WO2009105223A1 - Systems and methods of cancer staging and treatment - Google Patents

Abstract

Methods of assessing the sensitivity of a cancer cell to a tyrosine kinase inhibitor are disclosed. Such methods include assessing the expression of miR-497 and correlating reduced expression with sensitivity to the tyrosine kinase inhibitor. Also disclosed are methods of assessing the sensitivity of a cell to a tyrosine kinase inhibitor that includes assessing the expression of FGF1, HOXC10, and/or LHFP. Additionally disclosed are methods of treating patients with tyrosine kinase inhibitors such as sunitinib based on results obtained from the disclosed methods and kits that facilitate the methods.

Description

SYSTEMS AND METHODS OF CANCER STAGING AND TREATMENT

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application 61/029,656, filed 19 February 2008, the content of which is hereby incorporated by reference in its entirety.

FIELD

[0002] Provided herein are methods of treatment of cancer and more specifically methods of the differential treatment of cancer based upon micro RNA gene expression.

BACKGROUND

[0003] Worldwide, lung cancer is the leading cause of cancer-related mortality in both men and women. Although current treatments for advanced non-small cell lung cancer (NSCLC) are disappointing, there is growing promise using anti-angiogenic therapies. Some of these anti- angiogenic therapies, which include tyrosine kinase inhibitors (TKI) targeting VEGF receptor 2 and 3 (VEGFR-2/3), such as sunitinib and sorafenib, have been administered to patients in an unselected fashion.

[0004] MicroRNAs (also known as miRs or miRNAs) are a class of small non-coding RNAs having 21 to 25 nucleotides in length that have recently been implicated in cancer biology (Calin et al, Proc Natl Acad Sci USA 99:15524-15529, 2002). These RNA fragments post- transcriptionally regulate gene expression by binding to complementary sequences in the 3' untranslated region (3'UTR) of the target mRNA (Kumar et al., Nat Genet 39:673-677, 2007). This can ultimately lead to repression of protein translation and, as a result, of protein expression (Eder et al., N Eng J Med 352:2446-2448, 2005). Regulation of protein expression by miRs is emerging as an important area of study in carcinogenesis because their regulatory capabilities can drastically influence cell physiology (Scott et al, J Biol Chem 282:1479-1486, 2007). Therefore, there is a need for methods of predicting drug efficacy in individual patients by miR expression.

LAI-3002528vl

OOOOCO - OQOOQO - 1 - BRIEF SUMMARY

[0O05] In one aspect, provided is a method of evaluating the sensitivity of a tumor to a tyrosine kinase inhibitor using microRNA.

[0O06] In one embodiment, provided is a method of evaluating a cancer cell sensitivity to a tyrosine kinase inhibitor by assessing the expression of miR-497 in a patient sample and correlating a reduced expression of the miR-497 with a sensitivity to the tyrosine kinase inhibitor.

[0007] In another embodiment, provided is a method of predicting a cancer cell sensitivity to a tyrosine kinase inhibitor in order to apply a personalized medicine based therapy to cancer treatment.

[0008] In yet another embodiment, provided is a method of predicting a cancer cell sensitivity to a tyrosine kinased inhibitor in order to prevent a patent from suffering side effects of a drug that is unlikely to be efficacious.

[0009] In another embodiment, provided is a method of predicting a cancer cell sensitivity to a tyrosine kinase inhibitor in order to prevent a cancer patient from wasting time on a treatment that is unlikely to be efficacious.

[0010] In another embodiment, provided is a test that evaluates a sensitivity of a tumor to a tyrosine kinase inhibitor that does not require a tumor biopsy.

[0011] In one embodiment, a patient sample comes from blood. In another embodiment, a patient sample comes from a tumor biopsy.

[0O12] In another embodiment, an expression is assessed by mRNA detection methods such as RTPCR, microarray analysis, or Northern blot.

[0013] In another embodiment, a tyrosine kinase inhibitor specifically inhibits VEGFR2 alone, VEGFR3 alone, both VEGFR2 and VEGFR3, or either VEGFR2 or VEGFR3 in combination with any other molecule. In one embodiment, a tyrosine kinase inhibitor is sunitinib. In another embodiment, a tyrosine kinase inhibitor is sorafenib. [0014] In yet another embodiment, , the cancer cell is a non-small cell lung cancer cell. [0015] In another aspect, provided is a method involving slowing the expansion of a population of cancer cells including assessing the expression of miR-497 in a sample, correlating reduced expression of the miR-497 with sensitivity to a tyrosine kinase inhibitor, and treating the population of cancer cells with the tyrosine kinase inhibitor

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LAI-3002528vl [OO 16] In one embodiment, provided is a method of correlating positive expression of miR-497 with resistance to a tyrosine kinase inhibitor. In this case, the tyrosine kinase inhibitor is not administered.

[0017] In one embodiment, a sample is taken from a human. In another embodiment, the sample is a blood fraction. In yet nother embodiment, the sample is a tumor biopsy.

[0018] In one embodiment, cancer cells are shown to display a loss of heterozygosity in chromosomal region 17p.

[0019] In one embodiment, cancer cells are non-small cell lung cancer cells.

[0020] In one embodiment, a tyrosine kinase inhibitor specifically inhibits VEGFR2 alone,

VEGFR3 alone, both VEGFR2 and VEGFR3, or either VEGFR2 or VEGFR3 in combination with any other molecule.

[0021] In another embodiment, a tyrosine kinase inhibitor is sunitinib.

[0022] In yet another embodiment, the population of cancer cells comprises non-small cell lung cancer cells.

[0023] In yet another aspect, provided are kits that facilitate the assessment of the expression of miR-497 that includes a reagent that is capable of specifically recognizing the miR-497 itself or a product of the miR-497 gene. In one embodiment, the reagent comprises an oligonucleotide. In another embodiment, the reagent comprises an antisense nucleic acid. In yet another embodiment, the reagent is bound to a solid support.

[0024] In one embodiment, the kit contains a fluorescent label. In another embodiment, the kit contains a reagent capable of recognizing a gene other than the miR-497 gene or any product of that gene.

[0025] In another aspect, provided herein is a method involving an evaluation of cancer cells sensitivity to a tyrosine kinase inhibitor by assessing the expression of FGFl, HOXClO, and/or

LHFP, whether singly or in combination, and correlating the positive expression of these products with sensitivity to the tyrosine kinase inhibitor.

[0026] In one embodiment, a sample comes from a patient's blood. In another embodiment, a sample comes from a tumor biopsy.

[0027] In one embodiment, an expression is assessed by measuring a mRNA expression using methods such as RTPCR, microarray analysis, or Northern blot. In another embodiment, an expression is assessed by measuring a protein expression using methods that involve specific

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LAI-3002528vl ligands such as antibodies. Such methods include immunohistochemical methods, ELISA, and flow cytometry. In yet another embodiment, an expression is assessed using the methods of mass spectrometry.

[0O28] In one embodiment, a tyrosine kinase inhibitor is sunitinib.

[0029] In another embodiment, a cancer cell is a non-small cell lung cancer cell.

[0030] In another aspect, provided herein is a method involving slowing the expansion of a population of cancer cells comprising assessing the expression of FGFl, HOXClO, and/or

LHFP, alone or in combination in a sample, correlating positive expression of FGFl, HOXClO, and/or LHFP with sensitivity to the tyrosine kinase inhibitor, and treating the population of cancer cells with the tyrosine kinase inhibitor.

