tissue inhibitors of metalloproteases (TIMPs; Nagase & Woessner, 1999; Bode, 2003; Roy et al., 2006.

A related family of metzincins, disintegrin metalloproteases (also referred to as a disintegrin and metal lopeptidase domain (ADAM) proteases) have also been implicated in tumor growth and metastasis. Most ADAMs have the conserved Zn-binding catalytic domain similar to MMPs and are proteolytically active. ADAM family members are typically membrane bound, however, some can have alternatively spliced secreted isoforms as well (Roy et al., 2006). ADAM substrates include cell-surface associated type I or II integral membrane proteins however, like the MMPs, some of them are also considered matrix degrading enzymes. Based on their metalloprotease function and substrate specificity, MMPs and ADAMs are involved in normal developmental processes such as cardiac and neuronal development, mammary involution and bone turnover, but when dysregulated can lead to disease states such as cancer, inflammation, obesity and cardiac hypertrophy (Moali & Hulmes, 2009; Turner et al., 2009; Aiken & Khokha, 2010).

Overexpression of MMPs and/or ADAMs in tumor tissue and stroma can result in increased levels of MMP activity in various body fluids. Evidence is emerging that members of the MMP and/or ADAM family can serve not only as potential markers for diagnosis and prognosis, early detection and risk assessment but also as indicators of tumor recurrence, metastatic spread, and response to primary and adjuvant therapy for breast cancer (Roy et al., 2009). MMP-9 has been detected in the serum and plasma of tumor bearing rats and in humans with malignant tumors (Garbisa et al., 1992; Davies et al., 1993; Gohji et a/., 1996; Jung et al., 2003; Vasala & Turpeenniemi-Hujanen, 2007).

The instant co-inventors have previously reported that MMPs can be detected in urine from patients with a variety of cancers and are independent predictors of disease status (Moses et al., 1998; Yan et al., 2001 ; Fernandez et al., 2005; Smith et al., 2007; Roy ef al., 2008; Smith & Bentz, 2010). MMP-9 levels in tumor tissue as well as serum, plasma and urine are significantly elevated in breast cancer patients. Similarly, the detection of urinary ADAM12 in breast cancer patients is predictive of disease status and stage and ADAM 12 protein levels in urine increase with progression of disease (Roy et al., 2004). In addition, studies from our laboratory indicate that urinary MMP-9 and ADAM 2, in addition to being predictive markers for breast cancer, may also prove useful as non-invasive breast cancer risk assessment tools (Pories et al., 2008).

Oftentimes, activity of a particular enzyme can be more predictive of disease status rather than its mere presence as determined by ELISA or Western analysis. In addition, fluorescent and colorimetnc substrates can be used easily to assess enzymatic activities. Fluorescence resonance energy transfer (FRET) substrates have been used, for example, to detect stromelysin activity in synovial fluid (Beekman et al., 1997). A urine test could be simplified by using FRET substrates which would only require addition of the substrate to urine, and measurement of an increase in fluorescence over a short period of time in a fluorescence plate reader. In general, the detection of an amount of a biological activity of a particular enzyme in a biological sample can be a better predictor of disease status, and is more amenable tow the development of over the counter assays and/or other assays that can be performed quickly and easily.

Certain fluorescent substrates for MMPs and ADAMs were designed based on either known physiological substrates or results from substrate mapping experiments.

Disclosed herein is the use of fluorescent substrates to predict disease status of a representative cancer (i.e. , breast cancer) using an exemplary biological sample (i.e., urine) from patients with different forms of the disease. These fluorescent substrates are based in some embodiments on the peptides of SEQ ID NOs: 2-14, and have the following exemplary structures:

Table 2

Summary of Peptide Sequences and Targets

]L Definitions

All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

Following long-standing patent law convention, the terms "a", "an", and "the" mean "one or more" when used in this application, including the claims. Thus, the phrase "a cell" refers to one or more cells, unless the context clearly indicates otherwise.

As used herein, the term "and/or" when used in the context of a list of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase "A, B, C, and/or D" includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.

The term "comprising", which is synonymous with "including", "containing", and "characterized by", is inclusive or open-ended and does not exclude additional, unrecited elements and/or method steps. "Comprising" is a term of art that means that the named elements and/or steps are present, but that other elements and/or steps can be added and still fall within the scope of the relevant subject matter.

As used herein, the phrase "consisting of" excludes any element, step, and/or ingredient not specifically recited. For example, when the phrase "consists of" appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

As used herein, the phrase "consisting essentially of" limits the scope of the related disclosure or claim to the specified materials and/or steps, plus those that do not materially affect the basic and novel characteristic(s) of the disclosed and/or claimed subject matter. For example, the presently disclosed subject matter in some embodiments can "consist essentially of determining biological activity levels for one or more members of the matrix metalloprotease (MMP) and/or the a disintegrin and metallopeptidase domain (ADAM) family of polypeptides, which means that the recited gene(s) is/are the only genes for which biological activity levels are determined. It is noted, however, that in this case biological activity levels for various positive and/or negative control genes can also be determined, for example, to standardize and/or normalize the biological activity levels of the one or more member of the MMP and/or the ADAM family of polypeptides (if desired).

With respect to the terms "comprising", "consisting essentially of, and "consisting of", where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms. For example, the presently disclosed subject matter relates in some embodiments to methods comprising determining biological activity levels of one or more members of the MMP and/or the ADAM family of polypeptides. It is understood that the presently disclosed subject matter thus also encompasses methods that in some embodiments consist essentially of determining biological activity levels of one or more members of the MMP and/or the ADAM family of polypeptides; as well as methods that in some embodiments consist of determining biological activity levels of one or more members of the MMP and/or the ADAM family of polypeptides.

The term "subject" as used herein refers to a member of any invertebrate or vertebrate species. Accordingly, the term "subject" is intended to encompass any member of the Kingdom Animalia including, but not limited to the phylum Chordata (i.e., members of Classes Osteichythyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Aves (birds), and Mammalia (mammals)), and all Orders and Families encompassed therein. In some embodiments, the presently disclosed subject matter relates to human subjects.

Similarly, all genes, gene names, and gene products disclosed herein are intended to correspond to orthologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, the genes and/or gene products disclosed herein are also intended to encompass homologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds.

The methods and compositions of the presently disclosed subject matter are particularly useful for warm-blooded vertebrates. Thus, the presently disclosed subject matter concerns mammals and birds. More particularly provided is the use of the methods and compositions of the presently disclosed subject matter on mammals such as humans and other primates, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economic importance (animals raised on farms for consumption by humans) and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), rodents (such as mice, rats, and rabbits), marsupials, and horses. Also provided is the use of the disclosed methods and compositions on birds, including those kinds of birds that are endangered, kept in zoos, as well as fowl, and more particularly domesticated fowl, e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans. Thus, also provided is the application of the methods and compositions of the presently disclosed subject matter to livestock, including but not limited to domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.

The term "about", as used herein when referring to a measurable value such as an amount of weight, time, dose, etc., is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1 %, and in some embodiments ±0.1 % from the specified amount, as such variations are appropriate to perform the disclosed methods and/or to employ the presently disclosed arrays.

