JP2009532029A - Cancer prediction and prognostic methods and cancer treatment monitoring - Google Patents

Abstract

  The present invention relates to the use of biomarkers and biomarkers for cancer prediction and prognosis, and the use of biomarkers to monitor the effectiveness of cancer treatment.

Description

  The present invention relates to the use of biomarkers and biomarkers for cancer prediction and prognosis, as well as the use of biomarkers to monitor the effectiveness of cancer therapy.

  A number of pathological conditions can occur at different gene expression levels through changes in gene DNA copy number or through changes in transcription levels of specific genes (eg, through initiation control, provision of RNA precursors, RNA treatment, etc.). Characterized by differences. For example, gene loss and gain and malignant transformation. Play an important role in the progression. These gains and losses are thought to be driven by at least two genes, oncogenes and tumor suppressor genes. Oncogenes are positive regulators of tumor formation and tumor suppressor genes are negative regulators of tumorigenesis (Marshall, Cell 64: 313-326, 1991; Weinberg, Science 254: 1138-1146, 1991). Therefore, one mechanism for activating unregulated growth is to increase the number of genes encoding oncogene proteins or increase the expression levels of these genes (eg in response to cellular or environmental changes). Yes, and other mechanisms are to lose genetic material or reduce the expression level of genes encoding tumor suppressors. This model is supported by the loss and gain of genetic material associated with glioma (Mikkelson, et al., J. Cellular Biochem. 46: 3-8, 199). Thus, changes in expression (transcription) levels of specific genes (eg, oncogenes or tumor suppressor genes) serve as a guide to the presence and progression of various cancers.

  The present invention relates to the use of biomarkers and biomarkers for cancer prediction and prognosis, and the use of biomarkers to monitor the effectiveness of cancer treatment. The invention also relates to the use of soluble metalloproteinases (MMP) and interleukins (IL) or colony stimulating factor (CSF) as biomarkers for the effectiveness of treatment with sorafenib. The biomarker can be selected from the list listed in Table 1, for example MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen. Etc.

  In one embodiment of the invention, the biomarker comprises one or more genes and / or gene products that demonstrate altered expression after exposure to the drug. In a further embodiment, the drug is sorafenib and its derivatives, and in another embodiment, the bioacre is MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4Al, And / or a FOS-like antigen.

  Another embodiment of the present invention is a method of screening for the effect of a drug on a tissue or cell sample comprising the step of analyzing the expression level of one or more genes and / or gene products, wherein the tissue Alternatively, gene expression and / or gene product levels in a cell sample are analyzed before and after exposure to the drug, and variation in the expression level of the gene and / or gene product is an indication of drug effect or patient for the treatment Provide diagnostics or forecasts for responses. In further embodiments, the gene or gene product is MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or a FOS-like antigen.

  Another embodiment of the present invention is a novel drug discovery method comprising the step of analyzing the expression level of one or more genes and / or gene products, wherein the gene expression and / or gene products of a cell Levels are analyzed before and after exposure to the drug, and variations in gene and / or gene product expression levels are an indication of drug efficacy. In a further aspect, the gene or gene product is MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen.

  The invention further provides a method of identifying a compound useful for the treatment of cancer, the method comprising administering a test compound to a subject having cancer and measuring the activity of the polypeptide, wherein A change in activity is an indication that the test compound is useful in the treatment of cancer. In further embodiments, the polypeptide is MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen, and in other embodiments, The compound is sorafenib.

  The present invention can thus be used to identify compounds that can act as modulators or modulators such as agonists and antagonists, partial agonists, inverse agonists, activators, coactivators, and inhibitors, for example. Provide a method. Accordingly, the present invention provides reagents and methods for modulating the expression of polynucleotides or polypeptides associated with cancer. A reagent that modulates the expression, stability or amount of a polypeptide, or the activity of a polypeptide can be a protein, peptide, peptidomimetic, nucleic acid, nucleic acid analog (eg, peptide nucleic acid, locked nucleic acid), or small molecule. it can.

  The present invention also provides a method for providing patient diagnosis comprising the step of analyzing the expression level of one or more genes and / or gene products, wherein the gene expression and / or gene product level of normal and patient samples is A variation in the expression level of the gene and / or gene product in the patient sample that is analyzed is a diagnosis of the disease. Patient samples include, but are not limited to, blood, amniotic fluid, plasma, semen, bone marrow, and tissue biopsy. In further embodiments, the gene or gene product is MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or a FOS-like antigen.

  The present invention still further provides a method of diagnosing cancer in a subject comprising measuring the activity of a polypeptide in a subject suspected of having cancer, and a subject not suspected of having cancer If there is a difference in the activity of the polypeptide compared to the activity of the polypeptide in it, then the subject is diagnosed as having cancer. In further embodiments, the polypeptide is MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or a FOS-like antigen.

  In another embodiment, the present invention provides a method of detecting cancer in a patient sample, wherein an antibody against a protein is used to react with the protein in the patient sample. In further embodiments, the antibody is specific for MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen.

  Another aspect of the invention is a method for distinguishing between normal and pathological conditions comprising the step of analyzing the expression level of one or more genes and gene products, wherein the gene expression of normal and pathological tissues and Gene product levels are analyzed and variations in gene and / or gene product expression levels are indicative of a pathological condition. In a further aspect, the gene or gene product is MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or a FOS-like antigen.

  In another embodiment, the invention relates to a method for determining a phenotype of a cell comprising detecting differential expression compared to normal cells of at least one gene, wherein the gene is at least a factor of 2, at least a factor of 5. Hysteresis is expressed by at least a factor 20 or at least a factor 50. In further embodiments, the gene encodes MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen.

  For example, blood, urine, saliva, feces (for extracting nucleic acids, see for example US Pat. No. 6,177,251), swabs containing tissue, biopsy tissues, tissue sections, cultured cells, etc. Any sample that wishes to identify a polynucleotide or polypeptide can be used.

  Detection is other genes such as brain, heart, kidney, spleen, thymus, liver, stomach, small intestine, colon, muscle, lung, testis, placenta, pituitary, thyroid, skin, adrenal gland, pancreas, salivary gland, uterus, ovary, Prostate, peripheral blood cells (T cells, lymphocytes, etc.), embryo, normal breast fat, adult and fetal stem cells, endothelium, epidermis, myocytes, fat, luminal endothelium, basophilic epithelium, myoepithelial, stromal cells, etc. Other pathologic conditions such as cancer, etc., in combination with polynucleotide probes for genes expressed in cells.

  In yet another embodiment, the invention relates to a method for determining a cellular phenotype comprising detecting differential expression of at least one polypeptide compared to normal cells, wherein the protein has a factor of at least 2. , Differentially expressed up to at least coefficient 5, at least coefficient 20, and at least coefficient 50. In further embodiments, the polypeptide is MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or a FOS-like antigen.

  In another embodiment, the present invention provides a nucleic acid probe comprising a nucleotide sequence having at least about 10, at least about 15, at least about 25, or at least about 40 consecutive nucleotides, and a sample of cells from a patient And preparing a sample of cells, optionally substantially all non-cancerous, contacting a nucleic acid probe with each of said first and second cell samples under stringent conditions, and (a) a first Determining the phenotype of a cell from a patient by comparing the amount of hybridization of the probe with mRNA of a second cell sample with the amount of hybridization of the probe with mRNA of a second cell sample With respect to the method, wherein the amount of hybridization with the first cell sample mRNA is such that the second cell support Compared to the amount of hybridization of the pull mRNA, at least a factor 2, at least a factor 5, the difference of at least a factor 20 or at least a factor 50, an indication of the phenotype of the first cell sample. In further embodiments, the nucleic acid probe is a nucleotide sequence encoding MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen.

  In another embodiment, the present invention provides a test kit for identifying the presence of a cancer cell or tissue comprising a probe / primer for measuring the level of nucleic acid in a sample of cells isolated from a patient I will provide a. In some embodiments, the kit comprises instructions for using the kit, a solution for suspending or fixing the cell, a detectable tag or label, a solution for sensitizing the nucleic acid to hybridization, a cell A solution for dissolving or for purification of the nucleic acid. In further embodiments, the probe / primer comprises a nucleotide sequence (or to it) encoding MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or a FOS-like antigen. Complementary sequence).

  In one embodiment, the present invention provides a test kit for identifying the presence of cancer cells or tissues comprising an antibody specific for a protein. In some embodiments, the kit contains a solution for suspending or fixing cells, a detectable tag or label, a solution for sensitizing nucleic acids to hybridization, a solution for lysing cells, or a nucleic acid. A solution for purification can further be included. In further embodiments, the probe / primer comprises a nucleotide sequence (or to it) encoding MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or a FOS-like antigen. Complementary sequence).

  In other embodiments, the present invention demonstrates the efficacy of a compound or therapeutic agent in cancer cells or tissues that includes a probe / primer for measuring the level of nucleic acid in a sample of cells isolated from a patient. Provide a test kit for monitoring. In some embodiments, the kit comprises instructions for using the kit, a solution for suspending or immobilizing cells, a detectable tag or label, a solution for sensitizing nucleic acids to hybridization, a cell It may further comprise a solution for lysis or a solution for purifying the nucleic acid. In further embodiments, the probe / primer comprises a nucleotide sequence encoding MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen.

  The present invention also relates to a combination of a multikinase inhibitor (see Examples below) and a prodrug that is selectively activated in hypoxic tissue. For example, quinone bioreducible prodrugs are selectively activated in tissues where they can exert a cytotoxic effect. See, for example, Seow et al. Proc Natl Acad Sci USA, 2005, Jun 28; 102 (26): 9282-7.

  In one embodiment, the present invention provides a test kit for monitoring the effectiveness of a compound or therapeutic agent in a cancer cell or tissue comprising an antibody specific for a protein. In some embodiments, the kit further comprises instructions for using the kit. In some embodiments, the kit is a solution for suspending or fixing cells, a detectable tag or label, a solution for sensitizing a polypeptide to antibody binding, a solution for lysing cells, or A solution for purifying the polypeptide can further be included. In further embodiments, the antibody is specific for MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen.

  It is to be understood that the present invention is not limited to the particular methodologies, protocols, cell lines, animal species and genera, configurations, and reagents described, and may be varied. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention as recited in the claims.

  The singular forms used herein and in the claims also include the plural unless the context clearly dictates otherwise. Thus, a reference to one gene is a reference to one or more genes and includes equivalents known to those skilled in the art.

  Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods, devices and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, and the preferred methods, devices and materials are now described.

US provisional patent application no. All publications and patents mentioned herein, including 60 / 787,693, describe and disclose the configurations and methodologies described in the publications that may be used, for example, with respect to the invention described herein. So here is a reference. The publications discussed above and throughout the specification are provided for their disclosure prior to the filing date of the present application. The inventor should not be construed as accepting such disclosure as a prior invention.
[Definition]

  For convenience, the meaning of some terms and phrases used in the specification, examples, and claims are provided below.

  An “address” on an array (eg, a microarray) refers to the location where an element, eg, an oligonucleotide, is attached to the solid surface of the array.

  The term “agonist” as used herein refers to an agent that mimics or upregulates (eg, enhances or reinforces) the biological activity of a protein. The agonist can be a wild type protein or a derivative thereof having at least one biological activity of the wild protein. An agonist can also be a compound that upregulates the expression of a gene or increases at least one biological activity of a protein. An agonist can also be a compound that increases the interaction of a polypeptide with another molecule, such as a target peptide or nucleic acid.

  “Amplification” as used herein refers to the production of additional copies of a nucleic acid sequence. For example, amplification may be accomplished by polymerase chain reaction (PCR) techniques well known to those skilled in the art (see, eg, Jeffenbach and Dreksler (1955) PCR Primer, A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY). Can be implemented.

