WO2012077105A2

WO2012077105A2 - Biomarkers for detecting a cancerous state in a subject

Info

Publication number: WO2012077105A2
Application number: PCT/IL2011/000928
Authority: WO
Inventors: Ouriel Faktor; Dana Cohen; Tamar Peretz-Yablonsky; Rachel Bar-Shavit; Beatrice Uziely; Einat Weiss; Vardit Moshayoff
Original assignee: Bio-Marcare Technologies Ltd.; Hadasit Medical Research Services & Development, Ltd.
Priority date: 2010-12-07
Filing date: 2011-12-07
Publication date: 2012-06-14
Also published as: WO2012077105A3

Abstract

The present invention provides methods and kits for the early diagnosis of breast and colorectal cancer. The invention further provides diagnostic methods and kits for determining the severity of a cancerous state in a subject and for determining the effectiveness of a therapeutic treatment of a subject with an anti-cancer agent.

Description

BIOMARKERS FOR DETECTING A CANCEROUS STATE IN A SUBJECT

FIELD OF THE INVENTION

The present invention is directed to the field of cancer diagnosis and prognosis, particularly to detection of breast or colorectal cancer in a subject. More specifically, the diagnostic methods and kits of the invention involve determining the levels of at least two biomarkers, Protease Activated Receptor (PAR)-l and PAR2.

BACKGROUND OF THE INVENTION

Protease Activated Receptors (PARs), formerly known as thrombin receptors, are seven transmembrane G-coupled receptors (GPCR) that are uniquely activated by proteolytic cleavage. The activation mechanism involves a serine protease, which cleaves the receptor at a specific site in the extracellular N-terminus, thus revealing an N-tethered ligand domain that binds to the receptors and leads to its activation. The PARs act as sensitive sensors of extra cellular protease gradients to allow cells to respond to proteolytically modified environment. Four different PARs have been identified (PAR1-4), all responding to a highly select group of serine proteases. PARI, PAR3 and PAR4, are activated by thrombin and PAR2 is activated by trypsin and the mast cell protease tryptase.

The genes encoding the four PARs are conserved across species and have a similar structure with a single intron interrupting the sequence encoding the receptor amino terminus. The PARs are widely expressed and have been implicated in important processes such as homeostasis, tissue repair, angiogenesis and inflammation.

PARI

Thrombin receptor and use of said receptor as a diagnosis tool of cardiovascular diseases has been disclosed in International Patent Application No. WO 1992/14750. In certain aspects the 750 publication relates to the diagnosis of cardiovascular diseases by detection, in fluids such as blood or urine, of the peptide cleaved from the thrombin receptor when activated as a measure of thrombosis.

A 41 residue polypeptide released from PARI has been shown to be a strong platelet agonist (Claytor et al., Journal of Vascular Surgery, Volume 37, Issue 2, Pages 440-445 R, 2003). Brass et al. examined the localization of thrombin receptors in resting platelet activation and followed their redistribution during platelets activation, using, inter alia, antibodies directed against defined epitopes within the thrombin receptor amino terminus (Molino et al., J Biol Chem. 1997 Feb 28; 272(9):6011-7).

International Patent Application No. WO 1997/46879 relates to methods and kits for detecting thrombin-induced cell activation via a system of detection capable of determining the presence of the cleaved peptide fragment of the thrombin receptor.

While traditionally PARI plays a role in thrombosis, hemostasis and vascular biology, it emerges with particular assignment in tumor biology. This is supported by the pattern of PARI expression in normal and pathological epithelia. In addition, a cDNA expression library screen based on the loss of anchorage - dependent growth and focus forming activity in NIH3T3 cells led to the isolation of PARI as a novel oncogene. Thus, PARI joins a list of GPCRs that are oncogenes including mas and g2a. The oncogenic properties of PARI, along with ample evidence on the high expression levels of the human Pari (hParV) gene in tumor biopsy specimens and in differentially metastatic cell lines - point to a direct correlation between PARI expression and the degree of malignancy. PARI has been shown to be involved in a variety of primary human cancers including those of breast (Even-Ram S, et al. (1998) Nat Med. 4(8):909-14.); colon (Vergnolle N, et al. (2004) J Clin Invest.1 14 (10): 1444-56; Darmoul D, et al. (2004) Mol Cancer Res. 2(9):514-22.); prostate (Chay CH, et al. Urology (2002) 60(5):760-5; Salah Z, et al. (2005) FASEB J 19(l):62-72); ovary (Grisaru-Granovsky S, et al. 2005. Int J Cancer 1 13(3):372-8); and melanoma (Nierodzik ML, et al. (1998) Blood. 92(10):3694-700; Shi X, et al. (2004) Mol Cancer Res. 2(7):395-402).

International Patent Application No. WO 1997/07387, to an inventor of the present application, provides methods and kits for evaluating the metastatic tendency of tumor cells by determining the level of expression of the gene coding for the thrombin receptor or by determining the level of the thrombin receptor present on the membranes or within the tumor cells.

The fact that PARI gene and protein over expression are associated with the aggressiveness of tumors in vivo, reflect on its potential role in tumor dissemination and assigns it as an attractive target for anticancer therapy. In-fact, PARI plays a central role in breast tumor progression since introduction of an hParl antisense sequence (a plasmid of 462 base pairs antisense sequence containing part of the promoter and the start initiation site of the protein) reduces their ability to migrate through Matrigel coated filters, in vitro (Even-Ram S, et. al. (1998) Nat Med. 4(8):909-14).

International Patent Application Publication No. WO 2007/020645 provides nucleic acid molecules, vectors, compositions, and methods for modulating PARI gene expression via RNA interference. In particular, the '645 publication features small interfering RNA (siRNA) and short hairpin RNA (shRNA) molecules and methods for modulating the expression of protease-activated receptor 1 gene.

International Patent Application Publication No. WO 2004/081044 relates to a human PARI associated with the cardiovascular disorders, dermatological disorders, gastrointestinal and liver diseases, neurological disorders, cancer disorders and urological disorders; to assays for the identification of compounds useful in the treatment or prevention of these disorders and diseases and to compounds which bind to and/or activate or inhibit the activity of PARI as well as pharmaceutical compositions comprising such compounds.

International Patent Application Publication No. WO 2009/098689, to an inventor of the present application, provides methods and packages for determining a cancerous state in a subject, the method comprises determining binding of an antibody raised against PARI released peptide, or a fragment derived therefrom, to a marker within a fluid sample obtained from said subject, wherein binding of said antibody to said marker indicates a cancerous state. The '689 application further provides methods for determining severity of a cancerous state in a subject and methods for determining the effectiveness of a therapeutic treatment of a subject.

PAR2

PAR2 is distinguished from the other three PARs in that it is activated by trypsin and tryptase, while the other PARs are all activated by thrombin. Since its discovery, PAR2 receptor has been implicated in numerous physiological processes necessitating therapeutic intervention including inflammation, colitis, asthma, pruritis, tear secretion, bone remodeling, vascular tone, ischemia and nociception and in particular, PAR2 is thought to exert a protective effect in the airways, pancreas, gastrointestinal tract (GI) tract, and in ischemia in the brain and heart. PAR2 is expressed in the cardiovascular system, where it is suggested to play an important role in vascular tone and alterations in vascular function during inflammation with implications in, without limitation, hypertension. It is expressed in the gastrointestinal system, e.g., the small intestine and colon, as well as in the exocrine organs of the digestive tract, i.e., the stomach, pancreas and salivary glands. It is expressed in the myenteric and submucosal nerve plexuses, the signaling of which may alter the regulation of intestinal motility and secretion.

PAR2 is also expressed in the pulmonary system where its role in the modulation of inflammatory processes suggests it as a pharmacological target for pulmonary diseases such as, without limitation, asthma and chronic obstructive pulmonary disease. PAR2 has also been implicated in the pathology of skin diseases such as, without limitation, cutaneous neurogenic inflammation, pruritis, dry skin syndrome and other inflammatory skin diseases. Studies have suggested that PAR2 may play a key role in neurogenic inflammation and pain. PAR2 is also strongly expressed in human colon adenocarcinoma cells suggesting a role in cancer.

International Patent Application Publication No. WO 04/080373 relates to the regulation of human PAR2 for the treatment of cardiovascular disorders, gastrointestinal and liver diseases, neurological disorders, urological disorders, hematological diseases and respiratory diseases in mammals.

International Patent Application Publication No. WO 04/044178 provides methods and compositions for diagnosing dysplasia. The detection of PAR2 expression levels is disclosed, among an extensive list of genes, in the methods of the Ί 78 application.

Jahan et al. 2007 demonstrated up-regulation of PAR2 during ovarian cancers progression (Annals of Oncology 18: 1506-1512, 2007). In addition, Jahan et al. analyzed patient prognosis after surgery for ovarian cancers, with a 36-month survival rate.

No where in the art is it disclosed that a combination of biomarkers comprising PARI, PAR2, is particularly useful for early diagnosis and prognosis of cancer, as well as monitoring and managing a therapeutic treatment of a subject afflicted with cancer.

There remains an unmet need for reliable and accurate methods and kits providing early diagnosis of cancer in a subject and for determining disease staging and prognosis, the results of which can be used to manage the subject's treatment. There is also an unmet need for methods for determining the effectiveness of a therapeutic treatment of a subject with an anti-cancer agent. SUMMARY OF THE INVENTION

The present invention provides compositions, methods and kits for the early diagnosis, prognosis and monitoring cancer progression in a subject. More specifically, the invention provides simple assays, with high sensitivity and specificity for determining breast or colorectal cancerous state in a subject, wherein a combination of biomarkers comprising at least protease-activated receptor 1 (PARI) and PAR2, or fragments or derivatives thereof (e.g., PARI -released peptide and PAR2 -released peptide), are indicative of the subject's cancerous state. In some embodiment, the methods of the invention are non-invasive. The invention further provides methods and kits for determining the effectiveness of a therapeutic treatment of a subject with an anti-cancer agent.

The present invention is based, in part, on the unexpected discovery that the detection of a distinct set of biomarkers comprising PARI and PAR2 in a sample obtained from a subject (e.g., tissue or blood sample) reflects a cancerous state of the tested subject. Advantageously, the use of a combination of biomarkers provides particular sensitive and specific methods and kits for diagnosing cancer. Furthermore, the levels of the biomarkers in the fluid sample allow determining the severity of the cancer disease and the effectiveness of a therapeutic treatment. In particular, the methods of the invention advantageously enable determining the subject's disease free survival (DFS) term or overall survival (OS) as well as managing said subject's treatment.

Without wishing to be bound by any theory or mechanism of action, the cleavage of PARs N-terminus, expressed in malignant epithelial cells, results in the release of peptides into the bloodstream. Thus, the terms "PARI -released peptide" or "PAR2-released peptide" as used herein relates to the N-terminus extracellular peptide cleaved during PARI or PAR2 activation, respectively. In a particular embodiment of the invention, PARI -released peptide has the amino acid sequence as set forth in SEQ ID NO:l . In another particular embodiment of the invention, PAR2 -released peptide has the amino acid sequence as set forth in SEQ ID NO:7. While PARI -released peptide has been disclosed for the detection of a cancerous state in a subject, the present invention relates to determining the levels of a combination of biomarkers, comprising determination of at least two biomarkers thereby providing significantly more accurate and reliable assays than the PARI -released peptide biomarker alone. The present invention is also based, in part, on the identification of specific antibodies raised against particular epitopes within the PARI and PAR2 -released peptides. According to certain embodiments of the methods of the invention, a mixture of antibodies raised against different epitopes of the released peptides is used in the detection of said plurality of biomarkers. Advantageously, the use of a mixture of antibodies enables highly reliable and extremely accurate cancer diagnostics and prognostics. Furthermore, the biomarkers of the invention may be part of a complex with serum proteins (e.g., carboxypeptidase N and complement component 4), and therefore, the use of a mixture of antibodies in the methods of the present invention provides considerably more sensitive assays than using a single antibody.