As used herein the term "gene" refers to a hereditary unit including a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a particular characteristic or trait in an organism. Similarly, the phrase "gene product" refers to biological molecules that are the transcription and/or translation products of genes. Exemplary gene products include, but are not limited to mRNAs and polypeptides that result from translation of mRNAs. Any of these naturally occurring gene products can also be manipulated in vivo or in vitro using well known techniques, and the manipulated derivatives can also be gene products. For example, a cDNA is an enzymatically produced derivative of an RNA molecule (e.g., an mRNA), and a cDNA is considered a gene product. Additionally, polypeptide translation products of mRNAs can be enzymatically fragmented using techniques well known to those of skill in the art, and these peptide fragments are also considered gene products.

As used herein, the term "ADAM 8" refers to the ADAM metallopeptidase domain 8 (ADAM8) locus located on human chromosome 10 at 10q26.3. Exemplary, non-limiting ADAM8 gene products from humans are described in GENBANK® Accession Nos. NM_001109.4, NM_001 164489.1 , NM_001 164490.1 , NP_001 100.3, NP_ 001 157961.1 , and NP_001 157962.1. ADAM8 orthologs from Mus musculus, Danio rerio, Rattus norvegicus, Canis lupus familiaris, Gallus gallus, and other species are also present in the GENBANK® database, and each such entry and all annotations provided therewith are incorporated herein by reference in its entirety.

As used herein, the term "ADAM 9" refers to the ADAM metallopeptidase domain 9 (ADAM9) locus located on human chromosome 8 at 8p1 1.22. Exemplary, non-limiting ADAM9 gene products from humans are described in GENBANK® Accession Nos. NMJ 03816.2 and NP_003807.1. ADAM 9 orthologs from Pan troglodytes, Nomascus leucogenys, Pongo abelii, Macaca mulatta, Mus musculus, Rattus norvegicus, Canis lupus familiaris, Bos taurus, Gallus gallus, Danio rerio, Equus caballus, Sus scrofa, and several other species are also present in the GENBANK® database, and each such entry and all annotations provided therewith are incorporated herein by reference in its entirety.

As used herein, the term "ADAM 10" refers to the ADAM metallopeptidase domain 10 (ADAM 10) locus located on human chromosome 15 at 15q22. Exemplary, non-limiting ADAM 10 gene products from humans are described in GENBANK® Accession Nos. NM_001 1 10.2 and NPJD01101.1. ADAM10 orthologs from Pan troglodytes, Nomascus leucogenys, Mus musculus, Rattus norvegicus, Pongo abelii, Canis lupus familiaris, Bos taurus, Sus scrofa, Macaca mulatta, Equus caballus, and several other species are also present in the GENBANK® database, and each such entry and all annotations provided therewith are incorporated herein by reference in its entirety.

As used herein, the term "ADAM 12" refers to the ADAM metallopeptidase domain 12 (ADAM 12) locus located on human chromosome 10 at 10q26.3. Exemplary, non-limiting ADAM12 gene products from humans are described in GENBANK® Accession Nos. NM_0032474.4, NM_021641.3, NP_003465.3, and NP_067673.2. ADAM 12 orthologs from Mus musculus, Rattus norvegicus, Bos taurus, Danio rerio, Pan troglodytes, Equus caballus, and other species are also present in the GENBANK® database, and each such entry and all annotations provided therewith are incorporated herein by reference in its entirety.

As used herein, the term "ADAM 17" refers to the ADAM metallopeptidase domain 17 (ADAM 17) locus located on human chromosome 2 at 2p25. Exemplary, non-limiting ADAM17 gene products from humans are described in GENBANK® Accession Nos. NM_003183.4 and NP_003174.3. ADAM 17 orthologs from Pan troglodytes, Pongo abelii, Nomascus leucogenys, Callithrix jacchus, Macaca mulatta, Equus caballus, Bos taurus, Sus scrofa, Canis lupus familiaris, Mus musculus, Rattus norvegicus, Meleagris gallopavo, Gallus gallus, and other species are also present in the GENBANK® database, and each such entry and all annotations provided therewith are incorporated herein by reference in its entirety.

As used herein, the term "MMP-1 " refers to the matrix metalloproteinase 1 (MMP-1) locus located on human chromosome 1 1 at 1q22.3. Exemplary, non-limiting MMP-1 gene products from humans are described in GENBANK® Accession Nos. NM_002421.3, NM_001 145938.1 , NP_002412.1 , and NP_001 39410.1 . MMP-1 orthologs from Drosophila melanogaster, Bos taurus, Sus scrofa, Pan troglodytes, Macaca mulatta, Nomascus leucogenys, Pongo abelii, Equus caballus, Canis lupus familiaris, Rattus norvegicus, Mus musculus, rerio, and other species are also present in the GENBANK® database, and each such entry and all annotations provided therewith are incorporated herein by reference in its entirety As used herein, the term "MMP-2" refers to the matrix metalloproteinase 2 (MMP-2) locus located on human chromosome 16 at 16q13-q21. Exemplary, non-limiting MMP-2 gene products from humans are described in GENBANK® Accession Nos. NM_004530.4, NM_001 127891.1 , NP_004521.1 , and NP_001 121363.1. MMP-2 orthologs from Mus musculus, Rattus norvegicus, Bos taurus, Drosophila melanogaster, Danio rerio, Canis lupus familiaris, Equus caballus, and other species are also present in the GENBANK® database, and each such entry and all annotations provided therewith are incorporated herein by reference in its entirety

As used herein, the term "MMP-3" refers to the matrix metalloproteinase 3 (MMP-3) locus located on human chromosome 1 at 1 1q22.3. Exemplary, non-limiting MMP-3 gene products from humans are described in GENBANK® Accession Nos. NM_002422.3 and NP_002413.1. MMP-3 orthologs from Mus musculus, Rattus norvegicus, Bos taurus, Pan troglodytes, Nomascus leucogenys, Macaca mulatta, Pongo abelii, Canis lupus familiaris, Sus scrofa, Equus caballus, and other species are also present in the GENBANK® database, and each such entry and all annotations provided therewith are incorporated herein by reference in its entirety

As used herein, the term "MMP-8" refers to the matrix metalloproteinase 8 (MMP-8) locus located on human chromosome 1 1 at 1 1 q22.3. Exemplary, non-limiting MMP-8 gene products from humans are described in GENBANK® Accession Nos. NM_002424.2 and NP_002415.1. MMP-8 orthologs from Mus musculus, Rattus norvegicus, Bos taurus, Canis lupus familiaris, and other species are also present in the GENBANK® database, and each such entry and all annotations provided therewith are incorporated herein by reference in its entirety.

As used herein, the term "MMP-9" refers to the matrix metalloproteinase 9 (MMP-9) locus located on human chromosome 20 at 20q1 1.2-q13.1. Exemplary, non-limiting MMP-9 gene products from humans are described in GENBANK® Accession Nos. NM_004994.2 and NP_ 004985.2. MMP-9 orthologs from Macaca mulatta, Mus musculus, Rattus norvegicus, Pan troglodytes, Canis lupus familiaris, Bos taurus, Equus caballus, and other species are also present in the GENBANK® database, and each such entry and all annotations provided therewith are incorporated herein by reference in its entirety.