The term “antibody” as used herein is intended to include, for example, all antibodies of isotype (IgG, IgA, IgE, etc.) and fragments thereof that specifically react with vertebrate (eg, mammalian) proteins. To do. Antibodies can be fragmented using conventional techniques, and the fragments can be screened for utility in the same manner as described for whole antibodies. Thus, this term includes segments of an enzymatically or recombinantly produced portion of an antibody molecule that can selectively react with certain proteins. Non-limiting examples of such enzymatic degradation or recombinant fragments include Fab, F (ab ¹ ) 2, Fab ′, Fv and v [L] and / or v [H] domains joined by a peptide linker. Includes single chain antibodies (scFv). The scFv can be linked covalently or non-covalently to form an antibody having two or more binding sites. The present invention includes polyclonal, monoclonal or other purified preparations of antibodies and recombinant antibodies.

The term “array” or “matrix” refers to an addressable location or arrangement of addresses on a device. The locations can be arranged in a two-dimensional array, a three-dimensional array, or other matrix format. The number of positions can range from several to at least several hundred thousand. Most importantly, each position represents a completely independent reaction site. “Nucleic acid array” refers to an array containing nucleic acid probes, such as oligonucleotides or larger portions of genes. An array in which the probes are oligonucleotides is called an “oligonucleotide array” or “oligonucleotide chip”. A “microarray”, also referred to herein as a “biochip” or “biological chip”, is an array of regions having discrete region densities of at least about 100 / cm ² , and preferably at least about 1000 / cm ² . The area of the microarray has typical dimensions, for example a diameter in the range of about 10-250 μm, and is about the same distance away from other areas in the array.

  As used herein interchangeably, “biological activity” or “bioactivity” or “activity” or biological function ”refers to a polypeptide (in its native or denatured conformation) or by its subsequences. By effector or antigen function performed directly or indirectly. Biological activity includes binding to a polypeptide, binding to other proteins or molecules, activity as a DNA binding protein, activity as a transcriptional regulator, the ability to bind to damaged DNA, and the like. Bioactivity can be modulated by directly affecting the polypeptide of interest. Alternatively, bioactivity can be altered by modulating the level of the polypeptide, such as by modulating the expression of the corresponding gene.

  The term “biological sample” as used herein refers to a sample obtained from an organism or a component of an organism (eg, a cell). The sample can be any biological tissue or fluid. The sample can be a sample obtained from a patient, but saliva, blood, blood cells (eg, white blood cells), tissue or biopsy samples (eg, tumor biopsy), urine, ascites, and pleural fluid, or cells therefrom Including but not limited to. A biological sample can include a section of tissue, such as a frozen section taken for histological purposes.

  The term “biomarker” or “marker” encompasses a wide range of intracellular and extracellular events and all biophysiochemical changes. Biomarkers include virtually any aspect of cell function, including but not limited to the production level or rate of signal generating molecules, transcription factors, metabolites, gene transcripts, and post-translational modifications of proteins. Biomarkers can include whole genome analysis at the transcription level or whole protelome analysis at the protein level and / or modification.

  A biomarker can also refer to a gene or gene product that is up- or down-regulated in a diseased cell treated with a compound of interest in comparison to an untreated diseased cell. That is, a gene or gene product is sufficiently specific for a treated cell that can be used, optionally with other genes or gene products, to identify, predict or detect the effectiveness of a small molecule. Thus, a gene or gene product is a gene or gene product that is characteristic of the effectiveness of the compound in the diseased cell or the response of the diseased cell to treatment with the compound.

  A nucleotide sequence is “complementary” to another nucleotide sequence if the bases of the two sequences match, ie, can form a Watson-Crick base pair. The term “complementary strand” is used herein interchangeably with the term “complement”. The complement of nucleic acid strands can be the complement of a coding strand or the complement of a non-coding strand.

  “Gene detection agent” is used to specifically detect the gene, or other biological molecule related thereto, such as RNA transcribed from the gene or a polypeptide encoded by the gene. Refers to an agent capable of Exemplary detection agents are nucleic acid probes that hybridize to nucleic acids corresponding to the gene, and antibodies.

  “Baseline level” can refer to a standard control for normal levels (ie, patients without disease), but can also be relatively. That is, when the low baseline level is compared to the levels of other subjects with the disease.

  The term “cancer” includes solid cancers such as breast, respiratory, brain, genital, digestive, urinary, eye, liver, skin, head and neck, thyroid, parathyroid, and their distant metastases. Not limited to that. The term includes lymphoma, sarcoma and leukemia.

  Examples of breast cancer include, but are not limited to, invasive ductal malignancy, invasive lobular malignancy, in situ ductal malignancy, and in situ lobular malignancy.

  Examples of respiratory cancers include, but are not limited to, small and non-small cell lung malignancies, bronchial adenomas, and pleuropulmonary blastomas.

  Examples of brain cancer include, but are not limited to, brainstem and hypothalamic glioma, cerebellar and cerebral astrocytoma, medullary blastoma, ventricular ependymoma, and neuroexternal lobe and pineal tumor.

  Male genital tumors include, but are not limited to, prostate and testicular cancer.

  Gastrointestinal tumors include, but are not limited to, anus, colon, colorectum, esophagus, gallbladder, stomach, pancreas, rectum, small intestine and salivary gland cancer.

  Urological tumors include, but are not limited to, bladder, penis, kidney, renal pelvis (eg RCC), ureter and urethral cancer.

  Eye cancers include, but are not limited to intraocular melanoma and retinoblastoma.

  Examples of liver cancer include, but are not limited to, hepatocellular malignancies (hepatocellular malignancies with or without fibrous layered variants), hepatic duct cancer (intrahepatic bile duct cancer), and mixed hepatocyte and bile duct cancer .

  Skin cancer includes, but is not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel skin cancer, and non-melanoma skin cancer.

  Head and neck cancer includes, but is not limited to, maxilla / mandible / lower pharynx / nasopharynx / oropharyngeal cancer, and lip and oral cancer.

  Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphoma.

  Sarcomas include, but are not limited to, soft tissue sarcomas, malignant fibrous tissue tumors, lymphosarcoma, and rhabdomyosarcoma.

  Leukemia includes, but is not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

  A “cancer disease cell” refers to a cell present in a subject with cancer. That is, a modified form of normal cells that is significantly higher or lower in cells with cancer compared to cells that do not exist in subjects without cancer or who do not have cancer It is a cell.

  The term “equivalent” is understood to include nucleotide sequences that encode functionally equivalent polypeptides. Equivalent nucleotide sequences can include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants.

  The term “expression profile” used herein interchangeably with “gene expression profile” refers to a set of values representing the mRNA level of one or more genes in a cell. Expression levels preferably include values representing expression levels of at least about 10 genes, preferably at least 50, 100 or more. Expression profiles can also include mRNA levels of genes that are expressed at similar levels in multiple cells and conditions (eg, housekeeping genes such as GAPDH). For example, an expression profile of a cancerous pathological cell refers to a set of values that are representative of mRNA levels of 10 or more genes in the diseased cell.

  The term “gene” refers to a nucleic acid sequence that includes control and coding sequences necessary for the production of a polypeptide or precursor. A polypeptide can be encoded by a full-length coding sequence or by any portion of this coding sequence. A gene can be any or all known in the art, including plant, mold, animal, bacterial genome or episome, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA, or chemically synthesized DNA It can also be obtained from sources. A gene can include one or more modifications in the coding or untranslated region that can affect the aspect of the expression product, expression rate, or expression control. Such modifications include, but are not limited to, one or more nucleotide mutations, insertions, deletions and substitutions. A gene may comprise an uninterrupted coding sequence or may contain one or more introns joined by appropriate splice junctions.

  “Hybridization” refers to any process in which one strand of nucleic acid binds to a complementary strand by base pairing. For example, two single stranded nucleic acids “hybridize” when they form a double stranded duplex. The double-stranded region can comprise the full length of one or both of the single-stranded nucleic acids, or all of one single-stranded nucleic acid and the other single-stranded nucleic acid subsequence, or the double-stranded region can be A subsequence of each nucleic acid can be included. Hybridization can also include the formation of duplexes containing several mismatches as long as the two strands still form a double stranded helix. “Strict hybridization conditions” refers to hybridization in which specific hybridization occurs in nature.

  The term “isolated” as used herein with respect to nucleic acids such as DNA or RNA refers to molecules that have been separated from other DNA or RNA present in the natural source of the macromolecule. The term “isolated” also refers to chemical precursors or other chemicals that are free of cellular material, viral material, cell culture, or chemically synthesized when produced by recombinant DNA technology. Refers to nucleic acids or peptides that are substantially free of. Furthermore, an “isolated nucleic acid” can include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” also refers herein to polypeptides isolated from other cellular proteins and is meant to include both purified and recombinant polypeptides.

  The terms “label” and “detectable label” as used herein are radioactive isotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands ( For example, a molecule that can be detected, including but not limited to biotin or hapten. The term “fluorescer” refers to a substance or part thereof that exhibits fluorescence in a detectable range. Specific examples of labels that can be used in the present invention include fluorescein, rhodamine, dansyl, umbelliferone, Texas Red, luminol, NADPH, α-β-galactosidase, and horseradish peroxidase.

  The term “expression level” as used herein refers to a measurable level of a given nucleic acid. The expression level of the nucleic acid is determined by methods known in the art. The term “differentially expressed” or “differential expression” refers to an increase or decrease in a measurable level of a given nucleic acid. As used herein, “differentially expressed” or “differential expression” refers to a difference in nucleic acid expression level of at least 1.4-fold in the two samples used for comparison, both compared to a normal standard sample. That means that. “Heterologously expressed” or “differential expression” in accordance with the present invention also means that the difference in the level of nucleic acid expression in the two samples used for comparison is greater than 1.4 fold, 2 fold, 5 fold, 10 fold, Means double, 20 times, 50 times or more. If one of the two samples does not contain detectable expression of a given nucleic acid, the nucleic acid in the two samples will be “as long as the detectably expressed nucleic acid is expressed at least ± 1.4 fold. It should have been expressed differentially. The differential expression of nucleic acid sequences is “inhibited” if the difference in nucleic acid expression levels in the two or more samples used for comparison changes no longer at least 1.4 times. Absolute quantification of the level of nucleic acid expression creates a standard curve based on the amount of control nucleic acid, including known concentrations of one or more control nucleic acids, and extrapolates the expression level of “unknown” nucleic acid species with respect to the standard curve. Can be obtained.

  The term “nucleic acid” as used herein refers to polynucleotides such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA). This term also includes, as equivalent, RNA or DNA analogs made from nucleotide analogs, including single-stranded (sense or antisense) and double-stranded polynucleotides, as applicable to the described embodiments. Should be understood. Chromosomes, mRNA, rRNA and EST are representative examples of molecules that can be called nucleic acids.

  The term “oligonucleotide” as used herein refers to a nucleic acid molecule comprising, for example, from about 10 to about 1000 nucleotides. Oligonucleotides for use in the present invention are about 15 to about 150 nucleotides, more preferably about 150 to about 1000 in length. The oligonucleotide can be a naturally occurring oligonucleotide or a synthetic oligonucleotide. Oligonucleotides can be obtained using the phosphoramidite method (Beaucage and Carruthers, Tetrahedron Lett. 22: 1859-62, 1981), or the triester method (Mattucci, et al., J. Am. Chem. Soc. 103: 3185, 1981), Alternatively, it can be prepared by other chemical methods known in the art.

  The term “patient” or “subject” as used herein includes mammals (eg, humans and animals).