Thus, the present invention provides compositions, methods and kits for the early diagnosis of breast or colorectal cancer, for determining severity of the breast or colorectal cancer disease and for determining the effectiveness of a therapeutic treatment. According to certain embodiments, the methods of the invention comprise determining the presence or levels of a combination of biomarkers in a sample obtained from the subject. In various embodiments, the combination of biomarkers comprises PARI and PAR2. In certain embodiments, the combination of biomarkers consists of PARI and PAR2. In other embodiments, the combination of biomarkers comprises PARI -released peptide and PAR2 -released peptide. In certain embodiments, the combination of biomarkers consists of PARl-released peptide and PAR2-released peptide. In another embodiment, the combination of biomarkers further comprises at least one biomarker selected from the group consisting of estrogen receptor (ER), progesterone receptor (PR), H-19, carcinoembryonic antigen (CEA) cancer antigen 15-3 (CA 15-3), CA-125, TPS (tissue polypeptide specific antigen) and Her2/neu. In another embodiment, the combination of biomarkers comprises PARI, PAR2 and ER. In another embodiment, the combination of biomarkers consists of PARI, PAR2 and ER. In another embodiment, the combination of biomarkers comprises PARI, PAR2 and H-19. In another embodiment, the combination of biomarkers consists of PARI, PAR2 and H-19. Each possibility is a separate embodiment of the invention.

According to one aspect, the present invention provides a method for determining severity of breast or colorectal cancer or prognosis of breast or colorectal cancer in a subject comprising: (i) determining the levels of a plurality of biomarkers comprising PARI and PAR2, or fragments or derivatives thereof, in at least one sample obtained from the subject; and

(ii) comparing the levels of the plurality of biomarkers with the levels of at least one control value;

wherein a significant elevation in the levels of said plurality of biomarkers compared to the levels of at least one control value is indicative of the severity of breast or colorectal cancer or poor prognosis of said subject.

According to another embodiment of the methods of the invention, the at least one control value is selected from a value obtained from a statistically significant group of control individuals afflicted with defined severities of cancer, or a stored set of data (e.g., a reference value) corresponding to control individuals afflicted with defined severities of cancer. According to another embodiment, the at least one control value corresponds to the level of said biomarker in a healthy individual (e.g., not afflicted with cancer).

According to another embodiment of the methods of the invention, the control value is selected from the group consisting of a predetermined cutoff value, a value obtained from a healthy control individual, a panel of control values from a set of healthy individuals, and a stored set of data (e.g., a reference value) corresponding to control individuals that are not afflicted with cancer.

According to another embodiment of the methods of the invention, the predetermined cutoff value is obtained from said subject (suspected of having breast or colorectal cancer) at a prior-referenced time point. According to another embodiment, the at least one control value is determined by measuring the levels of said plurality of biomarkers in a sample obtained from said subject at at-least one prior-referenced time point. According to another embodiment, a significant increase in the level of said biomarkers compared to the prior-referenced time point indicates severity of the cancerous state. According to another embodiment, a significant increase in the level of said biomarkers compared to the prior-referenced time point indicates worsening of the cancerous state. According to another embodiment, a significant decrease in the level of said biomarkers compared to the prior-referenced time point indicates improvement of the cancerous state. Each possibility is a separate embodiment of the invention. In another embodiment, the method of prognosis of a malignant disease in a subject comprises determining the levels of a plurality of biomarkers comprising PARI , PAR2, or fragments or derivatives thereof, and estrogen receptor (ER). In another embodiment, the method of prognosis of a malignant disease in a subject comprises determining the levels of a plurality of biomarkers comprising PARI, PAR2, or fragments or derivatives thereof, and progesterone receptor (PR). In additional embodiment, the plurality of biomarkers further comprises at least one additional biomarker selected from the group consisting of: H-19, CEA, CA15-3, CA-125, TPS and Her2/neu. In certain embodiments, the prognosis is selected form overall survival (OS) or disease-free survival (DFS).

According to another aspect, the present invention provides a method for determining the effectiveness of a therapeutic treatment of a subject afflicted with breast or colorectal cancer, comprising:

(i) determining, at least at two successive time points, the level of a plurality of biomarkers comprising PARI and PAR2, or fragments or derivatives thereof, in at least one sample obtained from the subject, wherein at least one time point is during the therapeutic treatment;

(ii) comparing the level of the plurality of biomarkers at the at least two successive time points, wherein a significant difference in the levels of the biomarkers indicates the effectiveness of the therapeutic treatment.

According to another embodiment, said plurality of biomarkers further comprises at least one additional biomarker selected from the group consisting of: H-19, CEA, CA15-3, CA-125, TPS and Her2/neu, wherein each possibility represents a separate embodiment of the invention.

According to particular embodiments, the method comprises:

(i) determining, at least at two successive time points, the level of PARI and/or

PAR2, and at least one biomarkers selected from H-19, CEA, CA15-3, CA- 125, TPS and Her2/neu, in at least one sample obtained from the subject, wherein at least one time point is during the therapeutic treatment;

(ii) comparing the level of the biomarkers at the at least two successive time points, wherein a significant difference in the levels of the biomarkers indicates the effectiveness of the therapeutic treatment. In another embodiment, the method comprises determining the levels at least three biomarkers, at least four biomarkers, at least five biomarkers or at least six biomarkers. In another embodiment, the method comprises determining the levels of PARI, PAR2, CEA, CA15-3, CA-125, TPS and Her2/neu. In another embodiment, the method comprises determining the levels of PAR 1 , PAR2, H- 19, CEA, CA 15-3, CA- 125, TPS and Her2/neu.

According to another embodiment, at least one first sample is taken at a time point prior to initiation of said therapeutic treatment and at least one subsequent sample is taken at a time point during said therapeutic treatment, wherein a significant decrease in the level of said biomarkers in the at least one subsequent sample compared to that determined for the first sample indicates that said therapeutic treatment is effective.

According to another embodiment, the at least two successive time points are taken at time points during the therapeutic treatment, wherein a significant decrease in the levels of said biomarkers in a subsequent sample compared to the level in a first sample indicates that said therapeutic treatment is effective.

According to another embodiment, at least one first sample is taken at a time point during the therapeutic treatment and at least one subsequent sample is taken at a time point after the treatment has been discontinued, wherein a significant increase in the levels of said biomarkers in the at least one subsequent sample compared to the levels determined for the first sample indicates that said therapeutic treatment is effective.

According to another aspect, the present invention provides a method for diagnosing breast or colorectal cancer in a subject, comprising determining the levels of a plurality of biomarkers comprising PARI and PAR2, or fragments or derivatives thereof, in at least one sample obtained from the subject, wherein a significant elevation in the levels of the plurality of biomarkers compared to a control value is indicative of breast or colorectal cancer in said subject. In one embodiment, said method is for early diagnosis of breast or colorectal cancer. In another embodiment, said method is for monitoring breast or colorectal cancer recurrence in said subject. In another embodiment, said method is for monitoring breast or colorectal cancer relapse in said subject.

According to some embodiments of the methods of the invention, said cancer is breast cancer. According to additional embodiments of the methods of the invention, said cancer is colorectal cancer. According to some embodiments of the methods of the invention, said cancer is colon cancer. According to certain embodiments of the methods of the invention, the at least one sample obtained from the subject is a tissue sample. According to another embodiment, the tissue sample is derived from the breast of said subject. According to another embodiment, the tissue sample is obtained by biopsy. In one embodiment, the tissue sample is a breast biopsy. In another embodiment, the tissue sample is a colorectal biopsy.

According to another embodiment of the methods of the invention, the sample obtained from the subject is a fluid sample. According to another embodiment, the fluid sample is selected from the group consisting of: whole blood, plasma, serum, ascitic fluid or urine. According to another embodiment, the fluid sample is selected from blood, plasma or serum. In another embodiment, the sample contains peripheral blood mononuclear cells (PBMC). In another embodiment, the sample is obtained non-invasively.

With respect to the method for determining the levels of a plurality of biomarkers in a fluid sample obtained from a subject, the method comprises, in particular embodiments, determining the levels of PARI -released peptide and PAR2 -released peptide, fragments or derivatives thereof. In another embodiment, the method comprises determining the expression levels of PARI and PAR2 (such as using PCR assay as described herein below).

According to another embodiment, the plurality of biomarkers further comprises estrogen receptor (ER). With respect to the method for diagnosing breast cancer in a subject, the method comprises, in particular embodiments, determining the levels of at least two biomarkers selected from the group consisting of PARI, PAR2 and ER, in at least one sample obtained from the subject, wherein a significant elevation in the levels of the at least two biomarkers compared to at least one control value (e.g., corresponding to a healthy individual) is indicative of breast cancer in said subject. According to another embodiment, the method comprises determining the levels of PARI , PAR2 and ER, in at least one sample obtained from the subject, wherein a significant elevation in the levels of the biomarkers compared to at least one control value is indicative of breast cancer in said subject. It should be understood that according to the principles of the present invention, ER expression is determined in a tissue sample, such as a biopsy. In some embodiments, the levels of the plurality of biomarkers (e.g., ER and PARI, PAR2) are determined in a tissue sample. In another embodiment, the at least one sample obtained from the subject is two samples wherein one sample is a tissue sample and a second sample is a fluid sample. In a particular embodiment, the ER levels are determined in a tissue sample and PARI , PAR2 levels are determined in a fluid sample, e.g., via detection of PARI and PAR2 - released peptides, or via detection of PARI and PAR2 RNA expression levels. Each possibility is a separate embodiment of the invention.

According to another embodiment of the methods of the invention, said cancer is breast cancer, and said subject has been afflicted as having estrogen receptor positive breast cancer. According to another embodiment, said subject has been afflicted as having progesterone receptor positive breast cancer.

According to another embodiment of the methods of the invention, the plurality of biomarkers further comprises H-19. In a specific embodiment, said cancer is colorectal cancer. In yet another specific embodiment, the methods of the invention are useful for early diagnosis of colorectal cancer. In another embodiment, said colorectal cancer is a precancerous lesion such as adenomatous polyp.

According to certain embodiments of the methods of the invention, determining the levels of the plurality of biomarkers is performed by a method selected from immunoassay, tissue array, polymerase chain reaction (PCR), in situ hybridization (e.g., FISH). According to some embodiments, the PCR is selected from quantitative real-time PCR (qRT-PCR) and reverse transcriptase PCR (RT-PCR).

Determining the levels of the plurality of biomarkers, according to certain embodiments, relates to determining the expression levels of said biomarkers. In some embodiments, determining the expression levels is performed by determining the ribonucleic acid (RNA) expression levels. Said RNA is, in one embodiment, coding RNA (e.g., translated into protein). Various methods are known in the art for determining RNA expression levels, for instance, PCR, qRT-PCT, RT-PCR and FISH.

With respect to determining PARI RNA levels, the method comprises, in one embodiment, determining the levels of PARI having the polynucleotide sequence as set forth in SEQ ID NO: 14. With respect to determining PAR2 RNA levels, the method comprises, in one embodiment, determining the levels of PAR2 having the polynucleotide sequence as set forth in SEQ ID NO: 16.

In other embodiments, determining the expression levels is performed by determining the protein expression levels. With respect to determining PARI protein expression levels, the method comprises, in one embodiment, determining the levels of PARI having the amino acid sequence as set forth in SEQ ID NO: 13. With respect to determining PAR2 protein expression levels, the method comprises, in one embodiment, determining the levels of PAR2 having the amino acid sequence as set forth in SEQ ID NO: 15.