As used herein, the term "MMP-13" refers to the matrix metalloproteinase 13 (MMP-13) locus located on human chromosome 1 1 at 1 1q22.3. Exemplary, non-limiting MMP-13 gene products from humans are described in GENBANK® Accession Nos. NM_002427.3 and NP_002418.1. MMP-13 orthologs from Pan troglodytes, Nomascus leucogenys, Pongo abelii, Macaca mulatta, Mus musculus, Rattus norvegicus, Bos taurus, Canis lupus familiaris, Equus caballus, Gallus gallus, and other species are also present in the GENBANK® database, and each such entry and all annotations provided therewith are incorporated herein by reference in its entirety.

As used herein, the term "BTC" refers to the betacellulin (BTC) locus located on human chromosome 4 at 4q13-q21. Exemplary, non-limiting BTC gene products from humans are described in GENBANK® Accession Nos. NM_001729.2 and NP 001720.1 . BTC orthologs from Mus musculus, Rattus norvegicus, Equus caballus, Canis lupus familiaris, and other species are also present in the GENBANK® database, and each such entry and all annotations provided therewith are incorporated herein by reference in its entirety.

As used herein, the term "TGFa" refers to the transforming growth factor-alpha (TGFa) locus located on human chromosome 2 at 2p13. Exemplary, non-limiting TGFa gene products from humans are described in GENBANK® Accession Nos. NM_003236.2 and NP_003227.1 (transcript variant 1 ) and NM_001099691 .1 and NP_001093161.1 (transcript variant 2). TGFa orthologs from Mus musculus, Rattus norvegicus, Bos taurus, Canis lupus familiaris, and other species are also present in the GENBANK® database, and each such entry and all annotations provided therewith are incorporated herein by reference in its entirety.

III. Methods for Predicting Disease Status

In some embodiments, the presently disclosed subject matter provides methods for predicting disease status of a cancer in a subject. As used herein, the phrase "disease status of a cancer" refers to a relative measure of the severity of a cancer, including the presence or absence of cancer (for example, normal or cancerous). By way of example and not limitation, a disease status in the context of breast cancer can refer to distinguishing between whether a particular subject (and/or a sample isolated therefrom) is characterized normal, ductal carcinoma in situ, invasive breast cancer, and/or advanced metastatic breast cancer. In some embodiments, a disease status of a cancer is employed to distinguish between subjects that have invasive and/or metastatic breast cancer versus subjects that are normal and/or that have less severe forms of breast cancer.

In some embodiments, the presently disclosed methods comprise determining a level of biological activity of a member of the matrix metalloprotease (MMP) and/or the a disintegrin and metallopeptidase domain (ADAM) family of polypeptides in a biological sample isolated from or in the subject, wherein the determining provides a prediction of the disease status of the cancer in the subject. Biological samples that can be employed in the methods of the presently disclosed subject matter include any samples that would be expected to contain MMP and/or ADAM family polypeptides. Exemplary non-limiting such biological samples include, but are not limited to serum, plasma, urine, cerebral spinal fluid, blood, saliva, synovial fluid, broncheolar lavage, and biopsy samples of tumors and/or tissues isolated from a subject that has or that is suspected of having a cancer. In some embodiments, a biological sample is urine.

Any method that can be employed for determining a level of a biological activity of a polypeptide (e.g., an MMP and/or an ADAM family polypeptide) in a sample can be employed in the methods of the presently disclosed subject matter. An exemplary such method is the fluorescence resonance energy transfer (FRET) assay. Typically, a FRET assay employs a fluorescent reporter moiety and a quencher moiety in an arrangement wherein the two moieties are in sufficiently close physical proximity to each (e.g., less than about 1 nm or so) such that the fluorescence of the fluorescent reporter moiety is quenched (i.e., undetectable or less detectable) by the quencher moiety. One way to place these moieties in sufficiently close physical proximity is to incorporate them into the same molecule. In some embodiments of the presently disclosed subject matter, peptide substrates for MMP and/or ADAM family polypeptides are generated that include the following, non-limiting amino acid sequences:

1. FI₂₁: LAQA(homophenylalanine)RSK (SEQ ID NO: 2);

2. Fl₁₃: HGDQMAQKSK (SEQ ID NO: 3);

3. Flu: GPLGMRGK (SEQ ID NO: 4);

4. Fl₁₀: SPLAQAVRSSK (SEQ ID NO: 5);

5. Fl₈: P(Cha)^MCGHAK (SEQ ID NO: 6);

6. Fl₆₃: SNLAYYTAK (SEQ ID NO: 7);

7. FI_59A: APRWLLTAC (SEQ ID NO: 8);

8. APRWIQDK (SEQ ID NO: 11);

9. APFEMSAK (SEQ ID NO: 12);

10. APFEFSAK (SEQ ID NO: 13); and

11. EHADLLAWAK (SEQ ID NO: 14),

where (Cha) is cyclohexylalanine and C is methylcysteine. In some embodiments, these peptides can be labeled (e.g., at N-termini) with 4-((4- (dimethylamino)phenyl)azo)benzoic acid (Dabcyl) and/or can also be labeled (e.g., at their C-termini) with 5-carboxyfluorescein (5FAM) or fluorescein (FLU), although other combinations of fluorophores can also be used as would be understood by one of ordinary skill in the art. In some embodiments, the C-terminal lablel is attached to a lysine residue that is optionally present or absent on the C-termini of any of SEQ ID NOs: 2-7 and 9-14). Biological samples expected to contain MMP and/or an ADAM family polypeptides can be contacted with one or more of these peptides and the biological activities of the MMP and/or an ADAM family polypeptides contained therein can be assayed using standard FRET techniques, since the presence of MMP and/or an ADAM family polypeptides in the biological sample would be expected to cleave the peptide, thereby physically separating the fluorescent moiety from the quencher.

Additionally, any other technique in which the cleavage of a substrate peptide can be assayed can also be used. For example, peptides can be labeled with other detectable moieties (e.g., radioactive molecules) and size/molecule weight changes in substrates subsequent to cleavage can be assayed using standard molecule techniques, wherein the occurrence of a size/molecule weight change in a substrate is indicative of the presence of an MMP and/or an ADAM family polypeptide in a sample.

IV. Methods for Detecting Enzyme Activities

The presently disclosed subject matter also provides in some embodiments methods for detecting enzyme activity of a member of the MMP and/or the ADAM family of polypeptides. In some embodiments, the methods comprise contacting a sample suspected of comprising a member of the MMP and/or the ADAM family of polypeptides with a substrate comprising one or more of SEQ ID NOs: 2-14, and detecting the cleavage of the substrate, wherein the cleavage of the substrate is indicative of the enzyme activity of the member of the MMP and/or the ADAM family of polypeptides. The FRET technique can also be employed for this purpose, and thus in some embodiments the substrate is detectably labeled with two fluorescent molecules and the cleavage results in the two fluorescent molecules being present on different fragments of the substrate.

In some embodiments, the enzyme activities of ADAM8 and/or ADAM12 are detected, wherein the presence of enzyme activities of ADAM8 and/or ADAM12 are indicative of a disease state. In some embodiments, the enzyme activities of ADAM8 and/or ADAM 12 are employed to distinguish among various stages of breast cancer including, but not limited to early stage disease, ductal carcinoma in situ, locally invasive breast cancer, and advanced metastatic disease. By way of example and not limitation, in some embodiments the presence of enzyme activities for either or both of ADAM8 and ADAM12 in a biological sample (e.g. , a urine sample) isolated from a subject is indicative of the subject having locally invasive breast cancer and/or advanced metastatic disease.