  As used herein, nucleic acids or other molecules attached to an array are referred to as “probes” or “capture probes”. When the array contains several probes corresponding to one gene, these probes are called a “gene probe set”. The gene probe set can comprise, for example, about 2 to about 20 probes, preferably about 2 to about 10 probes, and most preferably about 5 probes.

  The “profile” of a cell's biological state refers to the levels of various components of the cell that are known to change in response to drug treatment and other perturbations of the cell's biological state. Cellular components include, for example, RNA levels, protein richness levels, or protein activity levels.

  The term “protein” is used interchangeably herein with the terms “peptide” and “polypeptide”.

  An expression profile of one cell is similar to the expression profile of another cell when the expression of the genes in the two profiles are sufficiently similar such that the similarity is an indication of a common feature, for example, the same type of cell. “Similar”. Thus, the expression profile of the first cell and the second cell is expressed at a level where at least 75% of the genes expressed in the first cell are within a factor of 2 with respect to the first cell in the second cell. Sometimes similar.

  As used herein, “small molecule” refers to a composition having a molecular weight of less than about 5 kD, and most preferably less than 4 kD. Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids, or other organic or inorganic compounds. Many pharmaceutical companies have extensive libraries of chemical and / or biological mixtures, often molds, bacteria or seaweed extracts, that can be screened with any of the assays of the invention to identify compounds that modulate bioactivity. ing.

  “Specific hybridization” of a probe to a target site of a template nucleic acid refers to hybridization of the probe that is dominant to the target so that the hybridization signal can be clearly deciphered. As further described herein, such conditions that result in specific hybridization will vary depending on the length of the homology region, the GC content of the region, and the melting point (Tm) of the hybrid. Hybridization conditions can vary in salt content, acidity, and hybridization solution and wash temperature.

  A “variant” of a polypeptide refers to a polypeptide having an amino acid sequence in which one or more amino acids are altered. Variants can have “conservative” changes in which the substituted amino acid has similar structural or chemical properties (eg, replacement of leucine with isoleucine). Variants can also have “nonconservative” changes (eg, replacement of glycine with tryptophan). Similar minor variations can include amino acid deletions or insertions or both. Guidelines for determining which amino acids can be substituted, inserted or deleted without destroying biological or immunological activity use computer programs well known in the art, such as LASERGEN software (DNASTAR). Can be identified.

  The term “variant” when used in the context of a polynucleotide sequence can encompass a polynucleotide sequence related to the sequence of a particular gene or its coding sequence. This definition may also include, for example, “conflict”, “splice” or “polymorph” variants. Splice variants can have significant identity to the reference molecule, but can generally have larger or smaller numbers of polynucleotides due to alternative splicing of exons during mRNA processing. Corresponding polypeptides can have additional functional domains or can be domain absent. A species variant is a polynucleotide sequence that varies from one species to another. The resulting polypeptides will generally have significant amino acid identity with respect to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants can also include “single nucleotide polymorphisms” (SNPs) in which the polynucleotide sequence has changed by one base. The presence of a SNP can be an indication of a trend for a particular population, pathological condition, or pathological condition, for example.

  One aspect of the present invention is directed to the identification of agents that can modulate the differentiation and proliferation of cells characterized by abnormal growth. A more particular aspect of the invention relates to a method of screening candidate compounds or substances for their ability to modulate the differential expression of nucleic acid sequences. That is, if certain nucleic acid sequences are overexpressed in cancer cells, then the candidate compounds are screened for their ability to reduce expression, and if the nucleic acid sequence is underexpressed in cancer cells, test Compounds are screened for their ability to increase expression. In addition, in other specific aspects, the invention relates to screening assays that identify test compounds or substances that modulate the activity of one or more polypeptides encoded by the differentially expressed sequences described herein. In this regard, the present invention provides assays for determining compounds that modulate the expression of marker nucleic acids and / or alter the bioactivity of the encoded polypeptide.

Any compound can be used in accordance with the present invention. Although the disclosure is described for example with respect to sorafenib, this is an example and the invention is not so limited. Other multi-kinase inhibitors can also be used including, for example, stents and other compounds described below. In addition, sorafenib (BAYER 43-9006) described in WO 00/42102, WO 00/41698, and PCT / US / 30542, which is hereby incorporated by reference in its entirety, is also included.
[Screening for modulation of differential expression]

  Drug screening is performed by adding a test compound (eg, sorafenib) to a sample of cells and monitoring the effect. Parallel samples without test compound are also monitored as controls. Treated and untreated cells are then compared by appropriate phenotypic criteria, including but not limited to microscopic analysis, viability testing, replication capability, histological examination, and the level of specific RNA or polypeptide associated with the cell Is done. Differences in effects between treated and untreated cells indicate what should be attributed to the test compound.

  The desired effect of the test compound is an effect on any phenotype conferred by the cancer associated marker nucleic acid sequence. Examples include test compounds that limit the overabundance of mRNA, limit the production of the encoded protein, or limit the functional effect of the protein. The effect of the test compound will be apparent when comparing results between treated and untreated cells.

  The invention also includes a method of screening for an agent that inhibits or enhances the expression of a nucleic acid marker in vitro (eg, sorafenib), wherein the method is to determine whether the agent can inhibit or enhance the production of mRNA. In addition, a cell or tissue in which marker nucleic acid mRNA (for example, MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen) can be detected in cultured cells Exposure to the agent and determining the level of mRNA in the exposed cells or tissues, wherein a decrease in the level of mRNA after exposure of the cell line to the agent is an indication of inhibition of marker nucleic acid mRNA production. And an increase in mRNA levels indicates enhanced marker mRNA production.

  Instead, screening methods expose cells or tissues in which marker protein is detectable in cultured cells to agents that appear to inhibit or enhance the production of marker protein, and determine the level of marker protein in the cell or tissue. A decrease in the level of marker protein after exposure of the cell or tissue to the agent is an indication of inhibition of marker protein production, and an increase in the level of marker protein is an indication of enhancement of marker protein production. is there.

  The invention also encompasses an in vivo screening method for agents that inhibit or enhance the expression of marker nucleic acid, wherein the method comprises subjecting a subject having tumor cells in which marker mRNA or protein is detectable to marker mRNA or protein. Exposure to agents suspected of inhibiting or enhancing production, and determining the level of marker mRNA or protein in the exposed mammalian tumor cells. A decrease in the level of marker mRNA or protein after subject to the agent is an indication of inhibition of marker nucleic acid expression, and an increase in marker nucleic acid is an indication of enhanced marker nucleic acid expression.

  The invention thus provides a method comprising incubating a cell expressing a marker nucleic acid with a test compound and measuring mRNA or protein levels. The invention further provides a method for quantitatively determining the expression level of a marker nucleic acid in a cell population and a method for determining whether an agent can increase or decrease the expression level of a marker nucleic acid in a cell population. I will provide a. A method for determining whether the expression level of a marker nucleic acid can be increased or decreased in a cell population comprises (a) preparing a cell extract from a control and agent-treated cell population, and (b) a marker from the cell extract. Isolating the polypeptide and (c) quantifying (eg, in parallel) the amount of immune complexes between the marker polypeptide and an antibody specific for said polypeptide. A marker polypeptide of the invention may be quantified by assaying for its bioactivity. Agents that induce increased marker nucleic acid expression can be identified by their ability to increase the amount of immune complex produced in the treated cells compared to the amount of immune complex produced in the control cells. In a similar manner, an agent that decreases the expression of a marker nucleic acid can be identified by its ability to reduce the amount of immune complexes produced in the treated cell extract as compared to control cells.

  The present invention provides isolated nucleic acid sequences that are differentially regulated in cancer and methods for identifying such sequences. The present invention provides a method for identifying a differentially regulated nucleic acid sequence in a subject having cancer, the method comprising extracting a nucleotide sample corresponding to RNA from the subject to one or more known identities. A nucleic acid sample obtained from a cancer-free subject, wherein the nucleic acid sample comprises hybridizing to a nucleic acid sample containing the nucleic acid molecule and measuring hybridization of one or more known identities of the nucleic acid sample to one or more nucleic acid molecules Compared to, a two-fold difference in hybridization of a nucleic acid sample with a known identity to one or more nucleic acid molecules is an indication of differential expression of nucleotide sequences in a subject with cancer.

In general, the present invention isolates messenger RNA from a subject, generates cRNA from an mRNA sample, hybridizes the cRNA to a microarray containing a plurality of nucleic acid molecules stably associated at discrete locations on the array, and A method for identifying differentially regulated nucleic acid sequences in a subject with cancer comprising identifying a pattern of hybridization to an array of cRNA is provided. In accordance with the present invention, nucleic acid molecules that hybridize to a given location on the array are hybridized at the same location on the same array where the hybridization signal is hybridized with a nucleic acid sample obtained from a subject that does not have cancer. It is said to be differentially adjusted if it is at least twice as high or lower than the signal.
[Microarray for determining gene expression level]

  Determination of gene expression levels can be accomplished using microarrays. In general, the following steps: (a) obtaining an mRNA sample from a subject and then preparing a labeled nucleic acid (target nucleic acid or target); (b) subjecting the target nucleic acid to a corresponding probe on the array and the target nucleic acid for example hybridization Or contact under conditions sufficient to bind by specific binding, (c) optionally remove unbound targets from the array, (d) detect bound targets, and (e) use a computer, for example Analyzing the results using the method can be included. As used herein, a “nucleic acid probe” or “probe” is a nucleic acid hybridized to an array.

  Nucleic acid specimens can be obtained from subjects to be tested by invasive or non-invasive sampling methods. A sampling means should be invasive if it involves the collection of nucleic acids from the skin or organ of an animal (including mice, humans, sheep, horses, cows, cats or dogs). Invasive methods include, for example, blood collection, semen collection, biopsy needle, pleural aspiration, umbilical cord biopsy. Examples of such methods are described in Kim, et al. (J. Virol. 66: 3879-3882, 1992); Biswas et al. (Ann. NY Acad. Sci. 590: 582-583, 1990); and Biswas, et al. (J. Clin. Microbiol. 29: 2228-2233, 1991).

  In contrast, a non-invasive sampling means is a means by which nucleic acid is recovered from the inner or outer surface of an animal. Examples of such non-invasive sampling means include, for example, tear, saliva, urine, fecal material, sweat, hair rod collection.

  In one embodiment of the invention, one or more cells from the subject to be tested are obtained and RNA is isolated from the cells. In a preferred embodiment, peripheral blood leukocytes (PBL) are obtained from the subject. It is also possible to obtain a cell sample from the subject and enrich the sample for the desired type. If the desired cells are in solid tissue, specific cells can be dissected, for example, by microdissection or laser capture microdissection (eg, Bonner et al., Science 278: 1481, 1997; Emmert-Buck (See, Fend, et al., Am. J. Path. 154: 61, 1999; Murakami, et al., Kidney Int. 58: 1346, 2000)), et al., Science 274: 998, 1996;

  RNA can be extracted from tissue or cell samples by various methods, such as guanidinium thiocyanate lysis followed by CsCl centrifugation (Chirgwin, et al., Biochemistry 18: 5294-5299, 1979). RNA from single cells can be obtained by the described methods for preparing cDNA libraries from single cells (eg, Dulac, Curr. Top. Dev. Biol. 36: 245, 1998; Jena, et al. al., J. Immunol. Methods 190: 199, 1996.)

