WO2007075672A2

WO2007075672A2 - Prognostic cancer markers

Info

Publication number: WO2007075672A2
Application number: PCT/US2006/048411
Authority: WO
Inventors: U. Margaretha Wallon; George C. Prendergast; Karen A. Knudsen
Original assignee: Lankenau Institute For Medical Research
Priority date: 2005-12-23
Filing date: 2006-12-20
Publication date: 2007-07-05
Also published as: WO2007075672A3

Abstract

Methods, kits, and compositions for diagnosing, assessing the prognosis of, or monitoring the progression of cancer, particularly breast cancer, in a subject are provided.

Description

PROGNOSTIC CANCER MARKERS

By U. Margaretha Wallon George C. Prendergast

Karen A. Knudsen

This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 60/753,619, filed on December 23, 2005. The foregoing application is incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to the fields of oncology and molecular biology. More specifically, the present invention provides methods for diagnosing cancer, particularly breast cancer, in a patient based on the expression level of certain nucleic acid and protein markers associated with cancer.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full. Decisions regarding treatment regimens and options are, in part, based on a patient's prognosis for disease-free survival. Predicting clinical outcome for cancer patients is largely based on the TNM score (tumor size, node status, and detectable distal metastasis). Traditionally, when tumors are smaller than 20 mm in diameter and there is no spread of the disease to other sites, patients have been given a good prognosis. However, with an increasing number of patients being diagnosed with small tumors it is apparent than TNM scores are insufficient for the accurate prognosis of cancers, such as small ductal carcinomas, the most common form of breast cancer. Additionally, the current use of the estrogen receptor and HER2 as predictive markers has produced less than ideal results. Using clinical survival information from breast cancer patients, it is evident that as many as 20% of those diagnosed with small ductal carcinomas are incorrectly identified as patients with good prognosis. Thus, a significant subgroup of patients who would be expected to be disease-free at 5 years after diagnosis will actually suffer a recurrence or succumb to their disease within that period. Due in part to the i acute and long-term morbidity associated with chemotherapy, there is a need to identify the lesser proportion of small tumors that are in fact dangerous, so that they can be treated more aggressively, as appropriate.

SUMMARY OF THE INVENTION

In accordance with the present invention, methods are provided for the diagnosis of cancers, particularly aggressive early-stage cancers, and/or the prognosis of cancer. An exemplary method entails providing a tumor biopsy sample from a cancer patient and determining the pattern of expression of a plurality of defined cancer markers in that sample, to gauge the future behavior of the disease and to assist decisions about suitable clinical treatment. The phrase "cancer markers," as used herein, refers to genes or gene products (e.g., RNA molecules or proteins) which are characteristic of some or all of the cells in a tumor or type of cancer. A cancer marker with diagnostic value can be a gene or gene product expressed in normal, non- cancerous cells, but is characteristic of a type or classification of cancer by, for example, its over-expression or under-expression as compared to its expression in normal, non-cancerous cells. A cancer marker with prognostic value is a gene or gene product for which the over-expression or under-expression confers predictive information about the future aggressiveness of a cancer and/or its response to therapy at the time of diagnosis. In a tumor sample, the patterns of expression of diagnostic and prognostic cancer markers allow one to accurately identify and determine the future course of the disease, respectively.

In a particular embodiment, the cancer markers employed in the methods of the instant invention include MTl-MMP, TIMP-4, P-cadherin, and Binl. The level of expression of the cancer markers may be determined by detecting the presence of the cancer marker protein or nucleic acid molecule. In a particular embodiment, the cancer marker protein is detected with an antibody or antibody fragment.

According to another aspect of the invention, kits for performing the methods described above are provided.

DETAILED DESCRIPTION OF THE INVENTION The instant invention provides a panel of cancer marker genes and their products which are useful for predicting the clinical outcome of a cancer patient. The amount of the gene or gene product (e.g., mRNA, protein) present in a biological sample from a subject can be detected by measuring the expression level of the protein, the mRNA level, or the copy number of the gene. Methods are provided for determining the amount of the marker in a biological sample by contacting the biological sample with a substance that binds or detects the DNA, mRNA, or protein of the marker. In a preferred embodiment, the protein levels of the cancer markers in the biological sample are detected.

In a particular embodiment, the panel of cancer markers of the instant invention comprises at least MTl-MMP, TIMP-4, P-cadherin, and Binl. In another embodiment, the panel of cancer markers comprises at least three cancer markers selected from the group consisting of MTl-MMP, TIMP-4, P-cadherin, and Binl . In yet another embodiment, the panel of cancer markers comprises at least two cancer markers selected from the group consisting of MTl-MMP, TIMP-4, P-cadherin, and Binl.

