EP2002264A2

EP2002264A2 - Methods for prediction and prognosis of cancer, and monitoring cancer therapy

Info

Publication number: EP2002264A2
Application number: EP07754481A
Authority: EP
Inventors: Scott Wilhelm
Original assignee: Bayer Healthcare LLC
Current assignee: Bayer Healthcare LLC
Priority date: 2006-03-31
Filing date: 2007-03-30
Publication date: 2008-12-17
Also published as: WO2007123722A3; WO2007123722A2; JP2009532029A; CA2648106A1

Abstract

The present invention also relates to biomarkers and the use of biomarkers for the prediction and prognosis of cancer as well as the use of biomarkers to monitor the efficacy of cancer treatment.

Description

METHODS FOR PREDICTION AND PROGNOSIS OF CANCER, AND MONITORING

CANCER THERAPY

FIELD OF THE INVENTION

[001] The present invention relates to biomarkers and the use of biomarkers for the prediction and prognosis of cancer as well as the use of biomarkers to monitor the efficacy of cancer treatment.

BACKGROUND OF THE INVENTION

[002] Many disease states are characterized by differences in the expression levels of various genes either through changes in the copy number of the genetic DNA or through changes in levels of transcription of particular genes (e.g., through control of initiation, provision of RNA precursors, RNA processing, etc.). For example, losses and gains of genetic material play an important role in malignant transformation and progression. These gains and losses are thought to be driven by at least two kinds of genes, oncogenes and tumor suppressor genes^'. Oncogenes are positive regulators of tumorgenesis, while tumor suppressor genes are negative regulators of tumorgenesis (Marshall, Cell 64:313-326, 1991; Weinberg, Science 254:1138-1146, 1991). Therefore, one mechanism of activating unregulated growth is to increase the number of genes coding for oncogene proteins or to increase the level of expression of these oncogenes (e.g., in response to cellular or environmental changes), and another mechanism is to lose genetic material or to decrease the level of expression of genes that code for tumor suppressors. This model is supported by the losses and gains of genetic material associated with glioma progression (Mikkelson, et at., J. Cellular Biochem. 46:3-8, 1991). Thus, changes in the expression (transcription) levels of particular genes (e.g., oncogenes or tumor suppressors) serve as signposts for the presence and progression of various cancers.

DESCRIPTION OF THE INVENTION

[003] The present invention relates to biomarkers and the use of biomarkers for the prediction and prognosis of cancer as well as the use of biomarkers to monitor the efficacy of cancer treatment. This invention also relates to the use of soluble metalloproteinases (MMP) and interleukins (IL) or colony stimulating factors (CSF) as biomarkers for the efficacy of treatment with sorafenib. Biomarkers can be selected from the list set forth in Table 1, including, e.g., MMP-1 , MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1 , and/or FOS-like antigen etc.

[004] In one embodiment of the present invention, the biomarkers comprise one or more genes and/or gene products that demonstrate altered expression following exposure to a drug. In a further embodiment, the drug is a sorafenib and derivatives thereof, and in another embodiment, the biomarker is MMP-1, MMP-10, IL-6, IL-8, IL- 10, CSF-2, SLCO4A1, and/or FOS-like antigen..

[005] Another embodiment of the present invention is a method for screening the effects of a drug on a tissue or cell sample comprising the step of analyzing the level of expression of one or more genes and/or gene products, wherein the gene expression and/or gene product levels in the tissue or cell sample are analyzed before and after exposure to the drug, and a variation in the expression level of the gene and/or gene product is indicative of a drug effect or provides a patient diagnosis or predicts a patient's response to the treatment. In a further embodiment, the drug is a sorafenib. In another embodiment, the gene or gene product is MMP-1, MMP-10, IL-6, IL-8, IL- 10, CSF-2, SLCO4A1 , and/or FOS-like antigen..

[006] Another Claim of the present invention is a method for discovering novel drugs comprising the step of analyzing the level of expression of one or more genes and/or gene products, wherein the gene expression and/or gene product levels of the cells are analyzed before and after exposure to the drug, and a variation in the expression level of the gene and/or gene product is indicative 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.

[007] The invention further provides a method for identifying a compound useful for the treatment of cancer comprising administering to a subject with cancer a test compound, and measuring the activity of the polypeptide, wherein a change in the activity of the polypeptide is indicative of the test compound being useful for the treatment of cancer. In a further embodiment, the polypeptide is MMP-1, MMP-10, IL- 6, IL-8, 1L-10, CSF-2, SLCO4A1 , and/or FOS-like antigen, and in another embodiment, the compound is a sorafenib.

[008] The invention, thus, provides methods which may be used to identify compounds which may act, for example, as regulators or modulators such as agonists and antagonists, partial agonists, inverse agonists, activators, co-activators, and inhibitors. Accordingly, the invention provides reagents and methods for regulating the expression of a polynucleotide or a polypeptide associated with cancer. Reagents that modulate the expression, stability, or amount of a polynucleotide or the activity of the polypeptide may be a protein, a peptide, a peptidomimetic, a nucleic acid, a nucleic acid analogue (e.g., peptide nucleic acid, locked nucleic acid), or a small molecule.

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

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

[011] In another embodiment, the invention provides a method for detecting cancer in a patient sample in which an antibody to a protein is used to react with proteins in the patient sample. In a still further embodiment, the antibody is specific for MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and/or FOS-like antigen. [012] Another aspect of the present invention is a method for distinguishing between normal and disease states comprising the step of analyzing the level of expression of one or more genes and/or gene products, wherein the gene expression and/or gene product levels of normal and disease tissues are analyzed, and a variation in the expression level of the gene and/or gene product is indicative of a disease state. 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.

[013] In another embodiment, the invention pertains to a method of determining the phenotype of cells comprising detecting the differential expression, relative to normal cells, of at least one gene, wherein the gene is differentially expressed by at least a factor of two, at least a factor of five, at least a factor of twenty, or at least a factor of fifty. In a further embodiment, the gene encodes MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1 , and/or FOS-like antigen.

[014] Any test sample in which it is desired to identify a polynucleotide or polypeptide thereof can be used, including, e.g., blood, urine, saliva, stool (for extracting nucleic acid, see, e.g., U.S. Pat. No. 6,177,251), swabs comprising tissue, biopsied tissue, tissue sections, cultured cells, etc.

[015] Detection can be accomplished in combination with polynucleotide probes for other genes, e.g., genes which are expressed in other disease states; tissues, cells, 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 gland, peripheral blood cells (T-cells, lymphocytes, etc.), embryo, normal breast fat, adult and embryonic stem cells, specific cell-types, such as endothelial, epithelial, myocytes, adipose, luminal epithelial, basoepithelial, myoepithelial, stromal cells, cancers thereof, etc.

[016] In yet another embodiment, the invention pertains to a method of determining the phenotype of cells, comprising detecting the differential expression, relative to normal cells, of at least one polypeptide, wherein the protein is differentially expressed by at least a factor of two, at least a factor of five, at least a factor of twenty, an up to at least a factor of fifty. In a further embodiment, the polypeptide is MMP-1, MMP-10, IL- 6, IL-8, IL-10, CSF-2, SLCO4A1 , and/or FOS-like antigen.

[017] In another embodiment, the invention pertains to a method for determining the phenotype of cells from a patient by providing 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, obtaining a sample of cells from a patient, optionally providing a second sample of cells substantially all of which are noncancerous, contacting the nucleic acid probe under stringent conditions with mRNA of each of said first and second cell samples, and comparing (a) the amount of hybridization of the probe with mRNA of the first cell sample, with (b) the amount of hybridization of the probe with mRNA of the second cell sample, wherein a difference of at least a factor of two, at least a factor of five, at least a factor of twenty, or at least a factor of fifty in the amount of hybridization with the mRNA of the first cell sample as compared to the amount of hybridization with the mRNA of the second cell sample is indicative of the phenotype of cells in the first cell sample. In a further embodiment, the nucleic acid probe comprises the nucleotide sequence encoding MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and/or FOS-like antigen.

[018] In another embodiment, the invention provides a test kit for identifying the presence of cancerous cells or tissues, comprising a probe/primer, for measuring a level of a nucleic acid in a sample of cells isolated from a patient. In certain embodiments, the kit may further include instructions for using the kit, solutions for suspending or fixing the cells, detectable tags or labels, solutions for rendering a nucleic acid susceptible to hybridization, solutions for lysing cells, or solutions for the purification of nucleic acids. In a further embodiment, the probe/primer comprises the nucleotide sequence encoding (or complementary to) MMP-1, MMP-10, IL-6, IL-8, IL- 10, CSF-2, SLCO4A1 , and/or FOS-like antigen. [019] In one embodiment, the invention provides a test kit for identifying the presence of cancer cells or tissues, comprising an antibody specific for a protein. In certain embodiments, the kit further includes instructions for using the kit. In certain embodiments, the kit may further include solutions for suspending or fixing the cells, detectable tags or labels, solutions for rendering a polypeptide susceptible to the binding of an antibody, solutions for lysing ce.lls, or solutions for the purification of polypeptides. In a still further embodiment, the antibody is specific for MMP-1, MMP- 10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1 , and/or FOS-like antigen.

[0201 In another embodiment, the invention provides a test kit for monitoring the efficacy of a compound or therapeutic in cancerous cells or tissues, comprising a probe/primer, for measuring a level of a nucleic acid in a sample of cells isolated from a patient. In certain embodiments, the kit may further include instructions for using the kit, solutions for suspending or fixing the cells, detectable tags or labels, solutions for rendering a nucleic acid susceptible to hybridization, solutions for lysing cells, or solutions for the purification of nucleic acids. In a further embodiment, the probe/primer comprises the nucleotide sequence encoding MMP-1, MMP-10, IL-6, IL- 8, IL-10, CSF-2, SLCO4A1, and/or FOS-like antigen.

[021] The present invention also relates to a combination of a multi-kinase inhibitor (see below for examples) and a prodrug that is selectively activated in hypoxic tissues. For example, quinone bioreductϊve prodrugs are selectively activated in tissues where they can exert a cytotoxic effect. See, e.g., Seow et al., Proc Natl Acad Sci U S A. 2005 Jun 28;102(26):9282-7.

[022] In one embodiment, the invention provides a test kit for monitoring the efficacy of a compound or therapeutic in cancer cells or tissues, comprising an antibody specific for a protein. In certain embodiments, the kit further includes instructions for using the kit. In certain embodiments, the kit may further include solutions for suspending or fixing the cells, detectable tags or labels, solutions for rendering a polypeptide susceptible to the binding of an antibody, solutions for lysing cells, or solutions for the purification of polypeptides. In a still further embodiment, the antibody is specific for MMP-1 , MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and/or FOS-like antigen.

[023] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, and reagents described and as such may vary. It is also to 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 present invention which will be limited only by the appended claims.

[024] It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" is a reference to one or more genes and includes equivalents thereof known to those skilled in the art, and so forth.

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

[026] All publications and patents including U.S. provisional application No. 60/787,693, filed March 31, 2006 mentioned herein are hereby incorporated herein by reference for the purpose of describing and disclosing, for example, the constructs and methodologies that are described in the publications which might be used in connection with the presently described invention. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

Definitions [027] For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below.

[028] An "address" on an array (e.g., a microarray) refers to a location at which an element, for example, an oligonucleotide, is attached to the solid surface of the array.

[029] The term "agonist," as used herein, is meant to refer to an agent that mimics or up-regulates (e.g., potentiates or supplements) the bioactivity of a protein. An agonist may be a wild-type protein or derivative thereof having at least one bioactivity of the wild-type protein. An agonist may also be a compound that up-regulates expression of a gene or which increases at least one bioactivity of a protein. An agonist can also be a compound which increases the interaction of a polypeptide with another molecule, for example, a target peptide or nucleic acid.