V, Compositions

The presently disclosed subject matter also provides compositions that can be employed in the methods disclosed herein. In some embodiments, the compositions comprise MMP and/or an ADAM family polypeptide substrates including, but not limited to substrates comprising any of SEQ ID NOs: 2-14. In some embodiments, the substrates are fluorescently labeled in order to be employed in a FRET assay. Exemplary such labeled substrates include the following:

1. Fl₂₁ Dabcyl-LAQA(homophenylalanine)RSK(5FAM)-NH₂;

2. Fl₁₃ Dabcyl-HGDQMAQKSK(5FAM)-NH₂;

Flu Dabcyl-GPLGMRGK(5FAM)-NH₂;

F o Dabcyl-SPLAQAVRSSK(5FAM)-NH₂;

5. Fl₈: Dabcyl-P(Cha) CGHAK(5FAM)-NH₂;

6. Fl₆₃: Dabcyl-SNLAYYTAK(5FAM)-K-NH₂;

7. FI₅9A: Dabcyl-APRWLLTAC(FLU)-NH₂;

8. Dabcyl-APRWIQDK(5FAM)-NH₂ (SEQ ID NO: 11);

9. Dabcyl-APFEMSAK(5FAM)-NH₂ (SEQ ID NO: 12);

10. Dabcyl-APFEFSAK(5FAM)-NH₂ (SEQ ID NO: 13); and

11. Dabcyl-EHADLLAWAK(5FAM)-K-NH₂ (SEQ ID NO: 14).

In some embodiments, a composition of the presently disclosed subject matter is selected from the group consisting of Fl₆3 and FI_59A.

It is noted that for any of the substrates of the presently disclosed subject matter, the N-terminus and C-terminus can be labeled with pairs of FRET labels other than Dabcyl and 5FAM/FLU and/or the positions of the Dabcyl and 5FAM/FLU can be reversed in each peptide, as desired.

In some embodiments of SEQ ID NOs: 2-7 and 9-14, the C-terminal lysine (K) residue can be present or absent. In some embodiments, the C- terminal lysine residue (K) is present and a detectable (e.g., fluorescent) moiety is conjugated thereto. In some embodiments, Any detectable moiety can be conjugated to a peptide of the presently disclosed subject matter either singly or multiply, as desired. Exemplary non-limiting detectable moieties include fluorescent moieties such as, but not limited to FAM, HEX, TET, VIC, YAKIMAYELLOW™, JOE, NED, a CY® dyes, Texas Red, Rhod, Rhod6G, ROX, TAMRA, Alexa dyes (e.g., ALEXA FLUOR® dyes), LC Red dyes, CASCADE BLUE®, MARINA BLUE®, PACIFIC BLUE®, and BODIPY® FL. Other detectable moieties can include colorimetric labels, radioactive labels, chemiluminescent labels, and tags that can be detected with specific antibodies (e.g., the hemagglutinin, Myc, HIS, VSV-G, HSV, V5, or FLAG® epitope tags, for which antibodies are available from Sigma- Aldrich Corp., St. Louis, Missouri, United States of America).

In some embodiments, one or more functional groups including, but not limited to amino acids, can be added to one or both termini of a peptide for coupling of detectable moieties, fixation to solid supports, and/or for solubility purposes. In some embodiments, linker and/or spacer groups can be added to the peptides to attach one or more detectable groups such as those exemplified herein above. In some embodiments, the function group can be used for attaching the peptide to a solid support such as a resin, glass (e.g., glass slides, beads, and/or membranes), paper (e.g., a dipstick that is coated with a peptide of the presently disclosed subject matter), or to any other desired surface, gel, and/or resin useful for a purpose including but not limited to facilitating detection of the peptide.

EXAMPLE

The following EXAMPLE provides illustrative, non-limiting embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following EXAMPLE is intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

Materials and Methods Employed

Study Population. Eighty-five samples were analyzed in this study, including samples from patients diagnosed with ductal carcinoma in situ (n = 24), invasive breast cancer (n = 22), and advanced metastatic breast cancer (n = 18) cancer. Age-matched controls (n = 21) were also analyzed. All diagnoses were biopsy proven. Specimens were obtained prior to surgical or other therapeutic intervention. Institutional Review Board approval for the study was obtained at each institution. All participants completed a detailed medical history form at the time of urine donation.

Urine sample collection and processing. For a pilot study, five normal and five metastatic age-matched urines were purchased from Bioreclamation, Inc. (Hicksville, New York, United States of America). Urine was collected according to the institutional bioethical guidelines pertaining to discarded clinical material (Moses et a/., 1998). Samples were collected in sterile containers and immediately frozen at -20°C. Urine was tested for presence of blood and leukocytes using MULTISTIX® 9 Urinalysis Strips (Bayer, Elkhart, Indiana, United States of America) and samples containing blood or leukocytes were excluded. Protein concentration of urine was determined by the Bradford method using bovine serum albumin as the standard (Moses et a/., 1998; Roy et al., 2004).

Screening of urines with fluorescence substrates. A pilot study composed of five normal and five metastatic age-matched urines was conducted. Fl₂₁ (Dabcyl-LAQAhomopheRSK(5FAM)-NH₂; SEQ ID NO: 2), Fl₁₃ (Dabcyl-HGDQMAQKSK(5FAM)-NH₂; SEQ ID NO: 3), Flu (Dabcyl- GPLGMRGK(5FAM)-NH₂; SEQ ID NO: 4), Fl₁₀ (Dabcyl- SPLAQAVRSSK(5FAM)-NH₂; SEQ ID NO: 5), Fl₈ (Dabcyl-P(Cha)- G ^eCHAK(5FAM)-NH₂; SEQ ID NO: 6), and Fl₆₃ (Dabcyl- SNLAYYTAK(5FAM)-KNH₂; SEQ ID NO: 7) were prepared as 10 mM stock solutions in dimethyl sulfoxide (DMSO). Substrates were diluted to 20 μΜ in buffer containing 50 mM Tris, pH 8, 10 mM CaCI₂, and 0.01 % BRIJ 35® non- ionic polyoxyethylene surfactant for assays measuring ADAM activity and 50 mM Tris, pH 7.5, 150 mM NaCI, 5 mM CaCI₂, 1 μΜ ZnSO₄, 0.01 % BRIJ 35® for assays measuring MMP activity. For the assay, 75 μΙ of urine was added to a Grenier 96 well black coated plate (Sigma-Aldrich, St. Louis, Missouri, United States of America).

To start the reaction, 25 μΙ of substrate was added via multipipettor. Fluorescence values were measured in a Labsystems Fluoroskan II fluorescence microplate reader at excitation and emission wavelengths of 485 nm and 530 nm, respectively. Control wells contained urine with substrate buffer alone or 25 μΙ of substrate buffer and 75 pi of phosphate buffered saline. The total running time for each assay was 2.5 hours, and readings were taken every 2 minutes. Ninety points were used to determine slope values which were linear. Fluorescence units (FU) versus time were plotted and slopes of the initial velocities were obtained by linear fit. Protein concentrations were determined using the Pierce Coomassie Plus (Bradford) protein assay. Absorbance was measured at 595 nm. Specific activities (U/mg) were determined by dividing the slopes from the FU versus time graphs by total protein present in the urine for each assay.