Binl (a BAR adapter encoding gene; also known as Amphiphysin 2) encodes a MYC interacting adaptor protein. The loss of expression of BINl has been shown to enhance the immune escape of cancer cells (Muller et al. (2005) Nat. Med., 11:312- 9). An exemplary amino acid sequence of human BINl is provided at GenBank Accession No. 000499.

Membrane-bound metal loproteinases (MT-MMPs) participate in the regulation of matrix metalloproteinases (MMPs), a family of enzymes which together are capable of degrading all of the components of the extracellular matrix and thereby facilitate cell migration through the surrounding tissue and vessels walls. As shown hereinbelow, the reduced expression of MTl-MMP is indicative of prognosis in cancer patients, particularly breast cancer patients. In a preferred embodiment, the panel of caner markers comprises MTl-MMP (also referred to as MMP-14). An exemplary amino acid sequence of MTl-MMP is provided at GenBank Accession No. NP_004986.

Tissue inhibitors of metalloproteinases (TIMPs) are capable of inhibiting MMPs. Specifically, TIMP-2 and TIMP-4 can interact with and inhibit MTl-MMP. As shown hereinbelow, the increased expression of TIMP-4 is indicative of a poor prognosis in cancer patient, particularly in breast cancer patients. In a preferred embodiment, the panel of cancer markers comprises TIMP-4. An exemplary amino acid sequence of TIMP-4 is provided at GenBank Accession No. NPJD03247. Cadherins are calcium-dependent cell-cell adhesion molecules. Placental cadherin (P-cadherin) expression in cancer patients has shown a correlation with poor prognosis for the patient. For example, 90% of breast carcinoma patients with P- cadherin negative tumors were alive five years after surgery while only 59% of breast carcinoma patients with P-cadherin positive tumors were alive five years after surgery (Soler et al. (1999) Cancer 86:1263-1272). An exemplary amino acid sequence of P- cadherin is provided at GenBank Accession No. NP_001784.

The panel of cancer markers provided in the instant invention may be used for the diagnosis and/or prognosis of any cancer. In a particular embodiment, the cancer may be selected from the group consisting of, without limitation, cancers of the prostate, colorectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck, skin, melanoma, basal carcinoma, mesothelial lining, white blood cells, lymphoma, leukemia, esophagus, breast, muscle, connective tissue, lung, small-cell lung carcinoma, non-small-cell carcinoma, adrenal gland, thyroid, kidney, or bone; glioblastoma, mesothelioma, renal cell carcinoma, gastric carcinoma, sarcoma, choriocarcinoma, cutaneous basocellular carcinoma, and testicular seminoma. In a particular embodiment, the cancer is breast cancer and, more specifically, small, node-negative breast cancer.

The panel of cancer markers as described above may further comprise other cancer markers. For example, the loss of expression (underexpression (e.g., at least two-fold)) of estrogen receptor, E-cadherin, and/or maspin have been associated with human breast tumor invasiveness, metastatic potential, and/or poor prognosis (see, e.g., Aamdal et al. (1984) Cancer, 53:2525-9; Clark et al. (1988) Semin. Oncol., 15:20-5; Thompson et al. (1992) J. Cell Physiol., 150:534-44; Vleminckx (1991) Cell, 66:107-19; Oka et al. (1993) Cancer Res., 53:1696-701; Zou et al. (1994) Science 263:526-9; Seftor et al. (1998) Cancer Res. 58:5681-5).

Additionally, the expression (or overexpression (e.g., at least two-fold)) of CLCA2 (Ca²⁺-activated chloride channel-2), IGF-IR (Type I insulin-like growth factor receptor), pl85 encoded by c-erbB-2 (HER2), cathepsin D, osteopontin, and/or vimentin have been associated with human breast tumor invasiveness, metastatic potential, and/or poor prognosis (see, e.g., Raymond et al. (1989) J. Pathol. 157:299- 306; Raymond et al. (1989) J. Pathol., 158:107-14; Thompson et al. (1992) J. Cell Physiol., 150:534-44; Gruber et al. (1999) Cancer Res. 59:5488-91; Surmacz et al. (1998) Breast Cancer Res. Treat., 47:255-67; Dunn et al. (1998) Cancer Res., 58:3353-61; Tan et al. (1997) Cancer Res. 57:1199-205; Dhingra et al. (1996) Semin Oncol., 23:436-45; and Revillion et al. (1998) Eur. J. Cancer, 34:791-808; Rochefort, H. (1990) Breast Cancer Res. Treat., 16:3-13; Johnson et al. (1993) Cancer Res., 53:873-7; Rochefort et al. (2000) Clin. Chim. Acta., 291:157-70; Denhardt et al. (1993) FASEB J., 7: 1475-82; Denhardt et al. (1998) J. Cell Biochem. Suppl., 30- 31:92-102; Tuck et al. (2000) J. Cell Biochem., 78:465-75; Tuck et al. (1999) Oncogene, 18:4237-46 (1999); and Singhal et al. (1997) Clin. Cancer Res., 3:605-11). In a particular embodiment, the cancer markers further comprises estrogen receptor and HER2. In a particular embodiment, the instant invention provides a method of diagnosing cancer, particularly aggressive cancers, and/or assessing the prognosis of a cancer patient comprising the steps of (i) providing a biological sample isolated from a subject, (ii) detecting the cancer markers in the biological sample, and (iii) providing diagnostic, prognostic, and/or predictive information based on the detection step. Exemplary detection methods are described hereinbelow.