[030] "Amplification," as used herein, relates to the production of additional copies of a nucleic acid sequence. For example, amplification may be carried out using polymerase chain reaction (PCR) technologies which are well known in the art. (see, e.g., Dieffenbach and Dveksler (1995) PCR Primer, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N. Y.)

[031] The term "antibody," as used herein, is intended to include whole antibodies, for example, of any isotype (IgG₁ IgA, IgM, IgE, etc.), and includes fragments thereof which are also specifically reactive with a vertebrate (e.g., mammalian) protein. Antibodies may be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. Thus, the term includes segments of proteolytically-cleaved or recombinantly-prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein. Non-limiting examples of such proteolytic and/or recombinant fragments include Fab, F(ab')2, Fab', Fv, and single chain antibodies (scFv) containing a V[L] and/or V[H] domain joined by a peptide linker. The scFv's may be covalently or non- covalently linked to form antibodies having two or more binding sites. The subject invention includes polyclonal, monoclonal, or other purified preparations of antibodies and recombinant antibodies.

[032] The terms "array" or "matrix" refer to an arrangement of addressable locations or "addresses" on a device. The locations can be arranged in two-dimensional arrays, three-dimensional arrays, or other matrix formats. The number of locations may range from several to at least hundreds of thousands. Most importantly, each location represents a totally independent reaction site. A "nucleic acid array" refers to an array containing nucleic acid probes, such as oligonucleotides or larger portions of genes. The nucleic acid on the array is preferably single-stranded. Arrays wherein the probes are oligonucleotides are referred to as "oligonucleotide arrays" or "oligonucleotide chips." A "microarray," also referred to herein as a "biochip" or "biological chip," is an array of regions having a density of discrete regions of at least about 100/cm², and preferably at least about 1000/cm². The regions in a microarray have typical dimensions, for example, diameters, in the range of between about 10-250 μm, and are separated from other regions in the array by about the same distance.

[033] "Biological activity" or "bioactivity" or "activity" or "biological function," which are used interchangeably, herein mean an effector or antigenic function that is directly or indirectly performed by a polypeptide (whether in its native or denatured conformation), or by any subsequence thereof. Biological activities include binding to polypeptides, binding to other proteins or molecules, activity as a DNA binding protein, as a transcription regulator, ability to bind damaged DNA, etc. A bioactivity can be modulated by directly affecting the subject polypeptide. Alternatively, a. bioactivity can be altered by modulating the level of the polypeptide, such as by modulating expression of the corresponding gene.

[034] The term "biological sample," as used herein, refers to a sample obtained from an organism or from components (e.g., cells) of an organism. The sample may be of any biological tissue or fluid. The sample may be a sample which is derived from a patient. Such samples include, but are not limited to, sputum, blood, blood cells (e.g., white cells), tissue or biopsy samples (e.g., tumor biopsy), urine, peritoneal fluid, and pleural fluid, or cells therefrom. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.

[035] The term "biomarker" or "marker" encompasses a broad range of intra- and extra-cellular events as well as whole-organism physiological changes. Biomarkers may be represent essentially any aspect of cell function, for example, but not limited to, levels or rate of production of signaling molecules, transcription factors, metabolites, gene transcripts as well as post-translational modifications of proteins. Biomarkers may include whole genome analysis of transcript levels or whole proteome analysis of protein levels and/or modifications.

[036] A biomarker may also refer to a gene or gene product which is up- or down- regulated in a compound-treated, diseased cell of a subject having the disease compared to an untreated diseased cell. That is, the gene or gene product is sufficiently specific to the treated cell that it may be used, optionally with other genes or gene products, to identify, predict, or detect efficacy of a small molecule. Thus, a biomarker is a gene or gene product that is characteristic of efficacy of a compound in a diseased cell or the response of that diseased, cell to treatment by the compound.

[037] A nucleotide sequence is "complementary" to another nucleotide sequence if each of the bases of the two sequences match, that is, are capable of forming Watson- Crick base pairs. The term "complementary strand" is used herein interchangeably with the term "complement." The complement of a nucleic acid strand may be the complement of a coding strand or the complement of a non-coding strand.

[038] "Detection agents of genes" refers to agents that can be used, to specifically detect the gene or other biological molecules relating to it, for example, RNA transcribed from the gene or polypeptides encoded by the gene. Exemplary detection agents are nucleic acid probes, which hybridize to nucleic acids corresponding to the gene, and antibodies. [039] Baseline levels can refer to a standard control for "normal" levels (i.e., patients without disease), but can also be comparative, e.g., where low baseline levels is compared to the levels of other subjects having the disease.

[040] The term "cancer" includes, but is not limited to, solid tumors, such as cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid, and their distant metastases. The term also includes lymphomas, sarcomas, and leukemias.

[041] Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.

[042] Examples of cancers of the respiratory tract include, but are not limited to, small-cell and non-small-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma.

[043] Examples of brain cancers include, but are not limited to, brain stem and hypophtalmic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumor.

[044] Tumors of the male reproductive organs include, but are not limited to, prostate and testicular cancer. Tumors of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus.

[045] Tumors of the digestive tract include, but are not limited to, anal, colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal, small-intestine, and salivary gland cancers. [046] Tumors of the urinary tract include, but are not limited to, bladder, penile, kidney, renal (e.g., RCC), pelvis, ureter, and urethral cancers.

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

[048] Examples of liver cancers include, but are not limited to, hepatocellular carcinoma (liver cell carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma.

[049] Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.

[050] Head-and-neck cancers include, but are not limited to, laryngeal / hypopharyngeal / nasopharyngeal / oropharyngeal cancer, and lip and oral cavity cancer.

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

[052] Sarcomas include, but are not limited to, sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.

[053] Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia. [054] "A diseased cell of cancer" refers to a cell present in subjects having cancer. That is, a cell which is a modified form of a normal cell and is not present in a subject not having cancer, or a cell which is present in significantly higher or lower numbers in subjects having cancer relative to subjects not having cancer.

[055J The term "equivalent" is understood to include nucleotide sequences encoding functionally equivalent polypeptides. Equivalent nucleotide sequences may include sequences that differ by one or more nucleotide substitutions, additions, or deletions, such as allelic variants.

[056] The term "expression profile," which is used interchangeably herein with "gene expression profile" and "fingerprint" of a cell refers to a set of values representing mRNA levels of one or more genes in a cell. An expression profile preferably comprises values representing expression levels of at least about 10 genes, preferably at least about 50, 100, 200 or more genes. Expression profiles may also comprise an mRNA level of a gene which is expressed at similar levels in multiple cells and conditions (e.g., a housekeeping gene such as GAPDH). For example, an expression profile of a diseased cell of cancer refers to a set of values representing mRNA levels of 10 or more genes in a diseased cell.

[057] The term "gene" refers to a nucleic acid sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence. The gene may be derived in whole or in part from any source known to the art, including a plant, a fungus, an animal, a bacterial genome or episome, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA, or chemically synthesized DNA. A gene may contain one or more modifications in either the coding or the untranslated regions which could affect the biological activity or the chemical structure of the expression product, the rate of expression, or the manner of expression control. Such modifications include, but are not limited to, mutations, insertions, deletions, and substitutions of one or more nucleotides. The gene may constitute an uninterrupted coding sequence or it may include one or more introns, bound by the appropriate splice junctions.

[058] "Hybridization" refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing. For example, two single-stranded nucleic acids "hybridize" when they form a double-stranded duplex. The region of double-strandedness may include the full-length of one or both of the single-stranded nucleic acids, or all of one single-stranded nucleic acid and a subsequence of the other single-stranded nucleic acid, or the region of double-strandedness may include a subsequence of each nucleic acid. Hybridization also includes the formation of duplexes which contain certain mismatches, provided that the two strands are still forming a double-stranded helix. "Stringent hybridization conditions" refers to hybridization conditions resulting in essentially specific hybridization.

[059] The term "isolated," as used herein, with respect to nucleic acids, such as DNA or RNA₁ refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. The term "isolated" as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an "isolated nucleic acid" may include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.

[060] As used herein, the terms ."label" and "detectable label" refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands (e.g., biotin or haptens), and the like. The term "fluorescer" refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range. Particular examples of labels which may be used in the present invention include fluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol, NADPH, alpha - beta -galactosidase, and horseradish peroxidase.

[061] As used herein, the term "level of expression" refers to the measurable expression level of a given nucleic acid. The level of expression of a nucleic acid is determined by methods well known in the art. The term "differentially expressed" or "differential expression" refers to an increase or decrease in the measurable expression level of a given nucleic acid. As used herein, "differentially expressed" or "differential expression" means the difference in the level of expression of a nucleic acid is at least 1.4-fold or more in two samples used for comparison, both of which are compared to the same normal standard sample. "Differentially expressed" or "differential expression" according to the invention also means a 1.4-fold, or more, up to and including 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or more difference in the level of expression of a nucleic acid in two samples used for comparison. A nucleic acid is also said to be "differentially expressed" in two samples if one of the two samples contains no detectable expression of a given nucleic acid, provided that the detectably expressed nucleic acid is expressed at +/- at least 1.4 fold. Differential expression of a nucleic acid sequence is "inhibited" the difference in the level of expression of the nucleic acid in two or more samples used for comparison is altered such that it is no longer at least a 1.4 fold difference. Absolute quantification of the level of expression of a nucleic acid may be accomplished by including a known concentration(s) of one or more control nucleic acid species, generating a standard curve based on the amount of the control nucleic acid and extrapolating the expression level of the "unknown" nucleic acid species from the hybridization intensities of the unknown with respect to the standard curve.

[062] As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and, as applicable to the embodiment being described, single-stranded (sense or antisense) and double-stranded polynucleotides. Chromosomes, cDNAs, mRNAs, rRNAs, and ESTs are representative examples of molecules that may be referred to as nucleic acids. [063] 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 preferably from about 15 to about 150 nucleotides, more preferably from about 150 to about 1000 in length. The oligonucleotide may be a naturally occurring oligonucleotide or a synthetic oligonucleotide. Oligonucleotides may be prepared by the phosphoramidite method (Beaucage and Carruthers, Tetrahedron Lett. 22:1859-62, 1981), or by the triester method (Matteucci, et al., J. Am. Chem. Soc. 103:3185, 1981), or by other chemical methods known in the art.

[064] The term "patient" or "subject" as used herein includes mammals (e.g., humans and animals).

[065] As used herein, a nucleic acid or other molecule attached to an array is referred to as a "probe" or "capture probe." When an array contains several probes corresponding to one gene, these probes are referred to as a "gene-probe set." A gene-probe set may consist of, for example, about 2 to about 20 probes, preferably from about 2 to about 10 probes, and most preferably about 5 probes.

[066] The "profile" of a cell's biological state refers to the levels of various constituents of a cell that are known to change in response to drug treatments and other perturbations of the biological state of the cell. Constituents of a cell include, for example, levels of RNA, levels of protein abundances, or protein activity levels.

[067] The term "protein" is used interchangeably herein with the terms "peptide" and "polypeptide."

[068] An expression profile in one cell is "similar" to an expression profile in another cell when the level of expression of the genes in the two profiles are sufficiently similar that the similarity is indicative of a common characteristic, for example, the same type of cell. Accordingly, the expression profiles of a first cell and a second cell are similar when at least 75% of the genes that are expressed in the first cell are expressed in the second cell at a level that is within a factor of two relative to the first cell.

[069] "Small molecule," as used herein, refers to a composition with a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids, or other organic or inorganic molecules. Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, which can be screened with any of the assays of the invention to identify compounds that modulate a bioactivity.

[070] The term "specific hybridization" of a probe to a target site of a template nucleic acid refers to hybridization of the probe predominantly to the target,, such that the hybridization signal can be clearly interpreted. As further described herein, such conditions resulting in specific hybridization vary depending on the length of the region of homology, the GC content of the region, and the melting temperature ("Tm") of the hybrid. Thus, hybridization conditions may vary in salt content, acidity, and temperature of the hybridization solution and the washes.