[0031] In one embodiment, provided is a method of correlating reduced expression of FGFl,

HOXClO, and/or LHFP with resistance to the tyrosine kinase inhibitor. In this case, the tyrosine kinase inhibitor is not administered.

[0032] In one embodiment, a sample is taken from a human. In another embodiment, a sample is a blood fraction. In another embodiment, a sample is a tumor biopsy. In yet another embodiment, cancer cells are non-small cell lung cancer cells. In another embodiemnt, the tyrosine kinase inhibitor is sunitinib.

[0033] In yet another aspect, provided are kits that facilitate the assessment of the expression of FGFl, HOXClO, and/or LHFP, alone or in combination, that include a reagent that is capable of specifically recognizing FGFl, HOXClO, and/or LHFP themselves or a product of the FGFl,

HOXClO, and/or LHFP genes. In one embodiment, the reagent comprises an oligonucleotide.

In another embodiment, the reagent comprises an antisense nucleic acid. In yet another embodiment, the reagent is bound to a solid support. In another embodiment, the kit contains a fluorescent label. In another embodiment, the kit contains a reagent capable of recognizing a gene other than the miR-497 gene or any product of that gene.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] A more complete understanding of the present invention may be derived by referring to the detailed description when considered in connection with the following illustrative figures. In the figures, like reference numbers refer to like elements or acts throughout the figures.

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LAI-3OQ₂528vl [0035] Figure 1 depicts a Western blot image showing expression of VEGFR2 and VEGFR3 in H358 cells transfected with miR-497 mimic or control mimic microRNA.

[0036] Figure 2 depicts the results of densitometry of Western blots showing expression of

VEGFR2 and VEGFR3 in H358 cells transfected with miR-497 inhibitor or mimic or control inhibitor or mimic microRNA.

[0O37] Figure 3 depicts the results of densitometry of Western blots showing expression of

VEGFR2 in H 1703 cells transfected with miR-497 inhibitor or mimic or control inhibitor or mimic microRNA.

[0O38] Figure 4 depicts the results of densitometry of Western blots showing expression of

VEGFR2 in H520 cells transfected with miR-497 inhibitor or mimic or control inhibitor or mimic microRNA.

[0O39] Figure 5 depicts the results of densitometry of Western blots showing expression of

VEGFR2 in Hl 57 cells transfected with miR-497 inhibitor or mimic or control inhibitor or mimic microRNA.

[0O40] Figure 6 depicts the results of densitometry of Western blots showing expression of

VEGFR2 in H 1703 cells transfected with miR-497 inhibitor or mimic or control inhibitor or mimic microRNA.

[0O41] Figure 7 depicts the results of densitometry of Western blots showing expression of

VEGFR2 and VEGFR3 in H 1703 cells transfected with miR-497 inhibitor or mimic or control inhibitor or mimic microRNA.

[0O42] Figure 8 depicts the results of sunitinib treatment on the viability of sunitinib sensitive (H520 and H1703) and sunitinib resistant (H322c, H358, H157, and A549) cell lines.

[0O43] Figure 9 depicts the results of qRT-PCR analysis of FGF 1 , LHFP, and HOXC 10 in sunitinib sensitive (H520 and H1703) and sunitinib resistant (H322c, H358, H157, and A549) cell lines.

DETAILED DESCRIPTION

Definitions

[0O44] Unless specifically noted, it is intended that the words and phrases in the specification and the claims be given their plain, ordinary, and accustomed meaning to those of ordinary skill in the applicable arts.

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LAl-3002528vl [0O45] In the following description, and for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the various aspects of the invention. It will be understood, however, by those skilled in the relevant arts, that the present invention may be practiced without these specific details. In other instances, known structures and devices are shown or discussed more generally in order to avoid obscuring the invention. The full scope of the disclosure is not limited to the examples that are described below.

Methods of Use

[0O46] Provided are methods of assessing the sensitivity of cancer cells to a drug using an miR-497 mimic in combination with a tyrosine kinase inhibitor. Also provided are methods of treating cancer with a tyrosine kinase inhibitor based upon the expression of miR-497 by the cancer cells. Additionally, provided are methods of assessing the sensitivity of cancer cells to a tyrosine kinase inhibitor on the basis of the expression of any of the genes FGFl, HOXClO, and LHPP, whether singly or in combination.

[0047] A target includes any molecular structure produced by a cell and expressed inside the cell, on the cell surface, or secreted by the cell. Targets include proteins, lipids, carbohydrates, nucleic acids, including RNA molecules and genomic DNA sequences, subcellular structures, glycoproteins, viruses and any other like structures known or yet to be disclosed whether alone or in combination. Illustrative examples of targets include, but are not limited to, VEGFR2, VEOFR3, miR-497, FGFl, HOXClO, LHFP and any products thereof including mRNA's and proteins.

[0048] Cancer cells include any cells derived from a tumor, neoplasm, cancer, precancer, cell line, or any other source of cells that have the potential to expand and grow to an unlimited degree. Cancer cells are derived from naturally occurring sources or are artificially created. Cancer cells are capable of invasion into other tissues and metastasis when placed into an animal host. Cancer cells further encompass any malignant cells that have invaded other tissues and/or metastasized. One or more cancer cells in the context of an organism may also be called a cancer, tumor, neoplasm, growth, malignancy, or any other term used in the art to describe cells in a. cancerous state.

[00-49] Cancers that serve as sources of cancer cells include, but are not limited to, solid tumors such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,

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LAI-3002528vl chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung carcinoma, bladder carcinoma, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, and retinoblastoma.

[0050] Additional cancers that serve as sources of cancer cells include, but are not limited to, blood borne cancers such as acute lymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblasts leukemia ,acute promyelocytic leukemia, acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, multiple myeloma, lymphoblastic leukemia, myelogenous leukemia, lymphocytic leukemia, myelocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, heavy chain disease, and polycythemia vera. [0051] In one embodiment, cancer cells are derived from non-small cell lung cancer (NSCLC.) NSCLC includes any carcinoma derived from lung tissues that does not include small cell lung cancers. Examples of non-small cell lung cancers include, but are not limited to, adenocarcenomas, large cell carcinomas, and squamous cell carcinomas of the lung. [0052] Expansion of a cancer cell includes any process that results in an increase in the number of individual cells derived from a cancer cell. Expansion of a cancer cell may result from mitotic division, proliferation, or any other form of expansion of a cancer cell, whether in vitro or in vivo. Expansion of a cancer cell further encompasses invasion and metastasis. A cancer cell may be in physical proximity to cancer cells from the same clone or from different clones that

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LAI-3002528vl may or may not be genetically identical to it. Such aggregations may take the form of a colony, tumor or metastasis, any of which may occur in vivo or in vitro. Slowing the expansion of the cancer cell may be brought about either by inhibiting cellular processes that promote expansion or by bringing about cellular processes that inhibit expansion. Processes that inhibit expansion include processes that slow mitotic division and processes that promote cell senescence or cell death. Examples of specific processes that inhibit expansion include caspase dependent and independent pathways, autophagy, necrosis, apoptosis, and mitochondrial dependent and independent processes and further include any such processes yet to be disclosed. [0053] Inhibition of the expansion of a cancer cell is achieved through the use of an outside agent applied to a cancer cell for the purpose of slowing the expansion of a cancer cell. Such agents include natural or synthetic ligands, blockers, agonists, antagonists or activators of receptors, immune cells, such as CD8+ T cells, viruses, inhibitors of gene or protein expression, such as siRNA or miR's, small molecules, pharmaceutical compositions, or any other composition of matter that when administered to a cancer cell results in slowing of the expansion of a cancer cell. The concept of agents that slow the expansion of a cancer cell encompasses restricting access to any natural or artificial agent necessary for cell survival including necessary nutrients, ligands, or cell-cell contacts. Examples of such agents and conditions include treatment with antiangiogenic inhibitors.