In a particular embodiment of the instant invention, the diagnosis methods may be repeated on a patient at various times in order to monitor the progression and/or regression of the cancer. The re-testing of the patient may be performed after a treatment in order to assess the efficacy of the treatment. In a further aspect of the instant invention, the diagnosis methods may be used to determine the efficacy of a test compound against cancer. For example, a patient can be treated with the test compound and then, at a later point in time, the diagnosis method of the instant invention can be performed to assess the aggressive character of the tumor following treatment with the test compound. In a particular embodiment, the diagnosis method is also performed prior to the administration of the test compound.

I. Definitions

The following definitions are provided to facilitate an understanding of the present invention.

"Nucleic acid" or a "nucleic acid molecule" as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5¹ to 3' direction. With reference to nucleic acids of the invention, the term "isolated nucleic acid" is sometimes used. This term, when applied to DNA, may refer to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism. Alternatively, this term may refer to a DNA that has been sufficiently separated from (e.g., substantially free of) other cellular components with which it would naturally be associated. "Isolated" is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification.

With respect to single stranded nucleic acids, particularly oligonucleotides, the term "specifically hybridizing" refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non- complementary sequence. Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art. For instance, one common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is set forth below (Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, New York):

T_m= 81.5°C + 16.6Log [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/#bp in duplex

As an illustration of the above formula, using [Na+] = [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T_m is 57°C. The T_m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.

The stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20-25⁰C below the calculated T_m of the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be approximately 12- 20⁰C below the T_n, of the hybrid. In regards to the nucleic acids of the current invention, a moderate stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42⁰C, and washed in 2X SSC and 0.5% SDS at 55°C for 15 minutes. A high stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42°C, and washed in IX SSC and 0.5% SDS at 65°C for 15 minutes. A very high stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42°C, and washed in 0.1X SSC and 0.5% SDS at 65°C for 15 minutes.

The term "primer" as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as appropriate temperature and pH, the primer may be extended at its 3' terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product. The primer may vary in length depending on the particular conditions and requirement of the application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able to anneal with the desired template strand in a manner sufficient to provide the 3' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5' end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.

The term "gene" refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences. The nucleic acid may also optionally include non coding sequences such as promoter or enhancer sequences. The term "intron" refers to a DNA sequence present in a given gene that is not translated into protein and is generally found between exons.

The term "promoter" or "promoter region" generally refers to the transcriptional regulatory regions of a gene. The "promoter region" may be found at the 5' or 3' side of the coding region, or within the coding region, or within introns. Typically, the "promoter region" is a nucleic acid sequence which is usually found upstream (5') to a coding sequence and which directs transcription of the nucleic acid sequence into mRNA. The "promoter region" typically provides a recognition site for RNA polymerase and the other factors necessary for proper initiation of transcription. A "vector" is a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element.

An "expression operon" refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.

As used herein, the term "biological sample" refers to a subset (e.g., portion or extract) of the tissues of a biological organism, its cells (or lysates thereof), or component parts (e.g. biological fluids such as, without limitation, blood, urine, serum, ascites, saliva, plasma, breast fluid, and peritoneal fluid). The biological sample may be freshly harvested or preserved (e.g., frozen, fixed, and/or paraffin embedded). The biological sample may be a surgical biopsy. In a preferred embodiment, the patient is human. In a particular embodiment, breast fluid can be obtained, for example, by nipple aspiration of the milk ducts or by ductal lavage of at least one breast milk duct by methods known in the art.

The term "patient" as used herein refers to human or animal subjects. In a particular embodiment, the patient is a human. As used herein, diagnostic information or information for use in diagnosis is any information that is useful in determining whether a patient has cancer and/or in classifying the cancer into a phenotypic category or any category having significance with regards to the prognosis of or likely response to treatment (either treatment in general or any particular treatment) of the cancer (e.g., aggressive versus non- aggressive).