[071] A "variant" of polypeptide refers to a polypeptide having an amino acid sequence in which one or more amino acid residues is altered. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). A variant may also have "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be identified using computer programs well known in the art, for example, LASERGENE software (DNASTAR).

[072] The term "variant," when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to that of a particular gene or the coding sequence thereof. This definition may also include, for example, "allelic," "splice," "species," or "polymorphic" variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative 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 also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.

[073] An aspect of the invention is directed to the identification of agents capable of modulating the differentiation and proliferation of cells characterized by aberrant proliferation. A more specific aspect of the invention relates to methods of screening candidate compounds or substances for their ability to regulate the differential expression of nucleic acid sequences. That is, if a nucleic acid sequence is overexpressed in cancer cells, then the candidate compounds are screened for their ability to decrease expression, and if a nucleic acid sequence is underexpressed in cancer cells, then a test compound is screened for its ability to increase expression. In addition, another specific aspect the invention relates to screening assays to identify test compounds or substances which modulate the activity of one or more polypeptides which are encoded by the differentially expressed sequences described herein. In this regard, the invention provides assays for determining compounds that modulate the expression of marker nucleic acids and/or alter the bioactivity of the encoded polypeptide.

[074] Any agent may be utilized in accordance with the present invention. Thus, although the disclosure may be written in terms of, e.g., sorafenib, the latter is an example, and the invention is not limited to it. For instance, other multi-kinase inhibitors can also be utilized, including Sutent and other compounds as mentioned below. In addition, derivatives of sorafenib (BAY 43-9006) are also included, e.g., as described in WO 00/42102, WO 00/41698, and PCT/US/30542 which are hereby incorporated by reference in their entirety.

Screening for modulation of differential expression

[075] Drug screening is performed by adding a test compound (e.g., sorafenib) to a sample of cells, and monitoring the effect. A parallel sample which does not receive the test compound is also monitored as a control. The treated and untreated cells are then compared by any suitable phenotypic criteria, including but not limited to microscopic analysis, viability testing, ability to replicate, histological examination, the level of a particular RNA or polypeptide associated with the cells, the level of enzymatic activity expressed by the cells or cell lysates, and the ability of the cells to interact with other cells or compounds. Differences between treated and untreated cells indicates effects attributable to the test compound.

[076] Desirable effects of a test compound include an effect on any phenotype that was conferred by the cancer-associated marker nucleic acid sequence. Examples include a test compound that limits the overabundance of mRNA, limits production of the encoded protein, or limits the functional effect of the protein. The effect of the test compound would be apparent when comparing results between treated and untreated cells.

[077] The invention thus, also encompasses methods of screening for agents (e.g., sorafenib) which inhibit or enhance the expression of the nucleic acid markers in vitro, comprising exposing a cell or tissue in which the marker nucleic acid mRNA (e.g., MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and/or FOS-like antigen) is detectable in cultured cells to an agent in order to determine whether the agent is capable of inhibiting or enhancing production of the mRNA; and determining the level of mRNA in the exposed cells or tissue, wherein a decrease in the level of the mRNA after exposure of the cell line to the agent is indicative of inhibition of the marker nucleic acid mRNA production and an increase in mRNA levels is indicative of enhancement of maker mRNA production. [078] Alternatively, the screening method may include in vitro screening of a cell or tissue in which marker protein is detectable in cultured cells to an agent suspected of inhibiting or enhancing production of the marker protein; and determining the level of the marker protein in the cells or tissue, wherein a decrease in the level of marker protein after exposure of the cells or tissue to the agent is indicative of inhibition of marker protein production and an increase on the level of marker protein is indicative of enhancement of marker protein production.

[079] The invention also encompasses in vivo methods of screening for agents which inhibit or enhance expression of the marker nucleic acids, comprising exposing a subject having tumor cells in which marker mRNA or protein is detectable to an agent suspected of inhibiting or enhancing production of marker mRNA or protein; and determining the level of marker mRNA or protein in tumor cells of the exposed mammal. A decrease in the level of marker mRNA or protein after exposure of the subject to the agent is indicative of inhibition of marker nucleic acid expression and an increase in the level of marker mRNA or protein is indicative of enhancement of marker nucleic acid expression.

[080] Accordingly, the invention provides a method comprising incubating a cell expressing the marker nucleic acids with a test compound and measuring the mRNA or protein level. The invention further provides a method for quantitatively determining the level of expression of the marker nucleic acids in a cell population, and a method for determining whether an agent is capable of increasing or decreasing the level of expression of the marker nucleic acids in a cell population. The method for determining whether an agent is capable of increasing or decreasing the level of expression of the marker nucleic acids in a cell population comprises the steps of (a) preparing cell extracts from control and agent-treated cell populations, (b) isolating the marker polypeptides from the cell extracts, and (c) quantifying (e.g., in parallel) the amount of an immunocomplex formed between the marker polypeptide and an antibody specific to said polypeptide. The marker polypeptides of this invention may also be quantified by assaying for its bioactivity. Agents that induce an increase in the marker nucleic acid expression may be identified by their ability to increase the amount of immunocomplex formed in the treated cell as compared with the amount of the immunocomplex formed in the control cell. In a similar manner, agents that decrease expression of the marker nucleic acid may be identified by their ability to decrease the amount of the immunocomplex formed in the treated cell extract as compared to the control cell.

[081] The present invention provides isolated nucleic acid sequences which are differentially regulated in cancer, and a method for identifying such sequences. The present invention provides a method for identifying a nucleotide sequence which is differentially regulated in a subject with cancer, comprising: hybridizing a nucleic acid sample corresponding to RNA obtained from the subject to a nucleic acid sample comprising one or more nucleic acid molecules of known identity; and measuring the hybridization of the nucleic acid sample to the one or more nucleic acid molecules of known identity, wherein a two-fold difference in the hybridization of the nucleic acid sample to the one or more nucleic acid molecules of known identity relative to a nucleic acid sample obtained from a subject without cancer is indicative of the differential expression of the nucleotide sequence in a subject with cancer.

[082] Generally, the present invention provides a method for identifying nucleic acid sequences which are differentially regulated in a subject with cancer comprising isolating messenger RNA from a subject, generating cRNA from the mRNA sample, hybridizing the cRNA to a microarray comprising a plurality of nucleic acid molecules stably associated with discrete locations on the array, and identifying patterns of hybridization pf the cRNA to the array. According to the present invention, a nucleic acid molecule which hybridizes to a given location on the array is said to be differentially regulated if the hybridization signal is at least two-fold higher or lower than the hybridization signal at the same location on an identical array hybridized with a nucleic acid sample obtained from a subject that does not have cancer.

Microarrays for Determining the Level of Expression of Genes

[083] Determining gene expression levels may be accomplished utilizing microarrays. Generally, the following steps may be involved: (a) obtaining an mRNA sample from a subject and preparing labeled nucleic acids therefrom (the "target nucleic acids" or "targets"); (b) contacting the target nucleic acids with an array under conditions sufficient for the target nucleic acids to bind to the corresponding probes on the array, for example, by hybridization or specific binding; (c) optional removal of unbound targets from the array; (d) detecting the bound targets, and (e) analyzing the results, for example, using computer based analysis methods. As used herein, "nucleic acid probes" or "probes" are nucleic acids attached to the array, whereas "target nucleic acids" are nucleic acids that are hybridized to the array.

[084] Nucleic acid specimens may be obtained from a subject to be tested using either "invasive" or "non-invasive" sampling means. A sampling means is said to be "invasive" if it involves the collection of nucleic acids from within the skin or organs of an animal (including murine, human, ovine, equine, bovine, porcine, canine, or feline animal). Examples of invasive methods include, for example, blood collection, semen collection, needle biopsy, pleural aspiration, umbilical cord biopsy. Examples of such methods are discussed by 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).

[085] In contrast, a "non-invasive" sampling means is one in which the nucleic acid molecules are recovered from an internal or external surface of the animal. Examples of such "non-invasive" sampling means include, for example, "swabbing," collection of tears, saliva, urine, fecal material, sweat or perspiration, hair.

[086] In one embodiment of the present invention, one or more cells from the subject to be tested are obtained and RNA is isolated from the cells. In a preferred embodiment, a sample of peripheral blood leukocytes (PBLs) cells is obtained from the subject. It is also possible to obtain a cell sample from a subject, and then to enrich the sample for a desired cell type. For example, cells may be isolated from other cells using a variety of techniques, such as isolation with an antibody binding to an epitope on the cell surface of the desired cell type. Where the desired cells are in a solid tissue, particular cells may be dissected, for example, by microdissection or by laser capture microdissection (LCM) (see, e.g., Bonner, et al., Science 278:1481, 1997; Emmert-Buck, et al., Science 274:998, 1996; Fend, et al., Am. J. Path. 154:61, 1999; and Murakami, et al., Kidney Int. 58:1346, 2000).

[087] RNA may be extracted from tissue or cell samples by a variety of methods, for example, guanidium thiocyanate lysis followed by CsCI centrifugation (Chirgwin, et al., Biochemistry 18:5294-5299, 1979). RNA from single cells may be obtained as described in methods for preparing cDNA libraries from single cells (see, e.g., Dulac, Curr. Top. Dev. Biol. 36:245, 1998; Jena, et al., J. Immunol. Methods 190:199, 1996).

fl)88] The RNA sample can be further enriched for a particular species. In one embodiment, for example, poly(A)+ RNA may be isolated from an RNA sample. In another embodiment, the RNA population may be enriched for sequences of interest by primer-specific cDNA synthesis, or multiple rounds of linear amplification based on cDNA synthesis and template-directed in vitro transcription (see, e.g., Wang, et al., Proc. Natl. Acad. Sci. USA 86:9717, 1989; Dulac, et al., supra; Jena, et al., supra). In addition, the population of RNA, enriched or not in particular species or sequences, may be further amplified by a variety of amplification methods including, for example, PCR; ligase chain reaction (LCR) (see, e.g., Wu and Wallace, Genomics 4:560, 1989; Landegren, et al., Science 241:1077, 1988); self-sustained sequence replication (SSR) (see, e.g., Guatelli, et al., Proc. Natl. Acad. Sci. USA 87:1874, 1990); nucleic acid based sequence amplification (NASBA) and transcription amplification (see, e.g., Kwoh, et al., Proc. Natl. Acad. Sci. USA 86:1173, 1989). Methods for PCR technology are well known in the art (see, e.g., PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, N.Y., N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila, et al., Nucleic Acids Res. 19:4967, 1991 ; Eckert, et al., PCR Methods and Applications 1:17, 1991; PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202). Methods of amplification 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. Ophtalmol. Vis. Sci. 40:3108, 1999); and Sakai, et al., (Anal. Biochem. 287:32, 2000). RNA amplification and cDNA synthesis may also be conducted in cells in situ (see, e.g., Eberwine, et al. Proc. Natl. Acad. Sci. USA 89:3010, 1992).

[089] The nucleic acid molecules may be labeled to permit detection of hybridization of the nucleic acid molecules to a microarray. That is, the probe may comprise a member of a signal producing system and thus, is detectable, either directly or through combined action with one or more additional members of a signal producing system. For example, the nucleic acids may be labeled with a fluorescently labeled dNTP (see, e.g., Kricka, 1992, Nonisotopic DNA Probe Techniques, Academic Press San Diego, Calif.), biotinylated dNTPs or rNTP followed by addition of labeled streptavidin, chemiluminescent labels, or isotopes. Another example of labels include "molecular beacons" as described in Tyagi and Kramer (Nature Biotech. 14:303, 1996). Hybridization may be also be determined, for example, by plasmon resonance (see, e.g., Thiel, et al. Anal. Chem. 69:4948, 1997).

