WO2016172216A1 - Use of soluble crkl compositions as breast cancer biomarkers and predictors of cancer metastasis - Google Patents

Abstract

Disclosed are assays suitable for quantitating soluble CRKL peptide from mammalian bodily fluids (including, for example, human plasma or serum), which are useful in the diagnosis, monitoring, and/or treatment of disease, including one or more mammalian cancers such as human breast cancer. Also disclosed are methods of using a soluble fraction of CRKL peptide as a biomarker for predicting the likelihood of metastasis in breast cancer patients.

Description

USE OF SOLUBLE CRKL COMPOSITIONS AS BREAST CANCER BIOMARKERS AND PREDICTORS OF CANCER METASTASIS

BACKGROUND OF THE INVENTION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to United States Provisional Patent Appl. No. 62/150,150; filed April 20, 2015 (pending; Arty. Dkt. No. 37182.184), the contents of which is specifically incorporated herein in its entirety by express reference thereto.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant Nos. U54- CA143837 and U54-CA151668-01 awarded by the National Institutes of Health, and Grant Nos. W81XWH-09-1-0212 and W81XWH-07-2-0101 awarded by the United States Department of Defense. The government has certain rights in the invention.

NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

[0003] Not Applicable. FIELD OF THE INVENTION

[0004] The present invention relates generally to the fields of molecular biology, oncology, and diagnostic laboratory medicine, and in particular, to the development of assays that are specific for the detection of cancers such as mammalian breast cancer. Disclosed are diagnostic methods and reagents suitable for quantitating soluble crk-like (CRKL) peptide in one or more body fluids, such as human plasma or serum, which has been shown to be predictive in diagnosing, monitoring, and/or treating one or more types of mammalian cancers, and in particular for determining the metastatic potential of breast cancer in humans. DESCRIPTION OF RELATED ART BREAST CANCER

[0005] Breast cancer is the second leading cause of death among women in the United States. Approximately one woman in every ten will develop breast cancer in her lifetime. Recent statistics estimate that 44,000 women will die of breast cancer, while 150,000 new female cases of breast cancer will be diagnosed in the next year.

[0006] It has been shown that screening for breast cancer can reduce breast cancer mortality. Among women aged 50 and older, studies have demonstrated a 20% to 40% reduction in breast cancer mortality for women screened by mammography and clinical breast examination. However, among women between 40 to 49 years of age, the mortality rate is reduced only 13% to 23%. These results suggest that further methods of screening could potentially reduce the mortality in the younger age group of women.

[0007] While physical examination and mammography are useful screening procedures for the early detection of breast cancer, they can produce a substantial percentage of false positive and false negative results especially in women with dense parenchymal breast tissue. For example, the probability of having a false negative mammographic examination is 20% to 25% among women between 40 to 49 years of age and 10% among women 50 to 69 years of age.

[0008] Consequently, screening will result in a number of negative biopsy results yielding a high percentage of false positives. There is also a demonstrated lack of sensitivity in detecting cancerous lesions in younger women yielding a significant percentage of false negatives.

[0009] There has also been a clear need for added modalities of screening to help diagnose cancer in younger women. Increased technology in the field of mammography has allowed more reliable detection of small lesions of the breast. Likewise, breast cancer researchers continue to seek additional adjunct diagnostic procedures to further enhance cancer screening and, thereby, to reduce mortality rates.

CRK-LiKE PROTEIN

[0010] CRKL, a member of the Crk (CT10 Regulator of Kinase) protein family, was originally identified as an intracellular adaptor protein in primary leukemic neutrophils from patients with chronic myeloid leukemia (ten Hoeve et al, 1993). The Crk group of proteins has been implicated in a wide variety of intracellular signaling pathways involving tumor cell migration, invasion, and survival (Birge et al, 2009). High expression levels of CRKL in the disease site have been observed in various solid tumors like gastric (Natsume et al, 2012), lung (Cheung et al, 2011; Wang et al, 2012), prostate (Mintz et al, 2009), hepatocellular carcinoma (Liu et al, 2013) and breast cancers (Fathers et al, 2012; Zhao et al, 2013). In these studies, over-expression of CRKL was associated with high-grade and proliferative breast cancers with poor prognosis and decreased survival. A recent study (Fathers et al, 2012) has demonstrated that Crk proteins play an important role in regulating aggressive basal breast cancers. Proteins located in various intra- and extracellular compartments can have multiple functions based on their location (Arnoys and Wang, 2007). When found intracellularly, CRKL has been implicated in integrin mediated signaling by binding to various tyrosine phosphorylated scaffold proteins such as C3G, paxillin and pl30Cas, thus affecting cell adhesion and migration. CRKL can also translocate to the nucleus and act as a nuclear adaptor protein for Stat5 regulating gene transcription through DNA binding (Fish et al, 1999). While most of the studies on CRKL have shown that it acts as an intracellular adaptor protein in cytoplasmic or nuclear compartments, recent studies have demonstrated that the un-phosphorylated form of the protein is secreted out either by ABC transporters or when the tumor cell dies and could be detected on the cell membrane (Mintz et al, 2009). The proposed mechanism of action of the secreted CRKL involves binding to the plexin-semaphorin-integrin (PSI) domain of βΐ integrin to activate the MAP kinase pathway causing nuclear transcription and eventual cell migration and proliferation (Ozawa et al, 2010).

SERUM PROTEIN BIOMARKERS FOR CANCER DETECTION

[0011] Currently used serum biomarker assays for breast cancer include carcinoembryonic antigens CA15-3 and CA27.29, circulating cytokeratins such as tissue polypeptide antigen (TP A), tissue polypeptide specific antigen (TPS) and cytokeratin 19 fragment (CIFRA-21-1), and the Proteolytically-cleaved ectodomain of the human epidermal growth factor receptor 2 (s-HER2) (Mirabelli and Incoronato, 2013).

BIOMARKERS FOR PREDICTING BREAST CANCER METASTASIS

[0012] Over-expressed and/or secreted proteins from cancer cells are easy to detect in the sera of cancer patients, and as such, have been used clinically as diagnostic and prognostic markers for cancers for a number of years (Wu et al, 2005). Examples of secreted proteins used for cancer diagnosis include prostate specific antigen (PSA) for prostate cancer (Balk et al, 2003), carcinoembryonic antigen, CA125, for ovarian cancer (Raamanathan et al, 2012) and colony stimulating factor-1 (CSF-1) (Kacinski et al, 1990) for endometrial carcinoma. A few soluble biomarkers for breast cancer including carcinoembryonic antigens, CA 15.3 and CA 27.59 and vascular endothelial growth factor (VEGF) are of prognostic value (Harris et al, 2007).

DEFICIENCIES IN THE PRIOR ART

[0013] Currently, CA 15-3 is the most widely used serum biomarker assayed in conjunction with diagnostic imaging for monitoring metastatic disease in breast cancer (Danova et al, 2011). However, this, and other existing breast cancer biomarkers (e.g., TP A/TPS, etc.) lack sensitivity for early disease, and are not very specific (Duffy, 2006). Thus, identification of more specific markers to predict metastasis would be of clinical significance, and would provide a significant advance over the prior art.

BRIEF SUMMARY OF THE INVENTION

[0014] The present disclosure overcomes these and other limitations inherent in the art by providing methods for the detection of soluble cancer biomarkers in serum, and their use in predicting, monitoring, and treating metastasis of cancers such as that of the human breast.

[0015] The present disclosure relates generally to the use of one or more soluble peptide biomarkers to diagnose breast cancer and, more particularly, to diagnostically differentiate between women with carcinoma of the breast, women with benign tumors, and healthy controls.

[0016] In a first embodiment, the present disclosure provides methods for assaying the level of soluble CRKL protein or peptide in a blood sample obtained from a mammalian test subject, in detection compositions, one or more pharmaceutical excipients, diluents, vehicles or buffers, and a set of instructions for using the compositions, for predicting, monitoring, and/or treating one or more metastatic mammalian cancers, including, without limitation, cancer of the human breast.