As used herein, "prognosis" refers to a forecast as to the probable outcome of a disease state (e.g., cancer) a determination of the prospect as to recovery from a disease as indicated by the nature and symptoms of a case, the monitoring of the disease status of a patient, the monitoring of a patient for recurrence of disease, and/or the determination of the preferred therapeutic regimen for a patient. A "good prognosis" may be a prognosis that the patient is expected to be disease free within five year period after diagnosis. A "poor prognosis" may be a prognosis that the patient has a high risk for developing a recurrence or succumbing to the disease within five year period after diagnosis. The term "immunologically specific" refers to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

An "antibody" or "antibody molecule" is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. The term includes polyclonal, monoclonal, chimeric, and bispecific antibodies. As used herein, antibody or antibody molecule contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule such as, without limitation, those portions known in the art as Fab, Fab', F(ab')2 and F(v). The term "detectable label" refers to agents which are capable of generating a measurable signal which allows for the visualization and/or quantification of the compound attached to the agent. Examples of detectable labels include, without limitation: biotin, avidin (e.g., streptavidin), chromophore, chemiluminescents, fluorescent compound, a radioisotope, and an enzyme. Typically, the enzyme yields a colored or fluorescent reaction product following the addition of a suitable substrate. Common enzymes include, without limitation, horseradish peroxidase, urease, alkaline phosphatase, glucoamylase, β-galactosidase, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, asparaginase, glucose oxidase, glucose oxidase plus peroxidase, ribonuclease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase acetylcholinesterase peroxidase, beta- glucuronidase, beta-D-glucosidase, galactose oxidase plus peroxidase, and acid phosphatase. Exemplary fluorescent compounds include, without limitation, fluorescein and derivatives thereof (e.g., fluorescein isothiocyanate), rhodamine and derivatives thereof, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, auramine, dansyl, umbel liferone, luciferin, and 2,3- dihydrophthalazinediones. Radioisotopes include, for example, ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P, and ³⁵S. Chemiluminescent compounds include, without limitation, luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester. A marker, as used herein, refers to a gene or product of gene expression (e.g., RNA or protein) which is characteristic of a particular cell type. In a particular embodiment, the marker is a cancer marker and is characteristic of some or all of the cells in a tumor or type of cancer. Notably, a marker (e.g., cancer marker) can be a gene or gene product expressed in normal (e.g., non-cancerous) cells, but is characteristic of a cell type (e.g., type of cancer) by, for example, its over-expression or under-expression.

Immunohistochemistry, as used herein, refers to methods using histochemical localization of immunoreactive substances, using antibodies as reagents, on or in cells or tissues, which may be frozen or paraffin-embedded samples, by technologies such as, but not limited to, flow cytometry, ELISA, Western and Southwestern blot, and microscopy.

The term protein microarray refers to a spatially defined and separated collection of individual proteins immobilized on a solid surface. As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition of the invention for performing a method of the invention. The instructional material of the kit of the invention can, for example, be affixed to a container which contains a kit of the invention to be shipped together with a container which contains the kit. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and kit be used cooperatively by the recipient.

II. Antibodies

Polyclonal and monoclonal antibodies directed toward a protein of interest (e.g., cancer markers such as those provided in the instant invention) may be prepared according to standard methods, such as those described in Harlow et al. (Using Antibodies: A Laboratory Manual (1998) Cold Spring Harbor Laboratory Press, Cold Spring Harbor), U.S. Patent 6,008,337, the general methods of general methods of

Kohler and Milstein (see, e.g., general methods of Kohler and Milstein (Nature (1975) 256:495-7), Coligan (Current Protocols in Immunology (1991) Wiley/Greene, New York); and Ausubel et al. (Current Protocols in Molecular Biology (2005) John Wiley and Sons, New York). Other techniques are taught by Mayer and Walker (Immunochemical Methods in Cell and Molecular Biology. (1987) Academic Press, London); U.S. Patent No. 4,376,110; Kosbor et al. (Immunol. Today (1983) 4:72); Cole et al. (Proc. Natl. Acad. Sci. USA (1983) 80:2026-2030); and Cole et al. (Monoclonal Antibodies And Cancer Therapy (1985) Alan R. Liss, Inc., pp. 77-96). In a preferred embodiment, the antibodies used in the methods of the instant invention are monoclonal.

Polyclonal or monoclonal antibodies that immunospecifically interact with a protein of interest can be utilized for identifying and purifying such proteins. For example, antibodies may be utilized for affinity separation of proteins with which they immunospecifically interact. Antibodies may also be used to immunoprecipitate proteins from a sample containing a mixture of proteins and other biological molecules.