Measurement of Fl?i and Fl-n cleavage activities. Fl₂i is Dabcyl- LAQA(homophe)RSK(Fam)-NH₂ (SEQ ID NO: 2) and Fl₁₃ is Dabcyl- HGDQMAQKSK(Fam)-NH₂ (SEQ ID NO: 3). Stock substrate solution was prepared at 5 mM concentration in DMSO. Substrates were stored at -80°C. Substrates were thawed and diluted into assay buffer (50 mM Tris, pH 7.5, 5 mM CaCI₂, 1 μΜ ZnSO₄) to obtain a working concentration of 20 μΜ for the assays.

Reactions were conducted in 96-well white polystyrene flat bottom plates (Whatman, GE Healthcare, Piscataway, New Jersey, United States of America) at room temperature. All assays were conducted in duplicate. The assay mixture consisted of 80 μΙ urine sample and 20 μΙ substrate (final substrate concentration 4 μΜ). To determine background fluorescent levels, control wells containing assay buffer only, substrate only, or urine sample only were used and background fluorescent levels subtracted before activity calculations. The enzyme, assay buffer, or urine samples were added to the wells first and subsequently the reaction was started by adding the substrate using a multipipettor. The reaction was monitored using a Wallace VICTOR²™ 1420 Multilabel counter (Perkin Elmer, Waltham, Massachusetts, United States of America). Excitation and emission filters were set to 485 nm and 530 nm, respectively. Readings were recorded every 15 minutes for 3 hours. Experimental data from the fluorimeter were imported into a Microsoft Excel spreadsheet for specific activity calculations. Net fluorescence was obtained by the subtraction of background fluorescence from each well. Slope was calculated using net fluorescence increase in the linear range versus time curves. Slope values were initially divided by an arbitrary number (1000). To obtain substrate cleavage activity (U/ml), slope values were multiplied by 12.5. Finally, specific activity (U/mg) was calculated using protein concentration values of each urine sample.

Statistical Analysis. Cleavage of FRET substrates Fl₂i and Fl ₃ were compared between breast cancer urine samples and controls using the nonparametric Mann-Whitney L/-test since these substrates displayed skewness as assessed by the Kolmogorov-Smimov test of normality (Altman, 1991). Data are presented as medians and interquartile ranges. Using an activity value or greater than 0 U/mg for Fl₂i and Fl₁₃ to identify possible breast cancer, sensitivity and specificity were calculated for breast cancer patients versus controls, and for invasive breast cancer (IBC)/metastatic disease versus controls with proportion of positive activity compared between cancer and controls groups using Fisher's exact test. Multivariable logistic regression was used to test whether Fl₂i and Fl ₃ (based on a positive or negative test results) were independently predictive of: (i) cancer versus control; and (ii) IBC/metastatic disease compared to controls with the odds ratio and 95% confidence interval (CI) for determining risk (Harrell, 2001 ; Katz, 2006). Receiver operating characteristic (ROC) curve analysis was applied to determine the diagnostic accuracy of each urinary substrate in differentiating cancer patients from controls using area under the curve (AUC) with a 95% CI as the measure of prediction (Pepe, 2004). Statistical analysis was performed using the SPSS software package (version 18.0, SPSS Inc./IBM, Chicago, Illinois, United States of America). Two-tailed values of P < 0.05 were considered statistically significant.

EXAMPLE

Screening of Fluorescent Substrates for MMP/ADAM Activity

Several different MMPs and ADAMs were screened for cleavage of a range of fluorescent substrates including Fl₈, F ₀, Flu, Fl₁₃, Fl₂i , and Fl₆3 (see Table 3). Substrates were chosen based on their ability to distinguish between MMP-9, MMP-2, ADAM 12 and ADAM8. For example, Fl₈ and Flu were chosen because of their selectivity for MMP-9 over MMP-2. In addition, these two substrates were not very reactive towards other MMPs with the exception of MMP-13, and ADAM family members, ADAM 8, -9, -10, -12, and -17. Since the presence of MMP-9 and not MMP-2 in urine was shown to correlate with breast cancer, it was reasoned that these two substrates could potentially be used successfully to predict disease status. Fl₆3 is a substrate selective for MMP-2 over MMP-9 and therefore served as a negative control. Finally, Fl ₀, Fl ₃, and FL₂i were fluorescent substrates that were employed to measure ADAM activity. F ₀ was not a selective substrate but was very sensitive for ADAM 17. Fl₂i was the best substrate known to date for ADAM12 (Moss & Rasmussen, 2007) although it can be cleaved by ADAM 17 and ADAM 10 as well. Fl ₃ was used typically to detect ADAM8 activity. Since a secreted isoform of ADAM8 has been reported and ADAM8 levels correlate with head and neck cancer (Stokes et al., 2010), Fl₁₃ was chosen in the event that ADAM8 was also found in urine. In addition, Fl ₃ was selective for ADAM8 over most of the MMPs.

Table 3

Screening of Fluorescent Substrate Cleavage Activity by MMPs and ADAMs

Fl₁₀ ^c Fl₂i^b Fi " Fl₆₃ ^a Fl₈ ^b Flu³

ADAM 8 33 100 53 2.5 1.7 2.4

ADAM 9 1 10 8.3 1.6 22 4.5 0.23

ADAM 10 3.6 6.2 0.27 14 0.19 0.01

ADAM 12 38 280 0.04 NA^d 3.0 NA^d

ADAM 17 960 430 ND^e 1.1 38 3.7

MMP- 51 28 ND^e 3.2 76 45

MMP-2 170 320 2.4 1300 29 480

MMP-3 39 4.0 ND^e 22 0.052 1.6

MMP-8 26 140 ND^e 340 24 36

MMP-9 600 220 ND^e 140 850 1400

MMP-13 1700 460 ND^e 1600 2100 3300 a Specificity values (x 10³) at 10 μΜ substrate concentration were measured by back calculation of enzyme concentrations after determination of k_Cat/K_m for known substrates. Enzyme concentrations were determined previously by active site titration as described in Rasmussen er a/., 2004.

b Values (x 10³) were taken from Moss & Rasmussen, 2007.

c Values (x 0³) were taken from Moss et al., 2010.

d Not attempted

⁶ No activity detected To ascertain whether these fluorescent substrates could be used to predict disease status, urine samples from patients with metastatic breast cancer and age- and sex-matched controls were initially screened with several substrates that were known to be efficiently cleaved by MMP-2, MMP-9, ADAM 8, and ADAM 12. Table 4 indicates the specific activity (fluorescent units (FU)/min.mg) calculated using the fluorescence units vs. time plots for each of the urine samples using six distinct substrates.

Table 4

Initial Screening of Urine Samples for Substrate Cleavage Activity³

Flu FI63 ie Fl-io FI21 FI-13 MMP-9 MMP-2 MMP-9 ADAM ADAM12 ADAM8

Normal

1 N 69^b 24 45 39 44 29

2N 0 0.8 0 15 0 0

3N 0 0 0 76 0 0

4N 15 0 87 0 0

5N 0 36 0 1.3 0 0

Average N 13.8 15 5 43.6 8.8 5.8

Metastatic

1 M 0 46 17 1000 4.7 4.8

2M 33 15 3.8 190 16 5.4

3M 47 13 100 1300 17 0

4M 15 7.9 21 3600 25 29

5M 0 10 0 1100 22 7.4

Average M 19 18 28 1438 17 9 a Reaction conditions are described hereinabove in the Materials and Methods section. Errors are less than 10%.

b Values are FU/min.mg.