[0054] In one embodiment, an agent that slows the expansion of a cancer cell comprises a tyrosine kinase inhibitor (TKI). A tyrosine kinase catalyzes the transfer of a phosphate group to the tyrosine residue of a specific protein. If a TKI inhibits an action of a kinase necessary for growth, differentiation or division of a cancer cell, expansion of a cancer cell is slowed. A TKI includes any agent that inhibits the action of one or more tyrosine kinases in a specific or nonspecific fashion. TKIs include small molecules, antibodies, peptides, or anything that directly, indirectly, allosterically, or in any other way inhibits tyrosine residue phosphorylation. [0055] Specific examples of tyrosine kinase inhibitors include N-(trifluoromethylphenyl)-5- methylisoxazol-4-carboxamide, 3-[(2,4-dimethylpyrτol-5-yl)methylidenyl)indolin-2-one, 17- (allylamino)-17-demethoxygeldanamycin, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3- (4-morpholinyl)propoxyl]quinazoline, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4- quinazolinamine, BIBX1382, 2,3,9,10,1 l,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9- methyl-9, 12-epoxy- 1 H-diindolo[ 1 ,2,3-fg:3',2', 1 '-k 1 ]pyrrolo[3,4-i] [ 1 ,6]benzodiazocin- 1 -one,

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LAI-3002528vl SH268, genistein, STI571, CEP2563, 4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo [2,3-d]pyrimidinemethane sulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6, 7- dimethoxyquinazoline, 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571 A, N-4-chlorophenyl-4-(4-pyridylmethyl)- 1 -phthalazinamine, N-[2-(diethylamino)ethyl]- 5-[(Z)-(5-fluoro-l,2-dihydro-2-oxo-3H-indol-3-ylidine)methyl]-2,4-dimethyl-lH-pyrrole-3- carboxamide (sunitinib), 4-[4-[[4-chloro-3 (trifluoromethyl)phenyl]carbamoylamino] phenoxy]- N-methyl-pyridine-2-carboxamide (sorafenib), and EMDl 21974. [0056] In another embodiment, a tyrosine kinase inhibitor has activity upon Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) and Vascular Endothelial Growth Factor Receptor 3 (VEGFR3). VEGFR2 and VEGFR3 are tyrosine kinases that phosphorylate proteins necessary for angiogenesis. Tyrosine kinase inhibitor may inhibit either VEGFR2 or VEGFR3 singly, both VEGFR2 and VEGFR3 to the exclusion of all other targets, or VEGFR2 and/or VEGFR3 in combination with one or more additional tyrosine kinases or other targets. . Tyrosine kinase inhibitors that inhibit VEGFR2 and/or VEGFR3 include sunitinib and sorafenib, but could be any inhibitor that targets VEGFR2 singly or in combination with any other tyrosine kinase, any inhibitor that targets VEGFR3 singly or in combination with any other tyrosine kinase, or any inhibitor that targets either VEGFR2 and VEGFR3 to the exclusion of or in combination with any other tyrosine kinase.

[0057] As used herein, VEGFR2 has the sequence provided below. gcagcatcgg acaagacccc cagcacttgg gggttcaggc ccggcagggc gggcagaggg ctggaggccc aggctgggaa ctcatctggt tgaactctgg tggcacagga gtgtcctctt ccctctctgc agacttccca gctaggaaga gcaggactcc aggcccaagg ctcccggaat tccgtcacca cgactggcca gggccacgct ccagctgccc cggcccctcc ccctgagatt cagatgtcat ttagttcagc atccgcaggt gctggtcccg gggccagcac ttccatggga atgtctcttt ggcgacctcc tttcatcaca ctgggtggtg gcctggtccc tgttttccca cgaggaatct gtgggtctgg gagtcacaca gtgttggagg ttaaggcata cgagagcaga ggtctcccaa acgccctttc ctcctcaggc acacagctac tctccccacg agggctggct ggcctcaccc acccctgcac agttgaaggg aggggctgtg tttccatctc aaagaaggca tttgcagggt cctcttctgg gcctgaccaa acagccaact agcccctggg gtggccacca gtatgacagt attatacgct ggcaacacag aggcagcccg cacacctgcg cctgggtgtt gagagccatc ctgcaagtct ttttcaacag aacttcacag actgttagag ctgctgagaa gaatttgctt tccgaattca gcctggaagg cgcccaggga cagctgtact gagtctagat gactctgacc cccgccccag gtcaaggcca gcagagcagt cagtgcctct ggagaaggcc cttgctctcc cacctggccc agactccgag gagcctgggt ctggagctgc cggtctggtt cttcccttta gagcccggat ctgccacctg cggcccctcc caagccgtga accagctcat gagagatgaa cactgtggga tccactcagg aaggctcggg gctggcacaa aggaccaccc agcattgccc tgtgccaccc agcactcagt ggacattctg gggacctgcc ttcagccttt tcctgccctg tgcctgacat cagcaccctg gctggtcaga atgccgccct cccagaggag cagccgagag atcccctgaa ggctggaggc attctgctca ggacccctat cccagctcac agtgcccaac catctcacca ggagaaagag ccacatcccc acgttaggac cacggagact gaccaccacc ctgacccccc aaacccacgc accagacgct tgcaggacag gcgccgcgca

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LAI-3002528vl gcgggcaggg gcttgcccgg ccgaccctcc cctccccacc tcccccactg cgcgttactc caggatatgc cgagtgcacg tataaggtca tcttcgtcgt ccccgtggac ctcccccttc ctctgcacgt cgtccaacgt gggactggcg tgtcaggctt ccctgggagg atctggaggt tgttctctgc agagaaccag cctggctcct ggcgcgcacc tctgctccct tctcctcact acccacccac gcatgtaccg ggaaaaaaac tactatgccc ttctagacca tgttctgaga aaagatcgaa aatatttaac aagagataat aataaatctg atgccggtct ttgtgtgtgt tgcgga (SEQ ID NO:2)