An exemplary BINl monoclonal antibody, 2Fl 1, is described in DuHadaway et al. (J. Cell. Biochem. (2003) 88:635-42) and is available from Santa Cruz Biochemicals (Santa Cruz, CA). An exemplary P-cadherin monoclonal antibody is described in Soler et al. (Cancer (1999) 86: 1263-1272) and is available from BD Transduction Laboratories (Lexington, KY). An exemplary polyclonal antibody against human MTl-MMP is described in Udayakumar et al. (Cancer Res. (2003) 63:2292-2299) and monoclonal antibodies are described in Aoki et al. (J. Immunoassay Immunochem. (2002) 23:49-68). Monoclonal and polyclonal antibodies to TIMP-4 are available from ACRIS Antibodies GmbH (Germany).

Polyclonal and monoclonal antibodies can be labeled with detectable labels by methods known in the art (see, e.g., Ausubel et al.(2005) (Current Protocols in Molecular Biology, John Wiley and Sons, New York).

III. Detection

The cancer markers of the instant invention may be detected by any method known in the art, such as, without limitation, immunohistochemistry, immunoblot, radioimmunoassays (RIA), enzyme-linked immunosorbent assay (ELISA), protein array, antibody array (see, e.g., Haab, B.B. (Proteomics (2003) 3:2116-2122), fluorescent resonance energy transfer (FRET) assays, and/or detecting modification of a substrate by the cancer marker. Immunofluorescence techniques employing a fluorescently labeled primary or secondary antibody may be used with microscopic, flow cytometric, or fluorimetric detection to detect the presence of the cancer markers. Typically, when performing the detection methods of the instant invention, a negative control (e.g., the biological sample from a normal patient) will also be analyzed in order to allow the comparison of test sample to a healthy individual.

In a particular embodiment, cancer markers may be detected using a variety of techniques that employ an antibody that recognizes the cancer marker polypeptide. These techniques include ELISA, immunoblot, immunohistochemistry, radioimmunoassay, and antibody arrays. Methods employing antibodies for detection may be used with any of the biological samples described hereinabove including, without limitation tissue portion or extract, cells or extracts thereof, and body fluids. Antibodies which are immunologically specific for a cancer marker may be directly detectably labeled. Alternatively, a secondary antibody or agent that recognizes the primary antibody (i.e., the antibody that binds to the cancer marker polypeptide being detected) is detectably labeled.

When the polypeptide to be detected is in a tissue sample (e.g., a biopsy sample), immunohistochemistry is a preferred detection method. Techniques for obtaining tissue and cell samples and performing immunohistochemistry are well known in the art. Indeed, such techniques are routinely used to detect estrogen receptor expression in breast tumor tissue or cell samples. In general, antibodies are employed histologically, e.g., in immunofluorescence or immunoelectron microscopy,

0 for in situ detection of cancer markers. In situ detection may be accomplished by isolating a biological sample (e.g., a surgical biopsy) from a patient and applying thereto an antibody which is optionally detectably labeled. The antibody may be applied by overlaying the antibody onto a biological sample. This procedure allows for the detection of a cancer marker as well as its location and/or distribution.

Various histological methods and staining procedures are known in the art which are particularly suited for certain types of tissues. In a particular embodiment, the biological sample (e.g., tissue or surgical biopsy) is embedded in paraffin and sliced into thin layers with a microtome for mounting on a slide. Preparatory to embedding, the biological sample may be pretreated in various solutions selected in accordance with the particular examination being conducted, e.g, the biological sample may be fixed, dehydrated, cleared, infiltrated with molten paraffin, and optionally stained.

Immunoassays for cancer markers typically comprise incubating a biological sample in the presence of a detectably labeled antibody capable of identifying the cancer marker and detecting the bound detectably labeled antibody by any of a number of techniques well-known to those of skill in the art. The biological sample for these assays may be immobilized onto a solid support such as, without limitation, nitrocellulose, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The solid support may then be washed with suitable buffers followed by treatment with the antibody specific the cancer marker. The solid support may then be washed with the buffer a second time to remove unbound antibody. The amount of antibody bound to the solid support may then be detected.

An exemplary ELISA method comprises: 1) binding the antibody to a substrate; 2) contacting the bound antibody with a biological sample; 3) washing and contacting the above with a secondary antibody bound to a detectable label; and 4) washing and contacting the above with the substrate for the detectable label, if necessary (see, generally, (Voller et al. (1978) J. Clin. Pathol., 31:507-520; Butler J. E. (1981) Meth. Enzymol., 73:482-523; Maggio, E. (ed.) (1980) Enzyme Immunoassay, CRC Press, Boca Raton, FIa.; Ishikawa et al. (eds.) (1981), Enzyme

Immunoassay, Kgaku Shoin, Tokyo; Ausubel et al. (eds.) (2005) (Current Protocols in Molecular Biology, John Wiley and Sons, New York).

When radiolabeled antibodies are employed, detection can be performed by such means as the use of a gamma counter, a scintillation counter, autoradiography and the like.