The MMP-9-specific substrates Fl₈ and Flu were only partially selective, (mean FU/min.mg of 5 vs. 28 and 13.8 vs. 19) for the normal and metastatic groups, respectively.

Mean activity values for the metastatic and control groups using the MMP-2 substrate Fl₆3 were very similar (see Table 4), and all the specific activities were above 1 with the exception of Sample 3N (see Table 4). Similar findings were obtained with the general ADAM substrate, Fli₀, as all the values were positive, even though there was a 33-fold mean difference change for the normal and metastatic groups.

Interestingly, fluorescent substrates Fl₁₃ and Fl₂i, with high specificity for ADAM8 and ADAM12, respectively, showed the most discrimination between the two test groups. The activity values for Fli₃ and Fl₂i were either negative or approached zero for all urine samples from normal controls with the exception of Sample 1 N (see Table 4). In contrast, urine samples from patients with metastatic disease all tested positive for Fl₂i cleavage activity, while 4 out of 5 samples tested were positive for Fl ₃ cleavage activity.

As disclosed herein, urine samples were tested using the fluorescent substrates Fl₂i and Fl₁₃ to determine whether there was a correlation between activity and disease status. A total of 85 urine samples were tested including 64 from breast cancer patients and 21 from age-/sex-matched controls. The breast cancer cohort included samples from across the disease spectrum such as patients at high risk of developing breast cancer, early stage disease, ductal carcinoma in situ (DCIS; n = 24), locally invasive breast cancer (IBC; n = 22), and advanced metastatic disease (n = 18; see Figures 1A and 1 B). Urine samples were tested in triplicate, and urinary Fl₂i and F ₃ cleavage activities for breast cancer patients and normal controls are presented in Table 5.

Table 5

Urinary Fl?i and F 113 Activity in Breast Cancer Patients and Controls

Substrate Controls Breast DCIS IBC Metastatic

(U/mg) (n = 21) Cancer (n = 24) (n = 22) (n = 18)

(n = 64)

Fl₂₁

Median 0 5.7 0 27.5 11.0

IQR 0-0 0-48 0-6 0-114 5-88

Range 0-94 0-4319 0-347 0-4319 0-362

P value <0.001 ^* 0.24 O.001^* <0.001^*

Median 0 35.0 0 73.7 43.0

IQR 0-25 0-101 0-40 32-270 9-137

Range 0-165 0-3702 0-242 0-3702 0-347

P value - 0.002^* 0.31 <0.001 ^* <0.001^{* *}Statistically significant, P values vs. controls, Mann-Whitney L/-test. IQR = interquartile range, DCIS = ductal carcinoma in situ, IBC = invasive breast cancer. In addition, IBC and metastatic groups were significantly higher than DCIS with respect to both FI21 and FI₁₃ (P < 0.001).

Using Fl₂i, only 19% of urines from normal controls displayed cleavage activity whereas 34%, 68%, and 89% samples from DCIS, IBC, and metastatic patients, respectively, displayed Fl₂i cleavage activity. For this substrate, the median specific activity values for samples from patients with DCIS were similar to normal controls; however, median specific activity levels were significantly higher in urine samples from invasive (P < 0.001) and metastatic breast cancer (P <0 .001 ; see Table 5). Similar trends were observed for FI13, with only 28% of samples from normal controls displaying any activity 37%, 77%, but 89% of samples from DCIS, IBC, and metastatic patients, respectively, having positive Fl₁₃ activity. In addition, the median specific activity values using FI13 were not very different for normal controls or DCIS samples, slightly (but not significantly) and significantly higher median specific activities were observed for urines from patients with IBC (P < 0.001) and metastatic breast cancer (P < 0.001).

These data were analyzed by univariate and multivariate statistics (see Figures 2A and 2B, and Tables 5 and 6).

Table 6

Diagnostic Performance Characteristics of the Substrates Breast Cancer vs. Controls IBC/Metastatic vs. Controls

AUC 95% CI P AUC 95% CI P value value

121 0.745 0.633- 0.001 0.842 0.735- <0.001

0.857 0.946

Fl 13 0.724 0.601- 0.002 0.81 1 0.692- <0.001

0.646 0.929

Sens Spec P Sens Spec P value value

Fl 21 41/64 17/21 0.001 31/40 17/21 <0.001

(64%) (81 %) (78%) (81 %)

Fl₁₃ 44/62=71 % 15/21 =71 % 0.002 33/38=87% 15/21 =71 % <0.001

IBC = invasive breast cancer. AUC = area under the curve based on ROC curve analysis. CI = confidence interval. Sens = sensitivity. Spec = specificity.

Multivariable logistic regression using binary cut-off values for the two substrates indicated that Fl₂i provided significant predictive information in differentiating breast cancer patients from controls (Table 6; odds ratio 7.7; 95% CI: 2.3 - 25.8; P < 0.001 ). Fl ₃ was not found to provide additional predictive information (P = 0.12). In a subgroup analysis considering the 40 IBC/metastatic disease patients and the 21 controls, multivariable logistic regression indicated that using binary cut-off values, Fl₂i (P = 0.009) and Fl ₃ (P = 0.028) were significant independent biomarkers. The odds of IBC/metastatic disease based on a value greater than 0 U/mg for Fl₂i were almost 8 times higher (odds ratio 7.9, 95% CI: 2.0 - 37.4), whereas the odds of IBC/metastatic disease were over 5 times higher in individuals having a positive Fl₁₃ activity (odds ratio 5.7, 95% CI: 1.3 - 27.2). Overall, there were significant differences in the median specific activity using both Fl₂i and F ₃ between all 64 cancer patients and 21 controls (see Table 5). Examining each group separately, it was determined that the substrates showed differences, although these differences were statistically significant at P < 0.05 only for the IBC and metastatic group compared to controls and did not reach this level of significance for the DCIS group. Medians, interquartile ranges (25^th - 75^th percentiles), and full ranges were used with the nonparametric Mann-Whitney C -test (due to skewness of the substrate data) for comparing groups. Multivariable analysis using logistic regression indicated that while both Fl₂i and Fl ₃ displayed some value in differentiating cancer versus controls, this was largely due to higher FI21 and FI13 values in the more advanced cancer subgroups (IBC and metastatic disease). Urinary FI21 and Fl ₃ activities were highly correlated and logistic regression confirmed that they were not independent markers. Therefore, in some embodiments one of these two substrates would likely be sufficient from the perspective of predicting the presence of any cancer.

Based on multivariable regression modeling using the two fluorescent substrates for predicting IBC and/or advanced metastatic disease, the model would predict a 20% probability if both substrates were negative, 55% probability if Fl ₃ was positive and FI21 was negative, 65% probability if FI₂1 was positive and Fl ₃ was negative, and a 90% probability of advanced breast cancer (IBC or metastatic disease) if urinary Fl₂₁ and Fl ₃ cleavage activities were both positive. In general, the value of these substrates would increase substantially in differentiating the more advanced breast cancer from normal controls or from the DCIS subgroup.