[0058] VEGFR3 has the sequence provided below. gcagcatcgg acaagacccc cagcacttgg gggttcaggc ccggcagggc gggcagaggg ctggaggccc aggctgggaa ctcatctggt tgaactctgg tggcacagga gtgtcctctt ccctctctgc agacttccca gctaggaaga gcaggactcc aggcccaagg ctcccggaat tccgtcacca cgactggcca gggccacgct ccagctgccc cggcccctcc ccctgagatt cagatgtcat ttagttcagc atccgcaggt gctggtcccg gggccagcac ttccatggga atgtctcttt ggcgacctcc tttcatcaca ctgggtggtg gcctggtccc tgttttccca cgaggaatct gtgggtctgg gagtcacaca gtgttggagg ttaaggcata cgagagcaga ggtctcccaa acgccctttc ctcctcaggc acacagctac tctccccacg agggctggct ggcctcaccc acccctgcac agttgaaggg aggggctgtg tttccatctc aaagaaggca tttgcagggt cctcttctgg gcctgaccaa acagccaact agcccctggg gtggccacca gtatgacagt attatacgct ggcaacacag aggcagcccg cacacctgcg cctgggtgtt gagagccatc ctgcaagtct ttttcaacag aacttcacag actgttagag ctgctgagaa gaatttgctt tccgaattca gcctggaagg cgcccaggga cagctgtact gagtctagat gactctgacc cccgccccag gtcaaggcca gcagagcagt cagtgcctct ggagaaggcc cttgctctcc cacctggccc agactccgag gagcctgggt ctggagctgc cggtctggtt cttcccttta gagcccggat ctgccacctg cggcccctcc caagccgtga accagctcat gagagatgaa cactgtggga tccactcagg aaggctcggg gctggcacaa aggaccaccc agcattgccc tgtgccaccc agcactcagt ggacattctg gggacctgcc ttcagccttt tcctgccctg tgcctgacat cagcaccctg gctggtcaga atgccgccct cccagaggag cagccgagag atcccctgaa ggctggaggc attctgctca ggacccctat cccagctcac agtgcccaac catctcacca ggagaaagag ccacatcccc acgttaggac cacggagact gaccaccacc ctgacccccc aaacccacgc accagacgct tgcaggacag gcgccgcgca gcgggcaggg gcttgcccgg ccgaccctcc cctccccacc tcccccactg cgcgttactc caggatatgc cgagtgcacg tataaggtca tcttcgtcgt ccccgtggac ctcccccttc ctctgcacgt cgtccaacgt gggactggcg tgtcaggctt ccctgggagg atctggaggt tgttctctgc agagaaccag cctggctcct ggcgcgcacc tctgctccct tctcctcact acccacccac gcatgtaccg ggaaaaaaac tactatgccc ttctagacca tgttctgaga aaagatcgaa aatatttaac aagagataat aataaatctg atgccggtct ttgtgtgtgt tgcgga (SEQ ID NO: 3)

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LAI-3002528vl [0059] However, contemplated is any sequence identifiable as VEGFR2 or VEGFR3 on the basis of its ability to be inhibited by one or more tyrosine kinase inhibitors, its ability to recognize a specific ligand, or its ability to perpetuate an intracellular signal. A sequence may display any one of these characteristics and any combination thereof. A sequence includes any mutation, truncation, or addition of extra nucleotides. A sequence having at least 60% 70%, 80%, or 90% identity to SEQ ID NO. 2 in the case of VEGFR2 and at least 60% 70%, 80%, or 90% identity to SEQ ID NO. 3 in the case of VEGFR3 at the genomic, mRNA, or protein level. [0O60] MicroRNA's (miR's) are non-coding RNAs having 18 to 36 nucleotides, in one embodiment 21 to 25 nucleotides in length that inhibit gene expression by binding to a sequence complementary to the miR sequence, often located in the 3' untranslated region (UTR) of the target mRNA. Mechanisms of gene silencing include repression of protein translation and downregulation of protein expression. [0061] As used herein, miR-497 has the sequence provided below.

cagcagcaca cugugguuug u (SEQ ID NO:1)

[0062] However, contemplated is any sequence identifiable as miR-497 including mutations, truncations, or additions of one or more nucleotides that is capable of binding the 3' UTR of VEGFR2 or VEGFR3 with such binding resulting in a reduced VEGFR2 or VEGFR3 expression. A sequence having at least 60%, 70%, 80%, or 90% identity to SEQ ID NO.l at the genomic or mRNA level. The concept of miR-497 includes one or more non-nucleotide small molecule compositions of matter derived from miR-497 capable of specifically binding to the 3' UTR of VEGFR2/3 such that VEGFR2/3 expression is silenced.

[0063] While a specific target is identified by a nucleic acid sequence, such as a cDNA, mRNA or protein sequence, a specific target is not limited to the products of that exact sequence. Rather, a specific target identified by a nucleic acid sequence encompasses all sequences that, when their expression is assessed, yield positive expression when assessed by the same method as the specific target.: In one embodiment, if expression of a specific target in a sample is assessed by immunohistochemical analysis, and if a sample expresses a sequence different from the sequence used to identify the specific target (e.g., a variation of one or more nucleic acid

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LAI-3002528vl molecules,) but positive expression is still determined, then the specific target encompasses the sequence expressed by the sample.

[0064] Expression encompasses all processes through which material derived from a nucleic acid template is produced. Expression thus includes RNA transcription, mRNA splicing, protein translation, protein folding, post-translational modification, membrane transport, associations with other molecules, addition of carbohydrate moeties to proteins, phosphorylation, protein complex formation and any other process along a continuum that results in biological material derived from genetic material. Expression also encompasses all processes through which the production of material derived from a nucleic acid template is actively or passively suppressed. Such processes include all aspects of transcriptional and translational regulation. Examples include heterochromatic silencing, transcription factor inhibition, any form of RNAi silencing, microRNA silencing, alternative splicing, protease digestion, post-translational modification, and alternative protein folding.

[0065] Expression is assessed by any number of methods used to detect material derived from a nucleic acid template used currently in the art and yet to be developed. Examples of such methods include any nucleic acid detection method including, but not limited to, microarray analysis, RNA in situ hybridization, RNAse protection assay, Northern blot, reverse transcriptase PCR, quantitative PCR, quantitative reverse transcriptase PCR, quantitative real-time reverse transcriptase PCR, or any other method of detecting a specific nucleic acid known or yet to be disclosed. Other examples include any process of detecting expression that uses an antibody including, but not limited to, flow cytometry, immunohistochemical methods, ELISA, Western blot, and immunoaffinity chromatograpy. Antibodies may be monoclonal, polyclonal, or any antibody fragment including an Fab, F(ab)2, Fv, scFv, phage display antibody, peptibody, multispecific ligand, or any other reagent with specific binding to a target. Such methods also include direct methods used to assess protein expression including, but not limited to HPLC, mass spectrometry, protein microarray analysis, PAGE analysis, isoelectric focusing, 2-D gel electrophoresis, and enzymatic assays. Samples from which expression is detected include single cells, whole organs or any fraction of a whole organ, whether in vitro, ex vivo, in vivo, or postmortem.

[0066] Other methods used to assess expression include the use of natural or artificial ligands capable of specifically binding a target, including a protein, carbohydrate, fat, nucleic acid,

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LAI-3002528vl catalytic site, or any combination of these such as an enzyme, glycoprotein, cell membrane, virus, cell, organ, organelle, or any other multimolecular structure that constitutes a target that is specifically bound by a ligand. Such ligands include antibodies, antibody complexes, conjugates, natural ligands, small molecules, nanoparticles, or any other molecular entity capable of specific binding to a target. Ligands are associated with a label such as a radioactive isotope or chelate thereof, dye (fluorescent or nonfluorescent,) stain, enzyme, metal, or any other substance capable of aiding a machine or a human eye from differentiating a cell expressing a target from a cell not expressing a target. Additionally, expression may be assessed by monomeric or multimeric ligands associated with substances capable of killing a cell. Such substances include protein or small molecule toxins, cytokines, pro-apoptotic substances, pore forming substances, radioactive isotopes, or any other substance capable of killing a cell.