Various methods are known in the art for determining protein expression levels, for instance, ELISA and immunohistochemical (IHC) methods. Conveniently, the methods of the invention are performed using an immunoassay. In one embodiment, the methods of the invention are performed using an enzyme-linked immunosorbent assay (ELISA). In another embodiment, the methods of the invention are performed using a bead flow cytometry assay. In another embodiment, the methods of the invention are performed using a radioimmunoassay (RIA). In another embodiment, the methods of the invention are performed using an antibody microarray chip. In another embodiment the methods of the invention are performed using lateral-flow immuno-chromatographic assay. Each possibility is a separate embodiment of the invention.

According to particular embodiments, determining the levels of the biomarkers of the invention comprises determining the levels of binding of the antibodies to said biomarkers. In one embodiment the method further comprises comparing the levels of binding of said antibodies to the biomarkers with the levels of control values, e.g., prior determined standards, which correlate the level of antibody binding to said biomarkers with the severity of the cancerous state. In another embodiment, determining the levels of the biomarkers, or alternatively determining the levels of binding of antibodies to the biomarkers of the invention, comprises determining a measurement selected from a quantitative measurement, a qualitative measurement or combinations thereof.

In one embodiment, the immunoassay is performed using an antibody raised against, and/or capable of specifically binding, a peptide epitope of a PARl-released peptide, or fragments or derivatives thereof. In another embodiment, the PARl-released peptide comprises SEQ ID NO:l, a fragment or derivative thereof. In another embodiment, the PARl-released peptide consists of SEQ ID NO:l . According to another embodiment, the fragment or derivative of said PARl-released peptide comprises the amino acid sequence selected from the group consisting of: SEQ ID NO:2 - SEQ ID NO:6 and SEQ ID NO: 10 - SEQ ID NO: l l . According to another embodiment, the fragment of said PARI- released peptide comprises the amino acid sequence selected from the group consisting of: SEQ ID NO:2 - SEQ ID NO:4.

According to one particular embodiment, the fragment of said PARl-released peptide consists of RLLLVAACFSLC (SEQ ID NO:2).

According to another particular embodiment, the fragment of said PARl-released peptide consists of CGPLLS ARTRARRPES (SEQ ID NO:3).

According to another particular embodiment, the derivative of said PARl-released peptide consists of CRRPESKATNATLDPR (SEQ ID NO:4).

According to another particular embodiment, the fragment of said PARl-released peptide consists of RRLLLV AACFSLCGPLLS AR (SEQ ID NO: 5).

According to another particular embodiment, the derivative of said PARl-released peptide consists of CSARTRARRPESKAT (SEQ ID NO: 6).

According to another particular embodiment, the fragment of said PARl-released peptide consists of RRPESKATNATLDPR (SEQ ID NO: 10).

According to another particular embodiment, the derivative of said PARl-released peptide consists of S ARTRARRPESKAT (SEQ ID NO: 1 1).

Each possibility is a separate embodiment of the invention.

In another embodiment, the immunoassay is performed using an antibody raised against, and/or capable of specifically binding, a peptide epitope of a PAR2-released peptide, a fragment or derivative thereof. In some embodiments the PAR2-released peptide comprises SEQ ID NO: 7, a fragment or derivative thereof. In another embodiment, the PAR2-released peptide consists of SEQ ID NO:7. According to another embodiment, the fragment or derivative of said PAR2-released peptide comprises the amino acid sequence selected from the group consisting of: SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 12.

According to one particular embodiment, the derivative of said PAR2-released peptide consists of MRSPSAGGSGC (SEQ ID NO:8).

According to another particular embodiment, the fragment of said PAR2 -released peptide consists of CSGTIQGTNRSSKGR (SEQ ID NO:9).

According to another particular embodiment, the fragment of said PAR2 -released peptide consists of MRSPSA (SEQ ID NO: 12). Each possibility is a separate embodiment of the invention.

In the methods of the invention, a "significant elevation" or a "significant increase" in the level or amount of a biomarker refers, in different embodiments, to a statistically significant elevation, or in other embodiments to a significant elevation as recognized by a skilled artisan. In the methods of the invention, a "significant reduction" or "significant decrease" in the level or amount of a biomarker refers, in different embodiments, to a statistically significant reduction, or in other embodiments to a significant reduction as recognized by a skilled artisan.

The marker panel of the present invention may be analyzed in a number of fashions well known to those of skill in the art. For example, each member of a panel may be compared to a "normal" value (e.g., corresponding to a healthy subject not afflicted with cancer), or a value indicating a particular outcome. A particular diagnosis/prognosis may depend upon the comparison of each marker to this value; alternatively, if only a subset of markers is outside of a normal range, this subset may be indicative of a particular diagnosis/prognosis. The skilled artisan will also understand that diagnostic markers, differential diagnostic markers, prognostic markers, time of onset markers, disease or condition differentiating markers, etc., may be combined in a single assay or device. Markers may also be commonly used for multiple purposes by, for example, applying a different threshold or a different weighting factor to the marker for the different purpose(s). In some embodiments, the cutoff value represents a value or range below or above which a majority or all subjects having a particular indication fall within. In other embodiments, the cutoff value represents a value or range below or above the value of the determined biomarker obtained from said subject (suspected of having breast or colorectal cancer) at a prior-referenced time point.

As the methods of the invention are amenable for automation and are thus suitable for medium and large scale screening, they may be used e.g. for screening subjects who may be at risk for developing cancer or subjects exposed to other risk factors.

According to further aspects the present invention provides kits for the prognosis, diagnosis or monitoring breast or colorectal cancer in a subject. In one embodiment the kits are suitable for determining the severity of breast or colorectal cancerous state in a subject (e.g., cancer prognosis). In another embodiment the kits are suitable for determining the effectiveness of a therapeutic treatment of a subject. Thus, in another embodiment, there is provided a diagnostic kit comprises means for determining the presence and/or levels of a plurality of biomarkers in a sample, wherein the plurality of biomarkers comprises PARI and PAR2, or fragments thereof. In another embodiment, the plurality of biomarkers comprises PARl-released peptide and PAR2-released peptide. In another embodiment, the plurality of biomarkers further comprises H- 19.

In another embodiment, the diagnostic kit further comprises instructions for performing the necessary steps for determining one or more of: the presence and/or levels of the biomarkers of the invention in the sample, difference between the level of binding of the biomarkers of the invention and level of prior determined standards, difference in the level of the biomarkers of the invention in two or more successive samples from the same subject. In another embodiment, the kit further comprises means for collecting a fluid sample from a subject.

Other objects, features and advantages of the present invention will become clear from the following description and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the expression of PARI and PAR2 biomarkers in early-stage breast tumor tissue biopsies. Figure 1A is a histogram representation of the percentage of breast cancer tumor biopsy tissues expressing PARI as opposed to tumor biopsy tissues expressing PARI and PAR2 (presented as patient percentage expressing said biomarkers). Results were obtained by using immunohistochemistry staining protocol adapted for anti- PARland anti-PAR2 polyclonal antibodies. Figure IB is a histogram representation of CA15.3 and CA125 biomarker expression in the blood of the same patients tested in Figure 1A.

Figure 2 demonstrates PARI and PAR2 prognosis of breast cancer. Figure 2A depicts Kaplan-Meier analysis showing Disease Free Survival (DFS) of ER positive breast cancer patients with negative PARI and PAR2 or with positive PARI or PAR2. Figure 2B depicts Kaplan-Meier analysis showing Overall Survival (OS) of ER positive breast cancer patients with negative PARI and PAR2 or with positive PARI or PAR2. Figure 2C depicts Kaplan-Meier analysis showing Overall Survival (OS) of lymph node positive breast cancer patients with negative PARI and PAR2 or with positive PARI or PAR2. (P denotes for statistical significance). Figure 3 demonstrates an ELISA competition assay directed to PARI -released peptide.

Figure 4 depicts PARI and PAR2 staining in breast cancer tissue array.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions, methods and kits for diagnosing cancer, particularly breast or colorectal cancer in a subject, wherein at-least PARI and PAR2, or fragments or derivatives thereof are used as biomarkers. The methods of the invention are useful for determining a cancerous state in a subject and particularly for determining the severity of the cancer disease. Further, the methods are useful for determining the effectiveness of a therapeutic treatment.

The present invention is based, in part, on the surprising finding that antibodies raised against a short amino acid peptide, derived from protease-activated receptor 1 (PARl)-released peptide, were capable of binding to a component of blood samples obtained from breast cancer patients, while no such binding was detected with respect to blood samples obtained from individuals which did not have breast cancer. The present invention is also based, in part, on the finding that the level of binding of the antibody to the blood component can be determined and this allows determination of the severity of the cancer disease. Further, the present invention is based on the finding that treatment of subjects having cancer with an anti-cancer agent affects the level of binding of the antibody to the blood component.

The present invention is also based, in part, on the unexpected discovery that the detection of a distinct set of biomarkers comprising at-least PARI and PAR2 in a sample obtained from a subject reflects a cancerous state of the tested subject. Remarkably, the use of a combination of biomarkers provides accurate and reliable methods for diagnosing cancer and determining severity of a cancerous state in a subject. Furthermore, the combination of the biomarkers of the invention advantageously enables more specific and sensitive than each biomarker alone.

In one embodiment, the human PARI amino acid has the amino acid sequence as set forth in SEQ ID NO: 13 (Accession No: NP_001983; UniProt No: P25116) and the polynucleotide sequence as set forth in SEQ ID NO: 14 (Accession No: NM_001992). The human PAR2 amino acid has, in another embodiment, the amino acid sequence as set forth in SEQ ID NO: 15 (Accession No: NP_005233; UniProt No: P55085), and the polynucleotide sequence as set forth in SEQ ID NO: 16 (Accession No: NM_005242).

The present invention particularly provides compositions, methods and kits for diagnosing cancer in a subject, wherein PARI and PAR2, or fragment thereof, (i.e., PARI and PAR2-released peptides) are used as biomarkers in a fluid sample. As described herein below, polyclonal and monoclonal antibodies were designed and raised against specific amino acid sequences derived from PARI and PAR2-released peptides. Therefore, the present invention, in certain embodiments, further provides specific amino acid sequences (e.g., SEQ ID NO: 2-4 raised against PARI -released peptide) useful as antibody-binding sites (peptide epitopes) for performing the methods of the invention.

Thus, the present invention provides compositions, methods and kits for diagnosing cancer, for determining severity of the cancer disease and for determining the effectiveness of a therapeutic treatment. According to certain embodiments, the invention provides methods for diagnosing breast or colorectal cancer in a subject, comprising determining the presence and/or levels of a combination of biomarkers in a fluid sample obtained from the subject. In various embodiments, the combination of biomarkers comprises PARI -released peptide and a PAR2 -released peptide. In another embodiment, the combination of biomarkers comprises at least one biomarker selected from the group consisting of: PARI - released peptide, PAR2 -released peptide, H-19, CEA, CA15-3, CA-125, TPS and Her2/neu.

According to another embodiment, the present invention provides a method for diagnosing breast or colorectal cancer (e.g., determining a cancerous state) in a subject in need thereof, comprising determining the levels of a plurality of biomarkers comprising PARI and PAR2, in a sample obtained from the subject, wherein a significant elevation in the levels of the plurality of biomarkers compared to a control value corresponding to a healthy individual indicates a said subject is afflicted with breast or colorectal cancer.

As used herein the term "diagnosing" or "diagnosis" refers to the process of identifying a medical condition or disease by its signs, symptoms, and in particular from the results of various diagnostic procedures, including e.g. detecting the expression of the nucleic acids according to at least some embodiments of the invention in a biological sample (e.g. in cells, tissue or serum, as defined below) obtained from an individual. Furthermore, as used herein the term "diagnosing" or "diagnosis" encompasses screening for a disease, detecting a presence or a severity of a disease, distinguishing a disease from other diseases including those diseases that may feature one or more similar or identical symptoms, providing prognosis of a disease, monitoring disease progression or relapse, as well as assessment of treatment efficacy and/or relapse of a disease, disorder or condition, as well as selecting a therapy and/or a treatment for a disease, optimization of a given therapy for a disease, monitoring the treatment of a disease, and/or predicting the suitability of a therapy for specific patients or subpopulations or determining the appropriate dosing of a therapeutic product in patients or subpopulations. The diagnostic procedure can be performed in vivo or in vitro. It should be noted that a "biological sample obtained from the subject" may also optionally comprise a sample that has not been physically removed from the subject.