[090] Another aspect of the present invention relates to methods and processes for detecting a differentially-expressed gene. Detection methods have a variety of applications, including for diagnostic, prognostic, forensic, and research applications. To accomplish gene detection, a polynucleotide in accordance with the present invention can be used as a "probe." The term "probe" or "polynucleotide probe" has its customary meaning in the art, e.g., a polynucleotide which is effective to identify (e.g., by hybridization), when used in an appropriate process, the presence of a target polynucleotide to which it is designed. Identification can involve simply determining presence or absence, or it can be quantitative, e.g., in assessing amounts of a gene or gene transcript present in a sample. Probes can be useful in a variety of ways, such as for diagnostic purposes, to identify homologs, and to detect, quantitate, or isolate a polynucleotide of the present invention in a test sample.

[091] Assays can be utilized which permit quantification and/or presence/absence detection of a target nucleic acid in a sample. Assays can be performed at the single- cell level, or in a sample comprising many cells, where the assay is "averaging" expression over the entire collection of cells and tissue present in the sample. Any suitable assay format can be used, including, but not limited to, e.g., Southern blot analysis, Northern blot analysis, polymerase chain reaction ("PCR") (e.g., Saiki et al., Science, 241:53, 1988; U.S. Pat. Nos. 4,683,195, 4,683,202, and 6,040,166; PCR Protocols: A Guide to Methods and Applications, lnnis et al., eds., Academic Press, New York, 1990), reverse transcriptase polymerase chain reaction ("RT-PCR"), anchored PCR, rapid amplification of cDNA ends ("RACE") (e.g., Schaefer in Gene Cloning and Analysis: Current Innovations, Pages 99-115, 1997), ligase chain reaction ("LCR") (EP 320 308), one-sided PCR (Ohara et al., Proc. Natl. Acad. ScL, 86:5673- 5677, 1989), indexing methods (e.g., U.S. Pat. No. 5,508,169), in situ hybridization, differential display (e.g., Liang et al., Nucl. Acid. Res., 21 :3269—3275, 1993; U.S. Pat. Nos. 5,262,311 , 5,599,672 and 5,965,409; WO97/18454; Prashar and Weissman, Proc. Natl. Acad. ScL, 93:659-663, and U.S. Pat. Nos. 6,010,850 and 5,712,126; Welsh et al., Nucleic Acid Res., 20:4965-4970, 1992, and U.S. Pat. No. 5,487,985) and other RNA fingerprinting techniques, nucleic acid sequence based amplification ("NASBA") and other transcription based amplification systems (e.g., U.S. Pat. Nos. 5,409,818 and 5,554,527; WO 88/10315), polynucleotide arrays (e.g., U.S. Pat. Nos. 5,143,854, 5,424,186; 5,700,637, 5,874,219, and 6,054,270; PCT WO 92/10092; PCT WO 90/15070), Qbeta Replicase (PCT/US87/00880), Strand Displacement Amplification ("SDA"), Repair Chain Reaction ("RCR"), nuclease protection assays, subtraction-based methods, Rapid-Scan (TM), etc. Additional useful methods include, but are not limited to, e.g., template-based amplification methods, competitive PCR (e.g., U.S. Pat. No. 5,747,251), redox-based assays (e.g., U.S. Pat. No. 5,871 ,918), Taqman-based assays (e.g., Holland et al., Proc. Natl. Acad, ScL, 88:7276-7280, 1991 ; U.S. Pat. Nos. 5,210,015 and 5,994,063), real-time fluorescence-based monitoring (e.g., U.S. Pat. 5,928,907), molecular energy transfer labels (e.g., U.S. Pat. Nos. 5,348,853, 5,532,129, 5,565,322, 6,030,787, and 6,117,635; Tyagi and Kramer, Nature Biotech., 14:303-309, 1996). Any method suitable for single cell analysis of gene or protein expression can be used, including in situ hybridization, immunocytochemistry, MACS, FACS, flow cytometry, etc. For single cell assays, expression products can be measured using antibodies, PCR, or other types of nucleic acid amplification (e.g., Brady et al., Methods MoI. & Cell. Biol. 2, 17-25, 1990; Eberwine et al., 1992, Proc. Natl. Acad. ScL,- 89, 3010-3014, 1992; U.S. Pat. No. 5,723,290). These and other methods can be carried out conventionally, e.g., as described in the mentioned publications. [092] In one embodiment, a plurality (e.g., 2, 3, 4, 5, or more) of sets of target nucleic acids are labeled and used in one hybridization reaction ("multiplex" analysis). For example, one set of nucleic acids may correspond to RNA from one cell and another set of nucleic acids may correspond to RNA from another cell. The plurality of sets of nucleic acids may be labeled with different labels, for example, different fluorescent labels (e.g., fluorescein and rhodamine) which have distinct emission spectra so that they can be distinguished. The sets may then be mixed and hybridized simultaneously to one microarray (see, e.g., Shena, et al., Science 270:467-470, 1995).

[093] Microarrays for use according to the invention include one or more probes of genes characteristic of small molecule efficacy. In a preferred embodiment, the microarray comprises probes corresponding to one or more of genes selected from the group consisting of genes which are up-regulated in cancer and genes which are down-regulated in cancer. The microarray may comprise probes corresponding to at least 10, preferably at least 20, at least 50, at least 100 or at least 1000 genes characteristic of small molecule efficacy.

[094] There may be one or more than one probe corresponding to each gene on a microarray. For example, a microarray may contain from 2 to 20 probes corresponding to one gene and preferably about 5 to 10. The probes may correspond to the full-length RNA sequence or complement thereof of genes characteristic of small molecule efficacy, or the probe may correspond to a portion thereof, which portion is of sufficient length to permit specific hybridization. Such probes may comprise from about 50 nucleotides to about 100, 200, 500, or 1000 nucleotides or more than 1000 nucleotides. As further described herein, microarrays may contain oligonucleotide probes, consisting of about 10 to 50 nucleotides, preferably about 15 to 30 nucleotides and more preferably about 20-25 nucleotides. The probes are preferably single- stranded and will have sufficient complementarity to its target to provide for the desired level of sequence specific hybridization. [095] Typically, the arrays used in the present invention will have a site density of greater than 100 different probes per cm². Preferably, the arrays will have a site density of greater than 500/cm², more preferably greater than about 1000/cm², and most preferably, greater than about 10,000/cm². Preferably, the arrays will have more than 100 different probes on a single substrate, more preferably greater than about 1000 different probes, still more preferably, greater than about 10,000 different probes and most preferably, greater than 100,000 different probes on a single substrate.

[096] A number of different microarray configurations and methods for their production are known to those of skill in the art and are disclosed in U.S. Patent 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; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681 ; 5,529,756; 5,545,531; 5,554,501; 5,561,071 ; 5,571,639; 5,593,839; 5,624,711 ; 5,700,637; 5,744,305; 5,770,456; 5,770,722; 5,837,832; 5,856,101 ; 5,874,219; 5,885,837; 5,919,523; 6,022,963; 6,077,674; and 6,156,501; Shena, et al., Tibtech 16:301, 1998; Duggan, et al., Nat. Genet. 21:10, 1999; Bowtell, et al., Nat. Genet. 21:25, 1999; Lipshutz, et al., 21 Nature Genet. 20-24, 1999; Blanchard, et al., 11 Biosensors and Bioelectronics, 687-90, 1996; Maskos, et al., 21 Nucleic Acids Res. 4663-69, 1993; Hughes, et al., Nat. Biotechol. (2001) 19:342; the disclosures of which are herein incorporated by reference. Patents describing methods of using arrays in various applications include: U.S. Pat. Nos. 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,848,659; and 5,874,219; the disclosures of which are herein incorporated by reference.

[097] Arrays preferably include control and reference nucleic acids. Control nucleic acids include, for example, prokaryotic genes such as bioB, bioC and bioD, ere from P1 bacteriophage or polyA controls, such as dap, lys, phe, thr, and trp. Reference nucleic acids allow the normalization of results from one experiment to another and the comparison of multiple experiments on a quantitative level. Exemplary reference nucleic acids include housekeeping genes of known expression levels, for example, GAPDH, hexokinase, and actin. [098] In one embodiment, an array of oligonucleotides may be synthesized on a solid support. Exemplary solid supports include glass, plastics, polymers, metals, metalloids, ceramics, organics, etc. Using chip masking technologies and photoprotective chemistry, it is possible to generate ordered arrays of nucleic acid probes. These arrays, which are known, for example, as "DNA chips" or very large scale immobilized polymer arrays ("VLSIPS™" arrays), may include millions of defined probe regions on a substrate having an area of about 1 cm² to several cm², thereby incorporating from a few to millions of probes (see, e.g., U.S. Patent No. 5,631,734).

[099] To compare expression levels, labeled nucleic acids may be contacted with the array under conditions sufficient for binding between the target nucleic acid and the probe on the array. In a preferred embodiment, the hybridization conditions may be selected to provide for the desired level of hybridization specificity; that is, conditions sufficient for hybridization to occur between the labeled nucleic acids and probes on the microarray.

[100] Hybridization may be carried out in conditions permitting essentially specific hybridization. The length and GC content of the nucleic acid will determine the thermal melting point and thus, the hybridization conditions necessary for obtaining specific hybridization of the probe to the target nucleic acid. These factors are well known to a person of skill in the art, and may also be tested in assays. An extensive guide to nucleic acid hybridization may be found in Tijssen, et al. (Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, P. Tijssen, ed. Elsevier, N.Y., (1993)).

[101] The methods described above result in the production of hybridization patterns of labeled target nucleic acids on the array surface. The resultant hybridization patterns of labeled nucleic acids may be visualized or detected in a variety of ways, with the particular manner of detection selected based on the particular label of the target nucleic acid. Representative detection means include scintillation counting, autoradiography, fluorescence measurement, colorimetric measurement, light emission measurement, light scattering, and the like. [102] One such method of detection utilizes an array scanner that is commercially available (Affymetrix, Santa Clara, CA), for example, the 417™ Arrayer, the 418™ Array Scanner, or the Agilent GeneArray™ Scanner. This scanner is controlled from a system computer with an interface and easy-to-use software tools. The output may be directly imported into or directly read by a variety of software applications. Preferred scanning devices are described in, for example, U.S. Patent Nos. 5,143,854 and 5,424,186.

[103] For fluorescent labeled probes, the fluorescence emissions at each site of a transcript array may be, preferably, detected by scanning confocal laser microscopy. Alternatively, a laser may be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores may be analyzed simultaneously (see, e.g., Shalon, et a!., Genome Res. 6:639-645, 1996). In a preferred embodiment, the arrays may be scanned with a laser fluorescent scanner with a computer controlled X-Y stage and a microscope objective. Fluorescence laser scanning devices are described in Shalon, et al., supra.

[104] Various algorithms are available for analyzing gene expression data, for example, the type of comparisons to perform. In certain embodiments, it is desirable to group genes that are co-regulated. This allows for the comparison of large numbers of profiles. A preferred embodiment for identifying such groups of genes involves clustering algorithms (for reviews of clustering algorithms, see, e.g., Fukunaga, 1990, Statistical Pattern Recognition, 2nd Ed., Academic Press, San Diego; Everitt, 1974, Cluster Analysis, London: Heinemann Educ. Books; Hartigan, 1975, Clustering Algorithms, New York: Wiley; Sneath and Sokal, 1973, Numerical Taxonomy, Freeman; Anderberg, 1973, Cluster Analysis for Applications, Academic Press: New York).

Biomarker Discovery

[105] Expression patterns may be used to derive a panel of biomarkers that can be used to predict the efficacy of drug treatment in the patients. The biomarkers may consist of gene expression levels from microarray experiments on RNA isolated from biological samples, RNA isolated from frozen samples of tumor biopsies, or mass spectrometry-derived protein masses in the serum.