  RNA samples can be further enriched for specific species. In one embodiment, for example, poly (A) + RNA can be isolated from an RNA sample. In other embodiments, the RNA population can be enriched for sequences of interest by primer-specific cDNA synthesis, or by multiple steps of linear amplification based on multiple steps of cDNA synthesis and template-directed in vitro transcription ( For example, see Wang, et al., Proc. Natl. Acad. Sci. USA 86: 9717, 1989; Dulac et al., Supra; see Jena et al., Supra). In addition, populations of RNA that are enriched or not enriched in a particular species or sequence can be, for example, PCR; ligase chain reaction (LCR) (eg, Wu and Wallace, Genomics 4: 560, 1989; Landegren, et al., Science 241 : 1077, 1988), self-sustained sequence replication (SSR) (see, eg, Guatell, et al., Proc. Natl. Acad. Sci: USA. 87: 1874, 1990); nucleic acid based sequence amplification (NASBA ) And transcription amplification (see, for example, Kwoh, et al., Pro. Natl. Acad. Sci. USA 86: 1173, 1989). PCR techniques are well known in the art (eg, PCR technology: Principles and Applications for DNA Application, ed.HA Erlich, Freeman Press, NY 1992, PCR Protocols: A Guide to Ms. Eds.Innis et al., Academic Press, San Diego, Calif., 1990; Mattilla et al., Nucleic Acids Pos. 19: 4967, 1991; Eckert, et al., PCR Methods and Applications 17: Ap. PCR, eds.McPherso n et al., IRL Press, Oxford; see US Pat. No. 4,683,202). Amplification methods are described, for example, by Ohyama et al. (BioTechniques 29: 530, 2000); Luo, et al. (Nat. Med. 5: 117, 1999); Hegde, et al. (BioTechniques 29: 548, 2000); Kacharmina, et al. (Meth. Enzymol. 303: 3, 1999); Livesey et al. (Curr. Biol. 10: 301, 2000); Spirin et al. (Invest. Ophthalmol. Vis. Sci. 40: 3108, 1998); Sakai, et al. (Anal. Biochem. 287: 32, 2000). RNA amplification and DNA synthesis can be performed in situ in cells (see, eg, Eberwine et al., Proc. Natl. Acad. Sci. USA 89: 3010, 1992).

  Nucleic acid molecules can be labeled to allow detection of hybridization of nucleic acids to the microarray. That is, the probe can include a member of the signal generation system, and thus can be detected directly or through a binding action with one or more additional members of the signal generation system. For example, the nucleic acid is fluorescently labeled dNTP (see, eg, Kricka, 1992, Nonisotopic DNA Probe Technologies, Academic Press, San Diego, Curif.). Labeling with biotinylated dNTPs or rNTPs and labeled streptavidin addition, chemiluminescence labeling, or radioisotope. Other examples of labels include “molecular beacons” as described in Tyagi and Kramer (Nature Biotech. 14: 303, 1996). Hybridization can also be determined, for example, by plasmon resonance (see, eg, Thiel, et al., Anal. Chem. 69: 4948, 1997).

  Another aspect of the invention relates to methods and processes for detecting differentially expressed genes. Detection methods have a variety of uses including diagnostic, prognostic, forensic and research applications. In order to achieve gene detection, the polynucleotide according to the invention can be used as a probe. The term “probe” or “polynucleotide probe” has its conventional meaning in the art, eg, a polynucleotide that is effective for identifying the presence of a designed target polynucleotide when used in an appropriate process. It is. Identification can simply include the determination of presence or absence, or can be quantified, for example, to assess the amount of a gene or gene transcript present in a sample. Probes are useful in a variety of embodiments, such as identifying homologs for diagnostic purposes and detecting, quantifying or isolating a polynucleotide of the invention in a test sample.

  Assays that permit quantification and / or presence / absence detection of the target nucleic acid in the sample can be utilized. The assay can be performed at the single cell level or in a sample containing a large number of cells, in which case the assay is an averaged expression across a collection of cells and whole tissues present in the sample. Any suitable assay format can be used without limitation, Northern blot analysis, Southern blot analysis, polymerase chain reaction (PCR) (eg, Saiki et al., Science 241: 53, 1988; US Pat. No. 4,683,195; 4,683,202; 6,040,166; PCR Protocols: A Guide to Methods and Applications, Innis et al. Eds., Academic Press, New York, 1990), reverse transcription polymerase. Chain reaction (RT-PCR), uncarded PCR, rapid amplification of cDNA ends (RACE) (eg, Schaefer in Gene Cloning and Analysis: Cur ent Innovations, Pages 99-115, 1997), ligase chain reaction (LCR) (EP 320308), one-sided PCRC (Ohara et al., Proc. Natl. Acad. Sci. 86: 5673-5677, 1989), indexing method ( For example, V.S. Pat. No. 5,508,169), in situ hybridization, hysteretic display (eg, Liang et al., Nucl. Acid Res. 21: 3269-3275, 1993; US Pat. Nos. 5,262,311, 5,599,672, 5,965,409; WO97 / 18454; Prashar and Weissman, Proc.Natl.Acad.Sci.93: 659-663, USP. No. 6, 010, 850, 5, 712, 126; Weish et al., Nucleic Acid Res. 20: 4965-4970, 1992, U.S. Pat. No. 5,487,985) and others. RNA fingerprint technology, nucleic acid sequence-based amplification (NASBA), and other transcription-based amplification systems (eg, US Pat. Nos. 5,409,818 and 5,554,527; WO88 / 10315), polynucleotides Arrays (eg, US Pat. Nos. 5,143,854, 5,424,186, 5,700,637; 5,874,219 and 6,054,270; WO92 / 10092; WO90 / 15070); Qbeta lipase (PCT / US87 / 00880), strand display Smentment amplification (SDA), repair chain reaction (RCR), nuclease protection assay, subtraction based method, rapid scan (TM), etc. Additional useful methods include, but are not limited to, for example, template-dependent amplification methods, competitive PCR (eg, US Pat. No. 5,747,251), redox-based assays (eg, US Pat. No. 5, 871, 918), Taqman-based assays (eg, Holland et al., Proc. Natl. Acad. Sci. 88: 7276-7280, 1991; US Pat. Nos. 5, 210, 015 and 5, 994,063), real-time fluorescence monitoring (eg US Pat. No. 5,928,907), molecular energy transfer label (eg US Pat. Nos. 5,348,853, 5,532,129). , 5,565,322, 6,030,787 and 6,117,635; Tyagi and Kra er, Nature Biotech, 14: 303-309,1996), including the. Any suitable method for single cell analysis of gene or protein expression can be used, including in situ hybridization, immunocytochemistry, MACS, FACS, flow cytometry, and the like. For single cell assays, the expression product may be antibody, PCR or other type of nucleic acid amplification (eg, Brady et al., Methods Mol. & Cell. Biol. 2: 7-25, 1990; Eberwine et al., 1992, Proc. Natl. Acad. Sci. 89: 3030-3014; US Pat. No. 5,723,290). These and other methods can be routinely performed, for example, as described above.

  In one embodiment, multiple sets of target nucleic acids (eg, 2, 3, 4, 5 or more) are labeled and can be used in a hybridization reaction (multiplex analysis). For example, a set of nucleic acids can correspond to RNA from other cells. Multiple sets of nucleic acids can be labeled with different labels, eg, different fluorescent labels (eg, fluorescein and rhodamine) having distinct emission spectra so that they can be distinguished. The set is then mixed and hybridized simultaneously to one microarray (see, eg, Shena et al., Science 270: 467-470, 1995).

  A microarray for use in accordance with the present invention includes one or more probes of genes characteristic of small molecule efficacy. In a preferred embodiment, the microarray comprises one or more genes selected from the group consisting of genes that are up-regulated in cancer and genes that are down-regulated in cancer. The microarray can comprise at least 10, preferably at least 20, at least 50, at least 100 or at least 1000 genes characteristic for small molecule efficacy.

  There can be one or more probes corresponding to each gene on the array. For example, the microarray can include 2 to 20 probes corresponding to a gene, preferably about 5 to 10 probes. The probe can correspond to a full-length RNA sequence, or the complement of a gene characteristic of small molecule efficacy, or the probe can correspond to those portions that are long enough to allow specific hybridization. be able to. Such probes can comprise from about 50 nucleotides to about 100, 200, 500 or 1000 nucleotides, or 1000 nucleotides or more. As described further herein, the microarray can contain oligonucleotide probes consisting of about 10-50 nucleotides, preferably about 15-30 nucleotides, and more preferably about 20-25 nucleotides. The probe is preferably single stranded and will have sufficient complementarity to the target to provide the desired level of sequence specific hybridization.

Typically, an array used in the present invention will have a site density per cm ² 100 or more different probes. Preferably it will have a site density of 500 / cm ² or more, more preferably about 1000 / cm ² or more, and most preferably about 10,000 / cm ² or more. Preferably, the array is a single substrate with 100 or more different probes, more preferably about 1000 different probes, still more preferably about 10,000 or more different probes, most preferably 100,000 or more different probes. Will have on the substrate.

  A number of different microarray structures and their methods of manufacture are known to those skilled in the art and are described in US Pat. S. Pat. Nos. 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,445,934; 5 556,752; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593 5,740,711; 5,744,305; 5,770,456; 5,770,722; 5,837,832; 5,856,101; 5,874,219; 5,885, 837; 5,919,523; 6,077,674; 6,156,501; Shena et al. Tibtech 16: 301, 1998; Duggan et al. Nat. Genet. 21:10, 1999; Bowtel et al. Nat. Genet. 21:25, 1999; Lipshutz et al. , Nature Genet. 21: 20-24, 1999; Blanchard et al. Biosensors and Bioelectronics 11; 687-90, 1996; Maskos et al. , Nucleic Acids Res. 21: 4663-69, 1993; Hughes et al. Nat. Biotechnol. 19: 342, 2001, the entire description of which is incorporated herein by reference. Patents describing methods of using arrays in various applications are described in US Pat. S. Pat. Nos. 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,806; 5,503,980; 5,510,270; 5,525,464; 547,839; 5,580,732; 5,661,028; 5,848,659; and 5,874,219, the disclosures of which are incorporated herein by reference.

  The array preferably includes control and reference nucleic acids. Control nucleic acids include, for example, prokaryotic genes such as bioB, bioC and bioD, cre from P1 bacteriophage, or poly A controls such as dap, lys, phe, thr and trp. The reference nucleic acid allows normalization of results from one experiment to another and comparison of multiple experiments at a quantitative level. Exemplary reference nucleic acids include housekeeping genes of known expression levels such as GAPDH, hexokinase and actin.

In one embodiment, an array of oligonucleotides can be synthesized on a solid support. Exemplary solid supports include glass, plastics, polymers, metals, metalloids, ceramics, organics, and the like. Using chip masking technology and photoprotection chemistry, it is possible to generate an ordered array of nucleic acid probes. These arrays, known as DNA chips or very large scale immobilized polymer arrays (VLSIPS arrays), can include millions of delimited probe regions on a substrate having an area of about 1 cm ^{2 to} several cm ² , It incorporates several to millions of probes (see, eg, US Pat. No. 5,631,734).

  To compare expression levels, the labeled nucleic acid can be contacted with the array under conditions sufficient for binding between the target nucleic acid and the probes on the array. In preferred embodiments, hybridization conditions can be selected to provide the desired level of hybridization specificity. That is, the conditions are sufficient for hybridization to occur between the labeled nucleic acid and the probe on the microarray.

  Hybridization can be carried out under conditions that inherently permit specific hybridization. The length and GC content of the nucleic acid will determine the thermal melting point and thus the hybridization conditions necessary to obtain specific hybridization of the probe to the target nucleic acid. These factors are well known to those skilled in the art and can be tested in assays. An extensive guide to nucleic acid hybridization is described in Tijsssen et al. , Laboratory Technologies in Binchemistry and Molecular Biology, Vol. 24; Hybridization With Nucleic Acid Probes, P. 24; Tijsssen, ed. Elsevier, N.M. Y. 1993.