According to another embodiment, the method further comprises determining severity of a cancerous state in a subject comprising determining the levels of the plurality of biomarkers in the sample obtained from said subject, and comparing the levels of said biomarkers with the levels of at least one control value that correlates the level of said biomarkers with the severity of the cancerous state.

According to certain embodiments of the methods of the invention, the sample is a fluid sample, and said PARI biomarker is a PARI -released peptide, and said PAR2 biomarker is a PAR2-released peptide. According to certain embodiments, the plurality of biomarkers further comprises at least one biomarker selected from: H-19, CEA, CA15-3, CA-125, TPS and Her2/neu. With respect to the method for determining the a cancerous state in a subject, the method comprises, in some embodiments, determining the levels of at least three biomarkers selected from the group consisting of PARI -released peptide, PAR2 -released peptide, H-19, CEA, CA15-3, CA-125, TPS and Her2/neu, in a fluid sample obtained from the subject, wherein detection of at least three biomarkers indicates a cancerous state in said subject.

With respect to the method for determining severity of a cancerous state or prognosis of breast or colorectal cancer, the method comprises, in some embodiments, determining the levels of at least three biomarkers, at least four biomarkers, or at least five biomarkers, selected from the group consisting of PARI -released peptide, PAR2 -released peptide, H-19, CEA, CA15-3, CA-125, TPS and Her2/neu, in a sample obtained from the subject, and comparing the levels of said biomarkers with the levels of at least one control value that correlates the level of said biomarkers with the severity of the cancerous state.

In certain embodiments of the invention, the methods comprise determining the levels of PARl-released peptide, PAR2-released peptide, H-19, CEA, CA15-3, CA-125, TPS and Her2/neu.

In one embodiment of the invention, PARl-released peptide is a 41 amino acid residue peptide derived from the N-terminal of PARI. In a particular embodiment the PARl-released peptide consists of the sequence:

Met-Gly-Pro-Arg-Arg-Leu-Leu-Leu-Val-Ala-Ala-Cys-Phe-Ser-Leu-Cys-Gly-Pro- Leu-Leu-Ser-Ala-Arg-Thr-Arg-Ala-Arg-Arg-Pro-Glu-Ser-Lys-Ala-Thr-Asn-Ala-Thr-Leu- Asp-Pro-Arg (SEQ ID NO:l).

According to the present disclosure, an antibody of the invention may be raised against a fragment derived from said PARl-released peptide. In a particular embodiment, the antibody specifically binds a peptide epitope within PARl-released peptide. In certain embodiments, a fragment of a PARl-released peptide may be any peptide or polypeptide comprising a sequence of at least 5, preferably at least 6, and more preferably, at least 7, amino acid residues derived from a PARl-released peptide. In some embodiments, the fragment comprises 5-41 amino acids, 7-35 amino acids, or alternatively 10-25 amino acids.

In another embodiment of the invention, PAR2-released peptide is a 36 amino acid residue peptide derived from the N-terminal of PAR2. In a particular embodiment the PAR2-released peptide consists of the sequence:

Μεΐ-Α^-86Γ-ΡΓθ-86Γ-Αΐ3-Αΐ3-Τ -Εευ-Εευ-0^-Α^-Αΐ3-Ιΐ6-Ε6υ-Ι.6υ-Αΐ3-Αΐ3-86Γ- Leu-Ser-Cys-Ser-Gly-Thr-Ile-Gln-Gly-Thr-Asn-Arg-Ser-Ser-Lys-Gly-Arg (SEQ ID NO:7).

According to the present disclosure, an antibody of the invention may be raised against a fragment derived from said PAR2-released peptide. In a particular embodiment, the antibody specifically binds a peptide epitope within PAR2-released peptide. In certain embodiments, a fragment of a PAR2-released peptide may be any peptide or polypeptide comprising a sequence of at least 5, preferably at least 6, and more preferably, at least 7, amino acid residues derived from a PAR2-released peptide. In some embodiments, the fragment comprises 5-36 amino acids, 7-30 amino acids, or alternatively 10-25 amino acids. It is noted that the biomarkers of the invention or fragment thereof may be part of a chimeric peptide, namely, the sequence being flanked by non-related sequence(s). In another embodiment, the biomarker is in a free form or in the form of a complex with another one, or more, polypeptide, peptide or protein present in the fluid sample. In one embodiment, the biomarker is in the form of a complex with one or more blood proteins.

The combination of biomarkers described herein, optionally includes any subcombination of biomarkers, wherein at least one biomarker is PARl-released peptide or PAR2-released peptide. Non limiting examples of sub-combination of biomarkers being determined according to the methods of the invention include PARl-released peptide, PAR2 -released peptide and H-19; PARl-released peptide, PAR2 -released peptide and ER; PARl-released peptide, PAR2-released peptide and CEA; PARl-released peptide, PAR2- released peptide, and CA15-3; PARl-released peptide, PAR2-released peptide, CA15-3 and CEA; PARl-released peptide, PAR2-released peptide and TPS; PARl-released peptide, PAR2-released peptide and TPS; PARl-released peptide, PAR2-released peptide and Her2/neu; wherein each possibility is a separate embodiment of the invention.

It should be understood that determining the presence or levels of the biomarkers of the invention, particularly of ER, PR, H-19, CEA, CA15-3, CA-125, TPS and Her2/neu, include using standard methods well known in the art.

CEA

Breast cancer is a prevalent and potentially fatal disease. It can develop from any of the cell types that make up the breast, and develop into a tumor that can travel throughout the body, in a process called metastasis. The National Cancer Institute reports that breast cancer led to more than 192,000 new cancer diagnoses and more than 40,000 deaths in the United States in 2009.

One blood test used to indicate the presence of breast cancer is the CEA test. CEA stands for carcinoembryonic antigen, a protein produced in some cancer cells found in normal adult tissue. CEA from cancer cells is released into the bloodstream, so analyzing the levels of CEA found in the blood can be used to indicate breast cancer. However, a test indicating high CEA levels on its own is not sufficient to diagnose breast cancer. CEA is produced by a number of cancer types, and some lifestyle factors such as smoking can increase CEA levels. The CEA test may be used to complement other breast cancer tests to confirm diagnosis. CEA testing during breast cancer therapy can indicate resistant breast cancer cases if CEA levels remain high over the course of cancer therapy.

CA15-3

CA15-3 is well known in the art as a biomarker indicative of the presence of breast cancer. CA15-3 stands for cancer antigen 15-3, a protein specifically found in breast cancer cells. CA15-3 is produced by the cancer cells, so high levels of CA15-3 in the blood indicate the presence of breast cancer. Lab Test Online UK indicates CA15-3 can be used to indicate the presence of metastatic cancer, since early forms of breast cancer do not lead to significantly higher blood CA15-3 levels.

Like CEA testing, CA 15-3 blood testing allows doctors to gauge a patient's response to breast cancer therapy. A series of CA 15-3 tests that show decreasing levels of CA 15-3 indicate breast cancer remission, while sustained high levels indicate resistant breast cancer. CA 15-3 alone cannot be used as a diagnostic tool in breast cancer, but it can be used in combination with other tests to indicate breast cancer. Estrogen Receptor (ER)

Estrogen receptors are over-expressed in around 70% of breast cancer cases, referred to as ER-positive (ER⁺). In women with ER-positive cancers, cancer cell growth is under the control of estrogen. Therefore, such cancers are often susceptible to treatment with tamoxifen, because tamoxifen works by blocking the interaction between estrogen and the estrogen receptor. In contrast, in ER-negative (ER^') breast cancers (cancers which do not possess ER) the growth of the cancer cells is not governed by estrogen, and therefore not treated with tamoxifen. Quantitative analysis of ER may be measured using methods and kits known in the art, e.g., the Abbott enzyme immunoassay kits (Abbott Laboratories, North Chicago, 111. 60064).

H-19

Studies of various tumors have demonstrated a re-expression or an upregulation of the HI 9 gene when compared to healthy tissues. Moreover in cancers of different etiologies and lineages, aberrant expression in allelic pattern was observed in some cases. While HI 9 shows mono-allelic expression in most tissues throughout development, with the exception of germ cells at certain stages of maturation, and in extra- villous trophoblasts, bi-allelic expression of this gene, referred as "relaxation of imprinting" or "loss of imprinting", have been found in an increasing number of cancers, for example, hepatocellular carcinoma, liver neoplasms and liver metastasis of various origins, lung adenocarcinoma, esophageal carcinoma, ovarian cancer, rhabdomyosarcoma, cervical cancer, bladder cancer , squamous cell carcinoma of the head and neck, colorectal carcinoma, pancreatic cancer, endometrial and in testicular germ cell tumors. Nearly 30 types of cancers show dysregulated expression of HI 9 gene as compared to healthy tissues, with or without loss of imprinting.

The specific expression of HI 9 gene in cancer cells has prompted its use in clinical applications for diagnosing cancer, for example, in U.S. Pat. No. 5,955,273, PCT Pub. No. WO 2004/024957 and PCT Pub. No. WO 2005/012569.

The methods of the present invention further comprise determining the presence and or levels of homologs or derivatives of the biomarkers disclosed herein. According to another embodiment, an antibody of the invention may be raised against homologs or derivatives of the biomarkers of the invention. The term "homolog" as used herein refers to a peptide having at least 70%, at least 80%, at least 90%, at least 95% and even at least 99% identity with one of the native biomarkers if the invention (e.g., PARI -released peptide or PAR2 -released peptide). In a particular embodiment, the identity is determined when the sequence of the homolog and the sequence of the biomarker are optimally aligned. Typically, an amino acid sequence of at least about 5-10 is required in order to elicit the production of antibodies. Thus, in one embodiment, the antibodies are raised against a peptide comprising at least 5-10 residues having at least 70%, at least 80%, at least 90%, at least 95% and even at least 99% identity with the native biomarker, when the sequence of the fragment (and/or homolog) and the sequence of the biomarker are optimally aligned.

The term "optimally aligned" is used to denote an alignment of two amino acids sequences (e.g. of the PARI -released peptide and the fragment thereof) giving the highest percent identity score.

In the context of the present disclosure, the fragment, being derived from the biomarkers of the invention is any amino acid sequence comprising at least 5 consecutive amino acid residues corresponding to consecutive amino acid residues in the biomarker, when the fragment and the biomarker are optimally aligned. In another embodiment, the fragment is any amino acid sequence comprising at least 5, 6, 7, 8, 9, or 10 consecutive amino acid residues corresponding to consecutive amino acid residues in the biomarker, when the fragment and the biomarker are optimally aligned. The at least 5, 6, 7, 8, 9, or 10 consecutive amino acid residues in the fragment may be identical to the corresponding consecutive amino acid residues in said biomarker (e.g., PARI or PAR2 -released peptides) or may comprise one or more conservative modifications of the original sequence, namely, of the naturally occurring sequence of said biomarker.

In another embodiment, the fragment is any amino acid sequence comprising at most 30, 25 or 20 consecutive amino acid residues corresponding to consecutive amino acid residues in the biomarker, when the fragment and the biomarker are optimally aligned.

Various fragments of PARI released peptide or PAR2 released peptide may be utilized in accordance with the present disclosure for designing and raising antibodies against said fragments.