[106] Although the precise mechanism for data analysis will depend upon the exact nature of the data, a typical procedure for developing a panel of biomarkers is as follows. The data (gene expression levels or mass spectra) are collected for each patient prior to treatment. As the study progresses, the patients are classified according to their response to the drug treatment; either as efficacious or non- efficacious. Multiple levels of efficacy can be accommodated in a data model, but a binary comparison is considered optimal, particularly if the patient population is less than several hundred. Assuming adequate numbers of patients in each class, the protein and/or gene expression data may be analyzed by a number of techniques known in the art. Many of the techniques are derived from traditional statistics as well from the field of machine learning. These techniques serve two purposes:

1. Reduce the dimensionality of data - In the case of mass spectra or gene expression microarrays, data is reduced from many thousands of individual data points to bout three to ten. The reduction is based upon the predictive power of the data points when taken as a set.

2. Training - These three to ten data points are then used to train multiple machine learning algorithms which then "learn" to recognize, in this case, patterns of protein masses or gene expression which distinguish efficacious drug treatment from non-efficacious. All patient samples can be used to train the algorithms.

[107] The resulting, trained, algorithms are then tested in order to measure their predictive power. Typically, when less than many hundreds of training examples are available, some form of cross-validation is performed. To illustrate, consider a ten-fold cross validation. In this case, patient samples are randomly assigned to one of ten bins. In the first round of validation the samples in nine of the bins are used for training and the remaining samples in the tenth bin are used to test the algorithm. This is repeated an additional nine times, each time leaving out the samples in a different bin for testing. The results (correct predictions and errors) from all ten rounds are combined and the predictive power is then assessed. Different algorithms, as well as different panels, may be compared in this way for this study. The "best" algorithm/panel combination will then be selected. This "smart" algorithm may then be used in future studies to select the patients that are most likely to respond to treatment.

[108] Many algorithms benefit from additional information taken for the patients. For example, gender or age could be used to improve predictive power. Also, data transformations such as normalization and smoothing may be used to reduce noise. Because of this, a large number of algorithms may be trained using many different parameters in order to optimize the outcome. If predictive patterns exist in the data, it is likely that an optimal, or near-optimal, "smart" algorithm can be developed. If more patient samples become available, the algorithm can be retrained to take advantage of the new data.

[109] As an example using mass spectrometry, plasma (1 μl) may be applied to a hydrophobic SELDI-target, washed extensively in water, and analyzed by the SELDI- Tof mass spectrometer. This may be repeated on 100 or more patient samples. The protein profiles resulting from the intensities of some 16,000 m/z values in each sample would be statistically analyzed in order to identify sets of specific m/z values that are predictive of drug efficacy. Identical experiments using other SELDI-targets, such as ion-exchange or IMAC surfaces, could also be conducted. These will capture different subsets of the proteins present in plasma. Furthermore, the plasma may be denatured and prefractionated prior to application onto the SELDI target.

Diagnostic & Prognostic Assays

[110] The biomarkers of the present invention can be utilized to determine and tailor treatment therapy in various classes of patients having cancer, including patients who have not received treatment, as well as patients already undergoing therapy, such as chemotherapy and radiation. In addition, the biomarkers can be used to monitor patients under active treatment to determine therapeutic efficacy and to indicate whether additional chernotherapeutic agents are needed, or whether the therapeutic course needs to be modified. For example, failure to modulate biomarkers, or achieve levels associated with therapeutic effects, can suggest treatment modification, including higher dosages of the drug and/or the additional of other therapeutic regimes. [111] Examples of useful chemotherapeutic agents include, but are not limited to, e.g., alkylating agents (e.g., cyclophosphamide, ifosfamide, melphalan, chlorambucil, aziridines, epoxides, alkyl sulfonates), cisplatin and its analogues (e.g., carboplatin, oxaliplatin), antimetabolitites (e.g., methotrexate, 5-fluorouracil, capecitabine, cytarabine, gemcitabine, fludarabine), toposiomerase interactive agents (e.g., camptothecin, irinotecan, topotecan, etoposide, teniposide, doxorubicin, daunorubicin), antimicrotubule agents (e.g., vinca alkaloids, such as vincristine, vinblastine, and vinorelbine; taxanes, such as paclitaxel and docetaxel), inteleukin-2, histone deacetylase inhibitors, monoclonal antibodies, estrogen modulators (e.g., tamoxifen, toremifene, raloxifene), megestrol, aromatase inhibitors (e.g., letrozole, anastrozole, exemestane, octreotide), octreotide, anti-androgens (e.g., flutamide, casodex), interferons (e.g., interferon-alpha, including subtypes thereof, such as interferon-alpha- 2a), etc. See, e.g. Cancer: Principles and Practice of Oncology, 7th Edtion, Devita et al, Lippincott Williams & Wilkins, 2005, Chapters 15, 16, 17, and 63.

[112] The present invention provides methods for determining whether a subject is at risk for developing a disease or condition characterized by unwanted cell proliferation by detecting biomarkers (e.g., MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1 , and/or FOS-like antigen), that is, nucleic acids and/or polypeptide markers for cancer.

[113] In clinical applications, human tissue samples may be screened for the presence and/or absence of biomarkers identified herein. Such samples could consist of needle biopsy cores, surgical resection samples, lymph node tissue, or serum. For example, these methods include obtaining a biopsy, which is optionally fractionated by cryostat sectioning to enrich tumor cells to about 80% of the total cell population. In certain embodiments, nucleic acids extracted from these samples may be amplified using techniques well known in the art. The levels of selected markers detected would be compared with statistically valid groups of metastatic, non-metastatic malignant, benign, or normal tissue samples. [114] In one embodiment, the diagnostic method comprises determining whether a subject has an abnormal mRNA and/or protein level of the biomarkers (e.g., MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and/or FOS-like antigen), such as by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR)₁ in situ hybridization, immunoprecipitatioπ, Western blot hybridization, or immunohistochemistry. According to the method, cells may be obtained from a subject and the levels of the biomarkers, protein, or mRNA level, are determined and compared to the level of these markers in a healthy subject. An abnormal level of the biomarker polypeptide or mRNA levels is likely to be indicative of cancer.

[115] Accordingly, in one aspect, the invention provides probes and primers that are specific to the unique nucleic acid markers disclosed herein. Accordingly, the nucleic acid probes comprise a nucleotide sequence at least 10 nucleotides in length, preferably at least 15 nucleotides, more preferably, 25 nucleotides, and most preferably at least 40 nucleotides, and up to all or nearly all of the coding sequence which is complementary to a portion of the coding sequence of a marker nucleic acid sequence.

[116] In one embodiment, the method comprises using a nucleic acid probe to determine the presence of cancerous cells in a tissue from a patient. Specifically, the method comprises:

1. providing a nucleic acid probe comprising a nucleotide sequence at least 10 nucleotides in length, preferably at least 15 nucleotides, more preferably, 25 nucleotides, and most preferably at least 40 nucleotides, and up to all or nearly all of the coding sequence which is complementary to a portion of the coding sequence of a nucleic acid sequence and is differentially expressed in tumors cells;

2. obtaining a tissue sample from a patient potentially comprising cancerous cells;

3. providing a second tissue sample containing cells substantially all of which are non-cancerous; 4. contacting the nucleic acid probe under stringent conditions with RNA of each of said first and second tissue samples (e.g., in a Northern blot or in situ hybridization assay); and

5. comparing (a) the amount of hybridization of the probe with RNA of the first tissue sample, with (b) the amount of hybridization of the probe with RNA of the second tissue sample; wherein a statistically significant difference in the amount of hybridization with the RNA of the first tissue sample as compared to the amount of hybridization with the RNA of the second tissue sample is indicative of the presence of cancerous cells in the first tissue sample.

[117] In one aspect, the method comprises in situ hybridization with a probe derived from a given marker nucleic acid sequence (e.g., MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1 , and/or FOS-like antigen). The method comprises contacting the labeled hybridization probe with a sample of a given type of tissue potentially containing cancerous or pre-cancerous cells as well as normal cells, and determining whether the probe labels some cells of the given tissue type to a degree significantly different (e.g., by at least a factor of two, or at least a factor of five, or at least a factor of twenty, or at least a factor of fifty) than the degree to which it labels other cells of the same tissue type.

[118] Also within the invention is a method of determining the phenotype of a test cell from a given human tissue, for example, whether the cell is (a) normal, or (b) cancerous or precancerous, by contacting the mRNA of a test cell with a nucleic acid probe at least 12 nucleotides in length, preferably at least 15 nucleotides, more preferably at least 25 nucleotides, and most preferably at least 40 nucleotides, and up to all or nearly all of a sequence which is complementary to a portion of the coding sequence of a nucleic acid sequence, and which is differentially expressed in tumor cells as compared to normal cells of the given tissue type; and determining the approximate amount of hybridization of the probe to the mRNA, an amount of hybridization either more or less than that seen with the mRNA of a normal cell of that tissue type being indicative that the test cell is cancerous or pre-cancerous.

[119] Alternatively, the above diagnostic assays may be carried out using antibodies to detect the protein product encoded by the marker nucleic acid sequence (e.g., MMP-1 , MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and/or FOS-like antigen). Accordingly, in one embodiment, the assay would include contacting the proteins of the test cell with an antibody specific for the gene product of a nucleic acid, the marker nucleic acid being one which is expressed at a given control level in normal cells of the same tissue type as the test cell, and determining the approximate amount of immunocomplex formation by the antibody and the proteins of the test cell, wherein a statistically significant difference in the amount of the immunocomplex formed with the proteins of a test cell as compared to a normal cell of the same tissue type is an indication that the test cell is cancerous or pre-cancerous. Preferably, the antibody is specific for MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1 , and/or FOS-like antigen.

[120] The method for producing polyclonal and/or monoclonal antibodies which specifically bind to polypeptides useful in the present invention is known to those of skill in the art and may be found in, for example, Dymecki, et al., (J. Biol. Chem. 267:4815, 1992); Boersma & Van Leeuwen, (J. Neurosci. Methods 51:317, 1994); Green, et al., (Cell 28:477, 1982); and Amheiter, et al., (Nature 294:278, 1981).

[121] Another such method includes the steps of: providing an antibody specific for the gene product of a marker nucleic acid sequence, the gene product being present in cancerous tissue of a given tissue type at a level more or less than the level of the gene product in non-cancerous tissue of the same tissue type; obtaining from a patient a first sample of tissue of the given tissue type, which sample potentially includes cancerous cells; providing a second sample of tissue of the same tissue type (which may be from the same patient or from a normal control, e.g. another individual or cultured cells), this second sample containing normal cells and essentially no cancerous cells; contacting the antibody with protein (which may be partially purified, in lysed but unfractionated cells, or in situ) of the first and second samples under conditions permitting immunocomplex formation between the antibody and the marker nucleic acid sequence product present in the samples; and comparing (a) the amount of immunocomplex formation in the first sample, with (b) the amount of immunocomplex formation in the second sample, wherein a statistically significant difference in the amount of immunocomplex formation in the first sample less as compared to the amount of immunocomplex formation in the second sample is indicative of the presence of cancerous cells in the first sample of tissue.

[122] The subject invention further provides a method of determining whether a cell sample obtained from a subject possesses an abnormal amount of marker polypeptide which comprises (a) obtaining a cell sample from the subject, (b) quantitatively determining the amount of the marker polypeptide in the sample so obtained, and (c) comparing the amount of the marker polypeptide so determined with a known standard, so as to thereby determine whether the cell sample obtained from the subject possesses an abnormal amount of the marker polypeptide. Such marker polypeptides may be detected by immunohistochemical assays, dot-blot assays, ELISA, and the like.

[123] Immunoassays are commonly used to quantitate the levels of proteins in cell samples, and many other immunoassay techniques are known in the art. The invention is not limited to a particular assay procedure, and therefore, is intended to include both homogeneous and heterogeneous procedures. Exemplary immunoassays which may be conducted according to the invention include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA)₁ nephelometric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELlSA), and radioimmunoassay (RIA). An indicator moiety, or label group, may be attached to the subject antibodies and is selected so as to meet the needs of various uses of the method which are often dictated by the availability of assay equipment and compatible immunoassay procedures. General techniques to be used in. performing the various immunoassays noted above are known to those of ordinary skill in the art.