  The method described above results in the production of a hybridization pattern of labeled target nucleic acids on the array surface. The hybridization pattern of the generated labeled nucleic acid can be visualized or detected in a variety of ways, with specific detection aspects selected based on the specific label of the target nucleic acid. Typical detection means include scintillation counting, autoradiography, fluorescence measurement, colorimetric measurement, luminescence measurement, light scattering, and the like.

  One such detection method utilizes a commercially available array scanner (Affymetrix, Santa Clara, Calif.), For example, 417 Arrayer, 418 Array Scanner, or Agilent Gene Array Scanner. The scanner is controlled from a system computer with an interface and easy-to-use software tools. The output can be directly installed or read directly by some software application. A preferred scanning device is e.g. S. Pat. Nos. 5,143,854 and 5,424,186.

  Because of the fluorescently labeled probe, fluorescence emission can be detected by a scanning confocal laser microscope at each site of the transfer array. Alternatively, a laser that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores can be used and the emission from the two fluorophores can be analyzed simultaneously (eg, Shalon et al. , Genome Res. 6: 639-645, 1996). In a preferred embodiment, the array can be scanned with a laser fluorescence scanner equipped with a computer XY stage and a microscope target. A fluorescence laser scanner is described in the above-mentioned Shalon et al. It is described in.

Various algorithms for analyzing gene expression data, eg, the type of comparison to be performed, may be utilized. In some embodiments, it is desirable to group genes that are co-regulated. This allows multiple profile comparisons. Preferred embodiments for identifying groups of such genes include clustering algorithms (for review of clustering algorithms, see, eg, Funkunaga, 1990, Statistical Pattern Recognition, 2nd Ed., Academic Press, San Diego; 1974, Cluster Analysis, London; Heinemann Educ. Books; Hartigan, 1975, Clustering Algorithms, New York, Wiley; Sueat and Axon, 1973, Numeric. is for Applications, Academic Press. New York).
[Biomarker discovery]

  Expression patterns can be used to derive a panel of biomarkers that can be used to predict the efficacy of drug treatment in a patient. Biomarkers can consist of RNA isolated from biological samples, gene expression levels from microarray experiments of RNA isolated from frozen samples of tumor biopsies, or mass spectrum derived protein amounts in serum.

Although the precise mechanism of data analysis will depend on the exact nature of the data, a typical procedure for developing a panel of biomarkers is as follows. Data (gene expression level or mass spectrum) is collected for pre-treatment patients. As the study progresses, patients are classified as valid or ineffective according to their response to drug treatment. Multiple levels of effectiveness can be accepted in the data model, but a two-way comparison is considered optimal if the patient population is less than a few hundred. Assuming each class has an appropriate number of patients, protein and / or genetic data can be compared by a number of techniques known in the art. Many such techniques are derived from the field of traditional statistics or machine learning. These technologies serve two purposes.
1. Reduce the number of dimensions of the data. -In the case of mass spectra or gene expression microarrays, the data is reduced from more than a few thousand data points to about 3-10. This reduction is based on the predictive power of the data points when taken as a set.
2. Training-These 3 to 10 data points are used to train a number of machine learning algorithms, which in this case then learn to recognize protein or gene expression patterns that distinguish effective drug treatment from ineffective. To do.

  The resulting trained algorithms are then tested to measure their predictive power. Typically, some form of cross-validation is performed when fewer than a few thousand training examples are available. To illustrate, consider 10-fold cross validation. In this case, patient samples are randomly assigned to one of 10 bins. In the first round of validation, 9 bin samples are used for training and the remaining samples in the 10th bin are used to test the algorithm. This is repeated nine additional times, leaving samples in different bins for each test. The results from all 10 rounds (correct predictions and errors) are combined and the predictive power is evaluated. Different algorithms and different panels can be compared in this way for consideration. The best algorithm / panel combination will then be selected. This clever algorithm can be used to select patients that are most likely to respond to treatment.

  Many algorithms benefit from additional information obtained from the patient. For example, gender or age can be used to improve forecasting power. Data transformations such as normalization and smoothing can also be used to reduce noise. Thus, many algorithms can be trained using many different parameters to optimize performance. If predictive power is present in this data, it seems that an optimal or near-optimal algorithm can be developed. If more patient samples are obtained, the algorithm can be retrained to benefit from new data.

As an example using mass spectra, plasma (1 μl) can be applied to a hydrophobic SELDI target, washed well with water and analyzed by a SELDI-Of mass spectrometer. This can be repeated for over 100 patients. The protein profile obtained from the 16,000 m / z value intensity in each sample will be analyzed statistically to identify a specific set of m / z values that are predictive of drug efficacy. The same experiment can be performed using other SELDI targets such as ion exchange or IMAC surfaces. These will capture different subsets of proteins present in the plasma. In addition, plasma can be denatured and pre-fractionated prior to application to the SEIDI target.
[Diagnosis and prognostic assay]

  The biomarkers of the present invention determine and tailor different classes of treatment therapies for patients with cancer, including patients who have not received treatment and those who have already received treatments such as chemotherapy and radiation. Can be used for. In addition, this biomarker will determine therapeutic efficacy for patients under active treatment and to indicate whether additional chemotherapeutic agents or treatment courses need to be modified Can be used for monitoring. For example, failure to achieve biomarker modulation or a level associated with a therapeutic effect may suggest treatment modifications including higher doses of drugs and / or the addition of other therapies.

  Examples of useful chemotherapeutic agents include, but are not limited to, for example, alkylating agents (eg cyclophosphamide, ifosfamide, melphalan, chlorambucil, aziridine, epoxide, alkyl sulfonate), cisputin and analogs thereof (eg carboplatin, oxaplatin) Antimetabolites (eg, methotrexan, 5-fluorouracil, capecitabine, cytarabine, chemcitabine, fludarabine), toposiomerase inactivators (eg, camptothecin, irinotecan, topotecan, etoposide, teniposide, doxorubicin, daunorubicin), antimicrotubule agents (eg, Vinca alkaloids such as vincristine, vinbiastin and vinorelbine, taxanes such as paclitaxel and docetaxel), interleukin-2, his Ndeacetylase inhibitors, monoclonal antibodies, estrogen modulators (eg, tamoxifen, toremifene, raloxifene), megestol, aromatase inhibitors (eg, flutamide, casodex), interferons (eg, interferon-alpha including subtypes such as interferon-alpha 2a), etc. including. See, for example, Cancer: Principles and Practice of Oncology, 7th Edition, Devita et al, Lipcottot Williams & Wilkins, 2005, Chapters 15, 16, 17, 18.

  The present invention provides biomarkers (eg, MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigens), ie nucleic acids for cancer and / or Methods are provided for determining whether a subject is at risk of developing a disease or condition characterized by unwanted cell proliferation by detecting a polypeptide marker.

  In clinical applications, human tissue samples can be screened for the presence and / or absence of the biomarkers identified herein. Such a sample can consist of a biopsy needle core, a surgically excised sample, lymph node tissue or serum. For example, these methods include obtaining a biopsy, which is optionally fractionated by cryostat incision to enrich the tumor cells to about 80% of the total cell population. In certain embodiments, nucleic acids extracted from these samples can be amplified by techniques well known in the art. The level of selected marker detected will be compared to metastatic, non-metastatic malignant, benign or normal tissue samples.

  In one embodiment, the diagnostic method is performed when the subject is biopsied, such as by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, immunoprecipitation, Western blot hybridization, or immunohistochemistry. Whether there is an abnormal mRNA and / or protein level of a marker (eg MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen) Including deciding. According to this method, cells can be obtained from a subject and the levels of biomarkers, proteins or mRNA are determined and compared to the levels of these markers in a healthy subject. An abnormal level of a biomarker polypeptide or mRNA can be an indication of cancer.

  Accordingly, in one aspect, the present invention provides probes and primers specific for the unique nucleic acid markers disclosed herein. Thus, the nucleic acid probe is at least 10 nucleic acids in length complementary to a portion of the coding sequence of the marker nucleic acid sequence, preferably at least 15 nucleotides, more preferably 25 nucleotides, and most preferably at least 40 nucleotides, and the entire coding sequence Or almost everything.

In one embodiment, the method includes using a nucleic acid probe to determine the presence of cancer cells in cells from a patient. Specifically, the method includes the following steps.
1. A length complementary to a portion of the coding sequence of a nucleic acid sequence comprises at least 10 nucleic acids, preferably at least 15 nucleic acids, more preferably 25 nucleotides, and most preferably at least 40 nucleotides, and all or nearly all of the coding sequence Providing a nucleic acid probe;
2. Obtaining a tissue sample from a patient that may contain cancer cells;
3. Providing a second tissue sample containing cells that are substantially all non-cancerous;
4). 4. Contact the nucleic acid probe with the first and second tissue samples under stringent conditions (eg, in a Northern blot or in situ hybridization assay); (A) comparing the amount of hybridization of the probe with mRNA of the first tissue sample to (b) the amount of hybridization of the probe with mRNA of the second tissue sample, wherein The statistically significant difference in the amount of hybridization with mRNA of the first tissue sample when compared to the amount of hybridization with mRNA of the two tissue samples is the presence of cancer cells in the first tissue sample Instructions.

  In one aspect, the method was derived from a given marker nucleic acid sequence (eg, MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen). In-situ hybridization with the probe. The method comprises contacting a labeled hybridization probe with a given type of sample and normal cells potentially containing cancer or precancerous cells, and a portion of the tissue type to which the probe is given. Determine whether cells are labeled to a statistically different extent (eg, at least a factor of 2, or at least a factor of 5, or at least a factor of 20, or at least a factor of 50) than to label other cells of the same tissue type Including doing.

  Also within the present invention, a length of at least 12 nucleotides that is complementary to a portion of the coding sequence of the nucleic acid sequence and differentially expressed compared to normal cells of a given tissue type, Preferably 15 nucleotides, more preferably at least 25 nucleotides, and most preferably at least 40 nucleotides, and contact the test cell mRNA with all or nearly all of the nucleic acid probes, and the approximate amount of hybridization of the mRNA probes By determining, for example, whether the cell is (a) normal, or (b) a cancer or a pre-cancerous state, from a given human tissue, wherein the tissue More or less hybridization than found in normal cell types of mRNA The amount of test cells is an indication of cancer or pre-cancerous condition.

  Instead, the diagnostic assay is encoded by a marker nucleic acid sequence (eg, MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen). It can be performed using an antibody directed to the protein product. Thus, in one embodiment, the assay contacts a test cell protein with an antibody specific for a gene product of a nucleic acid, wherein the marker nucleic acid is given in a normal cell of the same tissue type as the test cell. And determining the approximate amount of immune complex formation by the antibody and test cell protein, where the test cell protein is compared to normal cells of the same tissue type. A statistically significant difference in the amount of immune complex produced is an indication that the test cell is cancerous or precancerous. Preferably, the antibody is specific for MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen.

  Methods for producing polyclonal and / or monoclonal antibodies that specifically bind to polypeptides useful in the present invention are known to those skilled in the art and are described, for example, in Dymecki, et al. (J. Biol. Chem. 267: 4815, 1992); Boersma & Van Leewen (J. Neurosci. Methods 51: 317, 1994); Green, et al. (Cell 28: 477, 1982); Amheiter et al. (Nature 294: 278, 1981).

  Such other methods include a gene product of a marker nucleic acid sequence that is greater or less than the level of the gene product in a non-cancer tissue of the same tissue type in a cancer tissue of a given tissue type Obtaining a first sample of tissue of a given tissue type, comprising a normal cell, comprising an antibody specific for the gene product present in said, potentially containing cancer cells; Prepare a second sample of tissue of the same tissue type that is essentially free of cancer cells (this may be from the same patient or normal control, eg, another individual or cultured cell), and antibody and The protein of the first and second samples (which may be lysed but not fractionated or in situ) under conditions that allow immune complex formation between the marker nucleic acid sequence products present. The antibody can be partially purified) and (a) the amount of immune complex produced in the first sample compared to (b) the amount of immune complex produced in the second sample Wherein the statistically significant difference in the amount of immune complex produced in the first sample compared to the amount produced in the second sample is less than the amount produced in the first tissue sample. It is an indication of the presence of cancer cells.