According to certain embodiments, the peptide derived from PARl-released peptide are selected from the group consisting of:

RLLLVAACFSLC (SEQ ID NO: 2; corresponding to position 5-16 of PAR1- released peptide);

CGPLLSARTRARRPES (SEQ ID NO: 3; corresponding to position 16-31 of PARl-released peptide);

CRRPESKATNATLDPR (SEQ ID NO: 4; corresponding to position 27-41 of PARl-released peptide, with an additional N-terminal Cys residue) ;

RRLLLVAACFSLCGPLLSAR (SEQ ID NO: 5; corresponding to position 4-23 of

PARl-released peptide);

CSARTRARRPESKAT (SEQ ID NO: 6; corresponding to position 21-34 of PARl- released peptide with an additional N-terminal Cys residue).

RRPESKATNATLDPR (SEQ ID NO: 10; corresponding to position 27-41 of PAR 1 -released peptide) .

SARTRARRPESKAT (SEQ ID NO: 1 1 ; corresponding to position 21-34 of PARl- released peptide).

In another embodiment, the peptides derived from PAR2-released peptide are selected from the group consisting of: MRSPSAGGSGC (SEQ ID NO: 8; corresponding to position 1-6 of PAR2-released peptide with the addition of GGSGC in the C-terminus of said peptide, added for the purpose of antibody production);

CSGTIQGTNRSSKGR (SEQ ID NO: 9; corresponding to position 22-36 of PAR2- released peptide).

The term "derivative" or "conservative modification" is used to denote a modification to the original (or native) biomarker (e.g., having the naturally occurring sequence) including conservative replacement (substitution) of one or more naturally occurring amino acid with a non-naturally occurring amino acid, insertion or deletion of one or more amino acids as well as chemically modification of an amino acid, as appreciated by those versed in the art. The modification may also include alteration of a bond within the peptidic backbone.

The term "naturally occurring amino acid " refers to a moiety found within a peptide and is represented by -NH-CHR-CO-, wherein R corresponds to the side chain of the 20 naturally appearing amino acids. The term "non-naturally occurring amino acid " (amino acid analog) is either a peptidomimetic organic moiety, or is a D or L residue having the following formula: -NH-CHR-CO-, wherein R is an aliphatic group, a substituted aliphatic group, a benzyl group, a substituted benzyl group, an aromatic group or a substituted aromatic group and wherein R does not correspond to the side chain of a naturally-occurring amino acid. This term also refers to the D-amino acid counterpart of naturally occurring amino acids. Amino acid analogs are well known in the art; a large number of these analogs are commercially available.

The term "conservative replacement' in the context of the present invention refers to the replacement of an original amino acid residue present in the biomarkers of the invention with a naturally or non-naturally occurring amino having similar steric properties. Where the side-chain of the original amino acid residue to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid or a non-naturally occurring amino acid which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid); where the original amino acid to be replaced is charged, the conservative substitution should be with a naturally occurring amino acid, or a non-naturally occurring amino acid which are charged, or the original charged amino acid may be replaced with non-charged (polar, hydrophobic) amino acids that has the same steric properties as the side-chain of the replaced amino acid.

For example in accordance with the invention the following substitutions are considered as conservative: replacement of arginine by coralline; arginine by glutamine; aspartate by asparagine; glutamate by glutamine.

For producing conservative substitutions by non-naturally occurring amino acids it is also possible to use amino acid analogs (synthetic amino acids) well known in the art. A peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled practitioner. The following are some non-limiting examples of groups of naturally occurring amino acids or of amino acid analogs are listed bellow. Replacement of one member in the group by another member of the group will be considered herein as conservative substitutions:

Group I includes leucine, isoleucine, valine, methionine, phenylalanine, serine, cysteine, threonine and modified amino acids having the following side chains: ethyl, n- butyl, -CH₂CH₂OH, -CH₂CH₂CH₂OH, -CH₂CHOHCH₃ and-CH₂SCH₃. Preferably Group I includes leucine, isoleucine, valine and methionine.

Group II includes glycine, alanine, valine, serine, cysteine, threonine and a modified amino acid having an ethyl side chain. Preferably Group II includes glycine and alanine.

Group III includes phenylalanine, phenylglycine, tyrosine, tryptophan, cyclohexylmethyl, and modified amino residues having substituted benzyl or phenyl side chains. Preferred substituents include one or more of the following: halogen, methyl, ethyl, nitro, methoxy, ethoxy and -CN. Preferably, Group III includes phenylalanine, tyrosine and tryptophan.

Group IV includes glutamic acid, aspartic acid, a substituted or unsubstituted aliphatic, aromatic or benzylic ester of glutamic or aspartic acid (e.g., methyl, ethyl, n- propyl iso-propyl, cyclohexyl, benzyl or substituted benzyl), glutamine, asparagine, CO- NH-alkylated glutamine or asparagine (e.g., methyl, ethyl, n-propyl and iso-propyl) and modified amino acids having the side chain -(CH₂)₃COOH, an ester thereof (substituted or unsubstituted aliphatic, aromatic or benzylic ester), an amide thereof and a substituted or unsubstituted N-alkylated amide thereof. Preferably, Group IV includes glutamic acid, aspartic acid, glutamine, asparagine, methyl aspartate, ethyl aspartate, benzyl aspartate and methyl glutamate, ethyl glutamate and benzyl glutamate.

Group V includes histidine, lysine, arginine, N-nitroarginine, β-cycloarginine, μ- hydroxyarginine, N-amidinocitrulline and 2-amino-4-guanidinobutanoic acid, homologs of lysine, homologs of arginine and ornithine. Preferably, Group V includes histidine, lysine, arginine, and ornithine. A homolog of an amino acid includes from 1 to about 3 additional methylene units in the side chain.

Group VI includes serine, threonine, cysteine and modified amino acids having Ci- C₅ straight or branched alkyl side chains substituted with -OH or -SH. Preferably, Group VI includes serine, cysteine or threonine.

The term "deletion" as used herein includes exclusion of one or more amino acid residues (naturally occurring, non-naturally occurring, or peptidomimetic organic moiety) as compared to the original molecule from which it is derived.

The terms "insertion " or "addition " as used herein include the addition of one or more amino acid residues (naturally occurring, non-naturally occurring, or peptidomimetic (organic moiety) as compared to the original molecule from which it is derived.

The term "chemical modification" as used herein includes modification at the side chain of the amino acid residue, as well as modification of the peptidic bond. Accordingly, a functional group may be added to the side chain, deleted from the side chain or exchanged with another functional group. Typically, the modifications are conservative modifications resulting in conservative substitution. Examples of conservative modifications of this type include adding an amine or hydroxyl, carboxylic acid to the aliphatic side chain of valine, leucine or isoleucine, exchanging the carboxylic acid in the side chain of aspartic acid or glutamic acid with an amine or deleting the amine group in the side chain of lysine or ornithine. Other chemical modifications known in the art include carboxymethylation, acylation, phosphorylation, glycosylation or fatty acylation, and others.

The "chemical modification" also includes alteration of a bond within the peptidic backbone, i.e. that the bond between the N- of one amino acid residue to the C- of the next has been altered to non-naturally occurring bonds by reduction (to -CH₂-NH-), alkylation (methylation) on the nitrogen atom, or the bonds have been replaced by amidic bond, urea bonds, or sulfonamide bond, etheric bond (-CH₂-0-), thioetheric bond (-C¾-S-), or to -C- S-NH-; The side chain of the residue may be shifted to the backbone nitrogen to obtain N- alkylated-Gly (a peptidoid). Modification also includes cyclization of the amino acid molecule, e.g. by forming S-S bonds. S-S bonds may be formed via the inclusion of sulphur-containing amino acid residues, such as cysteine at each terminus of the amino acid molecule. Cyclic peptides have been shown to be more stable and with higher biological activity than the corresponding linear molecule (Jining L. et al. Eur. J. Biochem 271 :2873- 2886 (2004)).

Antibodies, immunoassays and kits

The present invention provides in some embodiments diagnostic methods involving determining the levels of the biomarkers of the invention (e.g., PARI, PAR2 or their respective released peptides). The methods of the invention may optionally and conveniently be affected using an immunoassay. The term "immunoassay" as used herein refers to a method of detecting or measuring antigens, in this case biomarkers of cancer, by using antibodies as reagents. The immunoassay is typically characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.

Antibodies, or immunoglobulins, comprise two heavy chains linked together by disulfide bonds and two light chains, each light chain being linked to a respective heavy chain by disulfide bonds in a "Y" shaped configuration. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains (CH). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at its other end, the light chain variable domain being aligned with the variable domain of the heavy chain and the light chain constant domain being aligned with the first constant domain of the heavy chain (CHI). The variable domains of each pair of light and heavy chains form the antigen binding site. The isotype of the heavy chain (gamma, alpha, delta, epsilon or mu) determines immunoglobulin class (IgG, IgA, IgD, IgE or IgM, respectively). The light chain is either of two isotypes (kappa, κ or lambda, λ) found in all antibody classes.

It should be understood that when the terms "antibody" or "antibodies" are used, this is intended to include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as proteolytic fragments thereof such as the Fab or F(ab')₂ fragments. Further included within the scope of the invention (for example as immunoassay reagents, as detailed herein) are chimeric antibodies; recombinant and engineered antibodies, and fragments thereof.

Exemplary functional antibody fragments comprising whole or essentially whole variable regions of both light and heavy chains are defined as follows: (i) Fv, defined as a genetically engineered fragment consisting of the variable region of the light chain and the variable region of the heavy chain expressed as two chains; (ii) single-chain Fv ("scFv"), a genetically engineered single-chain molecule including the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker; (iii) Fab, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain, which consists of the variable and CHI domains thereof; (iv) Fab', a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab' fragments are obtained per antibody molecule); and (v) F(ab')2, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab' fragments held together by two disulfide bonds).

Methods of generating monoclonal and polyclonal antibodies are well known in the art. Antibodies may be generated via any one of several known methods, which may employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries, or generation of monoclonal antibody molecules by continuous cell lines in culture. Antibody fragments may be obtained using methods well known in the art, including, but not limited to by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g., Chinese hamster ovary (CHO)) cell culture or other protein expression systems) of DNA encoding the fragment. Single-chain Fvs are prepared by constructing a structural gene comprising DNA sequences encoding the heavy chain variable and light chain variable domains connected by an oligonucleotide encoding a peptide linker. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two variable domains. For the production of polyclonal antibodies, various hosts including goats, rabbits, rats, mice, etc. may be immunized by injection with a biomarker of the present invention (e.g., PARI -released peptide). Depending on the host species, various adjuvants may also be used to increase immunological response. Such adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful adjuvants.

Monoclonal antibodies may also be used. Monoclonal antibodies may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (Nature 256:495-497, (1975)), the human B-cell hybridoma technique (Kosbor et al, Immunol. Today 4:72, (1983); Cote et al, Proc. Natl. Acad. Sci 80:2026-2030, (1983)) and the EBV-hybridoma technique (Cole, et al, Mol. Cell Biol. 62: 109-120, (1984); Chartrain M, Chu L. Development and production of commercial therapeutic monoclonal antibodies in Mammalian cell expression systems: an overview of the current upstream technologies. Curr Pharm Biotechnol. 2008;9(6):447- 67; Price PW et al. Engineered cell surface expression of membrane immunoglobulin as a means to identify monoclonal antibody- secreting hybridomas. J. Immunol. Methods. 2009; Fransson J, Borrebaeck CA. Selection and characterization of antibodies from phage display libraries against internalizing membrane antigens. Methods Mol Biol. 2009;480:1 13-27).

The term "antigen" as used herein is a molecule or a portion of a molecule capable of being bound by an antibody. The antigen is typically capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one or more epitopes. The specific reaction (or specific binding) referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

Thus, the immune reactivity of the antibody to the antigen, i.e. its ability to specifically bind the antigen, may be used to determine the amount of the antigen in the sample. In another embodiment, detection of the capacity of an antibody to specifically bind an antigen may be performed by quantifying specific antigen-antibody complex formation.