[0017] It is an object of the present disclosure to use a blood sample, or a component thereof, as a diagnostic medium and/or as part of a non-imvasive protocol for the detection and differential diagnosis of breast carcinomas, thereby overcoming various deficiencies and shortcomings of the prior art, including those outlined above.

[0018] It is another object of the present disclosure to identify one or more biomarkers present in mammalian blood, as having diagnostic value, and/or that can be used in prophylaxis, diagnosis, treatment, or post-treatment monitoring. Likewise, it is another object of the present disclosure to provide one or more CRKL peptide biomarkers as part of a diagnostic panel for the initial detection, follow-up screening for detection, reoccurrence of breast cancer in women, response to chemotherapy and/or surgical treatment of the disease state.

[0019] It would be understood by those skilled in the art that one or more aspects of the present disclosure can meet certain objectives, while one or more other aspects can meet certain other objectives. Each objective may not apply equally, in all instances, to every aspect of the present disclosure. As such, these objectives—in light of the prior art regarding diagnosis of breast cancer-can be viewed in the alternative with respect to any one aspect of the present disclosure.

[0020] In part, the present disclosure provides a method for using a serum peptide biomarker to differentially diagnose and/or detect reoccurrence of breast carcinoma. The method includes (1) using a human blood sample to provide a CRKL protein or peptide biomarker for that individual and diagnostic for carcinoma of the breast, (2) comparing the individual biomarker with a biomarker reference, and (3) differentially identifying the diagnosis for the individual as indicated by the biomarker comparison. The biomarker reference can be made up of a panel of constituents and can be developed using malignant tunor, benign tumor, and control group populations. Each referenced biomarker constituent can have associated with it a range of values comparable to a corresponding individual biomarker.

[0021] Each individual biomarker constituent can be associated with a concentration value, for comparison with a corresponding reference constituent. In one embodiment of the present disclosure, the concentration of CRKL peptide for an individual having a malignant breast tumor is at least about 100 percent higher than such a concentration for an individual having a benign tumor. Such diagnostic identification can be used alone, or in conjunction with one or more primary diagnostic methods for the testing and detection of breast carcinomas.

[0022] In part, the present disclosure provides a post-operative method of monitoring tumor growth. This method, in an overall and general sense includes at least the steps of: (1) providing an individual post-operative to the removal of a malignant tumor, (2) using a blood specimen from that individual to develop a post-operative biomarker panel that comprises at least a CRKL protein or CRKL peptide-derived biomarker, (3) comparing the post-operative biomarker panel with a pre-operative biomarker reference panel for the individual, and (4) determining the presence of malignancy by monitoring at least one constituent of the respective biomarker panels.

[0023] In part, the present disclosure also provides a method for using the concentration of an endogenously encoded protein to diagnose carcinoma of the breast. This method, in an overall and general sense involves at least the steps of: (1) using a blood specimen from an individual to provide a protein biomarker diagnostic for carcinoma of the breast, (2) comparing the individual protein biomarker with a reference protein, and (3) determining an elevated concentration of the individual protein biomarker over the referenced protein to diagnose the individual. In preferred embodiments, the biomarker protein is one constituent of a biomarker panel. Likewise, the reference protein can be one constituent of a reference panel. Regardless, any such protein can be developed as a reference using malignant tumor, benign tumor, and control group populations. In highly preferred embodiments, the individual protein biomarker is a CRKL-derived peptide biomarker.

[0024] The CRKL biomarkers described herein, and the related inventive methods disclosed are useful in detecting breast carcinoma, and provide an economical and logistical adjunct diagnostic test for conventional diagnostic tools, including, for example, mammography. Furthermore, the serum CRKL peptide biomarkers can also, in conjunction with physician- and/or self-performed breast examination, help to reduce morbidity and mortality rates for breast cancer and thereby reduce overall national health care expenditures. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] For promoting an understanding of the principles of the invention, reference will now be made to the embodiments, or examples, illustrated in the drawings and specific language will be used to describe the same. It will, nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein are contemplated as would normally occur to one of ordinary skill in the art to which the present disclosure relates.

[0026] The following drawings form part of the present specification and are included to demonstrate certain aspects of the present disclosure. The application contains at least one drawing that is executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. The present disclosure may be better understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements, and in which:

[0027] FIG. 1A and FIG. IB show the surface localization of CRKL in breast cancer cells in vitro. FIG. 1A shows the flow cytometry on non-permeabilized cells showing surface fraction of CRKL in breast cancer and epithelial cell lines. FIG. IB shows the immunofluorescence images of human breast cancer cells showing the difference in the staining pattern of CRKL (red) in permeabilized (bottom row) and non-permeabilized (top row) cells. The cells are counterstained with wheat germ agglutinin 488 for the cell membrane (green) and DAPI for the nucleus (blue) (Scale bar = 20 μιτι);

[0028] FIG. 2A, FIG. 2B, and FIG. 2C show CRKL expression in in vivo samples and clinical tumor biopsies: FIG. 2A shows immunohistochemistry results for CRKL in primary breast cancer xenografts (Scale bar = 50 μιτι); FIG. 2B shows human breast cancer tissue microarray showing (i) strong and (ii) weak staining of invasive ductal carcinoma tissues, (iii) normal breast tissue showing positive staining mainly in the ductal epithelium (Scale bar = 100 μm). FIG. 2C shows quantitation of intensity of staining through image analysis in tumor cores (early and advanced disease) and normal breast tissues; and

[0029] FIG. 3A, FIG. 3B, and FIG. 3C show the assessment of secreted CRKL levels in in vitro, in vivo and clinical samples - The CRKL concentration (CCRKL) was measured in (FIG. 3A) cell supernatant (FIG. 3B) serum from mice bearing patient derived breast tumor xenografts and (FIG. 3C) serum from breast cancer patients using ELISA.

BRIEF DESCRIPTION OF THE NUCLEIC ACID AND/OR AMINO ACID SEQUENCES

[0030] SEQ ID NO:l illustrates the amino acid sequence of a human CrkL protein in accordance with one embodiment of the present disclosure. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0031] Illustrative embodiments are described below. In the interest of clarity, not all features of an actual implementation are described in this specification. It will of course be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which will vary from one implementation to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

[0032] In the present study, it was shown that more than 40% of patients with localized or early stage disease and 100% of patients with metastatic disease have elevated serum levels of CRKL. Thus, soluble CRKL may serve as a stronger biomarker for predicting early disease and metastasis. Further, among patients analyzed in this study, four had not undergone treatment and had mean values 1.6-fold higher than the rest of the patients (n = 25) who were undergoing treatment, showing that CRKL levels may be very useful in monitoring the treatment of breast cancer.

EXEMPLARY DEFINITIONS

[0033] In accordance with the present disclosure, polynucleotides, nucleic acid segments, nucleic acid sequences, and the like, include, but are not limited to, DNAs (including and not limited to genomic or extragenomic DNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleic acid segments either obtained from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized in whole or in part by the hand of man.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and compositions similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions described herein. For purposes of the present disclosure, the following terms are defined below for sake of clarity and ease of reference:

[0035] In accordance with long standing patent law convention, the words "a" and "an," when used in this application, including the claims, denote "one or more." [0036] The terms "about" and "approximately" as used herein, are interchangeable, and should generally be understood to refer to a range of numbers around a given number, as well as to all numbers in a recited range of numbers (e.g., "about 5 to 15" means "about 5 to about 15" unless otherwise stated). Moreover, all numerical ranges herein should be understood to include each whole integer within the range.

[0037] As used herein, an "antigenic polypeptide" or an "immunogenic polypeptide" is a polypeptide which, when introduced into a vertebrate, reacts with the vertebrate's immune system molecules, i.e., is antigenic, and/or induces an immune response in the vertebrate, i.e., is immunogenic.