[124] In another embodiment, the level of the encoded product, or alternatively the level of the polypeptide, in a biological fluid (e.g., blood or urine) of a patient may be determined as a way of monitoring the level of expression of the marker nucleic acid sequence in cells of that patient. Such a method would include the steps of obtaining a sample of a biological fluid from the patient, contacting the sample (or proteins from the sample) with an antibody specific for an encoded marker polypeptide, and determining the amount of immune complex formation by the antibody, with the amount of immune complex formation being indicative of the level of the marker encoded product in the sample. This determination is particularly instructive when compared to the amount of immune complex formation by the same antibody in a control sample taken from a normal individual or in one or more samples previously or subsequently obtained from the same person.

[125] In another embodiment, the method may be used to determine the amount of marker polypeptide present in a cell, which in turn may be correlated with progression of a hyperproliferative disorder. The level of the marker polypeptide may be used predictively to evaluate whether a sample of cells contains cells which are, or are predisposed towards becoming, transformed cells. Moreover, the subject method may be used to assess the phenotype of cells which are known to be transformed, the phenotyping results being useful in planning a particular therapeutic regimen. For example, very high levels of the marker polypeptide in sample cells is a powerful diagnostic and prognostic marker for a cancer. The observation of marker polypeptide levels may be utilized in decisions regarding, for example, the use of more aggressive therapies.

[126] As set out above, one aspect of the present invention relates to diagnostic assays for determining, in the context of cells isolated from a patient, if the level of a marker polypeptide is significantly reduced in the sample cells. The term "significantly reduced" refers to a cell phenotype wherein the cell possesses a reduced cellular amount of the marker polypeptide relative to a normal cell of similar tissue origin. For example, a cell may have less than about 50%, 25%, 10%, or 5% of the marker polypeptide compared to that of a normal control cell. In particular, the assay evaluates the level of marker polypeptide in the test cells, and, preferably, compares the measured level with marker polypeptide detected in at least one control cell, for example, a normal cell and/or a transformed cell of known phenotype.

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

[128] Where tissue samples are employed, immunohistochemical staining may be used to determine the number of cells having the marker polypeptide phenotype. For such staining, a multiblock of tissue may be taken from the biopsy or other tissue sample and subjected to proteolytic hydrolysis, employing such agents as protease K or pepsin. In certain embodiments, it may be desirable to isolate a nuclear fraction from the sample cells and detect the level of the marker polypeptide in the nuclear fraction.

[129] The tissue samples are fixed by treatment with a reagent such as formalin, glutaraldehyde, methanol, or the like. The samples are then incubated with an antibody, preferably a monoclonal antibody, with binding specificity for the marker polypeptides. This antibody may be conjugated to a label for subsequent detection of binding. Samples are incubated for a time sufficient for formation of the immunocomplexes. Binding of the antibody is then detected by virtue of a label conjugated to this antibody. Where the antibody is unlabeled, a second labeled antibody may be employed, for example, which is specific for the isotype of the anti- marker polypeptide antibody. Examples of labels which may be employed include radionuclides, fluorescers, chemiluminescers, enzymes, and the like.

[130] Where enzymes are employed, the substrate for the enzyme may be added to the samples to provide a colored or fluorescent product. Examples of suitable enzymes for use in conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase, and the like. Where not commercially available, such antibody- enzyme conjugates are readily produced by techniques known to those skilled in the art. [131] In one embodiment, the assay is performed as a dot blot assay. The dot blot assay finds particular application where tissue samples are employed as it allows determination of the average amount of the marker polypeptide associated with a single cell by correlating the amount of marker polypeptide in a cell-free extract produced from a predetermined number of cells.

[132] It is well established in the cancer literature that tumor cells of the same type (e.g., breast and/or colon tumor cells) may not show uniformly increased expression of individual oncogenes or uniformly decreased expression of individual tumor suppressor genes. There may also be varying levels of expression of a given marker gerie even between cells of a given type of cancer, further emphasizing the need for reliance on a battery of tests rather than a single test. Accordingly, in one aspect, the invention provides for a battery of tests utilizing a number of probes of the invention, in order to improve the reliability and/or accuracy of the diagnostic test.

[133] In one embodiment, the present invention also provides a method wherein nucleic acid probes are immobilized on a DNA chip in an organized array. Oligonucleotides may be bound to a solid support by a variety of processes, including lithography. For example, a chip may hold up to 250,000 oligonucleotides. These nucleic acid probes comprise a nucleotide sequence at least about 12 nucleotides in length, preferably at least about 15 nucleotides, more preferably at least about 25 nucleotides, and most preferably at least about 40 nucleotides, and up to all or nearly all of a sequence which is complementary to a portion of the coding sequence of a marker nucleic acid sequence and is differentially expressed in tumor cells. The present invention provides significant advantages over the available tests for various cancers, because it increases the reliability of the test by providing an array of nucleic acid markers on a single chip.

[134] The method includes obtaining a biopsy, which is optionally fractionated by cryostat sectioning to enrich tumor cells to about 80% of the total cell population. The DNA or RNA is then extracted, amplified, and analyzed with a DNA chip to determine the presence of absence of the marker nucleic acid sequences. [135] In one embodiment, the nucleic acid probes are spotted onto a substrate in a two-dimensional matrix or array. Samples of nucleic acids may be labeled and then hybridized to the probes. Double-stranded nucleic acids, comprising the labeled sample nucleic acids bound to probe nucleic acids, may be detected once the unbound portion of the sample is washed away.

[136] The probe nucleic acids may be spotted on substrates including glass, nitrocellulose, etc. The probes can be bound to the substrate by either covalent bonds or by non-specific interactions, such as hydrophobic interactions. The sample nucleic acids can be labeled using radioactive labels, fluorophores, chromophores, etc.

[137] Techniques for constructing arrays and methods of using these arrays are described, for example, in EP No. 0 799 897; PCT No. WO 97/292 12; PCT No. WO 97127317; EP No. 0 785 280; PCT No. WO 97/02357; U.S. Pat. No. 5,593,839; U.S. Pat. No. 5,578,832; EP No. 0 728 520; U.S. Pat. No. 5,599,695; EP No. 0 721 016; U.S. Pat. No. 5,556,752; PCT No. WO 95/22058; and U.S. Pat. No. 5,631,734.

[138] Further, arrays may be used to examine differential expression of genes and may be used to determine gene function. For example, arrays of nucleic acid sequences may be used to determine if any of the nucleic acid sequences are differentially expressed between normal cells and cancer cells. Increased expression of a particular message in a cancer cell, which is not observed in a corresponding normal cell, may indicate a cancer-specific protein.

[139] In one embodiment, nucleic acid molecules may be used to generate microarrays on a solid surface (e.g., a membrane) such that the arrayed nucleic acid molecules may be used to determine if any of the nucleic acids are differentially expressed between normal cells or tissue and cancerous cells or tissue. In one embodiment, the nucleic acid molecules of the invention may be cDNA or may be used to generate cDNA molecules to be subsequently amplified by PCR and spotted on nylon membranes. The membranes may then be reacted with radiolabeled target nucleic acid molecules obtained from equivalent samples of cancerous and normal tissue or cells. Methods of cDNA generation and microarray preparation are known to those of skill in the art and may be found, for example, in Bertucci, et al., (Hum. MoI. Genet. 8:2129, 1999); Nguyen, et al., (Genomics 29:207, 1995); Zhao, et al., (Gene 156:207); Gress, et al., (Mammalian Genome 3:609, 1992); Zhumabayeva, et al., (Biotechniques 30:158, 2001); and Lennon, et al., (Trends Genet. 7:314, 1991).

[140] In yet another embodiment, the invention contemplates using a panel of antibodies which are generated against the marker polypeptides of this invention. Preferably, the antibodies are generated against MMP-1 , MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1 , and/or FOS-like antigen. Such a panel of antibodies may be used as a reliable diagnostic probe for cancer. The assay of the present invention comprises contacting a biopsy sample containing cells, for example, lung cells, with a panel of antibodies to one or more of the encoded products to determine the presence or absence of the marker polypeptides.

[141] The diagnostic methods of the subject invention may also be employed as follow-up to treatment, for example, quantitation of the level of marker polypeptides may be indicative of the effectiveness of current or previously employed cancer therapies as well as the effect of these therapies upon patient prognosis.

[142] In addition, the marker nucleic acids or marker polypeptides may be utilized as part of a diagnostic panel for initial detection, follow-up screening, detection of reoccurrence, and post-treatment monitoring for chemotherapy or surgical treatment.

[143] Accordingly, the present invention makes available diagnostic assays and reagents for detecting gain and/or loss of marker polypeptides from a cell in order to aid in the diagnosis and phenotyping of proliferative disorders arising from, for example, tumorigenic transformation of cells. [144] The diagnostic assays described above may be adapted to be used as prognostic assays, as well. Such an application takes advantage of the sensitivity of the assays of the invention to events which take place at characteristic stages in the progression of a tumor. For example, a given marker gene may be up- or down- regulated at a very early stage, perhaps before the cell is irreversibly committed to developing into a malignancy, while another marker gene may be characteristically up- or down-regulated only at a much later stage. Such a method could involve the steps of contacting the mRNA of a test cell with a nucleic acid probe derived from a given marker nucleic acid which is expressed at different characteristic levels in cancerous or precancerous cells at different stages of tumor progression, and determining the approximate amount of hybridization of the probe to the mRNA of the cell, such amount being an indication of the level of expression of the gene in the cell, and thus an indication of the stage of tumor progression of the cell; alternatively, the assay may be carried out with an antibody specific for the gene product of the given marker nucleic acid, contacted with the proteins of the test cell. A battery of such tests will disclose not only the existence and location of a tumor, but also will allow the clinician to select the mode of treatment most appropriate for the tumor, and to predict the likelihood of success of that treatment.

[145] The methods of the invention may also be used to follow the clinical course of a tumor. For example, the assay of the invention may be applied to a tissue sample from a patient; following treatment of the patient for the cancer, another tissue sample is taken and the test repeated. Successful treatment will result in either removal of all cells which demonstrate differential expression characteristic of the cancerous or precancerous cells, or a substantial increase in expression of the gene in those cells, perhaps approaching or even surpassing normal levels.

[146] The present invention also relates to methods of determining the efficacy of agents in treating a disease or disorder (e.g., a cancer, such as renal cell or heptatocellular carcinoma) in a subject who has been administered an agent, comprising, e.g., detecting the amounts of a mRNA, or polypeptide encoded thereby, which is selected from Table 1 (such as IL-6, IL-8, MMP-1 , and/or MMP-10) in a biological sample obtained from said subject, wherein said amount of said polypeptide is indicative of whether a therapeutically effective amount of said agent has been administered.

[147] Examples of agents include, multi-kinase inhibitors, especially inhibitors having VEGFR activity, such as sorafenib and derivatives thereof; multi-kinase inhibitors that have an activity profile similar to sorafenib (e.g., as described in Wilhelm et al., 2004); agents which have anti-angiogenic activity (e.g., VEGFR inhibitors, such as VEGF antibodies); and agents which have dual anti-angiogenic and tumor antiproliferative activities (e.g., sorafenib). In addition, combinations of the drugs are also included, e.g., a combination comprising a VEGFR2 inhibitor and a PDGFR-beta inhibitor; a combination of a VEGFR2 inhibitor and a cytotoxic or chemotherapeutic agent; a combination of sorafenib and chemotherapeutic agent, such as a quinone bioreductive prodrug (Jaffar et al., Curr Drug Deliv. 2004 Oct;1(4):345-50) or other drugs which are activated by increased hypoxia or that selectively kills cells under hypoxic conditions.