Table 6 summarizes results of ROC analysis and shows that while the area under the curve (AUC) was significant for substrates in differentiating all cancers (n = 64) compared to normal controls (n = 21), the diagnostic performance based on AUC values was much better when differentiating invasive and/or metastatic breast cancer from normal controls. Using any positive urinary activity measurement, Table 6 summarizes sensitivity and specificity for Fl₂i and Fl-|₃ for all breast cancer groups versus control and for IBC/metastatic versus controls (the sensitivity for each substrate was much higher).

In conclusion, the substrates Fl₂i and Fl₁₃ offer useful diagnostic characteristics as predictive biomarkers, particularly with respect to differentiating more advanced breast cancer disease from normal controls.

Discussion of the EXAMPLE

As disclosed herein, ADAM and MMP family members are found in biological fluids and were studied as potential biomarkers for cancer and inflammatory diseases. One of the most promising areas of research is the successful identification of MMPs and ADAMs in the urine of individuals with breast, prostate, and bladder cancer. Not only are elevated levels found in the urine of breast cancer patients, but also the amount of enzyme as assessed ELISA correlates with the severity of the disease.

As disclosed herein, fluorescent substrates were suitable to predict disease using urines from patients with breast cancer and normal controls. Fluorescent substrates specific for ADAM family member, but not MMP-2, MMP-9 and MMP-13, were predictive of disease status. Some ADAM substrates proved to be less useful, such as the selective TACE substrate F o and broad spectrum substrates described in Moss et a/., 2010). During the initial screening process, of all the substrates tested, those more selective for MMP-9 proved to be the most useful. The best MMP-9 substrate tested was Flu, which is slightly more specific than Fl₈ for MMP-9 over the ADAMs and other MMPs. F|₆3, which is an MMP-2-specific substrate, was not indicative of disease status, confirming previous findings that this enzyme is not a useful diagnostic marker for breast cancer.

The substrates that proved to be the most predictive for large-scale screening of urines were Fl₁₃ and Fl₂i . Fl₁₃, an ADAM8 substrate that is based on a cleavage sequence of CD23, was the most promising. F ₃ is unique in that it is not processed very well by any of the MMPs tested. Fl₂i is based on the processed site in TNF-a but has a homophenylalanyl moiety in place of valine at S1'. This substitution was shown previously to be helpful in increasing TACE activity (Lambert et al., 2005) although it is cleaved by a number of MMP and ADAM family members. The closely related Fl ₀, which has the native TNF-a substrate sequence, was not as effective as F½i , suggesting that the unnatural amino acid substitution was beneficial. Two other ADAM substrates, based on the TGF-a and BTC cleavage sites described previously (see Moss et a/., 2010) were also less effective. Therefore, substrates based on physiological cleavage sites of proteins that are shed by ADAMs could be used to detect ADAM family members and also to assess their abilities to determine disease status.

Also disclosed herein is the discovery that a combination of Fl₂i and Fli3 activity predicted with 90% accuracy individuals that had either invasive breast cancer or metastatic disease. While the substrates were less informative for less severe forms of breast cancer, the findings disclosed herein provide support for a simple screening of patients for either the presence or recurrence of breast cancer that could prove to be beneficial. Combinations of semi-selective substrates and the use of inhibitors as in the multiple enzyme multiple reagents assay system (MEMRAS) technique, to develop a fingerprint of metalloproteinase activities in urines, might ultimately also be useful in the clinic (Rasmussen er a/., 2004).

The present disclosure describes a proof-of-concept study in which fluorescent substrate cleavage activity was used to predict disease status in patients with breast cancer. Using a cohort of samples from across the disease spectrum such as patients at high risk of developing breast cancer, DCIS, locally invasive breast cancer, and advanced metastatic disease and comparing these to normal controls, it was determined that FI21 and Fl₁₃ cleavage activity could differentiate invasive and/or metastatic breast cancer from normal controls.

To assess the possibility of using enzymatic assays to measure urinary proteolytic activity, fluorescence resonance energy transfer substrates that have varying specificities for MMP-9 and MMP-2 and ADAM family members ADAM8, -9, -10, -12, and -17, were tested for their ability to be processed by urines from normal and breast cancer patients. Substrates that were semi-selective either for MMP-9 or MMP-2 were not as predictive of disease status as were substrates for ADAM family members. Two substrates, Fl₂i , a good substrate for ADAM 12 that can also be cleaved by other MMPs/ADAMs, and FI13, a selective substrate for ADAM8, were used. FI21 and Fl ₃ cleavage activity was detected in urine samples from patients with invasive and metastatic breast cancer at a significantly higher frequency as compared to urines from normal healthy controls. Using the cleavage activity of both Fl₂i and Fl ₃ to predict the presence of invasive and/or metastatic breast cancer, the presently disclosed model predicted a 20% probability when both substrates were negative, a 55% probability when Fl₁₃ was positive and Fl₂i was negative, a 65% probability when Fl₂i was positive and F ₃ was negative, and a 90% probability when both Fl₂i and Fl ₃ cleavage activity were positive. Taken together, these data supported the use of fluorescence resonance energy transfer substrates to non-invasively identify invasive and/or metastatic breast cancer in patients.

REFERENCES

All references listed in the instant disclosure, including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries (including but not limited to GENBANK® database entries including all annotations available therein) are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or compositions employed herein.

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NM_002421.3; NM_002422.3; NM_002424.2; NM_002427.3;

NM_003183.4; NM_003236.2; NM_0032474.4; NM_003816.2; NM 004530.4; NM_004994.2; NM_021641.3; NM_001099691.1 ;

NM_001 127891.1 ; NM_001145938.1 ; NM_001 164489.1 ;

NM_001 164490.1 ; NP_001 100.3; NP_001101.1 ; NP_001720.1 ;

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NP_001 121363.1 ; NP_001 139410.1 ; NP_001 157961.1 ;

NP_001 157962.1.

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It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

SEQUENCE LISTING

<110> Moss, arcia L.

Moses, Marsha A.

Roy, Roopali

<120> METHODS AND COMPOSITIONS FOR PREDICTING DISEASE STATUS CANCER

<130> 1682/4 PCT

<150> US 61/424,895

<151> 2010-12-20

<160> 14

<170> Patentln version 3.5

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His Glu Xaa Gly His Xaa Xaa Gly Xaa Xaa

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<221> MISC FEATURE

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<223> X is homophenylalanine

<221> MISC FEATURE

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<223> the K at residue 8 is present or

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Leu Ala Gin Ala Xaa Arg Ser Asn Lys

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Ala Pro Arg Trp Leu Leu Thr Ala Cys

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Val Asp Leu Phe Tyr Leu Gin Gin Pro Lys

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Claims

claimed is:

A method for predicting disease status of a cancer in a subject, the method comprising:

(a) determining the level of enzyme activity of a member of the matrix metalloprotease (MMP) and/or the a disintegrin and metallopeptidase domain (ADAM) family of polypeptides in a biological sample from a source isolated from or in the subject; and

(b) comparing the level of enzyme activity to a standard,

wherein the comparing provides a prediction of the disease status of the cancer in the subject.

The method of claim 1 , wherein the biological sample is selected from the group consisting of serum, plasma, urine, cerebral spinal fluid, blood, saliva, synovial fluid, broncheolar lavage, and a biopsy sample of a tumor and/or a tissue.