[0038] "Biocompatible" refers to a material that, when exposed to living cells, will support an appropriate cellular activity of the cells without causing an undesirable effect in the cells, such as a change in a living cycle of the cells, a change in a proliferation rate of the cells, or a cytotoxic effect.

[0039] The term "biologically-functional equivalent" is well understood in the art, and is further defined in detail herein. Accordingly, sequences that have about 85% to about 90%; or more preferably, about 91% to about 95%; or even more preferably, about 96% to about 99%; of nucleotides that are identical or functionally-equivalent to one or more of the nucleotide sequences provided herein are particularly contemplated to be useful in the practice of the methods and compositions set forth in the instant application.

[0040] As used herein, the term "buffer" includes one or more compositions, or aqueous solutions thereof, that resist fluctuation in the pH when an acid or an alkali is added to the solution or composition that includes the buffer. This resistance to pH change is due to the buffering properties of such solutions, and may be a function of one or more specific compounds included in the composition. Thus, solutions or other compositions exhibiting buffering activity are referred to as buffers or buffer solutions. Buffers generally do not have an unlimited ability to maintain the pH of a solution or composition; rather, they are typically able to maintain the pH within certain ranges, for example from a pH of about 5 to 7.

[0041] As used herein, the term "carrier" is intended to include any solvent(s), dispersion medium, coating(s), diluent(s), buffer(s), isotonic agent(s), solution(s), suspension(s), colloid(s), inert (s), or such like, or a combination thereof that is pharmaceutically acceptable for administration to the relevant animal or acceptable for a therapeutic or diagnostic purpose, as applicable. [0042] As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment obtained from a biological sample using one of the compositions disclosed herein refers to one or more DNA segments that have been isolated away from, or purified free from, total genomic DNA of the particular species from which they are obtained. Included within the term "DNA segment," are DNA segments and smaller fragments of such segments, as well as recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.

[0043] The term "effective amount," as used herein, refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect.

[0044] The term "for example" or "e.g., " as used herein, is used merely by way of example, without no limitation intended, and should not be construed as referring only those items explicitly enumerated herein.

[0045] As used herein, a "heterologous" is defined in relation to a predetermined referenced gene sequence. For example, with respect to a structural gene sequence, a heterologous promoter is defined as a promoter which does not naturally occur adjacent to the referenced structural gene, but which is positioned by laboratory manipulation. Likewise, a heterologous gene or nucleic acid segment is defined as a gene or segment that does not naturally occur adjacent to the referenced promoter and/or enhancer elements.

[0046] As used herein, "homologous" means, when referring to polynucleotides, sequences that have the same essential nucleotide sequence, despite arising from different origins. Typically, homologous nucleic acid sequences are derived from closely related genes or organisms possessing one or more substantially similar genomic sequences. By contrast, an "analogous" polynucleotide is one that shares the same function with a polynucleotide from a different species or organism, but may have a significantly different primary nucleotide sequence that encodes one or more proteins or polypeptides that accomplish similar functions or possess similar biological activity. Analogous polynucleotides may often be derived from two or more organisms that are not closely related (e.g., either genetically or phylogenetically).

[0047] As used herein, the term "homology" refers to a degree of complementarity between two or more polynucleotide or polypeptide sequences. The word "identity" may substitute for the word "homology" when a first nucleic acid or amino acid sequence has the exact same primary sequence as a second nucleic acid or amino acid sequence. Sequence homology and sequence identity can be determined by analyzing two or more sequences using algorithms and computer programs known in the art. Such methods may be used to assess whether a given sequence is identical or homologous to another selected sequence.

[0048] The terms "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to persons of ordinary skill) or by visual inspection.

[0049] As used herein, the phrase "in need of treatment" refers to a judgment made by a caregiver such as a physician or veterinarian that a patient requires (or will benefit in one or more ways) from treatment. Such judgment may made based on a variety of factors that are in the realm of a caregiver's expertise, and may include the knowledge that the patient is ill as the result of a disease state that is treatable by one or more compound or pharmaceutical compositions such as those set forth herein.

[0050] The phrases "isolated" or "biologically pure" refer to material that is substantially, or essentially, free from components that normally accompany the material as it is found in its native state. Thus, isolated polynucleotides in accordance with the present disclosure preferably do not contain materials normally associated with those polynucleotides in their natural, or in situ, environment.

[0051] As used herein, the term "kit" may be used to describe variations of the portable, self-contained enclosure that includes at least one set of reagents, components, or pharmaceutically-formulated compositions to conduct one or more of the assay methods described om the present disclosure. Optionally, such kit may include one or more sets of instructions for use of the enclosed compositions, such as, for example, in a laboratory or clinical application.

[0052] "Link" or "join" refers to any method known in the art for functionally connecting one or more proteins, peptides, nucleic acids, or polynucleotides, including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, electrostatic bonding, and the like. [0053] The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by the hand of man in a laboratory is naturally-occurring. As used herein, laboratory strains of rodents that may have been selectively bred according to classical genetics are considered naturally-occurring animals.

[0054] As used herein, the term "nucleic acid" includes one or more types of: polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases (including abasic sites). The term "nucleic acid," as used herein, also includes polymers of ribonucleosides or deoxyribonucleosides that are covalently bonded, typically by phosphodiester linkages between subunits, but in some cases by phosphorothioates, methylphosphonates, and the like. "Nucleic acids" include single- and double-stranded DNA, as well as single- and double-stranded RNA. Exemplary nucleic acids include, without limitation, gDNA; hnRNA; mRNA; rRNA, tRNA, micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snORNA), small nuclear RNA (snRNA), and small temporal RNA (stRNA), and the like, and any combination thereof.

[0055] The term "operably linked," as used herein, refers to that the nucleic acid sequences being linked are typically contiguous, or substantially contiguous, and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.

[0056] As used herein, the term "patient" (also interchangeably referred to as "host" or "subject"), refers to any host that can serve as a recipient of one or more of the therapeutic or diagnostic formulations as discussed herein. In certain aspects, the patient is a vertebrate animal, which is intended to denote any animal species (and preferably, a mammalian species such as a human being). In certain embodiments, a patient may be any animal host, including but not limited to, human and non-human primates, avians, reptiles, amphibians, bovines, canines, caprines, cavines, corvines, epines, equines, felines, hircines, lapines, leporines, lupines, murines, ovines, porcines, racines, vulpines, and the like, including, without limitation, domesticated livestock, herding or migratory animals or birds, exotics or zoological specimens, as well as companion animals, pets, or any animal under the care of a veterinary or animal medical care practitioner.

[0057] The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that preferably do not produce an allergic or similar untoward reaction when administered to a mammal, and in particular, when administered to a human.

[0058] As used herein, "pharmaceutically-acceptable salt" refers to a salt that preferably retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include, without limitation, acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like); and salts formed with organic acids including, without limitation, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic (embonic) acid, alginic acid, naphthoic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; salts formed with an organic cation formed from NN'-dibenzylethylenediamine or ethylenediamine; and combinations thereof.

[0059] As used herein, the term "plasmid" or "vector" refers to a genetic construct that is composed of genetic material (i.e., nucleic acids). Typically, a plasmid or a vector contains an origin of replication that is functional in bacterial host cells, e.g., Escherichia coli, and selectable markers for detecting bacterial host cells including the plasmid. Plasmids and vectors may be prepared that include one or more genetic elements as described herein arranged such that a cloned DNA sequence encoding one or more of the disclosed recombinant polypeptides may be transcribed and translated in a suitable expression cells. Additionally, such plasmids or vectors may further include one or more nucleic acid segments, genes, promoters, enhancers, activators, multiple cloning regions, or any combination thereof, including segments that are obtained from or derived from one or more natural and/or artificial sources.