[0O67] Positive expression includes any difference between a cell expressing a specific target and a cell that does not express a specific target. The exact nature of positive expression varies by the method, but is well known to those practicing a particular method. Positive expression is assessed by a detector, an instrument containing a detector, or by aided or uniaided human eye. Examples include, but are not limited to, specific staining of cells expressing a target in an IHC slide, binding of RNA from a sample to a microarray and detection by an instrument capable of detecting the binding to said microarray, a high rate of dye incorporation in real-time RTPCR, detection of fluorescence on a cell expressing a target by a flow cytometer, the presence of radiolabeled bands on film in a Northern blot, detection of labeled blocked RNA by RNAse protection assay, cell death measured by apoptotic markers, cell death measured by shrinkage of a tumor, or any other method by which expression is observed known or yet to be disclosed. [0068] Reduced expression constitutes a lack of positive expression such that there is not a significant difference between a cell expressing a particular target and a cell not expressing the particular target. The concept of reduced expression further encompasses insufficient expression to reach or exceed a threshold, cutoff, or level that results in a particular cellular or physiological response. For example, reduced expression includes the expression of a particular target in a test cell that is positive expression relative to a control cell known not to express the target. However, because the expression of the target in the test cell is insufficient to cause a particular physiological response (e.g., rendering the cell sensitive to a particular drug), the expression in

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LAI-3002528vl the test cell is still classified as reduced expression. Similarly, the concept of positive expression also encompasses expression sufficient to cause a physiological response. [0069] Also provided are methods of assessing the expression of a target in any biological sample from which the expression is assessed. One skilled in the art knows how to select a particular biological sample and how to collect said sample depending upon whether or not expression of germline DNA, tumor DNA, mRNA, or any form of protein is assessed. Examples of sources of samples include, but are not limited to, biopsy or other in vivo or ex vivo analysis of prostate, breast, skin, muscle, facia, brain, endometrium, lung, head and neck, pancreas, small intestine, blood, liver, testes, ovaries, colon, skin, stomach, esophagus, spleen, lymph node, bone marrow, kidney, placenta, or fetus tissues. In one embodiment, a sample comprises a fluid sample, such as peripheral blood, lymph fluid, ascites, serous fluid, pleural effusion, sputum, cerebrospinal fluid, amniotic fluid, lacrimal fluid, stool, or urine. In another embodiment, a sample comprises primary or metastatic NSCLC cells. In yet another embodiment, a sample comprises blood. MicroRNA is readily detectable in blood and blood compartments such as serum or plasma by a number of methods. (Chen X et al, Cell Research 18 983-984, October 2008).

[0070] Provided are kits that facilitate assessing the expression of a target. Such kits contain one or more reagents that indicate the presence of a target. Contents of such kits include one or more of the following alone or in combination: one or more oligonucleotide primers capable of hybridizing to sequences within the target which are further optimized for use in a PCR based method, an antisense probe to all or part of target sequence, a ligand with specificity to the target mRNA, protein or other measurable gene product, a label, a buffer, or any other reagent that is useful in a method that assesses the expression of a target whether known or yet to be disclosed.

EXAMPLES

Example 1.

[0071 J Expression of a microRNA (miR) with specificity to the 3 'UTR of VEGFR2 or VEGFR3 is capable of binding the UTR and silencing the VEGFR2 or VEGFR3 (VEGFR 2/3) expression. Positive expression of such miR indicates reduced VEGFR2/3 expression. Tumors with reduced VEGFR2/3 expression are resistant to VEGFR 2/3 specific tyrosine kinase inhibitors. Conversely, if a tumor displays reduced expression of a miR capable of

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LAI-3002528vl downregulating VEGFR-2/3, then the result is a more robust VEGFR-2/3 expression, indicating that the tumor is more sensitive to VEGFR2/3 specific tyrosine kinase inhibitors. Assessing VEGFR2/3 expression by miR has advantages over assessing VEGFR2/3 protein directly. Expression by miR is assessed quickly by PCR and high throughput sequencing methods. Further, miR is available in blood and the expression of an individual miR is easily assessed in plasma, serum, or other blood fractions. Such assays allow easy presymptomatic surveillance of a number of diseases, especially cancer.

[0072] Search of a publicly available database revealed that miRs, based on their sequence, potentially regulate VEGFR-2/3 expression (Targetscan Database, Whitehead Institute for Biomedical Research, 2006-2008). This comprised searching the 3' UTR of both genes for potential miR binding sites. One core predicted binding site GCTGCT was common for both the VEGFR2 and VEGFR3 3'UTR's. Using seed sequences, miR-497 was identified as possibly capable of regulating VEGFR2/3. MiR-497 is located on chromosome 17p. The fact that allelic loss in chromosome 17p is frequent in lung cancer ( Tonon et al, Proc Natl Acad Sci USA 102:9625-9630, 2005) further led to the selection of miR-497. Allelic loss of 17p occurs at a 50% rate in lung cancer. Additionally, chromosome 17p is situated in close proximity (<1 MB) to the TP53 gene locus, a frequent site of loss of heterozygosity in cancer generally (Chmara et al, Anticancer Res 24:4259-4263, 2004).

[0073] MiR-497 expression, VEGFR2/VEGFR3 protein and mRNA expression, and sensitivity of specific tyrosine kinase inhibitor sunitinib to the VEGFR2/3 were assessed in six cell lines derived from non-small cell lung cancer (Hl 703, A549, H520, H322C, H358, and Hl 57) and are summarized in Table 1 (below). Table 1

[0074] Positive expression of miR-497 is seen in the H1703, A549, and H520 cell_.lines, while reduced expression is seen in H322C, H358, and H157. The table further shows changes in

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LAI-3OO2528vl VEGFR2 and VEGFR3 mRNA expression after transfection with miR-497 inhibitor(I) or miR-497 mimic (M) (an RNA with a similar sequence to that of miR-497) normalized to the miR-497 expression with a control miR in which all three had been normalized to act in mRNA expression. ^~

[0075] Transfection of miR-497 mimic and inhibitor (an RNA with a sequence similar to the anti sense sequence of miR-497) into the indicated cell lines was performed, followed by Western blotting and qRT-PCR. Densitometry measurements were taken from Western blot images using Scion Image.

[0076] Table 2 summarizes the expression of VEGFR2 protein and VEGFR3 protein in the six listed above cell lines as the IC5₀ of each cell line to sunitinib (below). Table 2

[0077] The first two rows of Table 2 summarize the expression of VEGFR2 protein and VEGFR3 protein in the six listed cell lines by Western blot. The term "present" indicates positive expression of VEGFR2 or VEGFR3 protein.