In certain embodiments, the detection of the biomarker may be performed using an immunoassay such as an enzyme-linked immunosorbent assay (ELISA) testing kit. In such assays, for example, samples are typically incubated in the presence of an immobilized first specific binding agent (e.g. an antibody) capable of specifically binding the biomarker. Binding of the biomarker to said first specific binding agent may be measured using any one of a variety of known methods, such as using a labeled second specific binding agent capable of specifically binding the biomarker (at a different epitope) or the first specific binding agent.

Exemplary specific binding agents include e.g. monoclonal antibodies, polyclonal antibodies, and antibody fragments such as recombinant antibody fragments, single-chain antibodies (scFv) and the like.

In some embodiments, various conventional tags or labels may be used, such as a radioisotope, an enzyme, a chromophore or a fluorophore. A typical radioisotope is iodine^" ¹²⁵ or sulfur^'35. Typical enzymes for this purpose include horseradish peroxidase, horseradish galactosidase and alkaline phosphatase.

Alternately, other immunoassays may be used; such techniques are well known to the ordinarily skilled artisan and have been described in many standard immunology manuals and texts.

Many different detection systems are known in the art and can be utilized in the context of the present invention. These include competitive and non-competitive binding assays. Such systems include, without being limited thereto, techniques such as radioimmunoassays (RIA), enzyme immunoassays (EIA), enzyme linked immunosorbent assays (ELISA), "sandwich" immunoassays, immuno-precipitation reactions, gel diffusion reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, fluorescence polarization, protein A immunoassays, and Immunoelectrophoresis assays. All these different detection system may be used for direct detection of the marker or by competition reactions.

In one embodiment, the detection is based upon a "sandwich" immunoassay, where the antibody, preferably, a monoclonal antibody is bound to a solid support. The fluid sample is then brought into contact with the solid support and any marker in the fluid sample is captured by the bound antibody. A second antibody which will bind to the marker can then be placed in contact with the solid support. The amount of marker in the sample can then be determined by detecting the amount of the bound second antibody.

The second antibody can be labeled, with, for example, a fluorescent compound, such as, without being limited thereto, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine; with a chemiluminescent compound, such as, without being limited thereto, luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester; a bioluminescent protein such as, without being limited thereto, luciferin, luciferase and aequorin; and radionuclides.

Additional exemplary assays may be based on immunochromatography lateral-flow assays, or dipstick technology, as demonstrated, for example, in U.S. Pat. Nos. 4,632,901, 4,313,734 and 4,786,589 5,656,448 and EP 01251 18. For example, U.S. Pat. No. 4,632,901 , discloses a flow-through type immunoassay device comprising antibody (specific to a target antigen analyte) bound to a porous membrane or filter to which is added a liquid sample. As the liquid flows through the membrane, target analyte binds to the antibody. The addition of sample is followed by addition of labeled antibody. The visual detection of labeled antibody provides an indication of the presence of target antigen analyte in the sample. EP 01251 18 discloses a sandwich type dipstick immunoassay in which immunochemical components such as antibodies are bound to a solid phase. The assay device is "dipped" for incubation into a sample suspected of containing unknown antigen analyte. Enzyme-labeled antibody is then added, either simultaneously or after an incubation period. The device next is washed and then inserted into a second solution containing a substrate for the enzyme. The enzyme-label, if present, interacts with the substrate, causing the formation of colored products which either deposit as a precipitate onto the solid phase or produce a visible color change in the substrate solution.

For example, the method may be performed by the steps comprising:

a) collecting a fluid or tissue sample (e.g., a blood sample) from the subject;

b) contacting the sample, under conditions such that a specific antigen-antibody complex may be formed, with antibodies (being directed to the marker antigen of the invention (e.g., PARI and PAR2 -released peptides); c) determining the presence of antigen-antibody complex formed, wherein said presence is indicative of the presence of said marker in said sample; or quantifying the amount of antigen-antibody complex formed, wherein said amount is indicative of the amount of said marker in said sample.

Optionally, steps b) and c) may be repeated for one or more additional marker antigens of the invention as detailed herein.

In some embodiments, the methods of the invention are suitable for automated or semi-automated analysis, and may enable clinical, medium or high-throughput screening of multiple samples. For example, automated ELISA systems such as Biotest's Quickstep® ELISA Processor, Maxmat Automated microwell ELISA analyzer (Maxmat S.A., France), or DSX™ Four-Plate System (Dynex Technologies) may conveniently be used.

Other suitable assays include for example flow cytometry assays (such as singleplex and multiplex bead-based Luminex® assays (Invitrogen).

An antibody "directed to" an antigen, as used herein is an antibody which is capable of specifically binding the antigen. The term "specifically bind" as used herein means that the binding of an antibody to an antigen is not competitively inhibited by the presence of non-related molecules. Antibodies directed to PARI and PAR2 -released peptide (e.g., SEQ ID NO: l and SEQ ID NO: 7, respectively) may be prepared using well known methods, for example as detailed hereinabove. Alternatively, antibodies, or ELISA kits for determining the presence of these antigens, may be purchased from a variety of sources.

In the methods of the invention, a "significant elevation" in the level (or amount) of a marker refers, in different embodiments, to a statistically significant elevation, or in other embodiments to a significant elevation as recognized by a skilled artisan.

Typically, the sample is obtained or collected from the subject as is known in the art. In one embodiment, the sample is a tissue sample. In particular embodiments, the sample is selected from the group consisting of formalin-fixed paraffin-embedded (FFPE) tissue, fresh frozen (FF) tissue, and tissue comprised in a solution that preserves nucleic acid or protein molecules.

Diagnostic use

Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

Optionally, the method distinguishes a disease or condition with a sensitivity of at least 70% at a specificity of at least 85% when compared to normal subjects (e.g., a healthy individual not afflicted with cancer). As used herein, sensitivity relates to the number of positive (diseased) samples detected out of the total number of positive samples present; specificity relates to the number of true negative (non- diseased) samples detected out of the total number of negative samples present. Optionally, the method distinguishes a disease or condition with a sensitivity of at least 80% at a specificity of at least 90% when compared to normal subjects. Optionally, the method distinguishes a disease or condition with a sensitivity of at least 90% at a specificity of at least 90% when compared to normal subjects. Also optionally, the method distinguishes a disease or condition with a sensitivity of at least 70% at a specificity of at least 85% when compared to subjects exhibiting symptoms that mimic disease or condition symptoms.

The term "cancer" in accordance with the invention means any condition in which cells proliferate at an abnormally high and uncontrolled rate, the rate being more rapid than normal tissue growth. Generally, cancer may be broadly classified into three major types: Malignant tumors arising from epithelial structures (called carcinomas); malignant tumors that originate from connective tissues such as muscle, cartilage, fat or bone (called sarcomas); and malignant tumors affecting hematopoietic structures (structures pertaining to the formation of blood cells) including components of the immune system (called leukemias and lymphomas). Other abnormal tissue growth (neoplasms), include but are not limited to neurofibromatosis.

The term "cancer" as used herein should be understood to encompass any neoplastic disease (whether invasive or metastatic) which is characterized by abnormal and uncontrolled cell division causing malignant growth or tumor. Non-limiting examples of cancer which may be diagnosed according to at least some embodiments of the present invention are solid tumors, sarcomas, hematological malignancies, including but not limited to breast cancer (e.g. breast carcinoma), cervical cancer, ovary cancer (ovary carcinoma), endometrial cancer, melanoma, bladder cancer (bladder carcinoma), lung cancer (e.g. adenocarcinoma and non-small cell lung cancer), pancreatic cancer (e.g. pancreatic carcinoma such as exocrine pancreatic carcinoma), colon cancer (e.g. colorectal carcinoma, such as colon adenocarcinoma and colon adenoma), gastric cancer, urothelial cell carcinomas, prostate cancer including the advanced disease, hematopoietic tumors of lymphoid lineage (e.g. leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma, multiple myeloma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma), myeloid leukemia (for example, acute myelogenous leukemia (AML), chronic myelogenous leukemia), thyroid cancer, thyroid follicular cancer, myelodysplastic syndrome (MDS), tumors of mesenchymal origin (e.g. fibrosarcomas and rhabdomyosarcomas), melanoma, uveal melanoma, teratocarcinoma, neuroblastoma, glioma, glioblastoma, benign tumor of the skin (e.g. keratoacanthomas), renal cancer, anaplastic large-cell lymphoma, esophageal squamous cells carcinoma, hepatocellular carcinoma, follicular dendritic cell carcinoma, intestinal cancer, muscle-invasive cancer, seminal vesicle tumor, epidermal carcinoma, spleen cancer, head and neck cancer, stomach cancer, liver cancer, bone cancer, brain cancer, cancer of the retina, biliary cancer, small bowel cancer, salivary gland cancer, cancer of uterus, cancer of testicles, cancer of connective tissue, prostatic hypertrophy, myelodysplasia, Waldenstrom's macro globinaemia, nasopharyngeal, neuroendocrine cancer, myelodysplastic syndrome, mesothelioma, angiosarcoma, Kaposi's sarcoma, carcinoid, oesophagogastric, fallopian tube cancer, peritoneal cancer, papillary serous mullerian cancer, malignant ascites, gastrointestinal stromal tumor (GIST), and a hereditary cancer syndrome such as Li- Fraumeni syndrome and Von Hippel-Lindau syndrome (VHL).

The methods of the invention are of particular relevance to solid tumors. The term

"solid tumor", as is used herein, refers to any tumor which forms a mass. Tumor mass may show partial or total lack of structural organization and functional coordination with normal tissue and may be a primary tumor mass or a secondary tumor mass (e.g., as a result of cell migration from the original tumor site through the blood and lymph vessels). Examples of solid tumors include, but are not limited to, tumors of the brain, prostate, breast, colon, lung, kidney, bladder, liver, bone, head, neck, stomach, larynx, esophagus, ovary, cervix, hepatocellular carcinoma, lung carcinoma, rectum, colorectum and other sites in the gastrointestinal tract, uterus, ovary, skin (e.g., metastatic melanomas), endometrium, pancreas and testes, wherein each possibility is a separate embodiment of the invention..

In another embodiment, the solid tumor is an epithelial cancer. In another embodiment, the epithelial cancer is selected from the group consisting of breast carcinoma, prostate carcinoma, bladder carcinoma, ovarian carcinoma, choriocarcinoma, pancreas carcinoma, liver carcinoma, lung carcinoma, and colon carcinoma, wherein each possibility is a separate embodiment of the invention..

In a particular embodiment, the cancer is breast cancer. In another embodiment, the cancer is colon cancer. In another embodiment, the cancer is ovary cancer. In another embodiment, the cancer is bladder cancer. Each possibility is a separate embodiment of the invention.

In another embodiment, the subject is symptomatic. In another embodiment, the subject is asymptomatic.

In certain embodiment, the cancer is not related to hematological disorders, e.g., lymphoma or leukemia. In certain embodiment, the cancer is not related to gastrointestinal and liver diseases (e.g., colon cancer or rectum cancer). In another embodiment, the cancer is not kidney cancer. In certain embodiment, the cancer is not related to genitourinary disorders.

Thus, the present invention is applicable for determining both primary as well as secondary cancers. In one embodiment, the cancer is a primary cancer. In another embodiment, the cancer is a secondary cancer, i.e., a metastatic cancer.

The methods disclosed herein are applicable for determining a cancer state, cancer severity as well as treatment efficiency at any stage of cancer as well as for determining recurrent cancer.

"Cancer recurrence" or "cancer relapse" as used herein refers to the return of a sign, symptom or disease after a remission. The cancer cells may re-appear in the same site of the primary tumor or in another location, such as in secondary cancer.