Various other techniques for detecting the cancer markers identified herein are within the scope of the invention. For example, the cancer marker may be detected using an assay for a biochemical activity (e.g., enzymatic) of the cancer marker. Indeed, MTl-MMP could be detected by monitoring its cleavage of extracellular matrix proteins (see, e.g., Turk et al. (Nature Biotech. (2001) 19:661-667). Cleavage of the substrate may be monitored, for example, by detecting the loss of the substrate or gain of the cleavage products.

Although the detection of polypeptides using antibodies represents the most convenient and least expensive means of determining the level of expression of a cancer marker, the instant invention also encompasses the detection of polynucleotides (e.g., genes, cDNA, and mRNA) for this purpose. Suitable techniques for detecting and analyzing the level of polynucleotides include, without limitation, in situ hybridization, Northern blot, Southern blot, microarray analysis, single-stranded conformational polymorphism analyses (SSCP), and nucleic acid amplification techniques such as PCR (e.g., quantitative PCR) and RT-PCR.

IV. Therapeutics

The instant invention also encompasses the use of cancer marker genes and their expression products as targets for the development of therapeutics. The invention specifically encompasses agonists and antagonists to the cancer marker genes and their expression products. For example, as P-cadherin is overexpressed in cancers, agents which inhibit its activity are desired. Similarly, inasmuch as BINl expression is lowered or lost in cancers, agents which increase or induce its activity are desired. Such agents (e.g., antagonists and agonists) include antibodies, peptides, peptidomimetics, ligands, small molecules, and nucleic acid molecules encoding the cancer marker. Specific examples include, without limitation, nucleic acid molecules, preferably in a vector, encoding MTl-MMP; nucleic acid molecules, preferably in a vector, encoding BINl; small molecule or peptide inhibitors of TIMP-4; therapeutic antibodies against TIMP-4; small molecule or peptide inhibitors of P-cadherin; and therapeutic antibodies against P-cadherin.

Preferably antibodies suitable for use as antagonist therapeutics exhibit high specificity for the target polypeptide and low background binding to other polypeptides. Accordingly, monoclonal antibodies are generally preferred for therapeutic purposes. In the case of breast cancer, antibodies against the HER2/neu/ErbB2 polypeptide represent a paradigm in terms of the development of therapeutic antibodies. The HER2/neu/ErbB2 gene is overexpressed in approximately 25 to 30 percent of metastatic breast tumors, and an antibody against the HER2/neu/ErbB2 polypeptide, Herceptin™ (Trastuzumab), is approved for the treatment of certain patients with metastatic breast cancer. Thus, the utility of therapeutic antibodies directed against polypeptides that are specifically overexpressed in particular tumors subsets is evident.

In another embodiment, antibodies specific for a cancer marker may be used to deliver a toxic compound to the cell. These antibodies may be conjugated directly to a cytotoxic agent such as, without limitation, a toxin (e.g., ricin or diphtheria toxin), a radioactive moiety, and the like.

Antagonists of the instant invention may also function by affecting expression of the gene product (e.g., polypeptide). Reduction in expression of a cancer marker may be achieved by administering, for example, antisense nucleic acid molecules, ribozymes, siRNAs, shRNAs, and the like (see, generally, Ausubel et al.(2005) (Current Protocols in Molecular Biology. John Wiley and Sons, New York).

Small molecule modulators (e.g., inhibitors or activators) of gene expression are also within the scope of the invention and may be detected by screening libraries of compounds. Methods for identifying compounds capable of modulating gene expression are known in the art (see, e.g., U.S. Patent. 5,976,793). The instant invention also encompasses compounds (e.g., inhibitors or activators) that modulate the activity of a cancer marker protein.

As a further alternative, nucleic acid molecules encoding the cancer marker may be used in a method of gene therapy, to treat a patient. Vectors, such as viral vectors have been used in the prior art to introduce genes into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transformation can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide. The transfected nucleic acid may be permanently incorporated into the genome of each of the targeted cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically. A variety of vectors for gene therapy, both viral vectors and plasmid vectors, are known in the art.

The discovery of therapeutics against cancer markers facilitates the development of pharmaceutical compositions useful for the treatment of cancer. These pharmaceutical compositions may comprise at least one therapeutic agent (e.g. an agonist or antagonist) against a cancer marker of the instant invention (i.e., BINl, MTl-MMP, TIMP-4 and P-cadherin). In a preferred embodiment, the pharmaceutical composition comprises therapeutic agents against two of the cancer markers of the instant invention, against three of the cancer markers, or against all four of the cancer markers. When more than one therapeutic agent is to be administered, the therapeutic agents can be administered separately. Such pharmaceutical compositions further comprise at least one pharmaceutically acceptable carrier (e.g., sterile water, saline, buffered saline, or dextrose solution), excipient, carrier, buffer, antibiotic, or stabilizer. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, and intraperitoneal routes. General considerations in the formulation and manufacture of pharmaceutical compositions may be found, for example, in Remington's Pharmaceutical Sciences. 19^th ed., Mack Publishing Co., Easton, Pa., 1995.