[0060] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and includes any chain or chains of two or more amino acids. Thus, as used herein, terms including, but not limited to "peptide," "dipeptide," "tripeptide," "protein," "enzyme," "amino acid chain," and "contiguous amino acid sequence" are all encompassed within the definition of a "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with, any of these terms. The term further includes polypeptides that have undergone one or more post- translational modification(s), including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post- translation processing, or modification by inclusion of one or more non-naturally occurring amino acids. Conventional nomenclature exists in the art for polynucleotide and polypeptide structures.

[0061] For example, one-letter and three-letter abbreviations are widely employed to describe amino acids: Alanine (A; Ala), Arginine (R; Arg), Asparagine (N; Asn), Aspartic Acid (D; Asp), Cysteine (C; Cys), Glutamine (Q; Gin), Glutamic Acid (E; Glu), Glycine (G; Gly), Histidine (H; His), Isoleucine (I; He), Leucine (L; Leu), Methionine (M; Met), Phenylalanine (F; Phe), Proline (P; Pro), Serine (S; Ser), Threonine (T; Thr), Tryptophan (W; Trp), Tyrosine (Y; Tyr), Valine (V; Val), and Lysine (K; Lys). Amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form may be substituted for any L-amino acid residue provided the desired properties of the polypeptide are retained.

[0062] As used herein, the terms "prevent," "preventing," "prevention," "suppress," "suppressing," and "suppression" as used herein refer to administering a compound either alone or as contained in a pharmaceutical composition prior to the onset of clinical symptoms of a disease state so as to prevent any symptom, aspect or characteristic of the disease state. Such preventing and suppressing need not be absolute to be deemed medically useful.

[0063] "Protein" is used herein interchangeably with "peptide" and "polypeptide," and includes both peptides and polypeptides produced synthetically, recombinantly, or in vitro and peptides and polypeptides expressed in vivo after nucleic acid sequences are administered into a host animal or human subject. The term "polypeptide" is preferably intended to refer to any amino acid chain length, including those of short peptides from about two to about 20 amino acid residues in length, oligopeptides from about 10 to about 100 amino acid residues in length, and longer polypeptides including from about 100 amino acid residues or more in length. Furthermore, the term is also intended to include enzymes, i.e., functional biomolecules including at least one amino acid polymer. Polypeptides and proteins as set forth in the present disclosure also include polypeptides and proteins that are or have been post-translationally modified, and include any sugar or other derivative(s) or conjugate(s) added to the backbone amino acid chain.

[0064] "Purified," as used herein, means separated from many other compounds or entities. A compound or entity may be partially purified, substantially purified, or pure. A compound or entity is considered pure when it is removed from substantially all other compounds or entities, i.e. , is preferably at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure. A partially or substantially purified compound or entity may be removed from at least 50%, at least 60%, at least 70%, or at least 80% of the material with which it is naturally found, e.g., cellular material such as cellular proteins and/or nucleic acids.

[0065] The term "recombinant" indicates that the material (e.g., a polynucleotide or a polypeptide) has been artificially or synthetically (non-naturally) altered by human intervention. The alteration can be performed on the material within or removed from, its natural environment, or native state. Specifically, e.g., a promoter sequence is "recombinant" when it is produced by the expression of a nucleic acid segment engineered by the hand of man. For example, a "recombinant nucleic acid" is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other procedures, or by chemical or other mutagenesis; a "recombinant polypeptide" or "recombinant protein" is a polypeptide or protein which is produced by expression of a recombinant nucleic acid; and a "recombinant virus," e.g., a recombinant AAV virus, is produced by the expression of a recombinant nucleic acid.

[0066] The term "regulatory element," as used herein, refers to a region or regions of a nucleic acid sequence that regulates transcription. Exemplary regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, transcriptional control sequences, and such like.

[0067] The term "RNA segment" refers to an RNA molecule that has been isolated free of total cellular RNA of a particular species. Therefore, RNA segments can refer to one or more RNA segments (either of native or synthetic origin) that have been isolated away from, or purified free from, other RNAs. Included within the term "RNA segment," are RNA segments and smaller fragments of such segments.

[0068] The term "a sequence essentially as set forth in SEQ ID NO:X" means that the sequence substantially corresponds to a portion of SEQ ID NO:X and has relatively few nucleotides (or amino acids in the case of polypeptide sequences) that are not identical to, or a biologically functional equivalent of, the nucleotides (or amino acids) of SEQ ID NO:X.

[0069] The term "subject," as used herein, describes an organism, including mammals such as primates, to which treatment with one or more compositions can be provided. Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, apes; chimpanzees; orangutans; humans; monkeys; domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.

[0070] The term "substantially corresponds to," "substantially homologous," or "substantial identity," as used herein, denote a characteristic of a nucleic acid or an amino acid sequence, wherein a selected nucleic acid or amino acid sequence has at least about 70 or about 75 percent sequence identity as compared to a selected reference nucleic acid or amino acid sequence. More typically, the selected sequence and the reference sequence will have at least about 76, 77, 78, 79, 80, 81, 82, 83, 84 or even 85 percent sequence identity, and more preferably, at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequence identity. More preferably still, highly homologous sequences often share greater than at least about 96, 97, 98, or 99 percent sequence identity between the selected sequence and the reference sequence to which it was compared.

[0071] The percentage of sequence identity may be calculated over the entire length of the sequences to be compared, or may be calculated by excluding small deletions or additions which total less than about 25 percent or so of the chosen reference sequence. The reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome. However, in the case of sequence homology of two or more polynucleotide sequences, the reference sequence will typically comprise at least about 18-25 nucleotides, more typically at least about 26 to 35 nucleotides, and even more typically at least about 40, 50, 60, 70, 80, 90, or even 100 or so nucleotides.

[0072] When highly-homologous fragments are desired, the extent of percent identity between the two sequences will be at least about 80%, preferably at least about 85%, and more preferably about 90% or 95% or higher, as readily determined by one or more of the sequence comparison algorithms well-known to those of skill in the art, such as e.g., the FASTA program analysis described by Pearson and Lipman (1988).

[0073] As used herein, the term "structural gene" is intended to generally describe a polynucleotide, such as a gene, that is expressed to produce an encoded peptide, polypeptide, protein, ribozyme, catalytic RNA molecule, or antisense molecule.

[0074] The term "subject," as used herein, describes an organism, including mammals such as primates, to which treatment with the compositions according to the present disclosure invention can be provided. Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, humans, non-human primates such as apes; chimpanzees; monkeys, and orangutans, domesticated animals, including dogs and cats, as well as livestock such as horses, cattle, pigs, sheep, and goats, or other mammalian species including, without limitation, mice, rats, guinea pigs, rabbits, hamsters, and the like.

[0075] The term "therapeutically-practical period" means a period of time necessary for the active agent to be therapeutically-effective. The term "therapeutically effective" refers to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage.

[0076] A "therapeutic agent" may be any physiologically or pharmacologically active substance that may produce a desired biological effect in a targeted site in a subject. The therapeutic agent may be a chemotherapeutic agent, an immunosuppressive agent, a cytokine, a cytotoxic agent, a nucleolytic compound, a radioactive isotope, a receptor, and a pro-drug activating enzyme, which may be naturally occurring or produced by synthetic or recombinant methods, or any combination thereof. Drugs that are affected by classical multidrug resistance, such as vinca alkaloids (e.g., vinblastine and vincristine), the anthracyclines (e.g., doxorubicin and daunorubicin), RNA transcription inhibitors (e.g., actinomycin-D) and microtubule stabilizing drugs (e.g., paclitaxel) may have particular utility as the therapeutic agent. Cytokines may be also used as the therapeutic agent. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. A cancer chemotherapy agent may be a preferred therapeutic agent. For a more detailed description of anticancer agents and other therapeutic agents, those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician's Desk Reference and to Goodman and Gilman's "Pharmacological Basis of Therapeutics" tenth edition, Eds. Hardman et ctl, 2001.