The method of claim 1 , wherein the determining comprises employing a fluorescence resonance energy transfer (FRET) assay.

The method of claim 3, wherein the FRET assay employs a fluorescent substrate selected from the group consisting of FI₂₁ (Dabcyl-LAQA(homophenylalanine)RSK(5FAM)-NH₂; SEQ ID NO: 2), Fl₁₃ (Dabcyl-HGDQMAQKSK(5FAM)-NH₂; SEQ ID NO: 3), Flu (Dabcyl-GPLGMRGK(5FAM)-NH₂; SEQ ID NO: 4), Fl₁₀ (Dabcyl- SPLAQAVRSSK(5FAM)-NH₂; SEQ ID NO: 5), Fl₈ (Dabc l- P(cyclohexylalanine)G(methylcysteine)HAK(5FAM)-NH₂; SEQ ID NO: 6), Fl₆₃ (Dabcyl-SNLAYYTAK(5FAM)-K-NH₂; SEQ ID NO: 7), FI_59A (Dabcyl-APRWLLTAC(FLU)-NH₂; SEQ ID NO: 8), Fl ₅ (BTC sub; Dabcyl-VDLFYLQQPK(5FAM)-NH₂; SEQ ID NO: 9), and Fl ₄ (TGFa sub; Dabcyl-EHADLLAWAK(5FAM)-NH₂; SEQ ID NO: 10), Dabcyl- APRWIQDK(5FAM)-NH₂ (SEQ ID NO: 11); Dabcyl- APFEMSAK(5FAM)-NH₂ (SEQ ID NO: 12); Dabcyl- APFEFSAK(5FAM)-NH₂ (SEQ ID NO: 13), Dabcyl- EHADLLAWAK(5FAM)-K-NH₂ (SEQ ID NO: 14), or a combination thereof.

The method of claim 4, wherein the fluorescent substrate comprises FI-I3 and/or FI₂i and the biological sample comprises urine.

The method of claim 1 , wherein the standard comprises a panel of average levels of biological activity of the member of the matrix metalloprotease (MMP) and/or the a disintegrin and metallopeptidase domain (ADAM) family of polypeptides in the same source in normal subjects and/or from subjects with various stages of the cancer.

The method of claim 6, wherein the cancer is breast cancer and the standard comprises a panel of average levels of biological activity of the member of the matrix metalloprotease (MMP) and/or the a disintegrin and metallopeptidase domain (ADAM) family of polypeptides in the same source or in normal subjects, subjects with invasive breast cancer, subjects with advanced metastatic breast cancer.

The method of claim 1 , wherein the comparing comprises multivariable logistic regression analysis.

The method of claim 8, wherein the multivariable logistic regression is performed by a suitably programmed computer.

A method for detecting enzyme activity of a member of the MMP and/or the ADAM family of polypeptides, the method comprising contacting a sample suspected of comprising a member of the MMP and/or the ADAM family of polypeptides with a substrate comprising one or more of SEQ ID NOs: 2-14, and detecting the cleavage of the substrate, wherein the cleavage of the substrate is indicative of the enzyme activity of the member of the MMP and/or the ADAM family of polypeptides.

The method of claim 10, wherein the substrate is detectably labeled with two fluorescent molecules and the cleavage results in the two fluorescent molecules being present on different fragments of the substrate.

A peptide comprising, consisting essentially of, or consisting of any of the following amino acid sequences:

(a) SNLAYYTAKK (SEQ ID NO: 7);

(b) APRWLLTAC (SEQ ID NO: 8);

(c) APRWIQDK (SEQ ID NO: 11 );

(d) APFEMSAK (SEQ ID NO: 12);

(e) APFEFSAK (SEQ ID NO: 13); and

(f) EHADLLAWAK (SEQ ID NO: 14),

wherein the C-terminal lysine (K) of any of SEQ ID NOs: 7 and 11-14 is present or absent.

The peptide of claim 12, wherein the peptide further comprises one or more detectable labels conjugated thereto.

The peptide of claim 12, wherein the one or more detectable labels are selected from the group comprising of a fluorescent label, a radioactive label, a chemiluminescent label, a colorimetric label, and a tag labels, or a combination thereof.

The peptide of claim 12, wherein the peptide further comprises one or more tags conjugated thereto, where the tags are adapted for binding the peptide to a surface selected from the group consisting of glass, a membrane, a resin, a dip stick, plastic, and paper. The peptide of claim 12, wherein the peptide comprises two fluorescent labels conjugated thereto such that the two fluorescent labels are on opposite sides of a cleavage site for an enzyme selected from the group consisting of MMP family members and ADAM family members present in the peptide.

The peptide of claim 16, wherein a first of the two fluorescent labels comprises a dabcyl moiety or a dabsyl moiety conjugated at or near a first terminus of the peptide and a second of the two fluorescent labels comprises a fluorescein or a 5-carboxyflourescein moiety conjugated at or near the other terminus of the peptide.

The peptide of claim 12, wherein the peptide is adapted for use in a fluorescence resonance energy transfer (FRET) assay to test for a biological activity of a member of the MMP and/or the ADAM family of polypeptides.

A method for predicting disease status of breast cancer in a subject, the method comprising:

(a) determining an amount of an enzyme activity of an enzyme selected from the group consisting of a member of the disintegrin and metallopeptidase domain (ADAM) family of polypeptides, optionally, an ADAM8 polypeptide and/or an ADAM12 polypeptide, in a biological sample from a source isolated from or in the subject; and

(b) comparing the amount of the enzyme activity determined to a standard,

wherein the comparing provides a prediction of the disease status of the cancer in the subject.

The method of claim 19, wherein the biological sample is selected from the group consisting of serum, plasma, urine, cerebral spinal fluid, blood, saliva, synovial fluid, broncheolar lavage, and a biopsy sample of a tumor and/or a tissue.

The method of claim 19, wherein the determining step employs a substrate comprising two detectable moieties present on the substrate such that the enzyme activity, if present, cleaves the substrate resulting in the two detectable molecules being present on different fragments of the cleaved substrate. 22. The method of claim 21 , wherein the determining comprises employing a fluorescence resonance energy transfer (FRET) assay and the substrate comprises two fluorescent moieties present on the substrate such that the enzyme activity, if present in the biological sample, cleaves the substrate resulting in the two fluorescent moieties being present on different fragments of the cleaved substrate.

The method of claim 19, wherein the determining step employs a fluorescent substrate comprising F ₃ and/or Fl₂i and the biological sample comprises urine.

The method of claim 19, wherein the determining step employs a peptide substrate that is cleavable by the enzyme, if present, and further wherein the peptide substrate comprises one or more tags conjugated thereto, at least one of which is adapted for binding the peptide substrate to a surface selected from the group consisting of glass, a membrane, a resin, a dip stick, plastic, and paper.

The method of claim 19, wherein the standard is selected from the group consisting of an average level of enzymatic activity of ADAM8 and/or ADAM 12 in the same source isolated from normal subjects and an average level of enzymatic activity of ADAM8 and/or ADAM12 in the same source isolated from subjects with various stages of the cancer.

26. The method of claim 19, wherein the comparing comprises multivariable logistic regression analysis. 27. The method of claim 26, wherein the multivariable logistic regression is performed by a suitably programmed computer.