  The present invention provides a method for determining whether a cell sample obtained from a subject has an abnormal amount of a marker polypeptide comprising: (a) obtaining a cell sample from the subject; and (b) obtaining In order to quantitatively determine the amount of marker polypeptide in the sample, and (c) determine whether the sample obtained from the subject has an abnormal amount of marker polypeptide, the determined amount of marker polypeptide is known standard Comparing with Such marker polypeptides can be detected by immunohistochemical assays, dot blot assays, ELISA, and the like.

  Immunoassays are commonly used to quantify protein levels in cell samples, and numerous other immunoassay techniques are known in the art. The present invention is not limited to a particular assay operation and is therefore intended to include both homogeneous and heterogeneous operations. Exemplary immunoassays that can be performed in accordance with the present invention include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), turbidimetric inhibition immunoassay (NIA), enzyme linked immunoassay (ELISA), and radioimmunoassay (RIA). )including. Indicator moieties or label groups can be attached to the antibody of interest and are often selected to meet the demands of various applications of the method dictated by the availability of assay equipment and compatible immunoassay operations. The general techniques used to perform the various immunoassays described above are known to those skilled in the art.

  In other embodiments, the level of the encoded product in the patient's biological fluid (eg, blood or urine), or alternatively the level of the polypeptide, is the level of expression of the marker nucleic acid sequence in the patient's cells. It can be determined as a method of monitoring. Such a method involves taking a sample of biological fluid from a patient, contacting the sample (or protein from the sample) with an antibody specific for the encoded marker polypeptide, and immunoconjugate with the solid Determining the amount of somatic production, wherein the amount of immune complex produced is an indication of the level of the marker code product in the sample. This determination is particularly beneficial when compared to the amount of immune complex produced by the same antibody in a control sample taken from a normal individual or in one or more samples obtained before or after the same person.

  In other embodiments, the method can be used to determine the amount of marker polypeptide present in a cell that can be correlated to a hyperproliferative disorder. The amount of marker polypeptide can be used to predictively whether a sample of cells contains cells that have been transformed or pretreated towards it. In addition, this method can be used to assess the phenotype of cells known to be transformed, and the results of phenotyping are useful in the planning of specific treatment methods. For example, very high marker polypeptide levels in sample cells are powerful diagnostic and prognostic markers for cancer. Observation of the marker polypeptide can be used, for example, in decisions regarding the use of more aggressive treatments.

  As mentioned above, one aspect of the present invention relates to a diagnostic assay for determining whether the level of a marker polypeptide has been significantly reduced in a sample cell in the context of cells isolated from a patient. The term “significantly reduced” means that the cells have a reduced amount of marker polypeptide cell mass compared to normal cells of similar tissue source. For example, the cells can have less than about 50%, 25%, 10%, or 5% of the marker polypeptide compared to normal control cells. In particular, this assay assesses the amount of marker polypeptide in a test cell, and preferably detects the level detected in at least one control cell, such as a normal cell and / or a transformed cell of known phenotype. Compare with the level of polypeptide.

  Of particular importance to the present invention is the ability to quantify marker polypeptide levels as determined by the number of cells associated with normal or abnormal marker polypeptide levels. The number of cells with a particular marker polypeptide phenotype can then be correlated with patient prognosis. In one embodiment of the invention, the marker polypeptide phenotype of the lesion is determined as the percentage of cells in the biopsy that are found to have abnormally high / low levels of marker polypeptide. Such expression can be detected by immunohistochemical assays, dot blot assays, ELISA, and the like.

  If a tissue sample is used, immunohistochemical staining can be used to determine the number of cells having a marker polypeptide phenotype. For such staining, multiple blocks of tissue are taken from a biopsy or other tissue sample and subjected to proteolytic hydrolysis using agents such as protease K or pepsin. In certain embodiments, it may be desirable to isolate the fraction from the sample cells and detect the marker polypeptide in the fraction.

  Tissue samples are fixed by treatment with reagents such as formalin, glutaraldehyde, methanol and the like. The sample is then incubated with an antibody having binding specificity for the marker polypeptide, preferably a monoclonal antibody. This antibody can be conjugated to a label for detection after binding. The sample is incubated for a time sufficient for the formation of immune complexes. Antibody binding is then detected using a label conjugated to the antibody. If the antibody is not labeled, for example, a second labeled antibody specific for the isotype of the anti-marker polypeptide antibody can be used. Labels that can be used are radionuclides, fluorophores, chemiluminescent agents, enzymes and the like.

  If an enzyme is used, a substrate for the enzyme can be added to the sample to provide a colored or fluorescent product. Examples of suitable enzymes for use in the conjugate include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase and the like. If not commercially available, such antibody / enzyme conjugates can be readily produced by techniques known to those skilled in the art.

  In one embodiment, the assay is performed as a dot blot assay. The dot blot assay allows determination of the average amount of marker polypeptide associated with a single cell by correlating to the amount of marker polypeptide in the cell-free product obtained from a predetermined number of cells. Find special uses when tissue samples are used.

  In the cancer literature, tumor cells of the same type (eg, breast and / or colon tumor cells) do not show uniformly increased expression of individual oncogenes or uniformly decreased expression of individual tumor suppressor genes. There are facts established. There may also be varying levels of expression of a given marker gene among a given type of cancer cell, further emphasizing the need to rely on a set of tests rather than a single test. The Accordingly, in one aspect, the present invention provides a set of tests using multiple inventive probes to improve the reliability and / or accuracy of diagnostic tests.

  In one embodiment, the present invention also provides a method in which a nucleic acid probe is immobilized on a DNA chip in an organized array. Oligonucleotides can be bound to a solid support by a variety of operations including lithography. For example, one chip can hold 250,000 oligonucleotides. These nucleic acid probes are at least 12 nucleotides in length, preferably at least 15 nucleotides, more preferably at least about 25 nucleotides, and most preferably about 40 nucleotides, and are complementary to a portion of the coding sequence of the marker nucleic acid sequence; It contains all or nearly all of the sequences differentially expressed in tumor cells. The present invention provides significant benefits over available tests for various cancers. This is because reliability is increased by providing an array of nucleic acid markers on a single chip.

  This method is biopsyed and optionally fractionated by cryoincision to enrich tumor cells to about 80% of the total cell population. DNA or RNA is then extracted, amplified, and analyzed using a DNA chip to determine the presence or absence of the marker nucleic acid sequence.

  In one embodiment, the nucleic acid probes are spotted on a substrate in a two-dimensional matrix or array. The sample of nucleic acid can be labeled and then hybridized to the probe. Double-stranded nucleic acid containing labeled sample nucleic acid bound to the probe nucleic acid can be detected after washing away the unbound portion of the sample.

  The probe nucleic acid can be spotted on a substrate containing glass, nitrocellulose and the like. The probe can be bound to the substrate by covalent bonds or by non-specific interactions such as hydrophobic interactions. The sample nucleic acid can be labeled using a radioactive label, a fluorescent source, a chemiluminescence source, or the like.

  Techniques for constructing arrays and methods of using these arrays are described, for example, in EP0779897; WO97 / 29212; WO97 / 27317; EP0785280; WO97 / 02357; S. Pat. No. 5,593,839; S. Pat. No. 5,578,834; EP 0728520; S. Pat. No. 5,599,695; EP 0721016; S. Pat. No. 5,599,695; EP 0721016; S. Pat. No. No. 5,556,752; WO 95/22058; S. Pat. No. 5,631,734.

  Furthermore, the array can be used to examine the differential expression of genes and can be used to determine gene function. For example, an array of nucleic acid sequences can be used to determine whether any nucleic acid sequence is differentially expressed between normal and cancer cells. Increased expression of a particular message in a cancer cell that is not observed in the corresponding normal cell may indicate a cancer-specific protein.

  In one embodiment, the nucleic acid molecule uses a sequenced nucleic acid molecule to determine whether any of the nucleic acid is differentially expressed between a normal cell or tissue and a cancer cell or tissue. As can be used, it can be used to create microarrays on solid surfaces (eg membranes). In one embodiment, the nucleic acid molecules of the present invention can be cDNA or can be used to generate cDNA that is later amplified by PCR and spotted on a nylon membrane. The membrane can then be reacted with a radiolabeled target nucleic acid molecule obtained from an equal sample of cancer or normal cells or tissue. cDNA generation methods and microarray preparation are known to those skilled in the art and are described, for example, in Bertucci, et al. (Hum. Mol. Genet. 8: 2129, 1999); Nuguyen, et al. (Genomics 29: 209, 1995); Zhao, et al. (Gene 156: 207); Gress et al. (Mammalian Genome 3: 609, 1992); Zhumabayeva, et al. (Biotechniques 30: 158, 201); Lennon, et al. (Trends Genet. 7: 314, 1991).

  In yet other embodiments, the present invention contemplates the use of a panel of antibodies raised against the marker polypeptides of the present invention. Preferably, antibodies are raised against MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigens. Such a panel of antibodies can be used as a reliable diagnostic probe for cancer. The assay of the present invention involves contacting a biopsy sample containing cells, eg, lung cells, with a panel of antibodies directed against one or more encoded products and determining the presence or absence of a marker peptide.

  The diagnostic methods of the present invention can be used as a treatment follow-up, for example, quantification of marker polypeptide levels can be used to determine the effectiveness of current and previously adopted cancer therapies and the effects of these treatments on patient outcomes. Can be instructions.

  In addition, the marker nucleic acids or marker polypeptides can be used as part of a diagnostic panel for initial detection, follow-up screening, detection of recurrence, and post-treatment monitoring of chemotherapy or surgery.

  Thus, the present invention can provide diagnostic assays and reagents for detecting marker polypeptide gain and / or loss from cells, for example, to aid in the diagnosis and phenotyping of proliferative disorders arising from oncogenic transformation of cells And

  The diagnostic assays described above can be adapted for use in prognostic assays. Such an application takes advantage of the sensitivity of the assay of the present invention to events that occur at characteristic stages in tumor progression. For example, a given marker gene is very early stage, possibly up or down-regulated before cells are irreversibly committed to malignancy development, while other marker genes are up or down only at later stages. Can be adjusted down. Such methods express test cell mRNA at different characteristic levels in cancer or precancerous cells at different stages of tumor progression. Contacting with a nucleic acid probe derived from a given marker nucleic acid and determining an approximate amount of hybridization of the probe to cellular mRNA, such amount being indicative of the expression level of the gene in the cell And is therefore an indication of the tumor progression stage of the cell. Alternatively, the assay can be performed with an antibody specific for the gene product of a given marker nucleic acid in contact with the test cell protein. Such a set of tests does not disclose the presence and location of the tumor, but allows the physician to select the treatment mode that is most appropriate for the tumor and to predict the likelihood of successful treatment.

  The methods of the invention can also be used to follow a clinical course of tumors. For example, the method of the invention can be applied to a tissue sample from a patient after treatment of the patient for cancer, other tissue samples are taken, and the test repeated. Successful treatment results in the removal of all cells that exhibit differential expression characteristic of cancer or precancerous cells, or a substantial increase in gene expression in those cells, perhaps approaching or above normal levels Will bring about an increase.