[0077] As used herein, a "transcription factor recognition site" and a "transcription factor binding site" refer to a polynucleotide sequence(s) or sequence motif(s), which are identified as being sites for the sequence-specific interaction of one or more transcription factors, frequently taking the form of direct protein-DNA binding. Typically, transcription factor binding sites can be identified by DNA footprinting, gel mobility shift assays, and the like, and/or can be predicted based on known consensus sequence motifs, or by other methods known to those of ordinary skill in the art.

[0078] "Transcriptional regulatory element" refers to a polynucleotide sequence that activates transcription alone or in combination with one or more other nucleic acid sequences. A transcriptional regulatory element can, for example, comprise one or more promoters, one or more response elements, one or more negative regulatory elements, and/or one or more enhancers.

[0079] "Transcriptional unit" refers to a polynucleotide sequence that comprises at least a first structural gene operably linked to at least a first cis-acting promoter sequence and optionally linked operably to one or more other cis-acting nucleic acid sequences necessary for efficient transcription of the structural gene sequences, and at least a first distal regulatory element as may be required for the appropriate tissue-specific and developmental transcription of the structural gene sequence operably positioned under the control of the promoter and/or enhancer elements, as well as any additional cis sequences that are necessary for efficient transcription and translation (e.g., polyadenylation site(s), mRNA stability controlling sequence(s), etc.

[0080] As used herein, the term "transformation" is intended to generally describe a process of introducing an exogenous polynucleotide sequence (e.g., a viral vector, a plasmid, or a recombinant DNA or RNA molecule) into a host cell or protoplast in which the exogenous polynucleotide is incorporated into at least a first chromosome or is capable of autonomous replication within the transformed host cell. Transfection, electroporation, and "naked" nucleic acid uptake all represent examples of techniques used to transform a host cell with one or more polynucleotides.

[0081] As used herein, the term "transformed cell" is intended to mean a host cell whose nucleic acid complement has been altered by the introduction of one or more exogenous polynucleotides into that cell. [0082] Suitable standard hybridization conditions as applicable to the present disclosure include, for example, hybridization in 50% formamide, 5x Denhardt's solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS and 100 μg/mL of denatured salmon sperm DNA at 42°C for 16 hr followed by 1 hr sequential washes with 0.1 x SSC, 0.1% SDS solution at 60°C to remove the desired amount of background signal. Lower stringency hybridization conditions as set orth in the present disclosure may include, for example, hybridization in 35% formamide, 5x Denhardt's solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS and 100 μg/mL denatured salmon sperm DNA or E. coli DNA at 42°C for 16 hr followed by sequential washes with 0.8x SSC, 0.1% SDS at 55°C. Those of skill in the art will recognize that conditions can be readily adjusted to obtain the desired level of stringency.

[0083] Naturally, the present invention also encompasses nucleic acid segments that are complementary, essentially complementary, and/or substantially complementary to at least one or more of the specific nucleotide sequences specifically set forth herein. Nucleic acid sequences that are "complementary" are those that are capable of base- pairing according to the standard Watson-Crick complementarity rules. As used herein, the term "complementary sequences" means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to one or more of the specific nucleic acid segments disclosed herein under relatively stringent conditions such as those described immediately above.

[0084] As described above, oligonucleotide probes and/or primers useful in the practice of the disclosed embodiments may be of any conventional length. By assigning numeric values to a sequence, for example, the first residue is 1, the second residue is 2, etc. , an algorithm defining all probes or primers contained within a given sequence can be proposed:

[0085] n to n + y,

[0086] where n is an integer from 1 to the last number of the sequence, and y is the length of the probe or primer minus one, where n + y does not exceed the last number of the sequence. Thus, for a 25-basepair probe or primer (i.e., a "25-mer"), the collection of probes or primers correspond to bases 1 to 25, bases 2 to 26, bases 3 to 27, bases 4 to 28, and so on over the entire length of the sequence. Similarly, for a 35-basepair probe or primer (i.e., a "35-mer), exemplary primer or probe sequence include, without limitation, sequences corresponding to bases 1 to 35, bases 2 to 36, bases 3 to 37, bases 4 to 38, and so on over the entire length of the sequence. Likewise, for 40-mers, such probes or primers may correspond to the nucleotides from the first basepair to bp 40, from the second bp of the sequence to bp 41, from the third bp to bp 42, and so forth, while for 50-mers, such probes or primers may correspond to a nucleotide sequence extending from bp 1 to bp 50, from bp 2 to bp 51, from bp 3 to bp 52, from bp 4 to bp 53, and so forth.

[0087] In certain embodiments, it will be advantageous to employ one or more nucleic acid segments of the present invention in combination with an appropriate detectable marker (i.e., a "label,"), such as in the case of employing labeled polynucleotide probes in determining the presence of a given target sequence in a hybridization assay. A wide variety of appropriate indicator compounds and compositions are known in the art for labeling oligonucleotide probes, including, without limitation, fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, etc., which are capable of being detected in a suitable assay. In particular embodiments, one may also employ one or more fluorescent labels or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally less-desirable reagents. In the case of enzyme tags, colorimetric, chromogenic, or fluorigenic indicator substrates are known that can be employed to provide a method for detecting the sample that is visible to the human eye, or by analytical methods such as scintigraphy, fluorimetry, spectrophotometry, and the like, to identify specific hybridization with samples containing one or more complementary or substantially complementary nucleic acid sequences.

[0088] "Treating" or "treatment of as used herein, refers to providing any type of medical or surgical management to a subject. Treating can include, but is not limited to, administering a composition comprising a therapeutic agent to a subject. "Treating" includes any administration or application of a compound or composition of the invention to a subject for purposes such as curing, reversing, alleviating, reducing the severity of, inhibiting the progression of, or reducing the likelihood of a disease, disorder, or condition or one or more symptoms or manifestations of a disease, disorder, or condition. In certain aspects, the compositions of the present invention may also be administered prophylactically, i.e., before development of any symptom or manifestation of the condition, where such prophylaxis is warranted. Typically, in such cases, the subject will be one that has been diagnosed for being "at risk" of developing such a disease or disorder, either as a result of familial history, medical record, or the completion of one or more diagnostic or prognostic tests indicative of a propensity for subsequently developing such a disease or disorder.

[0089] The tern "vector," as used herein, refers to a nucleic acid molecule (typically comprised of DNA) capable of replication in a host cell and/or to which another nucleic acid segment can be operatively linked so as to bring about replication of the attached segment. A plasmid, cosmid, or a virus is an exemplary vector.

[0090] In certain embodiments, it will be advantageous to employ one or more nucleic acid segments of the present invention in combination with an appropriate detectable marker (i.e., a "label,"), such as in the case of employing labeled polynucleotide probes in determining the presence of a given target sequence in a hybridization assay. A wide variety of appropriate indicator compounds and compositions are known in the art for labeling oligonucleotide probes, including, without limitation, fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, etc., which are capable of being detected in a suitable assay. In particular embodiments, one may also employ one or more fluorescent labels or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally less-desirable reagents. In the case of enzyme tags, colorimetric, chromogenic, or fluorogenic indicator substrates are known that can be employed to provide a method for detecting the sample that is visible to the human eye, or by analytical methods such as scintigraphy, fluorimetry, spectrophotometry, and the like, to identify specific hybridization with samples containing one or more complementary or substantially complementary nucleic acid sequences. In the case of so-called "multiplexing" assays, where two or more labeled probes are detected either simultaneously or sequentially, it may be desirable to label a first oligonucleotide probe with a first label having a first detection property or parameter (for example, an emission and/or excitation spectral maximum), which also labeled a second oligonucleotide probe with a second label having a second detection property or parameter that is different (i.e., discreet or discernible from the first label. The use of multiplexing assays, particularly in the context of genetic amplification/detection protocols are well-known to those of ordinary skill in the molecular genetic arts.