[0078] As shown in Figures 1 to 5, reduced expression of the VEGFR2 protein with transfection of miR-497 mimic was observed in four of the six tested cell lines. Reduced expression of the VEGFR2 mRNA was not observed in any of the tested cell lines transfected with miR-497 mimic. Increased expression of the VEGFR2 protein was observed in three of the six lines transfected with miR-497 inhibitor. Increased expression of the VEGFR2 mRNA was observed in two of the six cell lines transfected with the miR-497 inhibitor. [0079] As shown in Figures 2 and 7, reduced expression of the VEGFR3 protein was observed with transfection of miR-497 mimic in one of the two cell lines in which it was assessed. Reduced expression of the VEGFR3 message was observed with transfection of miR-497 mimic in one of the six tested cell lines. Increased expression of the VEGFR3 protein was not observed in either of the cell lines transfected with the miR-497 inhibitor in which the

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LAI-300₂528vl VEGFR3 protein expression was assessed. Increased VEGFR3 message was observed in two of the six cell lines when those lines were transfected with the miR-497 inhibitor. [0080] As shown in Table 2 and Figure 8, untransfected cell lines Hl 703 and H520 were sensitive to sunitinib, displayed positive expression of miR-497 and both lacked the VEGFR3 protein expression. Additionally, H520 lacked the VEGFR2 protein expression. Untransfected cell lines A549, H322C, H358, and Hl 57 were resistant to sunitinb and displayed positive expression of both VEGFR2 and VEGFR3. Of these, only A549 displayed positive expression of miR-497. In general, in vitro exposure increased sensitivity to VEGFR-2/3 sunitinib correlated with a positive expression of microRNA-497.

[0081] As shown in Figure 9, microarray gene expression data from NCBI's GEO GSE 4342 were normalized by 'per chip normalization' and 'per gene normalization' using GeneSpring between resistant and sensitive NSCLC lines. Genes that were identified as having statistically significant differences (p<0.01) when grouped as sunitinib resistant (defined solely for the purposes of this example as having IC50 > 9 μM) and sunitinib sensitive (defined solely for the purposes of this example as having an IC50 < 3 μM), were validated by qRT-PCR. Genes meeting those criteria were confirmed by RTPCR. Using qRT-PCR, it was revealed that FGFl (SEQ ID NO:4), HOXClO (SEQ ID NO:5), and LHFP (SEQ ID NO:6) were present in NSCLC lines and were resistant to sunitinib. Surprisingly, pathway analysis revealed that these 3 genes were not part of canonical pathways of resistance to sunitinib.

[0082] Sequences corresponding to SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 are provided below. [0083] FGFl agctgcagta gcctggaggt tcagagagcc gggctactct gagaagaaga caccaagtgg attctgcttc ccctgggaca gcactgagcg agtgtggaga gaggtacagc cctcggccta caagctcttt agtcttgaaa gcgccacaag cagcagctgc tgagccatgg ctgaagggga aatcaccacc ttcacagccc tgaccgagaa gtttaatctg cctccaggga attacaagaa gcccaaactc ctctactgta gcaacggggg ccacttcctg aggatccttc cggatggcac agtggatggg acaagggaca ggagcgacca gcacattcag ctgcagctca gtgcggaaag cgtgggggag gtgtatataa agagtaccga gactggccag tacttggcca tggacaccga cgggctttta tacggctcac agacaccaaa tgaggaatgt ttgttcctgg aaaggctgga ggagaaccat tacaacacct atatatccaa gaagcatgca gagaagaatt ggtttgttgg cctcaagaag aatgggagct gcaaacgcgg tcctcggact cactatggcc agaaagcaat cttgtttctc cccctgccag tctcttctga ttaaagagat ctgttctggg tgttgaccac

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LAI-3OQ2528vl tccagagaag tttcgagggg tcctcacctg gttgacccaa aaatgttccc ttgaccattg gctgcgctaa cccccagccc acagagcctg aatttgtaag caacttgctt ctaaatgccc agttcacttc tttgcagagc cttttacccc tgcacagttt agaacagagg gaccaaattg cttctaggag tcaactggct ggccagtctg ggtctgggtt tggatctcca attgcctctt gcaggctgag tccctccatg caaaagtggg gctaaatgaa gtgtgttaag gggtcggcta agtgggacat tagtaactgc acactatttc cctctactga gtaaacccta tctgtgattc ccccaaacat ctggcatggc tcccttttgt ccttcctgtg ccctgcaaat attagcaaag aagcttcatg ccaggttagg aaggcagcat tccatgacca gaaacaggga caaagaaatc cccccttcag aacagaggca tttaaaatgg aaaagagaga ttggattttg gtgggtaact tagaaggatg gcatctccat gtagaataaa tgaagaaagg gaggcccagc cgcaggaagg cagaataaat ccttgggagt cattaccacg ccttgacctt cccaaggtta ctcagcagca gagagccctg ggtgacttca ggtggagagc actagaagtg gtttcctgat aacaagcaag gatatcagag ctgggaaatt catgtggatc tggggactga gtgtgggagt gcagagaaag aaagggaaac tggctgaggg gataccataa aaagaggatg atttcagaag gagaaggaaa aagaaagtaa tgccacacat tgtgcttggc ccctggtaag cagaggcttt ggggtcctag cccagtgctt ctccaacact gaagtgcttg cagatcatct ggggacctgg tttgaatgga gattctgatt cagtgggttg ggggcagagt ttctgcagtt ccatcaggtc ccccccaggt gcaggtgctg acaatactgc tgccttaccc gccatacatt aaggagcagg gtcctggtcc taaagagtta ttcaaatgaa ggtggttcga cgccccgaac ctcacctgac ctcaactaac ccttaaaaat gcacacctca tgagtctacc tgagcattca ggcagcactg acaatagtta tgcctgtact aaggagcatg attttaagag gctttggccc aatgcctata aaatgcccat ttcgaagata tacaaaaaca tacttcaaaa atgttaaacc cttaccaaca gcttttccca ggagaccatt tgtattacca ttacttgtat aaatacactt cctgcttaaa cttgacccag gtggctagca aattagaaac accattcatc tctaacatat gatactgatg ccatgtaaag gcctttaata agtcattgaa atttactgtg agactgtatg ttttaattgc atttaaaaat atatagcttg aaagcagtta aactgattag tattcaggca ctgagaatga tagtaatagg atacaatgta taagctactc acttatctga tacttattta cctataaaat gagatttttg ttttccactg tgctattaca aattttcttt tgaaagtagg aactcttaag caatggtaat tgtgaataaa aattgatgag agtgttagct cctgtttcat atgaaattga agtaattgtt aactaaaaac aattccttag taactgaact gtcatattta gaatggaagg aaaatgacag tttgtgaaag ttcaaagcaa tagtgcaatt gaagaattga cctaagtaag ctgacattat ggttaataat agtattttag atttgtgcag caaaataatt tcataacttt tttgtttttg ttacttggat aagatcaatc tgttttattt tagtaaatct ttgcaggcaa gttagagaaa atgcagtgtg gcttaacgtc tctttagtat gaagatttgg ccagaaaaag atacccagag aggaaatcta agataattat aatggtccat actttttatt gtatgaatca aactcaagca taacattggc caaggaaaat taaataccat tgctaacttg tgaaatggaa gtctgtgatt tcggagatgc aaagcattgt agtaaaaaca ccaatgtgac ctcgaccatc tcagcccaga tatcattcat atatctgttc aatgactatt aaggtgccta ctgtgtgcta ggcactgtac