The pharmaceutical compositions of this invention can be administered to humans and other animals, in need thereof, by a variety of routes including directly into a tumor (e.g., injection), oral, intravenous, intramuscular, intraarterial, subcutaneous, intraventricular, transdermal, rectal intravaginal, intraperitoneal, topical (as by powders, ointments, or drops), bucal, or as an oral or nasal spray or aerosol. In general the most appropriate route of administration will depend upon a variety of factors including the nature of the compound (e.g., its stability in the environment of the gastrointestinal tract), the condition of the patient (e.g., whether the patient is able to tolerate oral administration), the cancer being treated, etc. At present the intravenous route is most commonly used to deliver therapeutic antibodies and nucleic acids. However, the invention encompasses the delivery of the inventive pharmaceutical composition by any appropriate route taking into consideration likely advances in the sciences of drug delivery.

Whether it is a polypeptide, antibody, peptide, nucleic acid molecule, virus, small molecule or other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a "therapeutically effective amount," i.e., sufficient to show benefit (e.g. amelioration of symptoms, delay of progression, prevention of recurrence, cure, and the like) to the individual. More specifically, in accordance with the instant invention, the therapeutic agents or pharmaceutical compositions are administered to treat and or prevent cancer, particularly breast cancer, in a patient in a therapeutically effective amount and for such time as is necessary to achieve the desired result. In general, to determine the risk/benefit ratio of the pharmaceutical composition, therapeutic efficacy and toxicity may be determined by standard pharmacological procedures in cell cultures or with experimental animals. For example, the ED₅₀ (the dose that is therapeutically effective in 50% of the treated subjects) and the LD50 (the dose that is lethal to 50% of treated subjects) may be determined. The ED₅0ΛLD₅₀ may represent the therapeutic index of the compound. While it is preferred to have a large therapeutic index, smaller therapeutic indexes may be acceptable in the case of a serious disease such as cancer, particularly breast cancer. The ultimate selection of an appropriate range of doses for administration to humans is typically determined in the course of clinical trials with a physician determining the final specific amount to be administered.

V. Kits

Kits are provided for assessing the presence of cancer cells in a biological sample and thereby diagnosing the presence of aggressive early stage cancer in a patient. The kits of the instant invention comprise at least one agent capable of binding specifically with a cancer marker nucleic acid molecule or polypeptide. In a particular embodiment, the cancer markers are selected from the group consisting of MTl-MMP, TIMP-4, P-cadherin, and Binl. In a preferred embodiment, the agent for specifically binding with a cancer marker polypeptide is an antibody. The antibody may be detectably labeled or a secondary, detectably labeled antibody which recognizes the primary antibody may also be provided. Suitable reagents for specifically binding with a cancer marker nucleic acid molecule (e.g., genomic DNA, mRNA, cDNA, or the like) include complementary nucleic acids molecules. For example, the complementary nucleic acid reagents may include oligonucleotides (e.g., probes) which are optionally detectably labeled and optionally attached to a solid support, pairs of PCR primers, and the like.

The kit may contain further components such as buffers suitable for specifically binding complementary nucleic acid molecules or for binding an antibody with a protein with which it specifically binds. The kit may also further comprise at least one sample container. The kits may also further comprise instructional material.

Inasmuch as the instant invention encompasses the increased or decreased expression of cancer markers for the diagnosis of aggressive early stage cancer, the level of expression of the cancer marker in a normal (i.e. non-cancerous) and/or cancerous biological sample is helpful in determining the increased or decreased expression of a cancer marker in a biological sample from a patient. Accordingly, the instant kits may also further comprise biological samples from normal patients and/or cancer patients as negative and positive controls, respectively. These biological samples may be, for example, slides of surgical biopsies previously stained and fixed demonstrating the normal and/or cancerous expression of the cancer marker (e.g., paraffinized, archived human tissue sample). In another embodiment, the kits may comprise, in the alternative or in addition to the above biological samples, isolated cancer marker nucleic acid molecules and/or proteins at a known concentration. Such kits may further comprise information on the average range of expression for the cancer marker nucleic acid molecules and/or proteins in normal tissue and/or cancerous tissue for comparison to the level of expression of the cancer marker in a biological sample.

The examples set forth below are provided to better illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.