  The present invention also relates to a method of determining the effectiveness of an agent in the treatment of a disease or disorder (eg, renal cell or hepatocyte cancer) in a subject to whom an agent has been administered, said method comprising, for example, from said subject Detecting the amount of mRNA selected from Table 1 or a polypeptide encoded thereby (IL-6, IL-8, MMP-1 and / or MMP-10) in the obtained biological sample. Wherein the amount of the polypeptide is an indication of whether a therapeutically effective amount of the agent has been administered.

  Examples of such agents are multikinase inhibitors, particularly inhibitors having VEGFR activity such as sorafenib and derivatives thereof; multikinase inhibitors having an activity profile similar to sorafenib (see eg Wilhelm et al., 2004). An agent with both anti-angiogenic and anti-tumor proliferative activity (eg, sorafenib). In addition, combinations of drugs, such as a combination of a VEGFR2 inhibitor and a cytotoxin or chemotherapeutic agent; sorafenib and a quinone bioreducing prodrug (Jaffar et al. Curr Drug Deliv. 2004 Oct: 1 (4), 345-50 Or combinations with other drugs that are activated by increased hypoxia or that selectively kill cells under hypoxic conditions.

  Examples of multikinase inhibitors that induce biomarker production according to the present invention are, for example, ZD 6474, AZD 2171, AG-013736, AMG 706, BIBF-1120, XL999, MLN518, PKC412, OSI-930, OSI817, preferably Sutent (SU11248; sunitinib malate). Such agents include VEGFR (such as VEGFR2), PDGFR, and c-KIT activity; VEGFR (such as VEGFR2) and optionally RAF, PDGFR (such as PDGFR beta), c-KIT, RET, and Can have FLT3 activity. Examples of other multikinase inhibitors include, for example, MP-412, dasatinib, CEP-701, XL647, Tykerb, ST1571, AMN107 and AEE788.

  Chemotherapeutic agents include, but are not limited to, for example, alkylating agents (eg cyclophosphamide, ifosfamide, melphalan, chlorambucil, aziridine, epoxide, alkyl sulfonate), cisplatin and analogs (eg carboplatin, oxaliplatin), antimetabolites (eg Methotrexate, 5-florouracil, capecitabine, cytarabine, gemcitabine, fludarabine), toposiomerase inhibitors (eg camptothecin, linotecan, topotecan, etoposide, teniposide, doxorubicin, daunorubicin), anti-microtubule agents (eg vincristine and vinblastine, vinblastine, vinblastine, vinblastine) Vinca alkaloids; taxanes such as paclitaxel and docetaxel), interferon, interleukin-2, Stone deacetylase inhibitor, monoclonal antibody, estrogen modulator (eg tamoxifen, toremifene, raloxifene), megesterol, aromatase inhibitor (eg letrozole, anastrozole, exemestane, octreotide), antiandrogen (eg flutamide, casodex) Etc. See, for example, Cancer: Principles and Practice of Oncology, 7th Edition, Devita et al, Lipcottot Williams & Wilkins, 2005, Chapters 15, 16, 17 and 63. Also included are prodrugs, especially prodrugs activated in hypoxic tissues, such as quinone bioreductive compounds. For example, Jeffar et al. Curr. Drug Deliv. 2004, 1 (4): 345-350, 2004.

  The amount of mRNA, or polypeptide encoded thereby, can be routinely detected by any of the methods described herein. The term “detection” indicates that the presence of a target (ie, mRNA or polypeptide) is determined in the sample. The term “amount” can include any suitable measurement, value or scale, including quantitative and qualitative measurements, concentrations, absolute values, total amount present in a sample, and the like. The amount of mRNA (eg, including measurement of cDNA using target mRNA as a template) or the amount of polypeptide is described as an indication of whether a therapeutically or physiologically effective amount of solanifeb (or a derivative thereof) has been administered. be able to. Thus, when an amount of one or more of the biomarkers is detected, this is an indication that the agent (such as sorafenib) is achieving a physiological effect in the subject. Table 1 contains a complete list of biomarkers associated with sorafenib administration.

  “Therapeutic effect” indicates that the administered drug treats, fights, alleviates, reduces, ameliorates, ameliorates, etc. (eg, when administered in a therapeutically effective amount) at least one symptom of a disease or disorder. The therapeutic effects of sorafenib include, but are not limited to, for example, angiogenesis inhibition; cell proliferation inhibition; tissue necrosis or hypoxia; tumor growth, arrest or deceleration; tumor cell proliferation arrest or deceleration; eg tumor cells and / or endothelial cells Production of apoptosis; inhibition of tyrosine kinases; inhibition of VEGFR (like VEGFR2), PDGFR (like PDGFRbeta), FLT3, C-KIT, RET and RAF. For other activities, see, for example, Wilhelms et al, Canc. Res. 2004, 64 (19): 7099-109; Carlomagno et al, J. MoI. Nat1. Cancer Inst. See 2006 Mar 1:98 (5): 326-34. Sorafenib and other agents according to the present invention produce a biologically effective dose (BED) before obtaining a substantial therapeutic effect. The latter can occur, for example, when a dose of sorafenib is sufficient to interact with its kinase target, but is not suitable to effectively treat a disease or disorder. Absence of these conditions, ie modulation of one or more biomarkers (eg increased levels of IL-6, IL-8, MMP-1 and / or MMP-10) or if the amount of biomarkers is less than expected In circumstances, the dose of an agent (such as sorafenib) can be increased until the expected change in the amount of biomarker is observed.

  The amount of biomarker utilized as a diagnostic marker for drug effectiveness can be determined by various methods. For example, this amount can be determined using samples from the same subject before and after drug treatment, and a significant difference between the two time points determines that the drug has achieved its effect. Can be used to Such differences can be routinely determined and for example 2 × (times), 3 ×, 3.5 ×, 4 ×, 5 ×, 6 ×, 7 ×, 8 ×, 9 × 10 ×, 11 X, 15x, 20x or more, from about 3x-4x, from about 2x-5x, from about 2x-10x, and the like. As shown in Table 1, it was determined that 154 genes are differentially regulated in response to sorafenib treatment, and any of these genes, and the polypeptides they encode, are surrogate biologics for drug efficacy. Can be used as a marker. The term “differentially regulated” means that gene expression (eg, measured by mRNA or by a polypeptide) changes upon drug administration, but not all measured genes show such changes. Means.

  In addition to comparing amounts between samples from the same patient, standard or normal values can also be used. For example, a typical value or range of values for a particular biomarker can be established in a subject population, and such value can then be used as a threshold for drug efficacy. When values outside the typical range are observed, such values will indicate that the drug is having a therapeutic effect. On the other hand, the lack of significance of biomarker levels is to indicate that the disease (eg, cancer) is not responsive to the drug or that additional dose increases are required to be effective Can be used. In addition to comparing to standard or normal values, biomarker levels can be compared to samples from the subject prior to sorafenib administration. The negative (-) symbol in Table 1 indicates that the level of the biomarker has decreased, and if the symbol is not listed, it indicates that the biomarker has increased.

  The present invention may also be used when a biological sample is assayed for the effect of an agent (such as sorafenib) during a course of treatment of a subject, such as the differential gene or polypeptide expression of a biomarker during the course of treatment of the subject. In addition, a method for monitoring the effect of the agent is provided. When the detected amount indicates that an insufficient therapeutic amount of the agent has been administered, additional drug can be provided until an adequate biomarker response is reached and therapeutic levels are achieved.

  The increase in biomarkers can be observed at any time during treatment with the agent. For example, biomarker expression can be expressed in one or more doses (eg, multiple doses) to a subject, such as 2, 3, 6, 8, 10, 12, 14, 16, 20, 22, 26, 30, 36, 40 or It is not observed until after a dose has been administered, including higher doses (when the dose indicates the daily dose of the drug). Such an increase can occur, for example, about 4-24 hours, 1-3 days, 1-7 days, or 3-10 days after administration of the agent.

  In accordance with the present invention, any number of biomarkers can be detected, for example, to determine whether an agent has a therapeutic effect. These include 1, 2, 4, 10, 15, 20, 30, 60 and include the entire set of markers shown in Table 1. The amino acid and nucleotide sequences of the biomarkers in Table 1 are known and can be obtained from public databases such as GenBank.

  The present invention also provides a method of determining the effectiveness of the agent in treating a tumor in a subject that has been administered an agent (such as solanifeb or a stent), the method comprising tumor hypoxia in the subject. Measuring an amount, wherein said amount is an indication of whether a therapeutically effective amount of sorafenib has been administered. Tissue hypoxia can be defined as a decrease in oxygen use such that cells are experiencing anaerobic breathing. Various methods can be used to determine hypoxia. For example, systemic measurements include, for example, blood lactate, pH, oxygen transport / oxygen consumption, mixed venous oxygen saturation, venous arterial carbon dioxide gradient, and the like. In addition to systemic measurements, regional hypoxia can also be measured. These include, for example, blood oxygenation level dependent (BOLD) MRI (eg, Prasad et al., Noninvasive Evaluation of Internal Oxygenation with BOLD MRI, Circulation 94: 3271-3275, 1996), 18-F Misonidazole (18F-FMiSO) proton emission tomography (PET) (eg Lawrentschuk et al., BJU Int. 96 (4): 540-546, 2005) and other using 2-nitroimidazole hypoxia markers Includes non-invasive methods, such as methods. Because of other biomarker methods, sorafenib administration can be increased until hyperoxia is observed.

  The biomarker of the present invention can be used as an endogenous marker for tumor hypoxia, particularly when hypoxia occurs without new blood vessel formation. For example, sorafenib and stents are effective against tumor and blood vessel growth leading to tissue hypoxia when used to treat cancer. The presence and extent of tissue hypoxia in response to these and other agents are listed in Table 1, including, for example, IL-6, IL-8, MMP-1 and / or MMP-10 according to the present invention. It can be determined by measuring any biomarker. Like other methods, these biomarkers can be measured in blood, tumor cells, or other body fluids. The present invention therefore relates to biomarkers for tissue hypoxia. This can be achieved, for example, by MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen polypeptide or mRNA in a biological sample obtained from a subject. A method of determining the presence of tumor tissue hypoxia, comprising determining the amount of. The amount of the polypeptide or mRNA is an indication of the occurrence of hypoxia in the tumor tissue.

  The present invention also provides a method for determining whether a compound has pharmacological activity of sorafenib, the method comprising at least two differentially regulated mRNAs in cells exposed to sorafenib, or Detecting the amount of the polypeptide encoded thereby, wherein said amount is an indication of whether said compound has pharmacological activity of sorafenib and said differentially regulated mRNA is from Table 1. To be elected.

  The expression patterns of differentially expressed genes disclosed herein can be described as fingerprints in that they are unique patterns that are displayed by cells in response to sorafenib administration. Similar to fingerprints, an expression pattern can be used as a unique identifier to characterize its state with respect to the tissue sorafenib response, a point for comparing and characterizing cellular responses to multikinase inhibitors Can be used as For example, this fingerprint can be used to determine if the compound of interest has the same pharmacological action as sorafenib. Differential gene expression can be determined for certain test compounds. The closer the pattern is matched, the closer the compound mirrors the pharmacological profile of sorafenib. Such compounds can be used for any indication for which sorafenib is used.

The invention also provides a method of reducing an inflammatory event associated with sorafenib administration in a subject, the method comprising administering to such subject an effective amount of an anti-inflammatory agent. Any anti-inflammatory agent can be administered including, for example, non-steroidal anti-inflammatory agents (NSAIDs); cortisone and its derivatives and the like.
[Predictive assay]

  Laboratory assays that can predict the clinical benefit from a given anticancer agent will greatly enhance the clinical management of patients with cancer. In order to assess this effect, biomarkers associated with anti-cancer agents can be analyzed before, during and after treatment in a biological sample (eg, tumor sample, plasma).