[0091] The section headings used throughout are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are specifically incorporated herein in their entirety by express reference thereto. In the event that one or more of the incorporated references define a term in a manner that contradicts the definition of that term in this application, the definition used in the application controls.

EXAMPLE

[0092] The following example is included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the example that follows represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

SOLUBLE FRACTION OF CRKL AS A BIOMARKER FOR ADVANCED BREAST CANCER

[0093] The present example provides an assay for detecting secreted soluble CRKL, and its use as a biomarker for detection of breast cancers, and for correlating serum levels of CRKL to various clinical parameters. For this purpose, membrane-bound and secreted fractions of CRKL were evaluated in human cancer cell lines, orthotopic, breast- cancer xenografts in mice, and in clinical tissue and plasma samples from breast cancer patients and healthy donors. MATERIALS AND METHODS

[0094] Cell Culture. Human breast cancer cell lines MCF7, MDA-MB-231 and SUM159 were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Life Technologies, Grand Island, NY, USA) and 1% penicillin (100 U/mL) and streptomycin (100 μg/mL). Human breast epithelial cell line (MCF10A) was maintained in DMEM/F12 media supplemented with 5% horse serum, 20 ng/mL epidermal growth factor (EGF, PeproTech, Rocky Hill, NJ, USA), 500 μg/mL hydrocortisone (Sigma- Aldrich Co., St. Louis, MO, USA), 100 ng/mL cholera toxin (Sigma- Aldrich), 10 μg/mL insulin (Sigma- Aldrich) and 1% penicillin and streptomycin.

[0095] Animal Models. Patient-derived tumor xenograft (2665 A) and human cell line (MDA-MB-231) orthotopic xenograft sections were obtained from The Methodist Hospital Research Institute (Houston, TX, USA). Human cell line (SUM159) xenograft sections were also collected for CRKL staining analysis. The blood from nude mice grafted with patient derived breast tumors were collected for enzyme linked immunosorbent assay (ELISA). Animal procedures complied with all institutional, state, and federal guidelines.

[0096] Patient Samples. Progressive breast-cancer-tissue microarrays were purchased from the Cancer Diagnosis Program (National Cancer Institute, Bethesda, MD, USA). Ninety-four formalin-fixed and paraffin-embedded, tumor tissue blocks [corresponding to samples from different stages of disease progression classified using TNM (American Joint Committee on Cancer, VI^th Edition) with Tl, T2, T3 and T4] were studied. Twenty-three specimens of normal breast tissue surgically removed with the cancerous tissue were used as controls. Patient related data including the age, disease stage, tumor size, nodal involvement, metastatic status, and estrogen and progesterone receptor status for the included specimens were provided. lOx and 40x images were captured using a fluorescence microscope (Nikon Instruments, Inc.; Melville, NY, USA) and the intensity of the staining was measured using NIS Elements™ software (Nikon Instruments, Inc.).

[0097] Twenty-nine breast cancer patients' sera and nine healthy donor serum samples were purchased from ProMedDx, LLC (Norton, MA, USA) and tested for soluble CRKL fraction by ELISA. As in the tissue microarrays, the serum samples were from patients with different stages of disease progression classified using TNM staging. Twenty-five out of the twenty-nine patients were undergoing treatment. This study was performed using ProMedDx institutional guidelines; informed consent was obtained or implied by return of questionnaires at the physician's site.

[0098] Collection of Conditioned Media (CM) for Evaluation of Secreted CRKL.

Conditioned medium (CM) was collected from SUM159 and MCF10A cell lines. The cells were seeded at 50% confluency overnight, washed with PBS and incubated with serum free media for 24 hrs. The media was then collected, centrifuged at 5,000 rpm for 10 min, and the supernatant was stored at -80°C until the analysis by ELISA.

[0099] Evaluation of Surface-Bound CRKL by Flow Cytometry. Presence of CRKL on the surface of various cells was analyzed using CRKL antibody by flow cytometry analysis. Cells grown to 80% confluency in 6-well plates were harvested and incubated with CRKL antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 45 min followed by incubation with APC-labeled secondary antibody (Santa Cruz Biotechnology, Inc.) for 1 hr. They were then washed and re-suspended in PBS for flow-cytometric analysis. Cells incubated with the secondary antibody alone were used as controls. Cells were measured on a FACS Fortessa™ analyzer (BD Biosciences, San Jose, CA, USA) using 561-nm excitation laser. Twenty -thousand cells were counted for each sample. Analyses of flow cytometric data focused only on live cells using electronic gate to identify the APC-positive events using FACSDiva™ software (BD Biosciences). All studies were performed in triplicate.

[0100] Assessment of Cellular Localization of CRKL Using Immunofluorescence. The pattern of CPJ L staining in cells was analyzed by immunofluorescence. Cells were seeded in 8-chamber slides (BD Biosciences) with 80% confluency, and allowed to attach overnight. After fixation with 4% paraformaldehyde (PFA; Thermo Scientific, Waltham, MA, USA), they were randomly divided into two groups. In one group, the cells were permeabilized with 0.2% Triton™ X-100 (Sigma- Aldrich) for 5 min and washed extensively. In the second group, the cells were not permeabilized, to specifically analyze the membranal fraction of CRKL. Both categories were incubated with CRKL antibody (Santa Cruz Biotechnology, Inc.) at a 1 :500 dilution for 1 hr at room temperature, then washed and incubated with Dy light 594™-conjugated goat anti- rabbit secondary antibody (Thermo Scientific) for 45 min. After the cells were washed, they were fixed again with 4% PFA, mounted with Prolong™ gold-containing DAPI (Life Technologies) and visualized by fluorescence microscopy (Nikon Instruments).

[0101] Enzyme-Linked, Immunosorbent Assay (ELISA). The levels of secreted CRKL in cell culture supernatants, serum samples from mice bearing 2665A breast tumors and breast cancer patients sera were analyzed using commercially available ELISA kits (Cloud-Clone Corp.; Houston, TX, USA) according to manufacturer's instructions. The CRKL concentrations are expressed as pg/mL of blood plasma. The detection limit was 53 pg/mL.

[0102] Evaluation of CRKL Expression in Tumor Samples Using Immunohisto- chemistry. Immunohistochemical staining was performed on paraffin-embedded tumor sections to study the CRKL expression partem. After two washes, the slides were blocked for 10 min in peroxidase followed by 2.5% horse serum (Life Technologies) for lO min; reacted with anti-CRKL antibody (1 : 100) for 30 min; washed three times; incubated for 15 min with horse anti -rabbit IgG; reacted for 4 min with diaminobenzidine; rinsed; counter-stained with hematoxylin; mounted; and imaged. The intensity of staining was determined by image analysis using NIS Elements™ imaging software (Nikon Instruments).

RESULTS

[0103] CRKL Localization on Cell Surface. Based on previous studies, it was expected that the secreted CRKL from tumor cell lines would bind to the surface of the cells through interaction with βΐ integrin. To evaluate the membrane bound fraction of secreted CRKL, non-permeabilized breast cancer cells were analyzed by flow cytometry.

The percentage of cells with surface-bound CRKL varied among different cell lines.

Cancer cell lines (MCF7, MDA-MB-231 and SUM-159) showed 3- to 5-fold higher percentage of staining than mammary epithelial cell line (MCF10A). For instance 93.7% of MCF7 cells have surface bound CRKL when compared to 55.6% on SUM159 cells and only 17% on MCF10A cells (FIG. 1A).

[0104] Immunofluorescence staining of the various cell lines confirmed the presence of protein on the cell surface. The lack of co-localization of nuclear (DAPI) and CRKL staining in non-permeabilized cells confirmed that the observed staining is the surface- bound fraction of CRKL. On the other hand, permeabilization of the cells led to the staining of the cytoplasm and the nuclear region showing that, as expected, a large fraction of the protein is present in intracellular compartments (FIG. IB).