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LAI-3O02528vl tggatactgg ggaccttgtc tgtctggttt gctgctgtat cttctcccag ggcattatat ttatgatgaa agatgctgtg gattcaattc tttcagtcaa gaataaacac agactttgta ggttcctgct gaataaagca aatcccagaa acccagattt tggaagaatc agcaacccca gcataaaata aacccctatc aaaatgtcag aggacatggc aaggtaaact tagcattttc aactttagaa ccgggtcagc ttcaggggga ctgctttcaa atcagccaaa gagcctgtca gatcttctta gaaggaagag gttggtagtt ccctgctctg ttttgaacat gctctagttt attaacctgg ggacattccc attgctgtct taagtaagtc tcatagccag ctcctgtcac gtgactctca tatggattca ttttcgggcc agctctgaac aaagcatcat gaacatatgt gcttttggtc gtttgcaatg tgatggtggt ggaggtaggt attggtttcc ttggaaggca tgataagaaa gattcacaat ggccaacagt gtgtatgaac aaaaaactga ttggagcatc agctagtact gaaggtcctt gctttgtgtc agaggcaaag gaacccaagg cgccaagtcc tcagccttga gtgtactgct gacaactaaa ctcacaggct gcaaagcaga cctctgatga agatgcctgt tatttcacat cactgtcttt ttgtgtatca tagtctgcac cttacaaata ttaataaatg ttccaataat aggtgaaaaa aaaaa (SEQ ID NO: 4)

[0084] HOXClO cctcccctcc aaccgcgccc cccctcccgg atggggaaaa aaaaagatgt cagctcctcc gctgtagtat tgctccttaa aaacccctct ctctgaaaat gacatgccct cgcaatgtaa ctccgaactc gtacgcggag cccttggctg cgcccggcgg aggagagcgc tatagccgga gcgcaggcat gtatatgcag tctgggagtg acttcaattg cggggtgatg aggggctgcg ggctcgcgcc ctcgctctcc aagagggacg agggcagcag ccccagcctc gccctcaaca cctatccgtc ctacctctcg cagctggact cctggggcga ccccaaagcc gcctatcgcc tggaacaacc tgttggcagg ccgctgtcct cctgctccta cccacctagt gtcaaggagg agaatgtctg ctgcatgtac agcgcagaga agcgggcgaa aagtggcccc gaggcagctc tctactccca ccccttgccg gagtcctgcc ttggggagca cgaggtaccc gtgcccagct actaccgcgc cagcccgagc tactccgcgc tggacaagac gccccactgt tctggggcca acgacttcga agcccctttc gagcagcggg ccagtctcaa cccgcgcgcc gaacatctgg aatcgcctca gctggggggc aaagtgagtt tccctgagac ccccaagtcc gacagccaga cccccagccc caatgaaatc aagacggagc agagcctggc gggccctaaa gggagcccct cggagagcga aaaggagagg gccaaagctg ccgactccag cccagacacc tcggataacg aagcgaaaga ggagataaag gcagaaaaca ccacaggaaa ttggctgaca gcaaagagcg gaaggaagaa gaggtgcccc tatactaaac accagacgct ggaattggag aaagaatttc tgttcaatat gtatttgacg cgagagcgcc gcctggagat tagcaagacc attaacctta cagacagaca agtcaaaatc tggtttcaaa atcgcagaat gaaactcaag aaaatgaacc gagagaatcg gatccgggaa ctgacctcca attttaattt cacctgagag cgcggcctct cctcctccct tcccgctcct tcctctcccc gcccctcctc cctttgtgcc tggtgatata tttttttttc ctccctgagt ataaatgcaa tgcgactgca aaaaaggcaa agacctcaga ctctccttcc aagggacctg tggttcgtgc tgcgaagatg cttccactta aagcatgaga

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LAI-3O02528vl aatggggtgc cgggatgtgg ggtgtggtgt gtgccctcat agatgggggt gggagtgtgg ctggtgtgtg tgtcaagccc tcactcaccc acgcactcac acacagcatt ctgttctcca tgcaaagtta agatcgaatc catccgcttg taggggaaaa aaaggaaaaa aattaaccag agagggtctg taatctcgca gagcacaggc agaatcgttc cttccttgct gcatttcctc cttagactaa tagacgtttt ggaaagttcg gctagtgttc gtgtgtttgt cgtagcaccc agagcctcca ccaaaccctc tccatgtctt tacctcccag tcgctctaag aatctgcttg aagtctcgta tttgtactgc tttctgcttt tctcccaccc ctcctagcac ccccacatcc cccatctagt aacatctcag aaatttcatc cagaggaaca aaaaaattaa aaatagaaca tagcaaagca aagacagaat gccccccccc ccaaatattg tcctgtccct gtctgggagt tgtgttattt aaagatattc tgtatgttgt atcttttgca tgtagcttcc ttaatggaga aaaaaaaacc taataaattt ccagaatcat aatcctcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa (SEQ ID NO: 5)

[0085] LHFP aggaggcggt gcgtgcctcg cctgccaaag ggagatccgc tcctctgcgt gcgatccccg gcgcccgcgc gcgcccacag cgctccgcca gagctgccgc cgcggactcg ccgggagtgg gggtctccgc tggtgccagc ccgcttctgg agaccctccg cctcctgcca acccctgctc t^'tccaggtcg ggccccgggg ttctgcggct gttagggaca gaggcaaaga agggcaggac ggtccggttt cccgtggatg ttcccgcccg agaaagacag caagttgtgt gtgcgcccgg gacgcgggag ggaaggtagc cgccgcccgc cagccatgga ccatcatctt tagtgcagag gatggaaagt tgatgcccag taagactgaa gatccattct gcattacgga actgtggatt atctgtgggt ccctggtgat ttcacacctt cattcactcc tgcagtccct gaacacttac ttggggtcct cattgcccta tctggtgaaa gatggcatcc agcctgactt gtactggagt aatctgggct ttgctgtctt ttctttgtgc tgccacctcc tgcgtggggt tctttatgcc ttactggctc tggggatcac agctgggcaa gcctgtgtcc ttcggtacct tccggaggtg ctcatatcct gtgcatgatg agagtcggca gatgatggtg atggtggagg aatgtgggcg ctatgcctcc ttccagggca tccccagcgc agaatggagg atctgcacca tagtgaccgg cctgggttgt ggcctcctcc tcctggtggc gctcactgcc ctcatgggtt gctgtgtttc cgacctcatc tccaggacag tgggaagagt ggctggagga attcagtttc ttgggggctt gttgattggt gctggctgtg ccctctaccc cttgggctgg gacagtgagg aagtccggca gacttgtggc tacacttctg gccagtttga cctggggaag tgtgaaatcg gctgggccta ctactgcacg ggagcaggtg ccactgccgc catgctgctg tgcacgtggc tggcttgctt ttcgggcaag aaacagaagc actacccata ctgagatgga gctaccaaga gcagacagag gagaagatgg gccaaagggg cttggagagg tcaaaacatc cacctacctt caaaaggtgg gatagtagtt ctaatccaat acaatgctaa taaaatgaaa cccgataaaa tcaggaacat gatataggaa ggaaggattg taggagattt gtgggggaaa aaaaaggaga gtatagaatg atggagaaaa atggaccaaa ggctaaaaat attgcagggc atcgggtgtt tctattccac agagtattgt taatgtacaa cacacacaca cacacacaca cacacacaca cacacacaca

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LAI-3OO2528vl cacacaacaa atctacatat acaaacaagg gtttgggttt tagttttttt tttttaaggt gaggactcag aaaatcaaag ggctagtaga aacagtgtta tgttgggaag cagggtaccc ccaaagatgt tccctgtagg tcacggcact cccaaaagca cacaagcaca tacagacata tgcatcccca cacacgccta tgcacaaacg tggattatcg cacagactgg gaggtttagt ggtgcatttc tcctctgttt tctttttaat atacatttaa aatacagtat tatcact tta taaaacatac attaagccta ataaatggac caataagcca aactatcagt attttgtata tcctgcataa actctaattt agttcctcaa catattttca gtgtttatgc agacctttag agttaagcct ttgtatttcc atgttattcc acaatatgca atatttctct gagtagcttc tgctatgata ttcttatgaa gaaaaggggc aactttctgt ccactatagg agagaattca ccattattgt actgtgctgt accacattta tttctatatt cattttgtaa aaaatttaaa agtgctattt tgtttgtatt tgaaaatctc tgtgaataaa ttctctcttt gatcaataaa aaaaaaaaaa aaaaaaaaaa ( SEQ ID NO : 6)

[0086] All publications and patent applications cited in this application are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications made be made thereto without departing from the spirit or scope of the appended claims.