[148] Examples of multi-kinase inhibitors that elicit the production of biomarkers in accordance with the present invention include, e.g., ZD 6474, AZD 2171, AG-013736, AMG 706, BIBF-1120, XL999, MLN518, PKC412, OSI-930, OSI-817, preferably Sutent (SU11248; sunitinib malate). Such agents can have 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/or FLT3 activity. Examples of other multi- kinase inhibitors include, e.g., MP-412, dasatinib, CEP-701, XL647, Tykerb, STI571, AMN107, and AEE788.

[149] Examples of chemotherapeutic agents include, but are not limited to, e.g., alkylating agents (e.g., cyclophosphamide, ifosfamide, melphalan, chlorambucil, aziridines, epoxides, alkyl sulfonates), cisplatin and its analogues (e.g., carboplatin, oxaliplatin), antimetabolites (e.g., methotrexate, 5-fluorouracil, capecitabine, cytarabine, gemcitabine, fludarabine), toposiomerase interactive agents (e.g., camptothecin, irinotecan, topotecan, etoposide, teniposide, doxorubicin,. daunorubicin), antimicrotubule agents (e.g., vinca alkaloids, such as vincristine, vinblastine, and vinorelbine; taxanes, such as paclitaxel and docetaxel), interferons, inteleukin-2, histone deacetylase inhibitors, monoclonal antibodies, estrogen modulators (e.g., tamoxifen, toremifene, raloxifene), megestrol, aromatase inhibitors (e.g., letrozole, anastrozole, exemestane, octreotide), octreotide, anti-androgens (e.g., flutamide, casodex), etc. See, e.g. Cancer: Principles and Practice of Oncology, 7th Edition, Devita et al, Lippincott Williams & Wilkins, 2005, Chapters 15, 16, 17, and 63. Prodrugs can also be included, especially prodrugs that are activated in hypoxic tissues, such as quinone bioreductive compounds. See, e.g., Jaffar et al., Curr. Drug Deliv., 2004, 1(4):345-350, 2004.

[150] The amounts of mRNA, or polypeptide encoded thereby, can be detected routinely by any of the methods described herein. The term "detecting" indicates that the presence of the target (i.e., an 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, etc. The amount of the mRNA (e.g., including measurements of a cDNA using a target mRNA as a template) or polypeptide can be described as indicative of whether a therapeutically or physiologically effective amount of sorafenib (or derivatives thereof) has been administered. Thus, when a certain amount of one or more of the biomarkers are detected, this is an indication that agent (such as sorafenib) is achieving a physiological effect in the subject. Table 1 contains a complete list of the biomarkers associated with sorafenib administration.

[151] A "therapeutic effect" indicates that the administered drug treats, combats, alleviates, reduces, relieves, improves, etc., at least one symptom of the disease or disorder (e.g., when administered in a therapeutically effective amount). Therapeutic effects of sorafenib include, but are not limited to, e.g., inhibiting angiogenesis; inhibiting cell proliferation; producing tissue necrosis or hypoxia; stopping or slowing tumor growth; stopping or slowing tumor cell proliferation; stopping or slowing endothelial cell proliferation; producing apoptosis, e.g., in tumor and/or endothelial cells; inhibiting tyrosine kinases; inhibiting VEGFR (such as VEGFR2); PDGFR (such as PDGFR-beta); FLT3; c-KIT, RET, and RAF; etc. See, also Wilhelm et al., Cane. Res., 2004, 64(19): 7099-109 for other activities; Carlomagπo et al., J Natl Cancer Inst. 2006 Mar 1;98(5):326-34. Sorafenib, and other agents in accordance with the present invention, can also produce an biologically effective dose (BED) prior to achieving a substantial therapeutic effect. The latter can occur, e.g., when dosages of sorafenib are sufficient for it to interact with its kinase targets, but not adequate to effectively treat the disease or disorder. In these situations, the lack of modulation of one or more biomarkers (e.g., an increase in IL-6, IL-8, MMP-1, and/or MMP-10 levels), or if changes in a biomarker amount is smaller than expected, the agent (such as sorafenib) dosage can be increased until the expected change in biomarker amount is observed.

[152] The amount of biomarker which is utilized as a diagnostic marker for drug efficacy can be determined by a variety of methods. For example, the amount can be determined using samples obtained from the same subject prior to and after drug treatment, and a significant difference between the two points can be utilized to determine that the drug is achieving its effect. Such a difference can be determined routinely, and can include, e.g., a difference from about 2x (fold), 3x, 3.5x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11 x, 15x, 2Ox, or more, from about 3x-4x, from about 2x-5x, from about 2x-10x, etc. As shown in Table 1, 154 genes were determined to be differentially- regulated in response to sorafenib treatment, and any of these genes, and the polypeptide they encode, can be utilized as surrogate biomarker for drug efficacy. By the term "differential-regulation," it is meant that the gene expression (e.g., as measured by mRNA or by polypeptide) changes in response to drug administration, whereas not all genes measured showed such changes.

[153] In addition to comparing amounts between samples from the same patient, standard or normal values can also be used. For example, typical values or value ranges for a particular biomarker can be established in a subject population, and then such value can be used as threshold to determine drug efficacy. When a value outside the typical range is observed, then such value would indicate that drug is having a therapeutic effect. The lack of significant change in the biomarker levels, on the other hand, can be used to indicate that a disease (e.g., a cancer) is not responsive to the drug, or that an additional dosage increase of drug is needed to have efficacy. In addition to being compared to a standard or normal value, the levels of biomarker can be compared to a sample obtained from said subject prior to sorafenib administration. The presence of a negative sign (-) in Table 1 indicates that the levels of the biomarker are decreased; if no sign is listed, then the biomarker is increased.

[154J The present invention also provides methods for monitoring the effects of agent (such as sorafenib) during the course of a subject's treatment, e.g., where biological samples are assayed for differential gene or polypeptide expression of a biomarker during the course of a subject's treatment. When the detected amounts indicate that an insufficient therapeutic amount of the agent has been administered, additional drug can be provided until an appropriate biomarker response is reached, indicating that a therapeutic level has been achieved.

[165] The increase in biomarker can be observed at any time during therapy with the agent. For example, biomarker expression may not be observed until after one or more dosages (e.g., multiple dosages) have been administered to a subject, including, e.g., 2, 3, 6, 8, 10, 12, 14, 16, 20, 22, 26, 30, 36, 40, or more dosages (where "dosage" indicates the daily amount of drug). Such an increase can then occur, e.g., from about 4-24 hours, 1-3 days, 1-7 days, or 3-10 days after agent administration.

[156] Any number of biomarkers that can be detected in accordance with the present invention, e.g., to determine whether an agent is having a therapeutic effect. These include, 1 , 2, 4, 10, 15, 20, 30, 60, and including the entire set of markers indicated 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.

[157] The presenting invention also provides methods for determining the efficacy of an agent in treating a tumor in a subject who has been administered an agent (such as sorafenib or sutent), comprising, measuring the amount of tumor hypoxia in said subject, wherein said amount is indicative of whether a therapeutically effective amount of sorafenib has been administered. Tissue hypoxia can be defined as decrease in oxygen utilization, such that cells are experiencing anaerobic respiration. Various methods can be utilized to determine hypoxia. For example, global measurements include, e.g., blood lactate, pH, oxygen transport/oxygen consumption, mixed venous oxygen saturation, venous arterial carbon dioxide gradient, etc. In addition to global measurements, regional hypoxia can also be measured. These include non-invasive measurements, such as, e.g., blood-oxygenation level-dependent (BOLD) MRI (e.g., Prasad et al., Noninvasive Evaluation of lntrarenal Oxygenation with BOLD MRI, Circulation, 94:3271-3275, 1996); differential path-length spectroscopy; 18F- fluoromisonidazole (18F-FMISO) positron emission tomography (PET) (e.g., Lawrentschuk et al., BJU Int., 96(4):540-546, 2005) and other methods using 2- nitroimidazole hypoxia markers. As for the other biomarker methods, sorafenib administration can be increased until a hypoxic effect is observed.

[158] The biomarkers of the present invention can also be utilized as endogenous markers for the occurrence of tumor hypoxia, especially when the hypoxia occurs in the absence of new blood vessel formation. For example, Sorafenib and Sutent, when utilized to treat cancer, are effective against both tumor and blood vessel growth, resulting in tissue hypoxia. The presence and degree of tissue hypoxia in response to these agents, and any other, can be determined in accordance with the present invention, e.g., by measuring any of the biomarkers listed in Table 1, including IL-6, IL- 8, MMP-1, and/or MMP-10. As with other methods, the biomarkers can be measured in the blood, tumor tissue, or other body fluids. Accordingly, the present invention also relates to biomarkers for tissue hypoxia. This includes a method of determining the presence of tumor tissue hypoxia, comprising, e.g., detecting the amounts of a biomarker selected from Table 1, such as MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and/or FOS-like antigen polypeptide or polynucleotide or mRNA in a biological sample obtained from said subject, wherein said amount of said polypeptide or mRNA indicates the occurrence of hypoxia in a tumor tissue.

[159] The present invention also provides methods of determining whether a compound has the pharmacological activity of sorafenib, comprising detecting the amounts of at least two differentially-regulated mRNAs, or polypeptides encoded thereby, in a cell which has been exposed to said sorafenib, wherein said amount is indicative of whether said compound has the pharmacological activity of sorafenib, and wherein said differentially regulated mRNAs are selected from Table 1. [160] The expression patterns of the differentially expressed genes disclosed herein can be described as a "fingerprint", in that they are a distinctive pattern displayed by cells in response to sorafenib administration. Just as with a fingerprint, an expression pattern can be used as a unique identifier to characterize the status of the tissue with respect to its sorafenib response. It can be used as a point of reference to compare and characterize a cellular response to a multi-kinase inhibitor. For example, the fingerprint can be used to determine whether a compound of interest has the same pharmacological activity of sorafenib. A pattern of differential gene expression can be determined for a test compound. The closer the pattern match, the more closely the compound mirrors the pharmacological profile of sorafenib. Such compounds can be used for any indication that sorafenib is utilized for.

[161] The present invention also provides methods of reducing the inflammatory events associated with sorafenib administration in a subject, comprising: administering an effective amount of an anti-inflammatory agent to said subject. Any antiinflammatory agent can be utilized, including, e.g., non-steroidal anti-inflammatory (NSAID) agents; cortisone and derivatives thereof; etc.

Predictive Assays

[162] Laboratory-based assays, which can predict clinical benefit from a given anticancer agent, will greatly enhance the clinical management of patients with cancer. In order to assess this effect, a biomarker associated with the anti-cancer agent may be analyzed in a biological sample (e.g., tumor sample, plasma) before, during, and following treatment.

[163] For example, the expression of MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and/or FOS-like antigen mRNA and protein may be detected in plasma. Thus, changes in the baseline plasma concentration of MMP-1 , MMP-10, IL-6, IL-8, IL- 10, CSF-2, SLCO4A1, and/or FOS-like antigen may be monitored in patients with cancer. Additionally, MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and/or FOS-like antigen protein levels may also be monitored by quantitative immunohistochemistry using paraffin-embedded tumor biopsies. [164] Another approach to monitor treatment is an evaluation of serum proteomic spectra. Specifically, plasma samples may be subjected to mass spectroscopy (e.g., surface-enhanced laser desorption and ionization) and a proteomic spectra may be generated for each patient. A set of spectra, derived from analysis of plasma from patients before and during treatment, may be analyzed by an iterative searching algorithm, which can identify a proteomic pattern that completely discriminates the treated samples from the untreated samples. The resulting pattern may then be used to predict the clinical benefit following treatment.

[165] Global gene expression profiling of biological samples (e.g., tumor biopsy samples, blood samples) and bioinformatics-driven pattern identification may be utilized to predict clinical benefit and sensitivity, as well as development of resistance to an anti-cancer agent. For example, RNA isolated from cells derived from whole blood from patients before and during treatment may be used to generate blood cell gene expression profiles utilizing Affymetrix GeneChip technology and algorithms. These gene expression profiles may then predict the clinical benefit from treatment with a particular anti-cancer agent.