[0105] CRKL Expression in In Vivo and Clinical Tumor Biopsies. The patterns of CRKL expression was evaluated in in vivo breast tumor models originated from a human clinical specimen (2665 A) and human breast-tumor cell lines, MDA-MB-231 and SUM159. MDA-MB-231 xenografts exhibited cytoplasmic/membranal staining for CRKL, while SUM159 and patient derived xenograft 2665A showed stronger nuclear staining with mild cytoplasmic and extracellular staining (FIG. 2A).

[0106] To evaluate the CRKL expression in human breast cancer specimens, paraffin- embedded cancer progression tissue microarrays were immunostained with 94 tumor cores (FIG. 2B). It was noted that normal breast tissue stained positive for CRKL only in the ductal epithelium, therefore, during the pathological examination of the tumor tissue, the regions of ductal epithelium were excluded. More than 90% of the patients were Caucasian. Twenty-seven patients were estrogen receptor (ER) negative and 46 were progesterone receptor (PR)-negative. The average tumor size of the entire set of patients was 2.6 cm (range: 1 to 6.5 cm).

[0107] Similar to the in vivo studies, the staining pattern in the tumor tissue varied from nuclear to cytoplasmic to microenvironmental. Overall, about 85% of the breast cancer clinical samples stained positive for CRKL. The average intensity of CRKL (AU/pixel), as measured by image analysis, was ~12-fold higher in breast tumor biopsies than in the uninvolved breast tissue (46.8 ± 22.1 vs. 3.9 ± 1.9 AU/pixel, respectively, FIG. 2C). Table 1 summarizes the correlation of CRKL staining and the clinicopathological factors:

[0108]

TABLE 1

RELATIONSHIP BETWEEN CRKL EXPRESSION AND CLINICOPATHOLOGICAL

PARAMETERS

Staining intensity of CRKL in breast cancer tissue microarray

Negative Positive

Parameters Number % Number % P

Age

<60 years 10 19.6 41 80.4 0.86

>60 years 9 20.9 34 79.1

T stage

Tl 7 16.2 36 83.8 0.001

T2 8 20 32 80

T3 1 33.3 2 66.7

T4 3 37.5 5 62.5

Nodal involvement

Negative 3 9 30 91 0.007

Positive 11 24.4 34 75.6

Metastatic involvement

Negative 10 16.4 51 83.6 0.08

Positive 9 27.3 24 72.7

Tumor size

< 2 cm 7 15.9 37 84.1 0.22

> 2 cm 12 24 38 76

Estrogen receptor

Negative 4 14.8 23 85.2 0.27

Positive 15 22.4 52 77.6

Progesteron receptor Negative 7 15.2 39 84.8 0.11 Positive 12 25 36 75

[0109] Evaluation of Soluble CRKL in In Vitro and In Vivo. Conditioned medium from cell culture supematants and serum from breast tumor bearing mice was analyzed by ELISA for secreted CRKL. CM from human breast cancer cell line, SUM159, showed over a 20-fold higher CRKL concentration than that from normal breast epithelial cell line, MCFIOA (545 ± 7 vs. 26 ± 5 pg/mL, respectively) (FIG. 3A). Mean serum levels of secreted CRKL from mice bearing human-derived breast tumors were also significantly higher than that of control mice. In particular, mice grafted with patient-derived tumors had about 13-fold higher serum levels of CRKL than control mice (486 ± 130 vs. 37 ± 21 pg/mL), comparable to the results from in vitro studies (FIG. 3B).

[0110] Evaluation of Soluble CRKL in Clinical Samples. Soluble CRKL in sera from 29 breast cancer patients and 10 normal donors were measured by ELISA. The clinical parameters of the patients are given in Table 2.

[0111]

TABLE 2

RELATIONSHIP OF SERUM [CRKL] AND CLINICOPATHOLOGICAL PARAMETERS

Serum

Stage at Diagnosis Current

Age [CRKL]

Staging

N M (pg/mL)

49 Tib Nla M0 1 0

66 Tla NO MX 1 3.31

66 Tic N(i-) M0 1 6.94

56 T2 N0(i+) M0 2 0

52 T2a NO M0 2 0.67

47 Tic Nl N/A 2 1.41

75 T2 Nl M0 2 1.85

44 T2 Nl M0 2 1.91

48 T2 Nlmi M0 2 2.04

76 T2 NO M0 2 2.09

65 T2 NO M0 2 2.32

38 T2 NO M0 2 4.35

59 Tic Nl MX 2 4.36

50 T2 NO M0 2 5.03

55 T2 N/A N/A 2 5.94

69 T2 Nla N/A 2 6.51

63 T2 N2 M0 3 0 41 T3 Nla MO 3 0.04

49 T4 Ν3 MO 3 4.13

47 Τ2 N2a MX 3 7.97

48 Τ2 Ν3 MO 4 2.92

68 Τ2 Nl MO 4 3.19

42 ΤΧ NX Ml 4 4.11

49 Ν/Α N/A N/A 4 4.78

62 Ν/Α N/A N/A 4 5.29

40 Τ2 Nl Ml 4 6.38

63 Τ2 Nl MO 4 8.71

50 Τ2 N3a N/A 4 8.8

67 Τ3 NX Ml 4 8.86

[0112] The median CRKL concentrations in breast cancer patients' sera were more than 1.4-fold higher than that in healthy donors (4400 ± 2560 vs. 1860 ± 1840 pg/mL, respectively,/? < 0.05). Patients with advanced clinical disease (i.e., Stage 3 and Stage 4) exhibited a 2.8-fold (5300 ± 2300 pg/mL, p < 0.005) increase in serum CRKL concentration as compared to healthy donors (FIG. 3C). Serum levels of CRKL were elevated from the median value in more than 40% of patients with early disease (Tl and T2), in more than 90% of patients with advanced disease (T3 and T4), and in 100% of patients with metastatic disease. The sera from patients (n = 4) who had not undergone any treatment had higher values than those patients who were undergoing treatment (5800 ± 2000 pg/mL vs. 3600 ± 2700 pg/mL, respectively,/? = 0.13).

DISCUSSION

[0113] In breast tumors, the majority of clinically-used biomarkers are proteins that are over-expressed in tissues, and they can be detected histologically in specimens from biopsies or following tumor resection. Their expression prescribes various treatment settings and is correlated to disease prognosis (Harris et al, 2007), particularly in the early disease stages. One such biomarker, c-erbB2, led to the discovery of the monoclonal antibody, trastuzumab (Verma et al, 2010). However, the necessitation of invasive biopsies is a drawback that may be addressed by the detection of soluble biomarkers in body fluids. The present invention demonstrates that CRKL, generally considered as an intracellular protein, can serve as a soluble biomarker for breast tumors. The facility of this biomarker has been demonstrated in assays both in vitro and in vivo, and from analysis of clinical samples. [0114] It was previously demonstrated that CRKL is secreted from various cancer cell lines and binds to the extracellular PSI domain of βΐ integrins (Mintz et al, 2009). In agreement with this finding, an increased surface-bound fraction of CRKL was detected in various breast cancer cell lines (FIG. 1A and FIG. IB) when compared to non- cancerous epithelial cell lines. Immunofluorescence staining of breast cancer cell lines revealed CRKL on both the cell surface as well as in the intracellular compartments (FIG. 2A, FIG. 2B, and FIG. 2C)

[0115] In accordance with in vitro studies, CRKL was over-expressed in tumor sections (FIG. 3A, FIG. 3B, and FIG. 3C). Although immunohistochemical staining is not specific for surface-bound CRKL, it is in agreement with previously published data that CRKL is over-expressed in various tumor models, including gastric carcinomas (Natsume et al, 2012), head and neck squamous cell carcinomas (Yanagi et al, 2012) and lung carcinomas (Wang et al, 2012). Recent work has shown that CRKL is over- expressed in 37% of breast cancer samples studied, and it was correlated with progression and malignant proliferation (Zhao et al, 2013). The present invention provides further evidence that tissue-based expression of CRKL can serve as a biomarker for the diagnosis and/or prediction of metastasis of breast cancer in vivo.

