WO2007075672A2 - Marqueurs pour le pronostic du cancer - Google Patents

Marqueurs pour le pronostic du cancer Download PDF

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WO2007075672A2
WO2007075672A2 PCT/US2006/048411 US2006048411W WO2007075672A2 WO 2007075672 A2 WO2007075672 A2 WO 2007075672A2 US 2006048411 W US2006048411 W US 2006048411W WO 2007075672 A2 WO2007075672 A2 WO 2007075672A2
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cancer
expression
patient
biological sample
antibody
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PCT/US2006/048411
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English (en)
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WO2007075672A3 (fr
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U. Margaretha Wallon
George C. Prendergast
Karen A. Knudsen
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Lankenau Institute For Medical Research
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Publication of WO2007075672A2 publication Critical patent/WO2007075672A2/fr
Publication of WO2007075672A3 publication Critical patent/WO2007075672A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

Definitions

  • 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.
  • TNM scores are insufficient for the accurate prognosis of cancers, such as small ductal carcinomas, the most common form of breast cancer.
  • cancers such as small ductal carcinomas
  • 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.
  • 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.
  • cancer markers 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.
  • 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.
  • the cancer marker protein is detected with an antibody or antibody fragment.
  • kits for performing the methods described above are provided.
  • 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
  • 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.
  • the protein levels of the cancer markers in the biological sample are detected.
  • 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.
  • MT-MMPs Membrane-bound metal loproteinases
  • MMPs matrix metalloproteinases
  • 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 are capable of inhibiting MMPs.
  • TIMP-2 and TIMP-4 can interact with and inhibit MTl-MMP.
  • the increased expression of TIMP-4 is indicative of a poor prognosis in cancer patient, particularly in breast cancer patients.
  • 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.
  • the panel of cancer markers provided in the instant invention may be used for the diagnosis and/or prognosis of any cancer.
  • 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.
  • 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.
  • 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).
  • CLCA2 Ca 2+ -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.
  • the cancer markers further comprises estrogen receptor and HER2.
  • 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.
  • 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.
  • 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.
  • the diagnosis method is also performed prior to the administration of the test compound.
  • 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.
  • 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 5 1 to 3' direction.
  • 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.
  • 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.
  • a vector such as a plasmid or virus vector
  • 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.
  • 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”).
  • 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.
  • T m 81.5°C + 16.6Log [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/#bp in duplex
  • 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 20-25 0 C 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- 20 0 C below the T n , of the hybrid.
  • a moderate stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42 0 C, 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 ⁇ g/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 ⁇ g/ml denatured salmon sperm DNA at 42°C, and washed in 0.1X SSC and 0.5% SDS at 65°C for 15 minutes.
  • primer 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.
  • 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.
  • 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.
  • a non-complementary nucleotide sequence may be attached to the 5' end of an otherwise complementary primer.
  • 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.
  • 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.
  • intron refers to a DNA sequence present in a given gene that is not translated into protein and is generally found between exons.
  • 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.
  • 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.
  • 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.
  • 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.
  • the patient is human.
  • 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.
  • patient refers to human or animal subjects.
  • the patient is a human.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • detecttable 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.
  • detectable labels include, without limitation: biotin, avidin (e.g., streptavidin), chromophore, chemiluminescents, fluorescent compound, a radioisotope, and an enzyme.
  • 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, ⁇ -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, 99 Tc, 14 C, 131 I, 125 I, 3 H, 32 P, and 35 S.
  • Chemiluminescent compounds include, without limitation, luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.
  • a marker 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.
  • a marker e.g., cancer marker
  • Immunohistochemistry 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.
  • 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.
  • Polyclonal and monoclonal antibodies directed toward a protein of interest 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
  • 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.
  • 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).
  • 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.
  • a negative control e.g., the biological sample from a normal patient
  • a healthy individual will also be analyzed in order to allow the comparison of test sample to a healthy individual.
  • 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.
  • 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.
  • antibodies are employed histologically, e.g., in immunofluorescence or immunoelectron microscopy,
  • 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.
  • the biological sample e.g., tissue or surgical biopsy
  • the biological sample is embedded in paraffin and sliced into thin layers with a microtome for mounting on a slide.
  • 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
  • detection can be performed by such means as the use of a gamma counter, a scintillation counter, autoradiography and the like.
  • the cancer marker may be detected using an assay for a biochemical activity (e.g., enzymatic) of the cancer marker.
  • 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.
  • the instant invention also encompasses the detection of polynucleotides (e.g., genes, cDNA, and mRNA) for this purpose.
  • polynucleotides e.g., genes, cDNA, and mRNA
  • 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.
  • 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.
  • agents e.g., antagonists and agonists
  • Such agents include antibodies, peptides, peptidomimetics, ligands, small molecules, and nucleic acid molecules encoding the cancer marker.
  • 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 therapeutic antibodies against P-cadherin.
  • 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.
  • 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, HerceptinTM (Trastuzumab), is approved for the treatment of certain patients with metastatic breast cancer.
  • HerceptinTM Trastuzumab
  • antibodies specific for a cancer marker may be used to deliver a toxic compound to the cell.
  • 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
  • Small molecule modulators e.g., inhibitors or activators
  • 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.
  • 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.
  • compositions useful for the treatment of cancer 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).
  • a cancer marker of the instant invention i.e., BINl, MTl-MMP, TIMP-4 and P-cadherin.
  • 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.
  • a pharmaceutically acceptable carrier e.g., sterile water, saline, buffered saline, or dextrose solution
  • excipient e.g., sterile water, saline, buffered saline, or dextrose solution
  • carrier e.g., sterile water, saline, buffered saline, or dextrose solution
  • excipient e.g., sterile water, saline, buffered saline, or dextrose solution
  • carrier e.g., sterile water, saline, buffered saline, or dextrose solution
  • excipient e.g., sterile water, saline, buffer
  • 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.
  • 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.
  • the intravenous route is most commonly used to deliver therapeutic antibodies and nucleic acids.
  • 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.
  • 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.
  • 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.
  • 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.
  • therapeutic efficacy and toxicity may be determined by standard pharmacological procedures in cell cultures or with experimental animals.
  • the ED 50 the dose that is therapeutically effective in 50% of the treated subjects
  • the LD50 the dose that is lethal to 50% of treated subjects
  • the ED 5 0 ⁇ LD 50 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.
  • 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.
  • the cancer markers are selected from the group consisting of MTl-MMP, TIMP-4, P-cadherin, and Binl.
  • 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 include complementary nucleic acids molecules.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).