Overall, cancer stages may be referred to as Roman Numeral Staging. This system uses numerals I, II, III, and IV to describe the progression of cancer. Accordingly, cancer stages generally include: Stage I: cancers localized to one part of the body; Stage II or III: cancers locally advanced (staging will depend on type of cancer); Stage IV: cancers which have metastasized, or spread to other organs or throughout the body.

The level of binding may be determined by quantitative as well as qualitative measuring. In some embodiments, the phrase "qualitative" when in reference to differences in expression levels of a biomarker, refers to the presence versus absence of expression. In some embodiments, the phrase "quantitative" when in reference to differences in expression levels of a biomarker, refers to absolute differences in quantity of expression, as determined by any means, known in the art, or in other embodiments, relative differences, which may be statistically significant, or in some embodiments, when viewed as a whole or over a prolonged period of time, etc., indicate a trend in terms of differences in expression.

When referring to quantitative measurements, it may include determining the concentration of bound antibody, or of unbound antibody (determined e.g. by the use of a marked agent capable of binding to the free antibody). It is noted that this level correlates with the degree or severity of the disease. High level (above a predetermined threshold or in correlation with an a priori standard corresponding to a high cancer stage) is indicative of a sever state. With respect to the method for determining the effectiveness of treatment, and similarly, a decrease in the level is indicative in an improvement in the condition of a subject having cancer, e.g. as a result of an anti cancer treatment.

The terms "control value", "predetermined threshold" or "prior determined standards" are used herein to denote a reference value or range of values indicative of a healthy state or of a specific stage of a disease (degree of severity of the disease). When referring to a control value (or predetermined threshold or prior determined standards) of a healthy state, the control value may correlate with an averaged level of the biomarkers of the invention from a statistically significant group of healthy subjects or a value or range of values otherwise derived from the level biomarkers of the invention in a statistically significant group of healthy subjects. When referring to a control value, predetermined threshold or prior determined standards of a specific stage of a disease, the threshold (standards) may be determined based on levels of the biomarkers of the invention in a statistically significant group of patients diagnosed as having the disease in the specific stage of the disease. The manner of selecting the number of subjects used for a defining a group should be known to those versed in statistical analyses. Further, when referring to a control value of a specific stage of a disease, the value may be a reference point determined based on the level of the biomarkers of the invention in a sample taken from the examined subject at at-least one earlier time point, being when the subject was first diagnosed as having cancer or at a later time point. The level at that time point being the reference point for any follow up diagnosis of the subject's state making use of the method of the present invention.

According to another embodiment, the present invention provides a method for determining the effectiveness of a therapeutic treatment of a subject with an anti-cancer agent, comprising:

(i) determining, at least at two successive time points, the level of a combination of biomarkers comprising PARI -released peptide and PAR2 - released peptide in a fluid sample obtained from the subject, wherein at least one time point is during the therapeutic treatment;

(ii) comparing the level of the combination of biomarkers at the at least two successive time points, wherein a significant difference in the levels of the biomarkers indicates the effectiveness of the therapeutic treatment.

Thus, in accordance with this embodiment, one or more first samples are taken at a time point prior to initiation of the therapeutic treatment (this being a reference point) and one or more second samples is taken at a time point during the treatment, wherein a decrease in the level of the biomarker (e.g., binding of an antibody to said biomarker) exhibited in the at least one second sample as compared to that determined for the first sample is indicative that treatment is effective. In another embodiment, an increase in the level of the biomarker in the at least one second samples as compared to the first sample is indicative that the treatment is ineffective (or unsuccessful). Alternatively, one or more first samples are taken at a time point during the treatment and one or more second samples are taken at a time point during the treatment subsequent to the time point of the one or more first samples, such that a decrease in the level of the biomarker in the one or more second samples as compared to the one or more first samples is indicative that the treatment is effective. In another embodiment, an increase in the level of the biomarker in the one or more second samples as compared to the one or more first samples is indicative that the treatment is ineffective (or unsuccessful). Alternatively, one or more first samples are taken at a time point during the treatment and one or more second samples are taken at a time point after the treatment has been discontinued, wherein an increase in the level of binding in the one or more second samples as compared to the one or more first samples is indicative that the treatment is effective.

The term "managing subject treatment" refers to the behavior of the clinician or physician subsequent to the determination of the severity of the cancerous state (e.g., cancer status). For example, if the severity of the cancerous state indicates that surgery is appropriate, the physician may schedule the patient for surgery. Likewise, if the severity of the cancerous state is negative, e.g., late stage cancer or if the status is acute, no further action may be warranted. Furthermore, if the results show that treatment has been successful, no further management may be necessary. Alternatively, if the result of the methods of the present invention is inconclusive or there is reason that confirmation of status is necessary, the physician may order more tests.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES EXAMPLE 1

PARI and PAR2 have diagnostic value for early breast cancer detection

PARI and PAR2 gene expression was tested by using immuno-histochemical staining of breast tissues embedded in microarray. PARI and PAR2 expression results were compared to levels of CA15.3 and CA125 markers tested in the blood samples set that matched the biopsy collection.

Immuno-histochemical staining procedure:

Formalin - fixed, paraffin embedded breast carcinoma tissues from 299 non selected invasive breast carcinoma patients were obtained from the Departments of Oncology and Pathology, Hadassah-Hebrew University Medical Center, Jerusalem. The use of these specimens and data collection was approved by the Ethics Committee of the Hadassah Medical Center. The clinical and pathological characteristics of this patient cohort are presented in Table 1.

Sections (5μΜ) stained with H&E (hematoxylin and eosin stain) were obtained to confirm diagnosis and to identify typical representative areas of the specimen. From these defined areas, three tissue cores with a diameter of 0.6 mm were taken with a manual tissue arrayer MTA-1 (Beecher Instruments) as previously described (Kononen J. et al, Nat. Med. 1998. 4:844-7). From each specimen, three tissue cores with a diameter of 0.6 mm were taken from the different regions of the tumor and arrayed in triplicate on a recipient paraffin block (Hoos A. et al, Am. J. Path. 2001. 158: 1245-51). Sections of 5μΜ of the recipient blocks were cut and placed on charged poly-lysine coated slides. Immunodetection of PARI or PAR2 were performed following deparaffinization/ hydration of the slides (e.g., 3 washes of xylene for 3 minutes each, two washes of 100% ethanol, 2 minutes each; followed by 95% ethanol for 2 minutes, and 70% ethanol for 2 minutes; For rehydration: three washes in dH₂0 and incubation in d¾0 for 1 minutes; 3% hydrogen peroxidase for 5 minutes and finally three washes in d¾0; then, incubation in dH₂0 for 1 minute).

Next, antigen unmasking/retrieval was carried out by boiling of the slides in TE 10 mM Tris/1 mM EDTA, pH 9.0 then, maintaining at sub-boiling temperature for 15 minutes and cooling down for 15 minutes on ice. Prior to staining with specific antibody, the samples were washed three times in dH₂0 then incubated in dH₂0 for 1 minute. Next, sections were incubated in 3% hydrogen peroxidase for 5 minutes. This was followed by 3 washes in dH₂0 and incubation in dH₂0 for 1 minute. Finally, the sections were washed twice with Wash Buffer, 3 minutes. The tissue samples were then marked with PAP-PEN. The sections were then blocked with 200 μΐ blocking solution for 10 minutes at room temperature. After removing the blocking solution, 200 μΐ primary antibody (anti-Rabbit PARI antibody 1 :250; against peptide sequences CLLRNPNDKYEPFWED as also KSSPLQKQLPAFIC; or anti-Rabbit PAR2 antibody 1 :80 against peptide sequences CKVDGTSHVTGKGVT) diluted in CAS block were added and incubated overnight at 4°C. By the end of this period, the antibody was removed and the sections were washed in Wash-Buffer four times for 3 minutes each. Secondary antibody-HRP was then added (200 μΐ) and incubated for 30 minutes at room-temperature. The secondary antibody was then removed and the sections were washed five times with Wash-Buffer for 2 minutes each time. The intensity of staining was determined by the addition of DAB substrate to each section for 10 minutes. As soon as the color was developed the slides were immersed in tap water. The sections were then counterstained with hematoxylin for 2 minutes at room temperature, followed by dehydration (using 70% ethanol, 95% ethanol and two washes with 100% ethanol, followed by three rinses with Xylene) and careful mounting with cover slips.

Table 1: Clinical and pathological characteristics of breast cancer patients included in the tissue array biopsy study

Criteria Median (range)

Age 49 (20-82)

Tumor size (cm) 2 (0.2- 9)

Tumor type Number of cases (%)

Invasive ductal carcinoma (IDC) 220 (73.5)

Invasive lobular carcinoma (ILC) 33 (11.0)

IDC+ILC 13 (4.3)

Ductal carcinoma in-situ (DCIS) 9 (3.0)

DCIS + microinvasion 14 (4.6)

Mucinous 3 (1.0)

Medullary 7 (2.3)

Lymph node status

LN negative 159 (53.1)

LN positive 120 (40.1)

Grade (295 valid)

1-2 161 (54.5)

3 134 (45.4)

ER (282 valid

Negative 105 (37.2)

Positive 177 (62.7)

PR (279 valid}

Negative 235 (84.2)

Positive 44 (15.7)

Triple-neeative (273 valid)

TN 95 (34.79) Pathological evaluation of the tissue arrays resulted with the following outcome: more than seventy-seven percent (77.8%) of the tissue samples were positively stained with PARI , and more than 60 percent (61.7%) of the tissue samples were positively stained with PAR2 (Figure 1A and Figure 4, positive staining designated as "POS", negative staining designated as "0"). Remarkably, more than 85 percent (86.3%) of the tissue samples were positively stained with PARI or PAR2 (Figure 1 A), compared to 86.3% of patients positive for either PARI or PAR2, in the same patient cohort of CA15.3 expression was only 18% and CA125 expression was only 6% (Figure IB). Such comparison demonstrates the advantage of measuring PARI and PAR2 levels for early breast cancer detection.

EXAMPLE 2

Prognostic value of PARI and PAR2 for breast cancer patients with either tumor positive for estrogen receptor (ER) or with positive lymph-nodes

Significant portion of the patient cohort described in Example 1 was characterized as Estrogen Receptor positive (ER+). Statistical analyses of the tissue array staining for PAR genes in this specific group of patients (ER+) showed a significant difference in the Disease Free Survival (DFS) between PARI or PAR2 positive patients and PARI and PAR2 negative patients (Figure 2A, p=0.035). In the same analysis a significant difference was found for the Overall Survival (OS) in these two patient groups (Figure 2B, P=0.07). ( Therefore, PARI and PAR2 gene expression in ER(+) positive women may predict the disease outcome giving rise to poor prognosis of a significantly reduced Disease Free Survival period. Hence, PARI and PAR2 serve as a sensitive prognostic tool for ER(+) patients that receive hormone treatment (e.g., Tamoxifen). Based on this outcome the choice for treatment may be altered.