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LAI-3002528vl

Claims

WHAT IS CLAIMED:

1. A method of evaluating sensitivity of a cancer cell to a tyrosine kinase inhibitor comprising: assessing the expression of SEQ ID NO. 1 in a sample comprising a gene product; and correlating reduced expression of SEQ ID NO. 1 with sensitivity to the tyrosine kinase inhibitor.

2. The method of claim 1 wherein the sample comprises a tumor biopsy.

3. The method of claim 1 wherein the sample comprises blood.

4. The method of any one of claims 1 to 3 wherein the gene product comprises mRNA.

5. The method of claim 4 wherein assessing the expression of SEQ ID NO. 1 comprises using RTPCR.

6. The method of claim 4 wherein assessing the expression of SEQ ID NO. 1 comprises using a microarray.

7. The method of claim 4 wherein assessing the expression of SEQ ID NO. 1 comprises using Northern Blot.

8. The method of claim 1 wherein the tyrosine kinase inhibitor inhibits VEGFR2 (SEQ ID NO. 2).

9. The method of claim 1 wherein the tyrosine kinase inhibitor inhibits VEGFR3 (SEQ ID NO. 3).

10. The method of claim 1 wherein the tyrosine kinase inhibitor is sunitinib.

1 1. The method of claim 1 wherein the tyrosine kinase inhibitor is sorafenib.

12. The method of claim 1 wherein the cancer cell is a non-small cell lung cancer cell.

13. A method of slowing the expansion of a cancer cell comprising: assessing the expression of SEQ ID NO. 1 in a sample; correlating reduced expression of SEQ ID NO. 1 with sensitivity to a tyrosine kinase inhibitor; and administering an effective amount of the tyrosine kinase inhibitor.

14. The method of claim 13 wherein the sample is a human sample.

15. The method of claim 14 wherein the sample comprises a blood fraction.

16. The method of claim 14 wherein the sample is a tumor biopsy.

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LAI-30Q2528vl

17. The method of claim 13 wherein the cancer cell comprises a loss of heterozygosity in chromosomal region 17p.

18. The method of any one of claims 13 to 17 wherein the cancer cell is a non-small cell lung cancer cell.

19. The method of claim 18, wherein the tyrosine kinase inhibitor is sunitinib

20. A kit that facilitates the assessment of the expression of miR-497 comprising a reagent capable of specifically recognizing miR-497 or a product thereof.

21. The kit of claim 20 wherein the reagent comprises an oligonucleotide

22. The kit of claim 20 wherein the reagent comprises an antisense nucleic acid.

23. The kit of claim 20 wherein the reagent is bound to a solid support.

24. The kit of any one of claims 20 to 23 further comprising a fluorescent label.

25. The kit of any one of claims 20 to 24 further comprising a reagent capable of specifically recognizing a second gene or a product thereof.

26. A method of evaluating the sensitivity of a cancer cell to a tyrosine kinase inhibitor comprising: assessing the expression of a target gene product selected from the group consisting of FGFl (SEQ ID NO:4), HOXClO (SEQ ID NO:5) and LHFP (SEQ ID NO:6) in a sample; and correlating positive expression of the target gene product with resistance to the tyrosine kinase inhibitor.

27. The method of claim 26 wherein the sample comprises a tumor biopsy.

28. The method of claim 26 wherein the sample comprises blood.

29. The method of claim 26 wherein the cancer cell is a non-small cell lung cancer cell.

30. The method of any one of claims 26 to 29 wherein the gene product comprises mRNA.

31. The method of claim 30 wherein assessing the expression of the gene product comprises using RTPCR.

32. The method of claim 30 wherein assessing the expression of the gene product comprises using a microarray.

33. The method of claim 30 wherein assessing the expression of the gene product comprises using Northern Blot.

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LAI-3OO2528vl

34. The method of any one of claims 26 to 29 wherein the gene product comprises protein.

35. The method of claim 34 wherein assessing the expression of the gene product comprises using a ligand capable of specifically recognizing the gene product.

36. The method of claim 35 wherein the ligand comprises an antibody.

37. The method of claim 36 wherein assessing the expression of the gene product comprises using immunohistochemical methods.

38. The method of claim 36 wherein assessing the expression of the gene product comprises using ELISA.

39. The method of claim 36 wherein assessing the expression of the gene product comprises using flow cytometry.

40. The method of any one of claims 26 to 29 wherein assessing the expression of the gene product comprises using mass spectrometry.

41. The method of any one of claims 26 to 40 wherein the cancer cell is a non-small cell lung cancer cell.

42. The method of claim 41 wherein the tyrosine kinase inhibitor is sunitinib.

43. A method of slowing the expansion of a cancer cell in a subject comprising: assessing the expression of a target selected from the group consisting of FGFl (SEQ ID

NO:4), HOXClO (SEQ ID NO:5) and LHFP (SEQ ID NO:6) in a sample; correlating reduced expression of SEQ ID NO. 1 with sensitivity to a tyrosine kinase inhibitor; and administering an effective amount of the tyrosine kinase inhibitor to the subject.

44. The method of claim 43 wherein the sample is a human sample.

45. The method of claim 44 wherein the sample comprises a blood fraction.

46. The method of claim 44 wherein the sample is a tumor biopsy.

47. The method of any one of claims 43 to 46 wherein the cancer cell is a non-small cell lung cancer cell.

48. The method of claim 47, wherein the tyrosine kinase inhibitor is sunitinib

49. A kit that facilitates the assessment of the expression of a target selected from the group consisting of FGFl (SEQ ID NO:4), HOXClO (SEQ ID NO:5) and LHFP (SEQ ID NO:6) comprising a reagent capable of specifically recognizing a target selected from the group

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LAI-3002528vl consisting of FGFl (SEQ ID NO:4), HOXClO (SEQ ID NO:5) and LHFP (SEQ ID NO:6) or a gene product thereof.

50. The kit of claim 49 wherein the reagent comprises an oligonucleotide.

51. The kit of claim 49 wherein the reagent comprises an antisense nucleic acid.

52. The kit of claim 49 wherein the reagent comprises an antibody.

53. The kit of claim 49 wherein the reagent is bound to a solid support.

54. The kit of any one of claims 49 to 53 further comprising a fluorescent label.

55. The kit of any one of claims 49 to 54 further comprising a reagent capable of specifically recognizing a second gene or a product thereof.

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LAI-3002528vl