EXAMPLE I

The accuracy and reliability of prognostic evaluations was determined for patients with small ductal breast carcinomas without spread of the disease. As seen in Table I, the use of P-cadherin and TIMP-4 reduced the frequency of patients being given an inaccurate prognosis was reduced by about 50%. Better early detection allows for earlier treatment and increased long-term survival. % of patients given Prognostic Method incorrect prognosis

TNM score* 20%

P-cadherin marker 13%

P-cadherin and TIMP-4 10%

Table I: Proportion of patients given incorrect prognosis at time of diagnosis of early- stage breast cancer based on different predictive methods. * clinical standard.

EXAMPLE II The expression of the novel tumor suppressor and MYC-interacting adaptor protein BINl and the cell adhesion molecule P-cadherin in invasive breast carcinomas was studied. Using immunohistochemistry, archived breast tumors from 65 patients with invasive breast carcinomas were examined for loss of BINl expression and the presence of P-cadherin. The findings were related to tumor size, type, grade, node status, and estrogen and progesterone receptor expression.

Nineteen patients (30%) lost BINl expression. In comparison, 20 patients (31%) were estrogen/progesterone receptor (ER/PR) negative and 33 patients (51%) had positive P-cadherin expression. The outcome provided by these 3 markers (BINl, ER/PR, P-cadherin) provides further confidence in the prognostic evaluation. BINl expression was independent of node status, tumor grade, and tumor size, but BINl expression was significantly correlated with P-cadherin expression (p<0.001).

While the invention has been described in detail and with reference to specific examples thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims

What is claimed is:

1. A method of diagnosing and/or assessing the prognosis of cancer in a patient, the method comprising: a) providing a biological sample obtained from a patient, and b) comparing the level and/or pattern of expression of cancer markers in said biological sample from said patient with the level of expression of said cancer markers in a biological sample from a normal patient, wherein a difference in the level and/or pattern of expression of said cancer markers is indicative of said cancer in said patient, and wherein said cancer markers comprise MTl-MMP, TIMP-4, P-cadherin, and

Binl.

2. The method of claim 1, wherein said cancer is breast cancer.

3. The method of claim 2, where said breast cancer is small, node-negative breast cancer.

4. The method of claim 1, wherein the biological sample obtained from the patient is derived from the same tissue type as the biological sample obtained from a normal patient.

5. The method of claim 1, wherein an increase in the expression of P-cadherin is indicative of cancer.

6. The method of claim 1, wherein an increase in the expression of TIMP-4 is indicative of cancer.

7. The method of claim 1, wherein a decrease in the expression of MTl-MMP is ' indicative of cancer.

8. The method of claim 1, wherein a decrease in the expression of Binl is indicative of cancer.

9. The method of claim 1, wherein the biological sample is a surgical biopsy.

10. The method of claim 1, wherein the biological sample is obtained from the breast.

11. The method of claim 1, wherein the level of expression of said cancer markers is determined by detecting the presence of cancer marker proteins.

12. The method of claim 11, wherein the presence of said cancer marker proteins is detected using a reagent which specifically binds with said cancer marker proteins.

13. The method of claim 12, wherein the reagent which specifically binds with said protein is an antibody or an antibody fragment.

14. The method of claim 13, wherein said antibody or antibody fragment is detectably iabeled.

15. The method of claim 1, wherein said level of expression of said cancer markers is determined by detecting cancer marker nucleic acid molecules.

16. The method of claim 15, wherein said cancer marker nucleic acid molecules are rnRNA or cDNA.

17. The method of claim 1, further comprising determining the prognosis of said patient.

18. The method of claim 1, further comprising repeating steps a) and b) at a later time, thereby monitoring the progression of said cancer in said patient.

19. A method of assessing the efficacy of a test compound as a therapeutic agent against cancer, said method comprising: a) performing the method of claim 1; b) administering said test compound to said patient; and c) performing the method of claim 1; wherein a difference in the level of expression of said cancer markers is indicative the efficacy of said test compound against said cancer.

20. A kit for performing the method of claim 1, the kit comprising reagents for assessing the expression of said cancer markers in a biological sample from a subject.

21. The kit of claim 20, wherein the kit comprises at least one reagent which specifically binds to a cancer marker protein.

22. The kit of claim 21, wherein said at least one reagent is an antibody or antibody fragment.

23. The kit of claim 22, wherein said at least one reagent includes at least one antibody or antibody fragment immunologically specific for each of MTl-MMP, P- cadherin, TIMP-4, and Binl.

25. The kit of claim 22, wherein said antibody or antibody fragment is detectably labeled.

26. A method of assessing the prognosis of cancer in a patient, the method comprising: a) providing a biological sample obtained from a patient, and b) determining the level and/or pattern of expression of cancer markers in said biological sample from said patient, wherein the level and/or pattern of expression of said cancer markers is indicative of the prognosis of said cancer in said patient, and wherein said cancer markers comprise MTl-MMP, TIMP-4, P-cadherin, and

Binl.