Another portion of this patient cohort was characterized as lymph node positive patients. Statistical analyses of the tissue array staining for PARI and PAR2 genes expression in this specific group of patients showed a significant difference (P=0.023) in the Overall Survival between patients expressing PARI or PAR2 and patients that do not express either PARI or PAR2 (Fig. 2C). Surprisingly, lymph node positive patients that do not express PARI and PAR2 have prolonged survival. Based on these results lymph node positive patients that express PARI and PAR2 genes may predict poor prognosis and shorter overall survival. EXAMPLE 3

ELISA using a mixture of antibodies and competition with the 41 aa peptide

In order to develop a simple assay for measuring levels of PARI released-peptide the inventors of this application have developed an ELISA assay. The assay involves a competition between a mixture of polyclonal and monoclonal antibodies (towards the PARI released-peptide) and either synthetically added PARI released-peptide in solution or various blood samples of cancer patient containing the PARI released-peptide. Toward this purpose, 96 well plates were coated (overnight, at 4°C) with the entire released PARI peptide (41aa): MGPRRLLLVAACFSLCGPLLSARTRARRPESKATNATLDPR (SEQ ID NO: l) using 100 mM bicarbonate/carbonate coating buffer at pH 9.6. Next, the plates were blocked (3% BSA in PBS-Tween20) for additional 2 hr at room temperature (R.T.). For the competition assay, a mixture (1 : 1) of antibody 167 (CGPLLSARTRARRPES; SEQ ID NO:3, against the middle region of the released PARI peptide) and antibody 168 (CRRPESKATNATLDPR; SEQ ID NO: 4, against the carboxy terminal part of the peptide) was added to the synthetic 41 aa peptide or to a patient blood sample. The 3 components were incubated first for 30 minutes at room temperature, prior to application onto the coated plates. Noticeably, the peptide for competition was added at increased concentrations (10 ng/ml -200 ng/ml). The competition assay was carried out for 2 hours at R.T. By the end of this period, a secondary antibody in blocking solution was added (goat- anti Rabbit -HRP conjugate, Pierce, at dilution of 1 :40000) for 1 hour at R.T. This step was followed by the addition of a detection solution including TMB substrate per well, for 10 minutes (until appropriate color is formed). The reaction was then stopped by adding 0.5% H₂S0₄ stop solution. The color values were obtained at OD 450nm wavelength in a plate reader.

As seen in Figure 3, a dose dependent competition was observed using increased concentrations of the 41aa peptide in a solution. Further, the competition with increasing concentrations of the 41aa peptide spiked in serum of healthy individual was measured and a similar dose dependent competition was observed. EXAMPLE 4

qRT-PCR for determination of PARI, PAR2 and HI 9 RNA levels in blood samples

Plasma was collected from individuals that underwent colonoscopy and were either found to be healthy or at different stages of colorectal cancer. For plasma separation, blood was withdrawn from the volunteers in an EDTA vacuum blood tube, and was centrifuge for 10 min at 1500g in 4°c. Extraction of total RNA was performed by adding 3.5 to 4 volumes TRI Reagent to plasma. Plasma and TRI reagent were mixed thoroughly till homogenization. Then, manufacture protocol were followed till after phase separation. To each colorless phase, 350 μΐ of RLT buffer from RNaesy Plus Mini kit (Qiagen) were added. The mixture was loaded onto genomic DNA elimination spin column. Manufacture protocol was then followed. In the final step RNA was eluted with 35 μΐ RNase free water. RNA quantity was evaluated on Bioanalyzer 2100 Pico chip (Agilnet). 5 ng of total RNA were used for reverse transcription with SMART MMLV RT enzyme and random hexamers in a 20μ1 final volume reaction. After RT reaction was complete, each sample was diluted 2 times in Nuclease free water, of which 2μ1 were used for qPCR. Each qPCR reaction was done with primers and Taqman® probe specific for each gene (Table 3). All qPCR reactions were in duplicate. qPCR was performed in a 384 well PCR plate, in a final reaction volume of 7 μΐ for 52 cycles at ABI's default conditions, in ABI Prism 7900 system (Applied Biosystems, Foster City, CA). Relative quantification for each gene was calculated by DataAssist v2.0. The normalizing genes for said calculation are HPRT and TFRC.

As demonstrated in Table 2, PARI RNA levels are detected in early cancer stages as well as in all the tested carcinoma samples. PARI expression was detected in all blood from patient with carcinoma and advanced-polyps. However, low level was measured in three carcinoma samples and in all advanced polyp samples. The PRA2 levels in the same samples generated a different expression pattern with higher expression levels in some samples. In the non advanced-polyp, a sample negative for PARI was detected for PAR2 (but overall expression level was not high).

Thus, PARI and PAR2 expression detection may be combined to ensure a better detection rate. The different patterns of gene expression within the same set of samples provide the utility of gene combination as a way to increase the sensitivity of such diagnostic tool. Table 2 depicts RT-PCR for RNA of PARI, PAR2 and H19 in plasma samples from colonoscopy negative individuals or colorectal cancer patients

Table 3 depicts the primers and probe sets used for the RT-PCR reactions

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

A method for determining severity of breast or colorectal cancer in a subject or prognosis of breast or colorectal cancer in a subject, comprising:

(i) determining the levels of a plurality of biomarkers comprising PARI and PAR2, or fragments or derivatives thereof, in at least one sample obtained from the subject; and

The method of claim 1, wherein the at least one control value is selected from a predetermined cutoff value, a value obtained from a statistically significant group of control individuals afflicted with defined severities of cancer, or a stored set of data corresponding to control individuals afflicted with defined severities of cancer.

The method of claim 1 , wherein the at least one control value is determined by measuring the levels of said plurality of biomarkers in a sample obtained from said subject at at-least one prior-referenced time point.

The method of claim 1, wherein the prognosis of a malignant disease in a subject is a probability of survival.

A method for determining the effectiveness of a therapeutic treatment of a subject afflicted with breast or colorectal cancer , comprising:

(i) determining, at least at two successive time points, the level of a plurality of biomarkers comprising PARI and PAR2 in at least one sample obtained from the subject, wherein at least one time point is during the therapeutic treatment;

(ii) comparing the level of the plurality of biomarkers at the at least two successive time points;

wherein a significant difference in the levels of the biomarkers indicates the effectiveness of the therapeutic treatment.

6. The method of claim 5, wherein at least one first sample is taken at a time point prior to initiation of said therapeutic treatment and at least one subsequent sample is taken at a time point during said therapeutic treatment, wherein a significant decrease in the level of said biomarkers in the at least one subsequent sample compared to that determined for the first sample indicates that said therapeutic treatment is effective.

7. The method of claim 5, wherein the at least two successive time points are taken at time points during the therapeutic treatment, wherein a significant decrease in the levels of said biomarkers in a subsequent sample compared to the level in the first sample indicates that said therapeutic treatment is effective.

8. The method of claim 5, wherein at least one first sample is taken at a time point during the therapeutic treatment and at least one subsequent sample is taken at a time point after the treatment has been discontinued, wherein a significant increase in the levels of said biomarkers in the at least one subsequent sample compared to the levels determined for the at least one first sample indicates that said therapeutic treatment is effective.

9. A method for diagnosing breast or colorectal cancer in a subject, comprising determining the levels of a plurality of biomarkers comprising PARI and PAR2, or fragments or derivatives thereof, in at least one sample obtained from the subject, wherein a significant elevation in the levels of the plurality of biomarkers compared to a control value is indicative of breast or colorectal cancer in said subject.

10. The method of claim 9, for early diagnosis of breast or colorectal cancer.

11. The method of claim 9, for diagnosis of breast or colorectal cancer recurrence.

12. The method of claim 9, wherein said control value is selected from the group consisting of a predetermined cutoff value, a value obtained from a healthy control individual, a panel of control values from a set of healthy individuals, and a stored set of data corresponding to control individuals that are not afflicted with cancer.

13. The method of claim 12, wherein the predetermined cutoff value is obtained from said subject at at-least one prior-referenced time point.

14. The method of any one of claims 1, 5 or 9, wherein said cancer is breast cancer.

15. The method of any one of claims 1 , 5 or 9, wherein said cancer is colorectal cancer.

16. The method of claim 15, wherein said cancer is colon cancer.

17. The method of any one of claims 1, 5 or 9, wherein the at least one sample obtained from the subject is a tissue sample.

18. The method of claim 17, wherein the tissue sample is a breast biopsy.

19. The method of claim 17, wherein the tissue sample is a colorectal biopsy.

20. The method of any one of claims 1 , 5 or 9, wherein the at least one sample obtained from the subject is a fluid sample.

21. The method of claim 20, wherein the fluid sample is selected from the group consisting of: whole blood, plasma, serum, ascitic fluid or urine.

22. The method of claim 21 , wherein the fluid sample is selected from the group consisting of serum, plasma and blood.

23. The method of any one of claims 1 , 5 or 9, wherein the at least one sample obtained from the subject is two samples wherein one sample is a tissue sample and a second sample is a fluid sample.

24. The method of claim 1, 5 or 9, wherein the cancer is breast cancer, and wherein the plurality of biomarkers further comprises estrogen receptor (ER).

25. The method of claim 24, wherein the level of ER is determined in a tissue sample obtained from said subject and the levels of PARI and PAR2 are determined in a fluid sample obtained from said subject.

26. The method of any one of claims 1, 5 or 9, wherein said cancer is breast cancer, and wherein said subject has been afflicted as having estrogen receptor positive breast cancer.

27. The method of any one of claims 1, 5 or 9, wherein said plurality of biomarkers further comprises H-19.

28. The method of claim 27, wherein said cancer is colorectal cancer.

29. The method of any one of claims 20-22, wherein determining the levels of a plurality of biomarkers comprises determining the levels of PARI -released peptide and PAR2- released peptide, fragments or derivatives thereof.

30. The method of any one of claims 1, 5 or 9, wherein determining the levels of the plurality of biomarkers is performed by a method selected from immunoassay, tissue array, immunohistochemistry, polymerase chain reaction (PCR), in situ hybridization, fluorescent in situ hybridization (FISH).

31. The method of claim 30, wherein the PCR is selected from quantitative real-time PCR (qRT-PCR) and reverse transcription PCR (RT-PCR).

32. The method of claim 30, wherein determining the levels of said plurality of biomarkers is performed by an immunoassay.

33. The methods of claim 32, wherein the immunoassay is performed using an antibody raised against a PARI -released peptide, a fragment or derivative thereof.

34. The method of claim 33, wherein the PARl-released peptide comprises SEQ ID NO: 1 , a fragment or derivative thereof.

35. The method of claim 33, wherein the PARl-released peptide consists of SEQ ID NO.T .

36. The method of claim 33, wherein the fragment or derivative of said PARl-released peptide is selected from the group consisting of: SEQ ID NO:2 - SEQ ID NO:6.

37. The method of claim 33, wherein the fragment or derivative of said PARl-released peptide is selected from the group consisting of: SEQ ID NO:2 - SEQ ID NO:4.

38. The method of claim 37, wherein the fragment of said PARl-released peptide consists of RLLLVAACFSLC (SEQ ID NO:2).

39. The method of claim 37, wherein the fragment of said PARl-released peptide consists of CGPLLSARTRARRPES (SEQ ID NO:3).

40. The method of claim 37, wherein the derivative of said PARl-released peptide consists of CRRPESKATNATLDPR (SEQ ID NO:4).

41. The method of claims 32 wherein the immunoassay is performed using an antibody raised against a PAR2-released peptide, a fragment or derivative thereof.

42. The method of claim 41 , wherein the PAR2-released peptide comprises SEQ ID NO:7, a fragment or derivative thereof.

43. The method of claim 41, wherein the PARl-released peptide consists of SEQ ID NO:7.

44. The method of claim 42, wherein the fragment or derivative of said PAR2-released peptide is selected from the group consisting of: SEQ ID NO:8 and SEQ ID NO:9.

45. The methods of claim 44, wherein the derivative of said PAR2 -released peptide consists of MRSPS AGGSGC (SEQ ID NO:8).

46. The methods of claim 44, wherein the fragment of said PAR2-released peptide consists of CSGTIQGTNRSSKGR (SEQ ID NO:9).

47. A diagnostic kit for the prognosis, diagnosis or monitoring breast or colorectal cancer comprising means for determining the levels of a plurality of biomarkers comprising

PARI and PAR2, or fragments thereof, in a sample.

48. The diagnostic kit of claim 47, wherein the plurality of biomarkers comprises PARl- released peptide and PAR2-released peptide.

49. The diagnostic kit of claim 47, wherein the plurality of biomarkers further comprises H-19.