  For example, MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen mRNA and protein can be detected in plasma. Thus, changes in plasma concentrations of MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen can be monitored in cancer patients. . In addition, MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen protein levels are also quantitatively immunized using paraffin-embedded tumor biopsy. Can be monitored by histochemistry.

  Another approach to monitor treatment is assessment of serum proteomic spectrum. In particular, plasma samples can be subjected to mass spectra for analysis (eg, surface enhanced laser desorption and ionization), and proteomic spectra can be generated for each patient. The set of spectra obtained from the analysis of plasma from the patient before and during treatment is analyzed by an iterative search algorithm that can identify proteomic patterns that completely distinguish treated samples from untreated samples Can do. The resulting pattern can then be used to predict clinical benefit after treatment.

  Global gene expression profiles and bioinformatic induction pattern identification of biological samples (eg tumor biopsy samples, blood samples) predict and are resistant to clinical benefit and sensitivity to certain anti-cancer drugs Can be used to anticipate the development of For example, isolated cells obtained from whole blood from a patient before and during treatment can be used to generate a blood cell gene expression profile using Affymetrx GeneChip technology and algorithms. These gene expression profiles are predictive of clinical benefit from treatment with certain anticancer agents.

Analysis of the biochemical composition of urine by ¹ H-NMR (nuclear magnetic resonance) can also be used as a predictive assay. Pattern recognition techniques can be used to assess the metabolic response to treatment with anti-cancer agents, and this response can be correlated to a clinical endpoint. Biomarkers or endogenous metabolites excreted in the urine have been well characterized by proton NMR for normal subjects (Zuppi et al., Clin Chim Acta 265: 85-97, 1997). These metabolites (about 30-40) represent byproducts of major metabolic pathways such as citric acid and urea cycles. Drugs, illnesses, and genetic stimuli have been shown to give rise to changes in the metabolic specificity of the baseline urinary profile, which is an indication of the time line and magnitude of the metabolic response to this stimulus. These analyzes are multivariable and therefore use pattern recognition techniques to improve data interpretation. Urine metabolite patterns can be correlated to clinical endpoints to determine clinical benefit.

  The invention is described below with reference to the following non-limiting examples.

Affymetrix analysis

Total RNA was extracted from tumors using TRIzo1 reagent (Life Technologies, MD) according to a modified vendor protocol using the RNeasy protocol (Qiagen, CA). RNA (5 μg) was transcribed into cDNA using the Invitrogen Superscript III Choice system for RT-PCR according to the vendor protocol (Invitrogen, CA). The cDNA was used for in vitro transcription reactions incorporating biotinylated nucleotides using an RNA labeling kit (Enzo Diagnostics, NY). The resulting cRNA was purified, quantified, fragmented and loaded (10 μg) onto each array (Human Genome U133 Plus 2.0). Hybridization was allowed to proceed for 16-18 hours at 45 ° C. in an Affymetrix GemeChip hybridization oven 640 (Affymetrix; Santa Clara, Calif.). The array was stained with phycoerythrin-conjugated streptavidin using an Affymetrix Fluidics Station 450. Measurements were made after placing the array in an Affymetrix Scanner 3000. The intensity of the emitted light was digitally converted to a numerical value indicating the expression level. The relative fold change in gene expression was calculated from a comparison of sorafenib treated vehicle versus vehicle treated tumor.

  Based on Affymetrix analysis of tumor lysates, approximately 154 genes were up- or down-regulated 3.5-fold or more in sorafenib-treated tumors. At the 4 time points analyzed after sorafenib treatment, the most significant genetic change occurs at 4 hours after the third dose and begins to decrease by 24 hours. See Table 1.

Taqman analysis

  All genes of interest were MGB designed probes with a FAM fluorescent tag (FAM = 6-carboxyfluorescein). For normalization, an MGB probe against a ribosomal protein with a fluorescent tag (VIC amidite) was used.

  The PCR reaction was performed using cDNA prepared from RNA isolated from tumor cell samples using the Superscript III First Strand Synthesis kit (Invitrogen, Calif.), Using the ABI Prism 7900 Sequence Detector (PE Applied Biosystems on PE Applied Biosystems). It was done. The reaction included one pre-PCR cycle at 50 ° C. for 2 minutes and 95 ° C. for 10 minutes, followed by 40 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 minute.

  Estimated expression quality was based on C + values. Each individual sample was normalized to its own L37 value. Fold change was calculated using the ddCT method and treated samples were compared to vehicle control samples.

  Significant increases in mRNA levels of inflammation and vascular cytokines IL-6 and IL-8, and matrix metalloproteinases MMP-1 and MMP-10 were observed in treatment with sorafenib. The data generated on the Affimetrix gene chip was confirmed by Taqman analysis using human specific primers.

Measuring protein levels in tumors and plasma

  Tumor samples were snap frozen and lysis buffer (40 mM Tris, pH, 10% glycerol, 50 mM b-glycerol phosphate, 5 mM EGTA, 2 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 0.3% Triton X-100). , And in a protease inhibitor cocktail (Roche, Indianapolis, IN).

  IL-6, IL-8 and VEGF levels were determined using specific assay kits from Meso Scale Diagnostics (Gaithersburg, MD) according to the conditions from the manufacturer.

  MMP-1 and MMP-10 protein levels were determined in an immunoassay ELISA from R & D Systems (Minneapolis, Minn.) According to the vendor's protocol.

  Tumor protein levels of IL-6, IL-8, MMP-1 and MMP-10 were elevated by sorafenib treatment (QDX3) at the different doses tested. A statistically significant increase in the level of all four proteins was observed at the third dose (15, 30, 60 mg / kg dose) 24 hours. A trend for elevated levels of IL-6, IL-8, MMP-1 and MMP-10 in plasma was observed in plasma collected from sorafenib-treated animals.

Top probe set with multiples greater than 3.5 with 43-9006 treatment at some day / time point. Significant signals are> 1000.

An outline of gene regulatory protein expression studies is shown. Timeline for hypoxia assessment using sorafenib treatment and Hypoprobe in mice. One hour prior to tumor collection, mice were intravenously injected with Hypoxyprobe (pimonidazole hydrochloride 60 mg / kg, Chemicon International; Temecula, CA). Tumors were harvested 4 and 24 hours after the last sorafenib administration. For UV visualization, formalin-fixed paraffin-embedded tumor sections cut to 5 mm were incubated for 1 hour with FITC-conjugated Hypoprobe-1 primary antibody supplied with a kit (Hypoxyprobe-1 Plus Kit; Chemicon International: Temecula, Calif.). Sections were also incubated with goat anti-CD31 antibody (Santa Cruz: Santa Cruz, CA). Affymetrix analysis of genes regulated by sorafenib treatment is provided. Data are expressed as relative fold changes in gene expression when treated tumors are compared to corresponding control tumors. Here, bright red represents> 3.5-fold upregulation of gene expression and bright green represents> 3.5-fold downregulation of gene expression. Shows that the IL-8 and IL-6 genes are upregulated by sorafenib treatment as confirmed by taqman analysis. The MMP-1 and MMP-10 genes are up-regulated by sorafenib treatment as confirmed by taqman analysis. Sorafenib-treated tumors were evaluated for IL-6, IL-8, MMP-1 and MMP-10 protein levels. Data are mean ± SEM; animals per group n = 10 Panel (A) Tumor IL-6 (pg / ml) Panel (B) Tumor IL-8 (pg / ml) Panel (C) Tumor MMP-10 (pg / Ml) Panel (D) Tumor MMP-1 (pg / ml) Plasma from sorafenib treated animals was evaluated for IL-6, IL-8 and MMP-10 levels. Data are mean ± SE; n = 10 animals / group, * indicates P = 0.05. Panel (A) Plasma IL-6 (pg / ml) Panel (B) Plasma IL-8 (pg / ml) Panel (C) Plasma MMP-10 (pg / ml) VEGF levels were moderately assessed in sorafenib treated tumors. Data are mean ± SEM; n = 10 animals / group, * indicates P = 0.05.

Claims (20)

  A method for determining the effectiveness of sorafenib in a disease or disorder in a subject administered sorafenib, comprising MMP-1, MMP-10, IL-6, IL-8, in a biological sample obtained from said subject Detecting the amount of IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen polypeptide or polynucleotide, wherein said amount is an indication of whether therapeutically effective sorafenib has been administered And comparing the detected amount with a standard value.   2. The method of claim 1, wherein the amount is increased relative to a standard value that is a sample from the subject prior to sorafenib administration.   The method of claim 1, wherein the amount is increased compared to a standard value.   Standards are MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen in a biological sample obtained from the subject prior to sorafenib administration. 2. The method of claim 1 determined by detecting the amount of a polypeptide or polynucleotide.   MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or FOS-like antigen polypeptide or poly in biological samples obtained through a course of sorafenib administration The method of claim 1 comprising detecting the nucleotide.   2. The method of claim 1, wherein IL-6, IL-8 and / or MMP-1 are detected.   2. The method of claim 1, wherein the sample is a tumor tissue or tumor cell sample.   The method of claim 1, wherein the sample is a blood sample.   4. The method of claim 2 or 3, wherein the increase occurs about 4-24 hours, 1-3 days, 1-7 days, or 3-10 days after sorafenib administration.   2. The method of claim 1, further comprising administering sorafenib to the subject until the amount of IL-6, IL-8, MMP-1 and / or MMP-10 is increased compared to a control or standard level.   2. The method of claim 1, wherein the disease or disorder is a tumor.   2. The method of claim 1, wherein the disease or disorder is melanoma, hepatocellular carcinoma, non-small lung cell lung cancer, or renal cell cancer.   A method for determining the effectiveness of sorafenib in treating a disease or disorder in a subject administered sorafenib, comprising IL-6, IL-8, MMP-1 and / or in a biological sample obtained from said subject Detecting the amount of MMP-10 mRNA, wherein the amount of mRNA is an indication of whether a therapeutically effective amount of sorafenib has been administered.   A method for determining the effectiveness of sorafenib in the treatment of a disease or disorder in a subject administered sorafenib, comprising at least one differentially regulated mRNA in a biological sample obtained from said subject, or thereby Detecting the amount of encoded polypeptide, wherein the mRNA or polypeptide encoded thereby is selected from Table 1, and wherein the amount of the mRNA or polypeptide encoded thereby is sorafenib A method which is an indication of whether a therapeutically effective amount of was administered.   15. The method of claim 14, wherein the amount of at least 10 differentially regulated mRNA or polypeptide encoded by said mRNA is detected.   A method of determining the effectiveness of sorafenib in treating a tumor in a subject administered sorafenib, comprising measuring the amount of tumor hypoxia in said subject, wherein said amount is therapeutically of sorafenib A method, which is an indication of whether an effective amount has been administered.   A method of determining a course of sorafenib treatment for a subject having cancer, comprising: MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and / or Or administering a FOS-like antigen polypeptide or polynucleotide in an amount effective to induce an increase in a biological sample obtained from said subject relative to a control or normal value.   A method of determining whether a compound has pharmacological activity of sorafenib, the amount of at least two differentially regulated mRNAs or polypeptides encoded thereby in cells exposed to said sorafenib Wherein the amount is an indication that the compound has the pharmacological action of sorafenib, and the differentially regulated mRNA is selected from Table 1.   A method for determining the presence of tumor tissue hypoxia, wherein MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1 in a biological sample obtained from a subject And / or detecting the amount of FOS-like antigen polypeptide or polynucleotide or mRNA, wherein the amount of said polypeptide or mRNA is indicative of the occurrence of hypoxia in tumor tissue.   A method of reducing an inflammatory event associated with sorafenib administration in a subject comprising administering to said subject an effective amount of an anti-inflammatory agent.

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