[0116] It is generally agreed that intracellular signaling of CRKL is involved in tumor progression of various tumor types, however, the role of soluble (secreted) fraction of CRKL have not been explored. This is the first demonstration that the soluble fractions of CRKL can be detected in cell culture media, and in blood/serum. The results showed significantly-higher levels of soluble CRKL in the serum from breast-tumor xenograft- bearing mice, and breast-tumor patients compared to healthy donors, suggesting the facility of using CRKL as a soluble biomarker for breast cancer diagnosis, progression, and/or metastasis. The most widely used clinical breast cancer biomarker, CA 15.3, serves as a prognostic marker, to monitor response of breast cancer to treatment or detect early relapse (Daniele et al, 2013). However, CA 15-3 is elevated in only 3% of patients with localized early stage cancer, and up to 70% of patients with metastatic disease (Duffy, 2006).

[0117] In the present invention, more than 40% of patients with localized or early- stage disease, and 100% of patients with metastatic disease have elevated serum levels of CRKL. Thus, soluble CRKL has the potential to be a stronger biomarker for predicting metastasis than the conventional standard-of-care. Furthermore, among patients analyzed in this study, four had not undergone treatment, but had mean values that were 1.6-fold higher than the rest of the patients (ft = 25) who were already undergoing treatment. Albeit a small sample size, this clear trend (with a />-value of 0.13), suggested that CRKL levels also show potential in monitoring treatment outcomes of breast cancer.

[0118] In conclusion, the present invention provides a method for monitoring breast cancer using an assay that detects the level of secreted CRKL polypeptide in mammalian plasma. This invention provides the first evidence supporting the use of soluble fractions of CRKL in body fluids as a biomarker for cancer. In particular, the results herein suggest that CRKL can be used as a soluble serum biomarker in breast cancer patients, especially in advanced stages of the disease.

[0119] Amino acids 181-242 mapping within an internal region of Crk-L of human origin are shown in italics:

10 20 30 40 50 MSSARFDSSD RSAWYMGPVS RQEAQTRLQG QRHGMFLVRD SSTCPGDYVL

60 70 80 90 100

SVSENSRVSH YIINSLPNRR FKIGDQEFDH

] 7' 0 " n U 150

PAPRYPSPPM GSVSAPNLPT AEDNLEYVRT

1 16 D0 (~i 117700 1 " .:.8 Q O0 nu 190 200

VIIEKPEEQW WSARNKDGRV GMIPVPYVEK

? " .:. n 9 n n u 50

PEPAHAYAQP QTTTPLPAVS GSPGAAITPL

9 f O i~)i 9 7 n 9 o n u 300

YDKTALALEV GDIVKVTRMN INGQWEGEVN

303

ENE (SEQ ID NO : 1 )

REFERENCES

[0120] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference:

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[0152] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. All references, including publications, patent applications and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0153] The description herein of any aspect or embodiment of the invention using terms such as "comprising," "having," "including," or "containing," with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of," "consists essentially of," or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0154] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and/or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

CLAIMS: WHAT IS CLAIMED IS:

1. A method of diagnosing carcinoma of the breast in a human test subject, the method comprising: a) obtaining a blood sample from a human test subject suspected of having carcinoma of the breast to identify therein at least one soluble CRKL peptide- specific biomarker; and b) determining the concentration of the at least one soluble CRKL peptide- specific biomarker in the blood sample, wherein an increase in the level of the biomarker relative to a mean concentration of CRKL peptide biomarker in a control population of human subjects is indicative of a diagnosis of carcinoma of the breast in the human subject.

The method of claim 1, wherein the level of the at least one soluble CRKL peptide-specific biomarker in the blood of the test subject is at least 50% higher than the mean concentration of soluble CRKL peptide-specific biomarker in blood samples obtained from a control population of human subjects.

3. The method of claim 2, wherein the level of the at least one soluble CRKL peptide-specific biomarker in the blood of the test subject is at least 75% higher than the mean concentration of soluble CRKL peptide-specific biomarker in blood samples obtained from a control population of human subjects.

4. The method of claim 1, wherein an increase in the level of the at least one soluble CRKL peptide-specific biomarker is indicative of the test subject's having a malignant carcinoma of the breast.

The method of claim 4, wherein an increase in the level of the at least one soluble CRKL peptide is indicative of the subject's having an advanced stage of a malignant carcinoma of the breast.

The method of claim 1, wherein an increase in the level of the at least one soluble CRKL peptide-specific biomarker is predictive of an increased risk for metastasis of the carcinoma to one or more non-breast tissues within the body of the subject.

The method of claim 1, wherein determining the level of the at least soluble CRKL peptide-specific biomarker is performed as an adjunct to a standard-of- care clinical laboratory test for diagnosing carcinoma of the breast.

A post-operative method of monitoring the inhibition of breast tumor growth, the method comprising: a) obtaining a series of blood, serum, or plasma samples over time from a human test subject that is post-operative to the removal of a malignant tumor; and b) determining the concentration of a particular CRKL peptide-specific biomarker in each of the blood, serum, or plasma samples, wherein an increased level of the biomarker relative to a mean concentration of the same CRKL peptide-specific biomarker in a control population of human subjects over time is determinative of the degree of post-operative inhibition of the breast tumor growth.

The method of claim 8, further including administering a chemotherapeutic regimen to the subject post-operatively.

10. The method of claim 9, wherein the chemotherapeutic regimen includes the administration of at least one therapeutic dose of a composition selected from the group consisting of cyclophosphamide, methotrexate, fiuorouracil, and combinations thereof.

A method of using the concentration of an endogenously-encoded CRKL protein or peptide to diagnose carcinoma of the breast in a mammal, the method comprising: a) obtaining a blood sample from a mammalian test subject to provide an individual CRKL peptide biomarker diagnostic for carcinoma of the breast; b) comparing the level of the peptide biomarker in the sample from the test subject to a reference sample or that of a control subject; and c) determining an elevated concentration of the CRKL protein biomarker compared to the reference or the control subject sample to diagnose the subject, which is indicative of a diagnosis of carcinoma of the breast in the subject.

The method of claim 11 , wherein the CRKL peptide biomarker is one constituent of an individual panel of biomarkers.

The method of claim 12, wherein the reference is developed for each of a malignant tumor, a benign tumor, and a control group population.

The method of claim 12, wherein the reference sample comprises one or more constituent of a panel of biomarkers specific for cancer. The method of claim 12, wherein the diagnosis is an adjunct to a primary diagnostic test for a carcinoma of the breast.

The method of claim 12, wherein the blood sample is a serum sample or a plasma sample.

A diagnostic assay comprising: a) measuring or quantifying the polypeptide level of a mammalian CRKL protein or peptide biomarker in a test sample of blood or a fraction thereof; and b) comparing the measured or quantified level of the biomarker with a corresponding reference biomarker protein or peptide level, and if the level of CRKL protein or peptide in the test sample of blood or in the fraction thereof is higher than that of the reference biomarker protein or peptide level, identifying the subject as having an increased probability of carcinoma of the breast.

The diagnostic assay of claim 17, comprising an immunoassay or a mass spectrometry assay.

The diagnostic assay of claim 18, wherein the immunoassay is selected from the group consisting of a Western hybridization analysis, an ELISA, a sandwich ELISA, immunoprecipitation, an immunofluorescence assay, a radioimmunoassay, a dot blot assay, and fluorescence-activated cell-sorting. The diagnostic assay of claim 17, wherein the fraction comprises plasma serum.