Abstract

La présente invention concerne des procédés, des kits et des compositions permettant de diagnostiquer, d'évaluer le pronostic ou de surveiller la progression d'un cancer chez un sujet, en particulier d'un cancer du sein.
PCT/US2006/048411 2005-12-23 2006-12-20 Marqueurs pour le pronostic du cancer WO2007075672A2 (fr)

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EP2691776A1 (fr) * 2011-03-27 2014-02-05 Oncostem Diagnostics (Mauritius) Pvt. Ltd. Marqueurs pour l'identification de cellules tumorales, procédés et trousse correspondants
EP2765140A3 (fr) * 2012-12-13 2015-06-03 Oxford Bio Therapeutics Limited Molécules de liaison mucin-13 ou cadherin-2 ou pour le traitement du cancer
US9884921B2 (en) 2014-07-01 2018-02-06 Pfizer Inc. Bispecific heterodimeric diabodies and uses thereof
US10005836B2 (en) 2014-11-14 2018-06-26 Novartis Ag Antibody drug conjugates
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WO2010001585A1 (fr) 2008-06-30 2010-01-07 Oncotherapy Science, Inc. Anticorps anti-cdh3 marqués par un marqueur radioisotopique, et leurs utilisations
EP2313504A1 (fr) * 2008-06-30 2011-04-27 Oncotherapy Science, Inc. Anticorps anti-cdh3 marqués par un marqueur radioisotopique, et leurs utilisations
JP2011526583A (ja) * 2008-06-30 2011-10-13 オンコセラピー・サイエンス株式会社 放射性同位体標識で標識された抗ｃｄｈ３抗体およびその使用
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US8435749B2 (en) 2008-06-30 2013-05-07 Oncotherapy Science, Inc. Anti-CDH3 antibodies labeled with radioisotope label and uses thereof
US10260104B2 (en) 2010-07-27 2019-04-16 Genomic Health, Inc. Method for using gene expression to determine prognosis of prostate cancer
CN103562723A (zh) * 2011-03-27 2014-02-05 欧恩克斯特姆诊断学（毛里求斯）私人有限公司 用于识别肿瘤细胞的标记、方法及其试剂盒
EP2691776A4 (fr) * 2011-03-27 2015-04-15 Oncostem Diagnostics Mauritius Pvt Ltd Marqueurs pour l'identification de cellules tumorales, procédés et trousse correspondants
EP2691776A1 (fr) * 2011-03-27 2014-02-05 Oncostem Diagnostics (Mauritius) Pvt. Ltd. Marqueurs pour l'identification de cellules tumorales, procédés et trousse correspondants
EP2765140A3 (fr) * 2012-12-13 2015-06-03 Oxford Bio Therapeutics Limited Molécules de liaison mucin-13 ou cadherin-2 ou pour le traitement du cancer
US9884921B2 (en) 2014-07-01 2018-02-06 Pfizer Inc. Bispecific heterodimeric diabodies and uses thereof
US10005836B2 (en) 2014-11-14 2018-06-26 Novartis Ag Antibody drug conjugates
US10626172B2 (en) 2014-11-14 2020-04-21 Novartis Ag Antibody drug conjugates

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