[166] Analysis of the biochemical composition of urine by 1D ¹H-NMR (Nuclear Magnetic Resonance) may also be utilized as a predictive assay. Pattern recognition techniques may be used to evaluate the metabolic response to treatment with an anticancer agent and to correlate this response with clinical endpoints. The. biochemical or endogenous metabolites excreted in urine have been well-characterized by proton NMR for normal subjects (Zuppi, et al., Clin Chim Acta 265:85-97, 1997). These metabolites (approximately 30-40) represent the by-products of the major metabolic pathways, such as the citric acid and urea cycles. Drug-, disease-, and genetic-stimuli have been shown to produce metabolic-specific changes in baseline urine profiles that are indicative of the timeline and magnitude of the metabolic response to the stimuli. These analyses are multi-variant and therefore. use pattern recognition techniques to improve data interpretation. Urinary metabolic profiles may be correlated with clinical endpoints to determine the clinical benefit. BRIEF DESCRIPTION OF THE DRAWINGS

[167] Various features and attendant advantages of the present invention will be more fully appreciated as the same becomes better understood when considered in conjunction with the accompanying drawings, in which like reference characters designate the same or similar parts throughout the several views, and wherein:

FIG. 1 shows outline for gene regulation/protein expression studies.

FIG. 2 shows timeline of sorafenib treatment in mice and the assessment of hypoxia using hypoyprobe™ One hour before tumor collection, mice were injected i.v. with hyypoxyprobe™ [pimonidazole hydrochloride, 60 mg/kg; (Chemicon International; Temecula, CA )]. Tumors were harvested 4 and 24 h after final Sorafenib administration. For u.v. visualization, formalin-fixed, paraffin-embedded tumor sections cut at 5m were incubated for 1 h with the FITC-conjugated Hypoxyprobe-1 primary antibody supplied with the kit (Hypoxyprobe-1 Plus Kit; Chemicon International; Temecula, CA). Sections were also incubated with goat anti-CD31 Ab (Santa Cruz; Santa Cruz, CA).

FIG. 3 provides an affymetrix analysis of the genes that are regulated by sorafenib treatment. Data is represented as relative fold changes in gene expression when treated tumors are compared to corresponding control tumors, wherein:

• Bright red represents > 3.5 fold upregulation of gene expression

• Bright green represents > 3.5 fold down regulation of gene expression.

FIG. 4 shows IL-8 and IL-6 genes are upregulated upon sorafenib treatment as confirmed by taqman analysis.

FIG. 5 shows MMP-1 and MMP-10 genes are upregulated upon sorafenib treatment as confirmed by taqman analysis. FIG. 6 shows that sorafenib-treated tumors have elevated levels of IL-6, IL-8, MMP-10, and MMP-1 proteins. Data is average +/- SEM; n = 10 animals per group, * denotes p ≤ 0.05).

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)

FIG. 7. shows that plasma from sorafenib-treated animals has elevated levels of IL-6, IL-8 and MMP-10. Data is average +/- SEM; n = 10 animals/group, * denotes p ≤ 0.05.

Panel (A) Plasma IL-6 (pg/ml) Panel (B) Plasma IL-8 (pg/ml) Panel (C) Plasma MMP-10 (pg/ml)

FIG. 8 shows that VEGF levels are modestly elevated in sorafenib-treated tumors. Data is average +/- SEM; n = 10 animals/group, * denotes p ≤ 0.05.

Examples

[168] The invention will be explained below with reference to the following non-limiting examples.

[169] Affymetrix Analysis

[170] Total RNA was extracted from tumors using TRIzol reagent (Life Technologies, MD) according to a modified vendor protocol which utilizes the RNeasy protocol (Qiagen, CA). RNA (5 μg) was transcribed to cDNA using the Invitrogen Superscript III Choice System for RT-PCR according to vendor protocol (Invitrogen, CA). cDNA was used in an in vitro transcription reaction incorporating biotinylated nucleotides using an RNA labeling kit (Enzo Diagnostics, NY). The resulting cRNA was purified, quantified, fragmented and then loaded (10 μg) onto each array (Human Genome U133 Plus 2.0). Hybridization proceeded for 16-18 hours at 45⁰C in an Affymetrix GeπeChip Hybridization Oven 640 (Affymetrix; Santa Clara, CA). Arrays were stained with Phycoerythrin-conjugated Streptavidin using the Affymetrix Fluidics Station 450. Measurements were made after placing arrays in an Affymetrix Scanner 3000. Intensity of light emitted was digitally converted to a numeric value indicative of level of gene expression. The relative fold change in gene expression was calculated from a comparison of Sorafenib-treated tumors to vehicle-treated tumors.

[171] Based on Affymetrix analysis of tumor lysates, approximately 154 genes were up-regulated or down-regulated by greater than 3.5 fold in sorafenib-treated tumors. In the four time points analyzed after sorafenib treatment, the most significant changes in gene expression occurred 4 h after the third dose and began to decrease by 24 h. See Table 1.

[172] Taqman Analysis

[173] All genes of interest were MGB designed probes with a FAM fluorescence tag (FAM= 6 carboxy-fluorescein). For normalization, an MGB probe to ribosomal protein L37 with a VIC fluorescence tag (VIC Amidite) was used.

[174] The PCR reaction was performed on a ABI Prism 7900 Sequence Detector (PE Applied Biosystems, CA) using cDNA prepared from RNA isolated from tumor samples using a Superscript III First Strand Synthesis kit (InVitrogen; CA). Reactions included 1 pre-PCR cycle of 2 min at 50⁰C and 10 min at 95°C followed by 40 cycles of 15 sec at 95°C and 1 min at 6O⁰C.

[175] Estimation of expression quality was based on Ct value. Each individual sample was normalized to its own L37 value. Fold change was calculated using the dd CT method and compared treated samples to vehicle control samples.

[176] Significant increases in mRNA levels of the inflammatory and angiogenic cytokines, IL-6 and IL-8, as well as the matrix metalloproteinases, MMP-1 and MMP-10 were observed upon treatment with sorafenib. Data generated on Affymetrix gene chips was confirmed by Taqman analysis using human-specific primers. [177] Measurement of protein levels in tumors and plasma

[178] Tumor samples were snap-frozen and homogenized in lysis buffer [4OmM Tris, pH7.4, 10% glycerol, 5OmM b-glycerol phosphate, 5mM EGTA, 2mM EDTA, 1mM sodium orthovanadate, 1OmM sodium fluoride, 0.3% Triton-X-100, and a protease inhibitor cocktail (Roche; Indianapolis, IN)].

[179] Levels of IL-6, IL-8 and VEGF were determined using specific assay kits from MesoScale Diagnostics (Gaithersburg, MD) following conditions from the manufacturer.

[180] MMP-1 and MMP-10 protein levels were determined in an immunoassay ELISA from R&D Systems (Minneapolis, MN) following the protocols from the vendor.

[181] Tumor protein levels of IL-6, IL-8, MMP-1 , and MMP-10 were elevated by sorafenib treatment (QDX 3) at different doses tested. Statistically significant elevation of the levels of all four proteins was observed at 24 hours after the third dose (15, 30 and 60 mg/kg doses). Trends in elevation of II-6, IL-8 and MMP-10 levels in plasma were observed removed from Sorafenib-treated animals.

[182] The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

[183] From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. All publications and patents cited above and in the following list are incorporated herein by reference.

[184] Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the following invention to its fullest extent. The following specific preferred embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

[185] In the forgoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius and, all parts and percentages are by volume, unless otherwise indicated.

Claims

What is Claimed is

Claim 1. A method of determining the efficacy of sorafenib in treating a disease or disorder in a subject who has been administered sorafenib, comprising, detecting the amount of MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and/or FOS-like antigen polypeptide or polynucleotide in a biological sample obtained from said subject, wherein said amount of said polypeptide is indicative of whether a therapeutically effective amount of sorafenib has been administered and comparing the amount detected to a standard value.

Claim 2. A method of Claim 1, wherein said amount is increased in comparison to the standard value, which is a sample obtained from said subject prior to sorafenib administration.

Claim 3. A method of Claim 1, wherein said amount is increased in comparison to the standard value, which is a standard value.

Claim 4. A method of Claim 1 , wherein the standard is determined by detecting the amount of MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1 , and/or FOS-like antigen polypeptide or polynucleotide in a biological sample obtained from said subject prior to sorafenib administration.

Claim 5. A method of Claim 1 , further comprising detecting the amounts of MMP-1 , MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1 , and/or FOS-like antigen polypeptide or polynucleotide in biological samples obtained through the course of sorafenib administration.

Claim 6. A method of Claim 1, wherein IL-6, IL-8, and/or MMP-1 is detected.

Claim 7. A method of Claim 1, wherein said sample is a tumor tissue or tumor cell sample.

Claim 8. A method of Claim 1, wherein said sample is a blood sample.

Claim 9. A method of Claims 2 or 3, wherein said increase occurs from about 4-24 hours, 1-3 days, 1-7 days, or 3-10 days after sorafenib administration.

Claim 10. A method of Claim 1, further comprising administering sorafenib to said subject until said amount of IL-6, IL-8, MMP-1 , and/or MMP-10 increases relative to a control or standard value.

Claim 11. A method of Claim 1 , wherein said disease or disorder is a tumor.

Claim 12. A method of Claim 1, wherein said disease or disorder, is melanoma, hepatocellular carcinoma, non-small lung ceil lung cancer, or renal cell carcinoma.

Claim 13. A method of determining the efficacy of sorafenib in treating a disease or disorder in a subject who has been administered sorafenib, comprising, detecting the amounts of IL-6, IL-8, MMP-1 , and/or MMP-10 mRNA in a biological sample obtained from said subject, wherein said amount of said mRNA is indicative of whether a therapeutically effective amount of sorafenib has been administered.

Claim 14. A method of determining the efficacy of sorafenib in treating a disease or disorder in a subject who has been administered sorafenib, comprising, detecting the amounts of at least one differentially-regulated mRNA, or polypeptides encoded thereby, in a biological sample obtained from said subject, wherein said mRNA, or polypeptide encoded thereby, is selected from Table 1, and, wherein said amount of said mRNA, or polypeptide encoded thereby, is indicative of whether a therapeutically effective amount of sorafenib has been administered.

Claim 15. A method of Claim 14, wherein the amounts of at least 10 differentially- regulated mRNAs, or polypeptides encoded by said mRNA, are detected.

Claim 16. A method of determining the efficacy of sorafenib in treating a tumor in a subject who has been administered sorafenib, comprising, measuring the amount of tumor hypoxia in said subject, wherein said amount is indicative of whether a therapeutically effective amount of sorafenib has been administered.

Claim 17. A method of determining the course of sorafenib treatment for a subject having a cancer, comprising: administering an amount of sorafinib which is effective to elicit an increase relative to a control or normal sample of MMP-1, MMP-10, IL-6, IL-8, fL-10, CSF-2, SLCO4A1, and/or FOS-like antigen polypeptide or polynucleotide or mRNA in a biological sample obtained from said subject.

Claim 18. A method of determining whether a compound has the pharmacological activity of sorafenib, comprising detecting the amounts of at least two differentially-regulated mRNAs, or polypeptides encoded thereby, in a cell which has been exposed to said sorafenib, wherein said amount is indicative of whether said compound has the pharmacological activity of sorafenib, and wherein said differentially regulated mRNAs are selected from Table 1.

Claim 19. A method of determining the presence of tumor tissue hypoxia, comprising detecting the amounts of MMP-1, MMP-10, IL-6, IL-8, IL-10, CSF-2, SLCO4A1, and/or FOS-like antigen polypeptide or polynucleotide or mRNA in a biological sample obtained from said subject, wherein said amount. of said polypeptide or mRNA indicates the occurrence of hypoxia in a tumor tissue.

Claim 20. A method of reducing the inflammatory evens associated with sorafenib administration in a subject, comprising: administering an effective amount of an anti-inflammatory agent to said subject.